Viral vectors and producing cells

Abstract

Provided is a viral vector having a lipid bilayer envelope, the lipid bilayer envelope comprising an antibody binding domain displayed outside the envelope, the antibody binding domain being cell type specific; a viral envelope protein exhibited outside the envelope, the viral envelope protein being capable of promoting infection of the same cell type; and a nucleic acid molecule comprising a promoter capable of being expressed in the same cell type. Methods of making the viral vectors and methods of modifying cells and treating diseases / conditions using the viral vectors are also provided.

Description

[0001] This invention relates to the production of viral particles and their use as vectors for genetic modification of specific cell types. The invention also relates to the provision of such viral particles and their use in treating diseases.

[0002] Lentivirals are a family of retroviruses that infect cells by inserting their DNA into the genome of their host cells. Many of these viruses have formed the basis for research into the use of viruses in gene therapy, but what makes lentiviruses special is their ability to infect non-dividing cells.

[0003] Lentiviral vectors have been used to insert, modify, or delete genes in organisms. In particular, lentiviruses have been used as vectors to transfer genes into target cells, including transducing CD4 in humans. + CD8 + and / or CD4 + Regulatory T cells. Lentiviral infection and replication in host cells involves multiple steps. In the first step, the virus attaches to the outer surface of the cell using its surface glycoproteins. For example, lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) target a wide range of cell types because VSVG is specific for the receptor (LDLR) expressed on the surface of a large number of different cells. After the virus targets and binds to the target cell, it injects viral material into the cytoplasm of the host cell. In the cytoplasm, viral reverse transcriptase reverse transcribes the viral RNA genome to produce a viral DNA genome. The viral DNA is then transported to the nucleus of the host cell, where it is integrated into the host cell's genome with the help of the viral integrase enzyme. At this point, the host cell's systems are used to transcribe the entire viral RNA and express structural viral proteins, particularly those that form the viral capsid and envelope. The lentiviral RNA and viral proteins then assemble, and when enough newly formed viral particles are formed, they leave the host cell by budding or “bubbling” from the outer membrane of the infected cell.

[0004] In vitro and in vivo methods for genetic modification using lentiviruses have been considered. In the in vitro method, cells are extracted from a patient and then cultured. A lentiviral vector carrying a therapeutic transgene is then introduced into the culture to infect the cells and thus modify their genome. The modified cells are cultured until they are ready for infusion into a patient. The cells used in the in vitro method can be autologous (i.e., cells taken from and reinfused into the same individual) or allogeneic (i.e., cells taken from one individual and reinfused into a different individual).

[0005] In contrast, in vivo gene therapy involves injecting a genetically modified viral vector (such as a lentivirus) into the patient and subsequently modifying the genome.

[0006] Cellular immunotherapy for cancer is considered a method that utilizes the immune system of cancer patients to induce their own immune system to attack cancer cells in the patient's body, thereby promoting cancer remission.

[0007] One known form of cellular immunotherapy is the creation of CAR T cells. Chimeric antigen receptors (CARs) (also known as chimeric immune receptors, chimeric T-cell receptors, or artificial T-cell receptors) are receptor proteins engineered to specifically bind to target antigens. Therefore, when CAR proteins are expressed in genetically modified T cells that express CARs, the T cells target specific target antigens. These receptors are chimeric because they combine antigen-binding and T-cell activation functions into a single receptor.

[0008] Therefore, CAR T-cell therapy uses T cells engineered to express CARs to treat cancer by defining the target as a specific antigen displayed by cancer cells. In this way, an individual's immune system can be endowed with the ability to effectively target and destroy cancer cells within the individual.

[0009] After CAR T cells are infused into a subject (or patient), they come into contact with their target antigens on the cell surface. The CAR T cells bind to the target, and the T cells are activated, triggering T cell proliferation and making them cytotoxic. CAR T cells damage cells through several mechanisms, including: widespread stimulation of cell proliferation, increased cytotoxicity to other living cells, and increased secretion of factors that can affect other cells, such as cytokines, interleukins, and growth factors.

[0010] CAR T cells can be autologous, meaning they originate from the patient's own T cells, or allogeneic, meaning they originate from another healthy donor's T cells. Once isolated, the T cells are genetically engineered to express a specific CAR, thus programming them to target antigens present on the surface of cancer cells (e.g., tumors). For safety reasons, CAR cells are typically engineered to be specific for antigens expressed by cancer cells but not on healthy cells.

[0011] Cell therapies (such as CAR T-cell therapy) inevitably require complex reagent supply chains and time-consuming and expensive processing to obtain drug products. In addition to the complexity and difficulty of manufacturing cell-based therapies, obtaining cells as the basic material for providing therapeutic products is also challenging. Autologous cells are used because of the lower risk of patient side effects, but not all patients can provide enough autologous cells (such as T cells) to provide a useful therapy. Using allogeneic cells (such as T cells) carries a greater risk of side effects.

[0012] Therefore, there is a need for cell-based therapies that are cost-effective and have simplified application procedures. There is also a need for cell-based therapies that can effectively address a wider range of cancer types.

[0013] This invention provides a viral vector having a lipid bilayer envelope, the lipid bilayer envelope comprising:

[0014] a) An antibody-binding domain is displayed on the outside of the envelope, which is cell type specific;

[0015] b) Viral envelope proteins displayed on the outer surface of the envelope, which promote infection of the same cell types as in (a); and

[0016] c) A nucleic acid molecule containing a promoter that can be expressed in the same cell type as in (a).

[0017] The antibody-binding domain can be derived from scFv. Preferably, the antibody-binding domain is derived from a single-domain antibody (sdAb, i.e., VHH or Nanobody). ® ).

[0018] A lipid bilayer (or phospholipid bilayer) is a thin, polar membrane composed of two layers of lipid molecules. These membranes are flat plates that form a continuous barrier around all cells. The cell membranes of almost all organisms and many viruses are composed of lipid bilayers. Therefore, the viral vectors described herein can be based on enveloped viruses.

[0019] Single-domain antibodies (sdAbs or VHHs) are polypeptide or antibody fragments composed of a single monomeric variable antibody domain. Like antibodies composed of related polypeptide chains, they selectively bind to specific antigens. With a molecular weight of only 12kDa to 15kDa, single-domain antibodies (typically 12kDa to 15kDa) are much smaller than common antibodies (150kDa to 160kDa) composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (approximately 50kDa, one light chain and half a heavy chain) and single-chain variable region fragments (approximately 25kDa, two variable domains, one from the light chain and one from the heavy chain).

[0020] The first batch of single-domain antibodies were engineered from heavy-chain antibodies discovered in camels; these single-domain antibodies are called V. H H (VHH) fragment. Cartilaginous fish also possess heavy chain antibodies (IgNAR, i.e., "immunoglobulin neoantigen receptor"), from which a substance called V can be obtained. NAR Single-domain antibodies from fragments. An alternative method for generating single-domain antibodies is to split the dimeric variable domain of a common immunoglobulin G (IgG) from humans or mice into monomers.

[0021] Nanobodies are preferred over other antigen conjugates (e.g., scFv) because they typically offer higher solubility, stability, and smaller size. The smaller size of nanobodies allows them to penetrate and attach to areas of the therapeutic target that are normally inaccessible. Furthermore, nanobodies are less likely to induce adverse allergic reactions in individuals.

[0022] The VHH and its binding domain can bind to a domain via a linker, which connects it to or anchors it within a lipid bilayer membrane. Therefore, the VHH can be a membrane-anchored VHH. Preferably, the anchoring domain is a transmembrane polypeptide domain. Most preferably, the VHH and the anchoring domain are separate domains of a single fusion protein.

[0023] Advantageously, the linker portion between the VHH and its binding domain, as well as the domain that connects it to or anchors it within the lipid bilayer envelope, can modulate transduction efficiency. In particular, VHH presentation using a PDGFR / CD8a hinge scaffold can produce even higher transduction efficiency. Furthermore, increased CD69, CD25, and / or PD1 expression, and / or increased cell expansion and / or functional viral titer (VHH) can be achieved. These advantages can be considered by comparing with the IgG4 linker of the same VHH. Without being bound by theory, this technical effect likely stems from the better physical flexibility provided by the linker, thereby improving or optimizing binding to the target and / or achieving an improved distance between the two cells, thus improving transduction.

[0024] This invention offers the advantage that the amount (proportion) of VHHs displayed on the outer membrane can be controlled relative to the amount (proportion) of viral envelope proteins also displayed on the outer membrane. This ability to control the amount or proportion of these two displayed portions means that their proportions and scaffolds can be optimized (e.g., in the case of targeting VHHs), resulting in higher transduction efficiency. Therefore, it is preferable that the antibody domains acting as the targeting portions and the envelope proteins are displayed separately by the viral vector; that is, fusion / conjugation of the targeting portions and envelope proteins should not occur. Similarly, it is preferable that the antibody domains acting as the targeting portions and the envelope proteins are expressed separately by the vector-generated cells; that is, fusion / conjugation of the targeting portions and envelope proteins should not occur. It is undesirable to be bound by theory, as envelope proteins undergo very specific conformational changes during viral entry. Therefore, fusion of VHHs with viral envelope proteins may disrupt the viral entry function of ENV proteins. Furthermore, separating VHHs and envelope proteins provides the ability to optimize the VHH scaffold without the limitations associated with preserving the viral entry function of ENV proteins.

[0025] Therefore, the viral vector of the present invention has the advantage of cell type specificity. In the context of the present invention, "cell type specificity" may mean a specific cell type or a group of related cell types, such as immune cells, lymphocytes, tumor-infiltrating lymphocytes (TILs), monocytes, macrophages, T cells, or NK cells.

[0026] The viral envelope protein can be selected from VSV-G single mutant, double mutant, triple mutant, or quadruple mutant. Options include isoleucine-glutamic acid replacement at position 331 (I331E), alanine-glutamic acid replacement at position 51 (A51E), or isoleucine-glutamic acid replacement at position 182 (I182E). Other options for viral envelope proteins include wild-type or mutant Puerto Almendras virus glycoprotein, wild-type or mutant LeDantec virus glycoprotein, wild-type or mutant baculovirus Gp64 glycoprotein, wild-type or single mutant, double mutant, and triple mutant Lassa virus glycoprotein, such as having a tyrosine-to-alanine substitution at position 150 (Y150A), a histidine-to-alanine substitution at position 141 (H141A), or a phenylalanine-to-alanine substitution at position 147 (F147A), wild-type or mutant influenza HA protein, wild-type or mutant avian metapneumovirus glycoprotein, wild-type or mutant Sunshine virus glycoprotein, or wild-type or mutant Newcastle disease virus glycoprotein.

[0027] The amino acid sequence of the unmutated VSV-G viral envelope protein is given below as SEQ ID NO: 202 (Indiana vesicular stomatitis virus (San Juan strain) glycoprotein). The amino acid sequence of the 16 amino acids of the unmutated / wild-type VSV-G viral envelope protein, lacking the N-terminal signal peptide, is given below as SEQ ID NO: 203. Unless otherwise stated, the VSV-G mutants presented herein are described with reference to the coordinates of SEQ ID NO: 203.

[0028] The VSV-G viral envelope protein can be mutated at I331. The VSV-G viral envelope protein can contain mutants I331E, I331Q, I331L, I331M, I331W, and I331R. Preferably, the VSV-G viral envelope protein contains mutant I331E.

[0029] The VSV-G viral envelope protein can contain the mutant A51E. The VSV-G viral envelope protein can contain the mutant A51P.

[0030] VSV-G viral envelope proteins can contain mutants H8F, H8L, H8S, H8V, and H8Y.

[0031] The VSV-G viral envelope protein may contain mutants N9A, N9G, N9H, N9Q, N9R, and N9T.

[0032] VSV-G viral envelope proteins may contain the mutant Q10V.

[0033] VSV-G viral envelope proteins may include the mutant K11A.

[0034] VSV-G viral envelope proteins may include the mutant V33Y.

[0035] VSV-G viral envelope proteins may include the mutant M45L.

[0036] The VSV-G viral envelope protein can contain the mutant K47G. The VSV-G viral envelope protein can contain the mutant K47T. The VSV-G viral envelope protein can contain the mutant K47W.

[0037] VSV-G viral envelope proteins may contain the mutant H49A.

[0038] The VSV-G viral envelope protein can contain the mutant S48L. The VSV-G viral envelope protein can contain the mutant S48R.

[0039] The VSV-G viral envelope protein can contain the mutant K50H. The VSV-G viral envelope protein can contain the mutant K50S.

[0040] The VSV-G viral envelope protein can contain the mutant I52R. The VSV-G viral envelope protein can contain the mutant I52T.

[0041] The VSV-G viral envelope protein may contain the mutant Q53A. The VSV-G viral envelope protein may contain the mutant Q53I.

[0042] VSV-G viral envelope proteins may include the mutant T328S.

[0043] The VSV-G viral envelope protein can contain mutants R329T, R329H, R329K, and R329V.

[0044] VSV-G viral envelope proteins may include the mutant Y330M.

[0045] VSV-G viral envelope proteins may include the mutant V333Y.

[0046] VSV-G viral envelope proteins can contain mutants T351D, T351R, and T351S.

[0047] VSV-G viral envelope proteins can contain mutants T352D, T352E, T352I, T352Q, and T352S.

[0048] The VSV-G viral envelope protein can contain the mutant E353K. The VSV-G viral envelope protein can contain the mutant E353S.

[0049] The VSV-G viral envelope protein can contain the mutant R354K. The VSV-G viral envelope protein can contain the mutant R354M.

[0050] The VSV-G viral envelope protein can contain the mutant E355L. The VSV-G viral envelope protein can contain the mutant E355R.

[0051] VSV-G viral envelope proteins may include the mutant S408L.

[0052] VSV-G viral envelope proteins may include the mutant I502V.

[0053] The VSV-G viral envelope protein can be mutated at I182. The VSV-G viral envelope protein can contain the mutant I182E. Amino acid position 182 is an interesting but favorable position for point mutation because the amino acids surrounding positions 181, 183, and 184 either have side chains far from the CR domain of the LDLR (181, 183) or are located outside the CR domain binding pocket (184), making them unsuitable for mutagenesis.

[0054] The VSV-G viral envelope protein can be mutated at the following positions: I52, I331, and E355. The VSV-G viral envelope protein can contain mutations I52R, I331E, and E355L.

[0055] The VSV-G viral envelope protein can mutate at the following positions: N9, K47, I331, and T352. The VSV-G viral envelope protein can contain the mutations N9G, K47G, I331E, and T352S.

[0056] The VSV-G viral envelope protein can be mutated at the following positions: H8, A51, R329, and T352. The VSV-G viral envelope protein can contain mutations H8F, A51P, R329V, and T352S.

[0057] The VSV-G viral envelope protein can be mutated at the following positions: N9, K47, T328, and E355. The VSV-G viral envelope protein can contain mutations N9Q, K47T, T328S, and E355L.

[0058] The VSV-G viral envelope protein can mutate at the following positions: N9, K50, V333, and T351. The VSV-G viral envelope protein can contain mutations such as N9G, K50S, V333Y, and T351R.

[0059] The VSV-G viral envelope protein can mutate at the following positions: H8, S48, and R329. The VSV-G viral envelope protein can contain mutations H8F, S48L, and R329K.

[0060] The VSV-G viral envelope protein can be mutated at positions H8 and K47. The VSV-G viral envelope protein can contain mutations H8Y and K47G.

[0061] The VSV-G viral envelope protein can mutate at the following positions: N9, S48, R329, and T352. The VSV-G viral envelope protein can contain mutations N9H, S48R, R329T, and T352E.

[0062] The VSV-G viral envelope protein can mutate at the following positions: H8, M45, I331, and E355. The VSV-G viral envelope protein can contain mutations H8L, M45L, I331E, and E355R.

[0063] The VSV-G viral envelope protein can be mutated at the following positions: Q53, I331, and T352. The VSV-G viral envelope protein can contain mutations Q53A, I331E, and T352I.

[0064] The VSV-G viral envelope protein can be mutated at the following positions: N9, I52, R329, and T352. The VSV-G viral envelope protein can contain mutations N9T, I52T, R329K, and T352E.

[0065] The VSV-G viral envelope protein can mutate at the following positions: H8, H49, R329, and T352. The VSV-G viral envelope protein can contain the mutations H8S, H49A, R329H, and T352Q.

[0066] The VSV-G viral envelope protein can be mutated at the following positions: K11, S408, I331, and R354. The VSV-G viral envelope protein can contain mutations such as K11A, S408L, I331L, and R354K.

[0067] The VSV-G viral envelope protein can be mutated at the following positions: N9, Q53, I331, and R354. The VSV-G viral envelope protein can contain mutations N9R, Q53I, I331Q, and R354M.

[0068] The VSV-G viral envelope protein can be mutated at the following positions: H8, I502, V33, and T351. The VSV-G viral envelope protein can contain mutations H8S, I502V, V33Y, and T351S.

[0069] The VSV-G viral envelope protein can be mutated at positions I331 and E353. The VSV-G viral envelope protein can contain mutations I331E and E353K.

[0070] The VSV-G viral envelope protein can be mutated at the following positions: Q10, K50, I331, and T352. The VSV-G viral envelope protein can contain the mutations Q10V, K50H, I331M, and T352D.

[0071] The VSV-G viral envelope protein can mutate at the following positions: N9, I331, and E353. The VSV-G viral envelope protein can contain mutations N9A, I331E, and E353S.

[0072] The VSV-G viral envelope protein can be mutated at the following positions: H8, K47, Y330, and T351. The VSV-G viral envelope protein can contain the mutations H8V, K47W, Y330M, and T351D.

[0073] The viral envelope protein could be a Lassa virus envelope protein. The viral envelope protein could be Lassa virus GP1+2. The viral envelope protein could be a Lassa virus GP with a tyrosine-to-alanine substitution for Y150A. The viral envelope protein could be a LDV (Ledantec virus glycoprotein).

[0074] Using antibody domains pseudotyped with the mutant Lassa virus glycoprotein Y150A (LVGP1x) to target lentiviral vectors advantageously enables engineered vectors to target specific cell types expressing the antibody domain. Similarly, H141A F147A or Y150A H141A F147A Lassa virus (LVGP3X) glycoproteins enable engineered vectors to specifically target cells expressing VHH targets.

[0075] The antibody domain or VHH can target (bind) CD8, CD3, or T cell receptor (TCR), CD14, or CCR2. Preferably, VHH targets (binds) TCR. Therefore, T cell-specific targeting is preferably against cells expressing TCR, CD3, CD4, or CD8, and most preferably, specific targeting is preferably against cells expressing TCR. Preferably, the antibody-binding domain or VHH binds to TCR. Preferably, the antibody domain or VHH targets (binds) CD3, and most preferably, the antibody domain or VHH targets CD3ε (CD3e).

[0076] The antibody domain or VHH can target (bind) CD14 or CCR2. Preferably, the VHH targets (binds) CD14. Therefore, specific targeting of myeloid cells is preferably against cells expressing CD14 or CCR2, and most preferably, specific targeting is preferably against cells expressing CD14. Preferably, the antibody-binding domain or VHH binds to CD14.

[0077] Using antibody domains pseudotyped with LDV (Ledantec virus glycoprotein) to target lentiviral vectors advantageously enables engineered vectors to specifically target cells expressing antibody domain targets. Preferably, the antibody domain or VHH targets (binds) CD8.

[0078] Conversely, targeting lentiviral vectors with antibody domains of the present invention, which exhibit membrane-anchored VHHs on the surface and are pseudotyped with wild-type vesicular stomatitis virus glycoprotein (VSVG), is insufficient to achieve cell-specific transduction. Therefore, targeting lentiviral vectors with antibody domains pseudotyped with Lassa virus glycoprotein or LDV glycoprotein has advantageous characteristics compared to targeting lentiviral vectors with antibody domains pseudotyped with vesicular stomatitis virus glycoprotein.

[0079] However, targeting lentiviral vectors with antibody domains pseudotyped with A51E, I182E, and I331E mutants of the VSVG protein advantageously enables engineered vectors to specifically target cells expressing antibody domain targets. Preferably, the antibody domain targeting lentiviral vectors are pseudotyped with the I331E mutant of the VSVG protein. It is undesirable to be bound by theory, as the broad targeting tropism of VSVG is eliminated. Preferably, the antibody domain or VHH targets (binds to) the TCR. Lentiviral vectors pseudotyped with Lassa virus envelope protein or mutant VSVG protein and exhibiting envelope-anchored targeting of the TCR VHH stably transduc primary T lymphocytes. Lentiviral vectors as described herein can be pseudotyped with Lassa virus envelope protein having the Y150A mutation.

[0080] As an example, Lassa virus is advantageous for use in the context of this invention because only a small fraction of the general population is seropositive for Lassa virus. Furthermore, in affected individuals, Lassa virus infection produces low titers (titer values) of neutralizing antibodies.

[0081] The viral envelope may contain cell-cell fusion proteins, preferably glycoproteins. In the context of this invention, cell-cell fusion proteins are considered to be proteins that promote intercellular membrane fusion, particularly those that promote plasma membrane fusion between different cells. Most preferably, the fusion protein is viral in origin, but it can also be non-viral in origin.

[0082] The promoter is preferably cell type specific. The promoter may be cell type specific for cells expressing one or more of the following phenotypes: TCRab, TCRgd, CD3, CD25, CD4, CD8, CD11, CD14, CD16, CCR1, CCR2, CXCR4, CD56, CD20, and / or CD64 [Fcγ receptor I (FcγRI)] (preferably CD3 or CD14, CD56, or CD20). The promoter may be cell type specific for cells expressing one or more of the following: CD3, CD25, CD4, CD8, CD14, CD16, CCR1, CCR2, CXCR4, CD56, CD20, and / or CD64 [Fcγ receptor I (FcγRI)], CD56, CD19, CD20 (preferably CD3, CD14, CD56, or CD20).

[0083] The promoter can be cell type specific for cells with the phenotypes of CD3, CD25, CD4, CD8, CD11, CD14, CD16, CCR1, CCR2, CXCR4, CD56, CD20 and / or CD64 [Fcγ receptor I (FcγRI)], CD56, CD19, CD20 (preferably CD3, CD14, CD56 or CD20).

[0084] The promoter can be cell type specific for cells expressing CD3, CD25, CD4, CD8, CD11, CD14, CD16, CCR1, CCR2, CXCR4, CD56, CD20 and / or CD64 [Fcγ receptor I (FcγRI)], CD56, CD19, CD20 (preferably CD3, CD11, CD14, CD56 or CD20).

[0085] The present invention also has the advantage that using a target cell-specific promoter (thus implying its inactivity in the cells producing the viral vector particles) means that the producing cells do not have to bear the transcriptional and translational burden of expressing the "payload" of the viral vector particles. For example, if the "payload" is a chimeric antigen receptor (CAR), the CAR is not expressed by the producing cells but by the target cells. This has the advantage that using a cell-specific promoter allows producing cells in which the cell-specific promoter is inactive to produce a larger titer of vector. By way of example only, it is advantageous that restricting the expression of surface-expressed proteins and / or protein complexes during the production of viral vector particles increases the number of transduced competent lentiviral particles per milliliter (ml). Preferably, this increase is five-fold or greater, ten-fold or greater. Most preferably, this increase is five-fold or greater.

[0086] Nucleic acid molecules may contain open reading frames that can be expressed from a promoter. A promoter is a region in the nucleotide sequence of a gene that initiates gene transcription. A promoter may be a synthetic promoter containing promoter and enhancer elements, preferably, the promoter and enhancer elements are generally not associated with each other.

[0087] In the context of this disclosure, an enhancer is a region of a nucleotide sequence that regulates promoter activity. Preferably, the presence of an enhancer and / or its interaction with the promoter makes the activity of the synthetic promoter specific to a particular cell type or cell type subpopulation. Open reading frames may encode single or multiple polypeptides. These polypeptides may include engineered receptors, chimeric antigen receptors (CARs), chimeric autoantibody receptors (CAARs), engineered T-cell receptors, engineered B-cell receptors, universal CAR receptors, cytokines, growth factors, suicide genes, antibody fragments, enzymes, metabolic factors, and switch receptors. In some embodiments, the viral genome may also encode regulatory RNA elements, including interfering RNA, small hairpin RNA, microRNAs, and / or microRNA binding sites.

[0088] Nucleic acid molecules can contain promoter and enhancer elements on the same nucleic acid molecule.

[0089] The addition of enhancer sequences brings the advantage of increased expression from synthetic promoters. Therefore, synthetic constructs can achieve and / or exceed expression levels obtained from the ubiquitous promoter EF1a. T cell-specific synthetic promoters may have limited expression in non-T cell lineages, preferably compared to expression from the ubiquitous promoter EF1a (human elongation factor 1α) or PGK (promoter from mammalian phosphoglycerate kinase).

[0090] Preferably, the promoter is expressed only in T cells.

[0091] Preferably, the promoter is expressed only in monocytes, macrophages, or tumor-associated myeloid cells.

[0092] Preferably, the promoter is expressed only in natural killer (NK) cells.

[0093] Preferably, the promoter is expressed only in B cells.

[0094] A synthetic promoter may contain one, two or more enhancer elements, preferably two enhancer elements.

[0095] The synthetic promoter may contain one or more cell-specific microRNA elements. Preferably, the microRNA elements may have a sequence selected from SEQ ID NO 128 or 129.

[0096] Promoter elements for specific expression in T cells may contain a sequence selected from any of SEQ ID NO 11 to 20 or 121 to 127. The promoter may have a sequence selected from any of SEQ ID NO 11 to 20 or 121 to 127.

[0097] Preferably, the viral vector encodes a payload controlled by a T-cell-specific promoter, which includes or has the sequence of any one of SEQ ID NO 12 to 15.

[0098] Preferably, the viral vector encodes a payload under the control of a promoter that contains or has the sequence of SEQ ID NO. 15.

[0099] Promoter elements for specific expression in myeloid cells may comprise sequences selected from any of SEQ ID NO 130 to 152 and SEQ ID NO 193. Promoters may have sequences selected from any of SEQ ID NO 130 to 152, 193 and SEQ ID NO 197.

[0100] Preferably, the viral vector encodes a payload under the control of a promoter that contains or has the sequence of SEQ ID NO. 138, 139, or 193.

[0101] Promoter elements for specific expression in tumor-associated myeloid cells may comprise sequences selected from any of SEQ ID NO 141 to 152. Promoters may have sequences selected from any of SEQ ID NO 141 to 152 and SEQ ID NO 193.

[0102] Preferably, the viral vector encodes a payload under the control of a promoter that contains or has the sequence of SEQ ID NO. 143, 146 or 152.

[0103] The viral vector can be a retroviral vector, preferably a lentiviral vector.

[0104] The bioavailability of viral vectors can be increased by reducing phagocytosis, prolonging the vector's half-life, and / or defending against serum components. The viral vector envelope can display protein domains that protect the vector or protein modifications that act as markers of the vector itself. Displayed protein domains and modifications may include albumin-binding domains from G418, albumin-binding domains from Zag, and / or hypersialylation of lentiviral glycoproteins.

[0105] The viral vector can display CD47 on its envelope surface. Preferably, the CD47 level displayed on the envelope surface of the viral vector is at least 1.2 times the CD47 level on the envelope of a vector manufactured using production cells that do not express CD47. Preferably, the CD47 level displayed on the envelope surface of the viral vector is at least 1.2 times the CD47 level on the envelope of a vector produced by cells that have not been modified with additional CD47 expression or high-level CD47 expression. Expression of CD47 on the envelope has the advantages of reducing vector phagocytosis, reducing off-target transduction, and increasing the amount of vector available for targeted transduction.

[0106] The viral vector optionally displays high levels of CD47, agonist CD3 scFv, 41BBL, and / or CD80 on its envelope surface. Optionally, it displays anti-4-1BB scFv / VHH, anti-CD40 scFv / VHH, anti-CD28 scFv / VHH, CD86, CD86 immunoglobulin variable region-like domain, anti-CD40 scFv / VHH, anti-OX40 scFv / VHH, anti-ICOS scFv / VHH, IL2, and / or IL7.

[0107] Viral vectors can be protected against complement system-mediated degradation or inactivation. The viral vector envelope can display complement system modulators or protein domains or modifications that prevent complement system recognition. Displayed complement system modulators may include C1 protease inhibitors, C3 or C5 convertase inhibitors, C3b or C4b inactivators, membrane attack complex interfering agents, C8 or C9C1-INH binders, factor H, decay accelerators, DAF (CD55), MCP (CD46), CR1 (CD35), CD59, CFHR5, CR2, C4BP, kaposica (KSHV ORF4), EMICE, MOPICE, VCP, HVS-CCPH, and / or SPICE. Displayed protein domains and modifications may include albumin-binding domains from G418, albumin-binding domains from Zag, and / or hypersialylation of lentiviral glycoproteins.

[0108] Preferably, the viral vector envelope is substantially free of MHC class I. Most preferably, the viral envelope is free of MHC class I.

[0109] The envelope of viral vectors that are essentially free of MHC class I has the advantage of reducing the immunogenicity of the vector and reducing complement-mediated inactivation.

[0110] The viral vector can display a T-cell activator on its envelope surface. The T-cell activator can be an agonist-binding agent of CD3 or TCR. Agonist-binding agents of CD3 or TCR have the advantage of inducing T-cell activation, expansion, and enhancing resting T-cell transduction. Particularly preferred is the VHH binding agent, TCRab VHH. Optionally, the T-cell activator can be combined with co-stimulatory ligand expression for further advantages. The co-stimulatory ligand can be OKT3, for example via scFv. The co-stimulatory ligand can be CD28, for example via scFV. The co-stimulatory ligand can be CD80. Preferably, the co-stimulatory ligand is 4B1-4.

[0111] The viral vector can display a binder or protein that induces T cell co-stimulation signals on its envelope surface. Preferably, the binder or protein induces co-stimulation via at least one of OX40 or 41BB, CD28, ICOS, HVEM, CD27, DR3, GITR, CD30, SLQM, CD2, 2B4, TIM1, TIM2, TNFRSF15, CD40L, or CD226. Binding agents or proteins that induce T cell co-stimulation have the advantages of inducing T cell growth, memory, and survival of activated T cells.

[0112] Antibody-binding domains can be specific to T cells.

[0113] The antibody-binding domain can be specific for CD3ε. VHH can contain the amino acid sequence of any one of SEQ ID NO. 1 to 4. Preferably, VHH contains the amino acid sequence of SEQ ID NO. 3.

[0114] The antibody-binding domain can be specific to either the CD8a or b subunit. VHH can contain the amino acid sequence of any one of SEQ ID NO. 5 to 8. Preferably, VHH contains the amino acid sequence of SEQ ID NO. 7.

[0115] The antibody-binding domain may have an amino acid sequence of any one of SEQ ID NO: 1 to 8, 9, 10, 29, 34, 41, 44 to 46, 48, 49 or 54 to 65.

[0116] The antibody-binding domain can be specific to the T-cell receptor (TCR), preferably specific to the TCR constant region. Advantageously, the viral vector described herein efficiently transduces T cells, preferably primary T cells, expressing endogenous levels of TCR. The antibody-binding domain specific to the T-cell receptor (TCR) may comprise the amino acid sequence of any one of SEQ ID NO. 9 to 10 or 54 to 65. The antibody-binding domain specific to the T-cell receptor (TCR) may be the VHH of SEQ ID NO. 9, 56, or 62. Preferably, the VHH of the T-cell receptor (TCR) has SEQ ID NO. 9.

[0117] Antibody-binding domains can be specific to monocytes or macrophages.

[0118] The antibody-binding domain can be specific to CD14, CD16, CCR1, CCR2, CXCR4 or CD64, preferably specific to CD14 or CCR2.

[0119] The antibody-binding domain may be specific for CD14 or CCR2, preferably specific for CD14. Advantageously, the viral vector described herein efficiently transduces monocytes / macrophages expressing endogenous levels of CD14 or CCR2, preferably primary monocytes / macrophages. The antibody-binding domain specific for CD14 or CCR2 may comprise the amino acid sequence of any one of SEQ ID NOs 66 to 120. The antibody-binding domain specific for CD14 may be the VHH of SEQ ID NOs 70, 72, 76, 107, or 111. Preferably, the VHH for CD14 comprises SEQ ID NO. 76.

[0120] Antibody-binding domains can be specific to natural killer (NK) cells.

[0121] The antibody-binding domain can be specific to CD56, NKp46, or NKG2C.

[0122] Antibody-binding domains can be specific to B cells.

[0123] The antibody-binding domain can be specific for CD19, MS4A1, FCRL1, or CNR2.

[0124] Antibody-binding domains can be specific to dendritic cells.

[0125] The antibody-binding domain can be specific for CD19, MS4A1, FCRL1, or CNR2.

[0126] In all cases, the antibody-binding domain is preferably in the form of a VHH or VHH-binding domain.

[0127] The present invention also provides a viral vector for therapeutic use according to any of the preceding claims.

[0128] In one particular embodiment, the viral vector described herein comprises a VHH-binding domain specific to CD8 or TCRab and a VHH / scFvir ligand-binding domain specific to CD3, CD28, CD86, 41BB, CD40, OX40, IL2, or IL7 receptors, which are displayed on the exterior of the envelope. Using this viral vector has produced advantageous characteristics for transducing resting T cells.

[0129] Embodiments of the present invention have the advantage that the viral vector of the present invention is particularly suitable for in vivo administration. In vivo administration of the viral vector allows for the genetic modification of target cells to express the nucleic acid sequence encoded therein. Preferably, this is for the expression of a specific protein, such as a chimeric antigen receptor (CAR).

[0130] Suitablely, the viral vector of the present invention can be administered via systemic or local injection, after which the vector delivers the "payload" gene contained within the vector to target cells in an individual or patient. In this way, the target cells are genetically modified in vivo rather than in vitro.

[0131] In vivo administration of viral vectors offers several advantages: it eliminates the need to collect or donate target cells from a patient or another individual for in vitro modification. This significantly simplifies the production of viable modified cells in cell therapy, as well as the generation and administration of pharmaceutically effective substances. When in vitro processing and re-administration are required, the following steps are eliminated: cell collection (abortion), cell processing (e.g., centrifugation and cryopreservation), transportation, propagation, manufacturing, product packaging and cryopreservation, distribution to treatment sites, processing and preparation for infusion to individuals, and further administration or re-administration of cells to individuals.

[0132] The viral vector of the present invention can be used for in vitro modification of autologous or allogeneic target cells, which can be administered to individuals or patients. Using this viral vector produces advantageous characteristics, for example in T cells: it can transduce T cell subsets while reducing the likelihood of transduction of transformed cells from circulating peripheral blood cells collected from patients. The T cell subsets can be resting T cell populations.

[0133] The present invention also provides a viral vector for diagnosing, preventing, or treating a disease. Preferably, the disease is cancer.

[0134] Cancer can be leukemia. Cancer can be stomach cancer. Cancer can be lung cancer. Cancer can be colorectal cancer. Cancer can be breast cancer.

[0135] Cancer can be multiple myeloma.

[0136] This invention also provides a viral vector for use in therapies. Therapies may include cancer therapies. Optionally, the therapy is used for autoimmune diseases. Cancer therapies may be used for cancers selected from: bladder cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0137] Cancers can be selected from acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adrenocortical carcinoma; astrocytoma, childhood (brain cancer); atypical teratoma-like / rhabdoid tumor, childhood, central nervous system (brain cancer); basal cell carcinoma of the skin - see skin cancer; bile duct cancer; cholangiocarcinoma; bladder cancer; bone cancer (Ewing sarcoma and osteosarcoma and malignant fibrous histiocytoma); brain tumor; breast cancer; bronchial tumor (lung cancer); Burkitt lymphoma; central nervous system cancer (atypical teratoma-like / rhabdoid tumor, childhood) Stage (brain cancer); Cervical cancer; Extracranial germ cell tumors in children; Hemangioma (soft tissue sarcoma) in children; Bile duct cancer; Chordoma (bone cancer); Chronic lymphocytic leukemia (CLL); Chronic myeloid leukemia (CML); Chronic myeloproliferative neoplasms; Colorectal cancer; Craniopharyngioma (brain cancer); Cutaneous T-cell lymphoma (mycosis fungoides and Cezari syndrome); Ductal carcinoma in situ (DCIS); Endometrial cancer (uterine cancer); Ependymoma, childhood (brain cancer); Olfactory neuroblastoma (head and neck cancer); Ewing sarcoma (bone cancer); Extracranial tumors Germ cell tumors; extragonadal germ cell tumors; ocular cancer (intraocular melanoma; fallopian tube cancer; gallbladder cancer; gastric cancer; gastrointestinal neuroendocrine tumors; gastrointestinal stromal tumors (GIST) (soft tissue sarcoma); germ cell tumors (childhood central nervous system germ cell tumors (brain cancer); gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; cardiac tumors, childhood; hepatocellular carcinoma; Hodgkin's lymphoma; hypopharyngeal cancer (head and neck cancer); intraocular melanoma (eye); islet cell tumors, pancreatic neuroendocrine tumors; Kaposi's precipitate. Western sarcoma (soft tissue sarcoma); renal cell carcinoma; Langerhans cell histiocytosis; laryngeal cancer (head and neck cancer); leukemia; lip and oral cavity cancer (head and neck cancer); liver cancer; lung cancer (non-small cell, small cell, pleural pulmonary blastoma, myofibroblastic pulmonary tumor, and tracheobronchial tumor); lymphoma; medulloblastoma and other CNS embryonal tumors, childhood (brain cancer); melanoma; Merkel cell carcinoma (skin cancer); mesothelioma; metastatic squamous cell carcinoma of the neck (head and neck cancer); midline carcinoma; mouth cancer. Cancer (head and neck cancer); Multiple endocrine adenoma syndrome; Multiple myeloma / plasma cell tumor; Mycosis fungoides (lymphoma); Myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm; Chronic myeloid leukemia (CML); Acute myeloid leukemia (AML); Chronic myeloproliferative neoplasm; Nasal cavity and paranasal sinus cancer (head and neck cancer); Nasopharyngeal carcinoma (head and neck cancer); Neuroblastoma; Neuroendocrine tumors (gastrointestinal); Non-Hodgkin lymphoma; Non-small cell lung cancer; Esophageal cancer; Oral cancer, lip and oral cavity cancer, and oropharyngeal cancer (head and neck cancer); Oropharyngeal cancer; Osteosarcoma (bone cancer); Osteosarcoma and undifferentiated pleomorphic sarcoma of bone; Ovarian cancer; Ovarian germ cell tumors; Pancreatic cancer; Pancreatic neuroendocrine tumors (islet cell tumors); Papilloma (pediatric larynx)Paraganglioma; Sinus and nasal cavity carcinoma (head and neck cancer); Parathyroid carcinoma; Penile cancer; Pharyngeal cancer (head and neck cancer); Pheochromocytoma; Pituitary adenoma; Plasma cell tumor / multiple myeloma; Pleuroblastoma (lung cancer); Pregnancy and breast cancer; Primary central nervous system (CNS) lymphoma; Primary peritoneal cancer; Prostate cancer; Myofibroblastoma (lung cancer); Rectal cancer; Renal cell (kidney) carcinoma; Retinoblastoma; Rhabdomyosarcoma, childhood (soft tissue sarcoma); Salivary gland cancer (head and neck cancer); Sarcoma (childhood rhabdomyosarcoma (soft tissue sarcoma); Cezari syndrome ( Lymphoma); skin cancer; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma of the skin; occult primary metastatic squamous neck cancer (head and neck cancer); gastric (stomach) cancer; cutaneous T-cell lymphoma (mycosis fungoides and Cezari syndrome); testicular cancer; laryngeal cancer (head and neck cancer); thymoma and thymic carcinoma; thyroid cancer; tracheobronchial tumors (lung cancer); transitional cell carcinoma of the renal pelvis and ureter (renal (renal cell) carcinoma); transitional cell carcinoma of the ureter and renal pelvis (renal (renal cell) carcinoma); uterine cancer; uterine sarcoma; vaginal cancer; hemangioma (soft tissue sarcoma); vulvar cancer; and Wilms' tumor.

[0138] The therapy can be used for inflammatory diseases, including but not limited to autoimmune diseases, alopecia areata, pemphigus, scleroderma, Sjögren's syndrome, and / or vitiligo. Most preferably, autoimmune diseases include lupus, arthritis diseases such as rheumatoid arthritis (RA), osteoarthritis, gouty arthritis, spondylitis, and reactive arthritis; Behçet's syndrome; sepsis; septic shock; endotoxin shock; Gram-negative sepsis; Gram-positive sepsis; toxic shock syndrome; multi-organ injury syndrome secondary to sepsis (septicemia), trauma, or hemorrhage (hemoptysis); ophthalmic conditions, including but not limited to allergic conjunctivitis, vernal conjunctivitis, uveitis, and thyroid-associated ophthalmopathy; eosinophilic granuloma; and pulmonary or respiratory conditions, including but not limited to... Asthma, chronic bronchitis, allergic rhinitis, adult respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), chronic inflammatory lung diseases (e.g., chronic obstructive pulmonary disease), silicosis, pulmonary sarcoidosis, pleurisy, alveolitis, vasculitis, pneumonia, bronchiectasis, hereditary emphysema, and pulmonary oxygen toxicity; ischemia-reperfusion injury, such as ischemia-reperfusion injury of the myocardium, brain, or limbs; fibrosis, including but not limited to cystic fibrosis; keloid formation or scar tissue formation; atherosclerosis; autoimmune diseases, including but not limited to systemic lupus erythematosus (SLE). E) Lupus nephritis, autoimmune thyroiditis, multiple sclerosis, certain forms of diabetes and Raynaud's syndrome; tissue or organ transplant rejection, including but not limited to graft-versus-host disease (GvHD) and allogeneic transplant rejection; chronic or acute glomerulonephritis; inflammatory bowel disease, including but not limited to Crohn's disease, ulcerative colitis and necrotizing enterocolitis; inflammatory dermatitis, including but not limited to contact dermatitis, atopic dermatitis, psoriasis and urticaria; fever and myalgia due to infection; inflammatory conditions of the central or peripheral nervous system, including but not limited to meningitis (e.g., acute meningitis). Purulent meningitis, encephalitis, and brain or spinal cord injury due to minor trauma; Sjögren's syndrome; diseases involving leukocyte exudation; alcoholic hepatitis; bacterial pneumonia; community-acquired pneumonia (CAP); Pneumocystis carinii pneumonia (PCP); antigen-antibody complex-mediated diseases; hypovolemic shock; type I diabetes; acute and delayed-type hypersensitivity reactions; disease states due to leukocyte cachexia and metastasis; thermal injury; granulocyte infusion-related syndrome; cytokine-induced toxicity; stroke; pancreatitis; myocardial infarction; respiratory syncytial virus (RSV) infection; and spinal cord injury.

[0139] Chimeric antigen receptors (CARs) for targeting epitopes displayed by cancer cells of one, more, or any of the aforementioned cancers may be used in the context of this invention. A CAR may be a B-cell maturation antigen (BCMA) CAR. A CAR may be a CD19 CAR. A CAR may be a sealing protein 18.2 CAR; optionally, the sequence encoding the sealing protein 18.2 CAR is derived from SEQ ID NO: 53. A CAR may be a HER2 CAR. A CAR may be a mesothelin CAR.

[0140] Engineered TCRs for targeting epitopes displayed by cancer cells of one, more, or any of the aforementioned cancers are applicable within the context of this invention. The TCR may be specific to cancer-testis antigens. Cancer-testis antigens may be NY-ESO, MAGEA4, or PRAME. The TCR may be specific to viral antigens. Viral antigens may be derived from HPV. The TCR may be specific to WT1.

[0141] Engineered TCRs can be "domain-exchanged" TCRs. Exogenous human TCR αβ transduced in human CD3+ T lymphocytes may mismatch with endogenous TCR αβ chains, potentially leading to the mismatched TCR recognizing self-antigens and causing toxicity. To reduce this risk of mismatch between exogenous and endogenous TCR chains, regions of the constant domain (Calpha; Cα) of the α chain and regions of the constant domain (Cbeta; Cβ) of the β chain are "exchanged" between the two chains. Using this "domain-exchanged" approach, the α receptor consists of an α variable region (Vα) and a Cβ domain (denoted as VaCb), while the β receptor consists of a β variable region (Vβ) and a Cα domain (denoted as VbCa). The two domain-exchanged receptors can still form heterodimers together, but no longer pair with any wild-type TCR α or β chains, thus preventing TCR chain mismatch.

[0142] The present invention also provides a method for preparing a viral vector, the method comprising the following steps:

[0143] (a) Engineering cells to express cell type-specific antibody-binding domains (preferably membrane-anchored).

[0144] (b) The cell was engineered to express a viral envelope protein that promotes infection of the same cell type as in (a);

[0145] (c) Replication of a nucleic acid vector in a cell, the nucleic acid vector containing a promoter that can be expressed in the same cell type as in (a); and

[0146] (d) Harvest viral vectors in vesicle form released from cells.

[0147] The present invention also provides a cell modified to produce viral vector particles as disclosed herein. In the context of this invention, a cell modified to produce viral vector particles as disclosed herein is referred to as a "production cell".

[0148] Vesicles are structures composed of liquid or cytoplasm surrounded by a lipid bilayer. Vesicles released from a cell can be called vesicles. In cell biology, a vesicle is a protrusion of the cytoplasmic membrane, characterized by a spherical, "bubble-like," or swollen shape. Bubbling or vesicle formation is the process by which enveloped viral particles are released from the infected cells that produced them.

[0149] Nucleic acid vectors can be plasmids.

[0150] This method may include the step of engineering production cells to knock down the expression of MHC class I. Preferably, the method includes the step of engineering production cells to eliminate the expression of MHC class I.

[0151] Most preferably, the method includes a step of modifying MHC class I expression by introducing changes in the nuclear genome sequence. Therefore, the viral vector envelope can be engineered to be free of MHC class I.

[0152] The absence of MHC class I on the cell membrane produces a vector without MHC class I on its surface, which in turn reduces the immunogenicity of the vector and decreases complement-mediated inactivation.

[0153] Protein editing tools in the form of nucleases, base editors, and prime editors can be packaged in vectors to mediate the introduction of nuclear genome sequence changes.

[0154] In the current context, “mediation” is defined as all factors that enable the first stage or substrate production of a physiological pathway or process.

[0155] The endonuclease can be an RNA-directed DNA-specific endonuclease. Preferably, the endonuclease is a CRISPR-Cas ribonucleoprotein complex.

[0156] Endonucleases can be TALENs. Zinc finger endonucleases are also an option.

[0157] The production cell line can be the 293T cell line or a cell line derived from it.

[0158] Production cells can be engineered to express or overexpress cell surface or intracellular proteins. In some embodiments, this protein is CD47. Advantageously, expression of CD47 on the membrane reduces phagocytosis of vectors produced by production cells, reduces off-target transduction, and increases the amount of vector available for targeted transduction.

[0159] In some embodiments, the producing cells can express a protein destined to be packaged into budding viral particles. In some embodiments, this protein can be Vpx. Advantageously, delivery of Vpx to transduced cells in vivo can enhance the efficiency of target cell reprogramming through Vpx-mediated inhibition of cellular antiviral defenses. In another embodiment, the protein can be a gene-editing protein, including Cas9, Cas12, Cpf1, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), base editors, or leader editors. Gene-editing proteins can be modified to enhance packaging within viral particles. Advantageously, delivery of gene-editing proteins using the vectors described herein provides a mechanism for cell-specific gene editing in vivo.

[0160] In any case, it should be understood that production cells can be modified in a suitable manner to allow the production of viral vectors according to the invention, as outlined above and below.

[0161] The present invention also provides a method for modifying cells, the method comprising the step of exposing cells to a viral vector as described herein.

[0162] The cells to be modified can display antigens on their exterior, which can be bound by antibody-binding domains (preferably VHH binding domains) displayed on the envelope of the viral vector.

[0163] Cell modification is preferably performed in vivo. Advantageously, this invention achieves this objective through a combination of viral vector features that produce highly specific and effective reagents.

[0164] In some embodiments, vectors targeting different cell types can be administered together or sequentially for short periods to achieve in vivo reprogramming of different cell types. Different cell-specific vectors can carry the same cargo or transgene, or each can carry different cargo or transgene. Advantageously, this provides a method for reprogramming different cell types to possess potentially synergistic and non-redundant disease-fighting mechanisms for more effective disease treatment. The cells to be modified can be T cells. Modified T cells are used in various therapies, such as CAR-T cell therapy; however, the ability to successfully modify T cells in vivo has been limited to date, making ex vivo modification of T cells a preferred option. Therefore, the present invention overcomes these limitations, meaning that an efficient method for generating modified T cells in vivo is provided.

[0165] The cells to be modified are preferably immune effector cells.

[0166] The cells to be modified can be T cells.

[0167] The antigen is preferably an antigen that is highly conserved in T cells and expressed on the cell membrane.

[0168] An antigen can be a T-cell receptor (TCR).

[0169] Antigens can have the CD3ε phenotype.

[0170] The cells to be modified can be monocytes or macrophages. The cells to be modified can be B cells. The cells to be modified can be dendritic cells (DCs).

[0171] The antigen is preferably an antigen that is highly conserved in monocytes and / or their differentiated forms and expressed on the cell membrane.

[0172] The antigen may be selected from CD14, CD16, CCR1, CCR2, CXCR4, and CD64. Preferably, the antigen is selected from CD16, CXCR4, and CCR1.

[0173] The cells to be modified can be natural killer (NK) cells.

[0174] The antigen is preferably an antigen that is highly conserved in NK cells and expressed on the cell membrane.

[0175] It can be greater than 10 6 Greater than 10 7 Greater than 10 8 Greater than 10 9 Greater than 10 10 or greater than 10 11 Carrier particles, preferably more than 10 8 The amount of viral vector applied is one vector particle.

[0176] The viral vector can be administered at a frequency of less than two weeks, that is, a second or supplementary administration of the viral vector is performed less than two weeks after the first administration. Preferably, a second or supplementary administration of the viral vector is performed less than one week after the first administration.

[0177] A first course of treatment with the viral vector (one or more doses) may be administered, followed by a second or supplemental dose of the viral vector after a period selected from the following time periods: six months, one year, eighteen months, two years, three years, four years, five years, or six years.

[0178] In another embodiment, the present invention provides a virus-like particle having a lipid bilayer envelope, the lipid bilayer envelope comprising:

[0179] (a) A VHH-binding domain is displayed on the outer membrane, which is cell type specific; and

[0180] (b) Exhibits an envelope protein on the outside of the envelope that promotes infection of the same cell type as in (a), wherein the envelope protein is as described elsewhere in this document.

[0181] Virus-like particles are suitable for delivering materials contained within them to specific types of cells, as described elsewhere in this document.

[0182] Therefore, the present invention provides a highly specific viral vector or virus-like particle, a method for using the viral vector or virus-like particle in a therapy, a method for preparing the viral vector or virus-like particle, and a method for modifying cells in vitro and in vivo using the viral vector or virus-like particle. Attached Figure Description

[0183] The invention will now be illustrated with reference to the following figures, in which:

[0184] Figure 1 An exemplary example of a carrier according to the invention is shown.

[0185] Figure 2 depicts a flow cytometry scatter plot of engineered 293T packaged monoclonal cell lines used to generate engineered lentiviral vectors in a specific embodiment. Parental 293T cells overexpressing surface hCD47 (middle plot) and depleted MHCI (e293T) were transduced with a γ-retrovirus (gRV) encoding CD3e or CD8a membrane-anchored VHH, and gated (P3) was performed to isolate stably expressing cells via fluorescence-activated cell sorting (A). The resulting polyclonal cell lines (B) were expanded and frozen for long-term storage and use. (C) In another iteration, parental e293T cells were transduced with a lentivirus (LV) encoding TCRab membrane-anchored VHH, and monoclonal cell lines were isolated by limiting dilution clones. e293T B619 clone 38 is a monoclonal production cell line derived through this method, and the expression of B2M, CD47, and membrane-anchored nanobodies (VHH) was confirmed by flow cytometry. In contrast, the reference wild-type cell line HEK 293T is known to be B2M positive, CD47 at baseline level, and VHH negative.

[0186] Figure 3 shows flow cytometry scatter plots of Jurkat wild-type cells (WT), Sup-T1 cells, Jurkat cell lines stably expressing hCD8a (Jurkat CD8+), and primary human PBCM stained with antibodies against CD3, CD4, CD8, and TCR. The results indicate that, unlike Sup-T1 cells which express surface CD3 and CD8, Jurkat WT cells do not express CD8 but do express TCR, while Jurkat cells stably expressing CD8 show surface expression of CD3, CD4, TCR, and transgenic CD8a.

[0187] Figure 4 This figure shows a flow cytometry graph quantifying the percentage of transduction determined by GFP reporter expression in Jurkat WT and Sup-T1 cells transduced with different types of wild-type vesicular stomatitis virus (VSVG) pseudotyped lentiviral vectors. Jurkat WT and Sup-T1 cells were transduced with VSVG pseudotyped lentiviral vectors expressing GFP reporters and displaying CD8a VHH (CD8 VSVG) or CD3e VHH (CD3 VSVG) or not displaying VHH (VSVG) on their envelopes. GFP reporter expression in the cells was analyzed by flow cytometry 3 days post-transduction. As shown in the figure, transduction of VSVG wild-type pseudotyped vectors displaying CD3e or CD8a membrane-anchored nanobodies was equivalent between Jurkat WT (which does not express CD8) and Sup-T1 (which does not express CD3). These results indicate that displaying membrane-anchored VHHs on the surface of a lentiviral vector pseudotyped with wild-type vesicular stomatitis virus glycoprotein is insufficient for cell-specific transduction.

[0188] Figure 5 A flow cytometry-quantified plot of the transduction percentage determined by GFP reporter expression is shown, comparing the transduction percentage in Jurkat WT and Jurkat CD8+ cells transduced with different types of engineered lentiviruses. Jurkat cells (Jurkat WT) or Jurkat cells stably expressing human CD8a (Jurkat CD8+) were transduced with GFP-expressing lentiviral vectors pseudotyped with mutant Lassa virus glycoprotein Y150A (LVGP1x) or wild-type rdanovirus glycoprotein (LDV), displaying membrane-anchored CD8a VHHs on the viral envelope. This result indicates that, unlike the broadly wild-type VSVG lentiviral particles, CD8a VHH-targeting lentiviral particles LVGP1x-CD8 and LDV-CD8 specifically target the engineered vectors to Jurkat cells (Jurkat CD8+) expressing the VHH target.

[0189] Figure 6 This diagram shows a flow cytometry-quantified plot of the transduction percentages of the targeted (LVGP1x-CD3) and non-targeted (VSVG) vectors to target (Jurkat WT) and non-targeted (Sup-T1) cells. Sup-T1 and Jurkat WT cells were transduced with either a broadly tropism-dependent VSVG lentiviral vector or a vector displaying CD3 VHH pseudotyped with the LVGP1x glycoprotein. The results show that, unlike the VSVG vector, the LVGP Y150A lentiviral particle displaying CD3e VHH transduced only CD3-expressing cells. This demonstrates that target-specific transduction can also be achieved using a VHH pseudotyped vector targeting LVGP1x, which differs from CD8a.

[0190] Figure 7 The graph shows a flow cytometry-quantified plot of the percentage of transduction efficiency of targeted (LVGP1x-TCR2 and LDV-VHHTCR2) and non-targeted (VSVG) vectors to target (JurkatWT) and non-targeted (Sup-T1) cells. Sup-T1 cells and Jurkat WT cells were transduced with broadly tropism-dependent VSVG lentiviral vectors (VSVG) or TCR VHH-expressing vectors pseudotyped with LVGP1x or LDV (LVGP1x-TCR2 and LDV-VHHTCR2). The results show that, unlike the VSVG vector, TCR VHH targeting LVGP1x or LDV lentiviral particles only transduced TCR-expressing cells (Jurkat WT). This demonstrates that target-specific transduction can be achieved by targeting LVGP1x and LDV pseudotyped vectors with nanobodies targeting different T cell markers.

[0191] Figure 8 The graph shows the percentage of transduction efficiency of the targeted (LVGP1x-CD8a and LDV-CD8a) and non-targeted (VSVG) vectors to target cells over time, quantified by flow cytometry. Jurkat CD8+ cells stably expressing human CD8a were transduced with VSVG, LVGP1x-CD8a, or LDV-CD8a GFP expression vectors, and GFP expression was quantified by flow cytometry within 14 days of culture. This result indicates that, similar to VSVG lentiviral particles, the LVGP1x and LDV pseudotyped CD8a VHH-targeting lentiviral particles stably transduced target cells.

[0192] Figure 9Flow cytometry quantification of the percentage of transduction efficiency of the targeted (LVGP1x-CD3) and non-targeted (VSVG) vectors in target cells over time, as well as graphs showing the quantification of viral genome insertion by quantitative PCR, are presented. Cultured Jurkat WT cells were transduced with either a broadly tropist VSVG vector or a target-specific vector pseudotyped with LVGP1x linked to either of two different CD3e nanobodies (G7 and H6). GFP transgene expression was monitored over time using flow cytometry, and vector insertion into the host genome was assessed by quantitative PCR amplification of the inserted vector sequence on day 21. The results show that transduction using the LVGP1x-CD3e VHH targeting vector was stable over 21 days (A.) and resulted in viral genome insertion into the host DNA (B.).

[0193] Figure 10 shows a graph plotting the percentage of transduction efficiency of the targeted (LVGP1x-CD8) and non-targeted (VSVG) vectors to target (Sup-T1) and non-targeted (Jurkat WT) cells on day 3 (A.) and over time (B.), as well as the quantification of viral genome insertion by quantitative PCR (C.). Cultured Jurkat WT and Sup-T1 cells were transduced with either a broadly tropism-dependent VSVG vector or a target-specific vector displaying CD8a VHH pseudotyped with LVGP1x. GFP transgene expression was monitored over time using flow cytometry, and genome insertion was assessed by quantitative PCR amplification of the inserted vector sequence on day 21. The results show that transduction using the LVGP1x-CD8 VHH targeting vector was specific to CD8a-expressing target cells (A.), stable over 21 days (B.), and resulted in viral genome insertion into the host DNA (C.).

[0194] Figure 11 This diagram shows a flow cytometry-quantified graph of the percentage of transduction efficiency of the target (JurkatWT) over time using targeted (LVGP1x-TCR and LDV-TCR) and non-targeted (VSVG) vectors. Cultured Jurkat WT cells were transduced with either a broadly tropism-loving VSVG vector or a target-specific vector displaying a TCR-targeting VHH pseudotyped with LVGP or LDV. GFP transgene expression was monitored over time using flow cytometry. The results show that transduction using LVGP or LDV TCR VHH targeting vectors was stable within 16 days.

[0195] Figure 12A graph showing the percentage of transduction efficiency of targeted (LVGP1x-CD8) and non-targeted (VSVG) vectors in human primary CD4+ and CD8+ T cells is presented by flow cytometry. Human PBMCs were activated in culture and transduced using either the LVGP1x-CD8 or VSVG vectors, and GFP expression was quantified by flow cytometry 7 days post-transduction. These results indicate that, unlike the VSVG pseudotyped vector, the LVGP1x-CD8 vector specifically transduces primary T cells expressing endogenous levels of the CD8a VHH target (i.e., CD8a).

[0196] Figure 13 shows flow cytometry plots and graphs of the percentage of transduction efficiency of targeted (LVGP1x-TCR) and non-targeted (VSVG) vectors in human primary CD4+ and CD8+ T cells. Human PBMCs were activated in culture and transduced with either LVGP1x-TCR or VSVG vectors. GFP expression was quantified by flow cytometry at 4 days (A.) and 20 days (B.) post-transduction. These results indicate that the LVGP1x-TCR vector effectively transduces primary T cells expressing endogenous levels of TCRs, and that LVGP1x-TCR transduction is stable and more effective than the VSVG pseudotyped vector under the same conditions.

[0197] Figure 14 shows flow cytometry plots of BCMA CAR-transduced Jurkat-CD8+ cells co-cultured with cells expressing or not expressing BCMA, or transduced cells alone. Jurkat-CD8 cells were transduced with LVGP1x-CD8 or LDV-CD8 or a broadly tropism-dependent VSVG lentiviral vector encoding the BCMA CAR. Transduced cells were cultured alone (without a target), with BCMA-expressing cells (NCI-H929), or with a cell line lacking BCMA expression (K562). Surface expression of CD69 (a marker of T cell activation) was assessed by flow cytometry (A.), and a coordinate plot summarizing the data is presented in (B.). The results show that co-culturing BCMA CAR-expressing Jurkat-CD8 cells with NCI-H929 cells resulted in increased CD69 surface expression compared to using T cells alone or co-culturing T cells with non-target cells. These results indicate that transducing Jurkat-CD8 with the LVGP1x-CD8 or LDV-CD8 vector encoding BCMA CAR leads to functional expression of the CAR construct and signaling consistent with T cell activation, comparable to cells transduced with the widely tropism-dependent VSVG vector.

[0198] Figure 15 shows flow cytometry plots of BCMA CAR-transduced primary T cells or T cells alone, co-cultured with cells expressing or not expressing BCMA. Activated primary human T cells were transduced with a LVGP1x-CD3-targeting or broadly tropism-dependent VSVG lentiviral vector encoding the BCMA CAR. Transduced cells were cultured alone (without a target), with BCMA-expressing cells (NCI-H929), or with a cell line lacking BCMA expression (K562). Surface expression of CD137 (a marker of T cell activation) was assessed by flow cytometry (A.), and a coordinate plot summarizing the data is presented in (B.). This figure shows that co-culturing BCMA CAR-expressing T cells with NCI-H929 cells resulted in increased CD137 surface expression compared to culturing BCMA CAR-T cells alone or with non-target cells. These results indicate that transduction of primary T cells with the LVGP1x-CD3 vector encoding BCMA CAR resulted in functional expression of the CAR construct and signal transduction consistent with T cell activation, comparable to cells transduced with the widely tropism-dependent VSVG vector.

[0199] Figure 16 shows a graph summarizing the flow cytometry quantification of target and non-target cell numbers in BCMA CAR-T co-cultures. Activated human PBMCs were transduced with LVGP1x-CD8 or a broadly tropism-dependent VSVG lentiviral vector encoding the BCMA CAR, controlled by a T cell-specific synthetic promoter (SYN 17; for the LVGP1x-CD8 vector) or a constitutive promoter (EF1a; for the VSVG vector). Transduced cells were expanded and co-cultured with NCI-H929 (BCMA-expressing cells) or K562 non-target cells (not expressing BCMA) at a ratio of 1:1 or 1:2 (the ratio of effector T cells to target / non-target cell lines). The number of target / non-target cells was quantified by flow cytometry in 50 μl of culture medium stained with Cell Trace Violet and summarized in graphs A and B. The figure shows that, compared with co-cultures of VSVG-GFP-transduced or untransduced T cells, co-cultures of CD19 CAR-T cells significantly reduced the number of target cells (NCI-H929), but did not significantly reduce the number of non-target cells (K562). This result indicates that transducing primary T cells with LVGP1x-CD8 encoding the BCMA CAR leads to functional expression of the CAR construct and specific cytotoxicity of the transduced cells against the target cells, comparable to T cells transduced with a widely tropism-inducible VSVG vector.

[0200] Figure 17 shows a graph summarizing the flow cytometry quantification of target (Nalm6) and non-target (K562) cell numbers in CD19 CAR-T cocultures. Activated human PBMCs were transduced with LVGP1x-CD8 or a broadly tropism-dependent VSVG lentiviral vector encoding the CD19 CAR, controlled by a T cell-specific synthetic promoter (SYN 17; for the LVGP1x-CD8 vector) or a constitutive promoter (EF1a; for the VSVG vector). Transduced cells were expanded and co-cultured with Nalm6 (CD19-expressing cells) or K562 non-target cells (not expressing CD19) at a ratio of 1:2 or 1:5 (the ratio of effector T cells to target / non-target cell lines). The number of target or non-target cells stained with Cell Trace Violet in 50 μl of medium was quantified by flow cytometry and summarized in graphs A and B, respectively. The figure shows that, compared with co-cultures of untransduced T cells, co-cultures of CD19 CAR-T cells significantly reduced the number of target cells (Nalm6) but did not significantly reduce the number of non-target cells (K562). This result indicates that functional transduction using the LVGP1x-CD8 vector can be applied beyond BCMA CARs, and that transduction of primary T cells with LVGP1x-CD8 encoding CD19CAR leads to functional expression of the CAR construct and specific cytotoxicity of the transduced cells to the target cells, comparable to T cells transduced with the widely tropism-dependent VSVG vector.

[0201] Figure 18 shows a graph summarizing the flow cytometry quantification of target and non-target cell numbers in BCMA CAR-T co-cultures. Activated human PBMCs were transduced with LVGP1x-TCRs encoding BCMA CARs controlled by a T cell-specific synthetic promoter (SYN19). Transduced cells were expanded and co-cultured with NCI-H929 (BCMA-expressing cells) or K562 non-target cells (not expressing BCMA) at ratios of 1:1, 1:2, or 1:5 (the ratio of effector T cells to target / non-target cell lines). The number of target / non-target cells stained with Cell Trace Violet in a fixed volume of culture medium was quantified by flow cytometry and summarized in graphs A and B. The graph shows that, compared with co-cultures of untransduced T cells, co-cultures of BCMA CAR-T cells significantly reduced the number of target cells (NCI-H929) but did not significantly reduce the number of non-target cells (K562). These results indicate that transducing primary T cells with LVGP1x-TCR encoding BCMA CAR under the control of a T cell-specific promoter leads to functional expression of the CAR construct and specific cytotoxicity of the transduced cells to the target cells.

[0202] Figure 19 shows flow cytometry plots of human CD8 or CD4 T cells in peripheral blood of humanized NSG mice injected with the LVGP1x-CD8 vector, and the persistence of CD8+ transduced cells over 27 days. On day 0, activated human PBMCs were injected intraperitoneally into immunodeficient NSG mice, followed one day later by intraperitoneal injection of a viral vector encoding GFP under the control of the EF1a promoter. Human T cell transduction was assessed by detecting GFP and cell-specific markers in mouse peripheral blood at days 4, 7, 14, 21, and 27. The figure shows that injection of 25 × 10⁶ or 100 × 10⁶ transduction units (TU) of LVGP1x-CD8 resulted in specific transduction of CD8+ human T cells (A.), and the expression of the transgene and transduced CD8 T cells persisted over 27 days (B.). These results demonstrate that target-specific and stable transduction of human CD8 T cells was achieved in vivo using the CD8 VHH targeting vector LVGP1x-CD8.

[0203] Figure 20 (A, B) shows flow cytometry graphs summarizing the levels of transduced human CD8 and CD4 T cells and CD20 B cells in humanized NSG mice transfected with activated human PBMCs and viral vectors via intraperitoneal injection. On day 0, activated human PBMCs were intraperitoneally injected into immunodeficient NSG mice. One day later, VSVG vectors encoding GFP or CD19CAR under the control of the EF1a promoter (VSVG GFP and VSVG EF1a CD19CAR) or LVGP1x-CD8 vectors encoding CD19CAR under the control of a synthetic T cell-specific promoter (LVGP1x-CD8 SYN CD19CAR) were injected intraperitoneally. On day 7 post-viral injection, CD19CAR expression in human T cells in peripheral blood was assessed by flow cytometry (A). On day 7 post-viral injection, the B cell marker CD20 was detected by flow cytometry in dissociated spleen, bone marrow, or peripheral blood to assess B cell aplasia (B). The figure shows that injection of 100e6 transduction units of LVGP1x-CD8 resulted in specific transduction of CD8+ human T cells, while injection of the same dose of the VSVG vector resulted in broad transduction of CD4 and CD8 cell populations. Furthermore, compared to animals injected with the GFP-encoded vector, mice injected with the CD19 CAR-encoded vector showed a significantly reduced percentage of B cells in the spleen, bone marrow, and peripheral blood. These results indicate that LVGP1x-CD8, using a CAR-encoded construct, achieves target-specific transduction of human CD8 T cells in vivo, leading to a level of target cell depletion similar to that observed in mice injected with a broadly tropism-dependent VSVG vector encoding the CAR.

[0204] (C) A flow cytometry graph shows the percentage of circulating human B cells in hCD34+ humanized NCG mice intravenously injected with the eLV-LVGP1X-CD8a viral vector. Healthy hCD34+ stable humanized NCG mice were intravenously injected with the eLV-LVGP1X-CD8a vector expressing GFP or CD19 CAR constructs under the control of the T cell-specific synthetic promoter #19. The percentage of CD20+ (B cells) in peripheral blood was assessed by flow cytometry. Data are presented as mean ± SEM from 4 to 6 animals per condition. These results demonstrate that CD19 CAR-T cells reprogrammed in vivo by a single injection of the eLV-LVGP1X-CD8a targeting vector expressing BCMA CAR under the control of the T cell-specific promoter #19 possess potent and durable anti-target cell activity.

[0205] (D to F) show coordinate plots summarizing flow cytometry quantifications of human CD20+ (B cells), CD3+, CD4+, and CD8+ (T lymphocytes) cells in the peripheral blood of humanized NSG mice injected with vectors targeting LVGP1x-CD8-CD19 CAR (10E+06 TU), LVGP1x-TCRab-CD19 CAR (10E+06 TU), or LVGP1x-TCRab-GFP (50E+06 TU) or a broadly tropism-dependent VSVG LV vector (50E+06 TU) expressing CD19-CAR. On day 0, activated human PBMCs were injected intraperitoneally into immunodeficient NSG mice. One day later, a CD19 CAR construct encoded by the T cell-specific synthetic promoter SYN23 or a viral vector encoding GFP encoded by the EF1a promoter was injected intraperitoneally. On day 7, the levels of human B cells and T cell transduction in mouse peripheral blood were assessed by flow cytometry. (D.) CD19 CAR T cells were detected in animals injected with the CD19 CAR vector as early as day 7 post-vector administration. The LVGP1x-TCRab-CD19 CAR vector efficiently transduced both CD4+ and CD8+ T cells, while the LVGP1x-CD8-CD19 CAR vector transduction was specific for CD8+ T cells. The transduction efficiency of LVGP1X-TCRab was significantly higher than that of the broadly tropism-dependent VSVG LV, achieving the same level of T cell transduction at 1 / 5 of the dose. (E.) In the LVGP1x-TCRab-CD19 CAR group, B cell regeneration impairment was complete on day 7 post-vector infusion, while it was nearly complete for LVGP1x-CD8. These results indicate that the use of the LVGP1X T cell targeting vector results in potent anti-target activity in reprogrammed CD19 CAR-T cells in vivo. (F.) Eleven days post-vector injection, the percentage of CAR+ cells in different immune cell types in peripheral blood (mouse CD45+ cells, hCD45+ / CD56+ human NK cells, hCD45+ / CD3+ / CD56+ human NKT cells, hCD45+ / CD14+ human myeloid cells, hCD45+ / CD3+ / CD4+, and CD8+ human T cells) was analyzed by flow cytometry. Results are expressed as mean ± SEM from eight animals. Note: B cell expression of CD19CAR was not assessed due to B cell regeneration impairment.

[0206] Figure 21A graph showing the flow cytometry quantification of the transduction percentage of CD8a target-negative cell lines (Jurkat WT and 293T) and CD8a-positive cell line (Sup-T1) using a widely tropist VSVG lentiviral vector and a CD8a-targeting lentiviral vector pseudotyped with Y150A (LVGP1X) and Y150A H141AF147A Lassa virus (LVGP3X) glycoprotein is presented. CD8a-negative cell lines Jurkat WT and 293T cells and CD8a-positive Sup-T1 cells were transduced with VSVG and LVGP1x-CD8 or LVGP3x-CD8 lentiviral vectors, and GFP expression was quantified by flow cytometry 3 days post-transduction. The results show that, compared with the broadly tropism-dependent VSVG lentiviral vectors, the LVGP1x and LVGP3x-CD8 vectors specifically transduced target cells, demonstrating that LVGP envelope glycoprotein mutations targeting residues important for binding to Lassa virus targets can be used in combination with membrane-anchored nanobodies to target and transduce cells of specific cell types.

[0207] Figure 22 A flow cytometry graph shows the percentage of human PBMCs transduced in vitro using GFP-expressing CD8-targeting lentiviral vectors pseudotyped with WT Lassa virus GP protein or Lassa virus GP mutants (Lassa virus Y150A; Lassa virus H141A, F147A; Lassa virus Y150A, F446L). In vitro transduction was performed using the vectors at MOI 10 (G, Lassa virus Y150A; Lassa virus H141A, F147A; Lassa virus Y150A, F446L) or MOI 2 (WT) based on SupT1 titration. Results are expressed as mean ± SEM of PBMC transduction from three different human donors. These results indicate that although the F446L mutation eliminates the fusion ability of Lassa virus GP protein, Lassa virus WT, Lassa virus Y150A, or Lassa virus H141A, F147A pseudotyped enables specific and persistent transduction of CD8+ cells via CD8-targeting vectors.

[0208] Figure 23 shows data supporting the selection and testing of VSVG mutants, designed to identify point mutations that eliminate VSVG binding to LDLR and promote target-specific transduction of VHH to eLV. (A) Table of mutation locations in the original Indiana strain vesicular stomatitis virus glycoprotein (VSV-G, UniProtKB accession number P03522). Mutations were made at 24 locations identified on the available crystal structures of VSV-G to spatially prevent VSV-G from binding to its natural ligand LDL-R. Single-letter amino acid codes were used (G: Gly, A: Ala, L: Leu, M: Met, F: Phe, W: Trp, K: Lys, Q: Gln, E: Glu, S: Ser, P: Pro, V: Val, I: Ile, C: Cys, Y: Tyr, H: His, R: Arg, N: Asn, D: Asp, T: Thr, del: deletion). (B). Flow cytometry analysis of wild-type Jurkat cells (CD8 negative) or Jurkat CD8+ (stable transduction of CD8α / β heterodimers) transducing GFP-expressing lentiviruses with either wild-type or VSV-G mutant envelope proteins pseudotyped individually (grey) or with VHH pseudotyped targeting CD8 protein (blue). Jurkat CD8+ cells transducing GFP-expressing lentiviruses with both wild-type or VSV-G mutant envelope proteins and VHH pseudotyped targeting CD8 protein (orange; the last item in the legend is labeled 8G_Jk-CD8). The coordinate plot depicts wild-type VSV-G normalized to 100% GFP+. (C). A graph showing the percentage of transduction of CD8a target-negative cell line (Jurkat WT) and CD8a positive cell line (Sup-T1) using a widely tropism-dependent VSVG lentiviral vector (WT) and a CD8a-targeting lentiviral vector pseudotyped with a VSVG mutant designed to eliminate VSVG protein tropism.

[0209] Figure 24 shows a graph plotting the percentage of transduction of CD8a target-negative and positive cell lines and primary CD4+ and CD8+ human T lymphocytes using CD8aVHH targeting vectors pseudotyped with different VSVG mutants designed to eliminate VSVG protein tropism. Activated primary CD4+ and CD8+ human T lymphocytes were transduced using (A) the CD8a target-negative cell line (Jurkat WT) and the CD8a positive cell line (Sup-T1) or (B) the CD8a target-negative cell line (Sup-T1) using a widely tropistic VSVG lentiviral vector (WT) and CD8a targeting lentiviral vectors pseudotyped with VSVG mutants, and GFP expression was quantified by flow cytometry 3 days post-transduction. The results show that the three CD8 lentiviral vectors pseudotyped with mutant VSVG glycoproteins specifically transduced the target cells compared to the widely tropistic VSVG lentiviral vector. This demonstrates that the A51E, I182E, and I331E mutations in the VSVG protein eliminate the broad tropism of VSVG targeting and support VHH-mediated targeting to transduce marker-positive cell types. (C) shows a flow cytometry-quantified plot of the transduction percentages of CD8a or TCRab-targeting lentiviral vectors pseudotyped with the targeting-deficient mutant VSVGI331E (G1x) to CD8a or TCRab. CD3 / 28-activated human CD4 / CD8 primary T cells, isolated primary monocytes, and T-cell lymphoblast lines (Jurkat and SupT1) were transduced using incremental amounts of non-targeting G1x eLV, G1x CD8 eLV, or G1x TCRab eLV vector particles (all pseudotyped with VSV-GI331E). The percentage of transduced cells was assessed by flow cytometry 7 days post-transduction. The results showed that targeting eLV vectors (ENaBL-T8 and ENaBL-TT, respectively) with VSV-GI331E pseudotyped CD8a VHH or TCRab VHH induced specific transduction of CD8a-expressing cells (CD8+ T cells, SupT1) and TCRab-expressing cells (CD4+ T cells, CD8+ T cells, Jurkat), respectively, while the transduction level of target-negative cells was <1%. Importantly, transduction of the non-targeted (VHH-deficient) ENaBL vector pseudotyped with VSV-GI331E was undetectable in target-positive cells. Results are expressed as mean ± SEM of two separate studies using two separate human PBMC donors.

[0210] Figure 25 illustrates the binding of TCRab VHH VSV-GI331E eLV vector particles to a Retrogenix cell microarray of HEK293T cells expressing surface human proteins to assess potential off-target binding of the vector. TCRabVHH VSV-GI331E eLVs encoding BCMA CARs were seeded at three different physical MOIs (6000, 8000, and 11000 vector particles / cell, A., B., and C.) onto immobilized HEK293 microarrays expressing six different targets (SIRPa, CD19, CD8A+CD8B2 heterodimer, CD4, LDLR, and EGFR). Binding of the vector to the selected targets was detected using a fluorescently labeled anti-VHH antibody. No signal was detected when the array was probed with the vector (PBS, D.), and expression of each target was confirmed by a transfection control (E.). The Retrogenix cell microarray did not include HEK293 cells expressing the TCRab complex (which requires co-expression of multiple proteins for adequate expression on the membrane). SIPRa (CD47 receptor on the vector surface) was used as a positive control. Results showed that the TCRabVHH VSV-GI331E eLV bound to HEK293 cells expressing SIRPa, but no binding was observed with CD19, CD8A+CD8B2 heterodimer, CD4, or EGFR. A slightly elevated LDLR signal was detected, but this signal was not reproduced in full-text screening, further supporting the targeting defect of the VSV-GI331E mutant. (F to G) Based on these results from A to E and the signal intensity compared to background, 6000 vector particles / cells were selected for full-text screening. The binding of TCRab VHH VSV-GI331E eLV was tested on a whole-cell microarray of HEK293 cells expressing 6105 full-length human plasma membrane proteins and cell surface tethered human secretory proteins, plus heterodimers of an additional 400 human surface proteins (Retrogenix Cell Microarray Technology). As shown in Figures 25 F and G, the results of two replicates of full-library screening and confirmatory screening indicate that TCRab VHH VSV-GI331E eLV specifically binds to SIRPa (isotypes 1, 2, and 4), with very weak / weak binding to CD7, VSIG8, SIRPG, EPHB2, and EPHA7. In summary, the data obtained from the Retrogenix cell microarray indicate that the TCRab VHH VSV-GI331E eLV vector has minimal off-target binding.

[0211] Figure 26 shows a coordinate plot illustrating the generation of functional BCMA CAR-T cells from resting human PBMCs via transduction with TCRab_VHH and VSV-GI331E eLV in vitro. (A) At a fixed multiplicity of infection of 0.4 transduction units per cell, 1 × 10⁶ unstimulated PBMCs from two independent donors were transduced using a lentiviral vector expressing BCMA CAR, targeted by TCRab_VHH and pseudotyped with VSV-GI331E. CAR expression was assessed by flow cytometry using BCMA-Fc on days 7 and 11 post-transduction to reveal CAR+ cells. (B) T cell expansion was also assessed by flow cytometry and compared with PBMCs treated with anti-CD3 / CD28 (UTD TransAct) or untreated (UTD unactivated) as positive and negative controls for cell activation and expansion, respectively. CAR-T cell counts are shown in (C.), where approximately 10-fold CAR+ T cell counts were obtained by day 11 post-transduction compared to the initial PBMC count. On day 11 post-transduction, cells were subsequently co-cultured with various GFP-expressing target cells at an effector-to-target (E:T) ratio based on CAR+ cells. CAR-T cells were normalized with untransduced PBMCs from each donor to achieve equal T cell counts at each E:T ratio between BCMA_CAR, BCMA_CAR_2, and untransduced (UTD) TransAct-treated PBMC controls, and the cytotoxicity of target cells was assessed by flow cytometry. Two experiments were performed separately, one using BCMAHigh NCI-H929 target cells and BCMANeg K562 cells (D to F), and the other using BCMALow Nalm6 target cells and BCMANeg K562 cells (G to H). BCMA_CAR and BCMA_CAR_2 T cells showed no background cytotoxicity against K562 cells at a non-critical E:T ratio (D), but effectively lysed BCMAHigh NCI-H929 cells (E). Under more critical conditions of low E:T ratios with BCMAHigh NCI-H929 cells (F) or with BCMALow Nalm6 cells (H), the more effective BCMA CAR_2 demonstrated control over target cell growth. (I) IL2 production in the co-culture supernatant on day 1 after co-culture was also assessed by titration of HEK_Blue_IL2 cells, showing significant IL2 production when co-cultured with BCMAHigh NCI-H929 target cells. Results are expressed as mean ± SEM of linear scale plots or geometric mean ± geometric SD of logarithmic scale plots for two independent PBMC donors (n=2).Overall, these results demonstrate that resting human PBMCs were transduced using the TCRab VHH VSV-GI331E eLV vector, resulting in multifunctional CAR-T cells. (K to N) shows a coordinate graph illustrating the generation of functional CLDN18.2 CAR-T cells using the TCRab VHH VSV-GI331E eLV vector. One x 10⁶ unstimulated PBMCs from two independent donors were transduced using a lentiviral vector carrying the TCRab_VHH targeting molecule and pseudotyped with VSVgI331E (ENabL-GTT). Alternatively, 1 x 10⁶ cells were stimulated with TransAct. 6 Two PBMCs were transduced with a lentiviral vector expressing CLDN18.2 CAR pseudotyped with VSVg (G.WT) 24 hours later. Various target cells expressing luciferase were then co-cultured with CAR+ cells at various effector-to-target (E:T) ratios based on different CAR+ cell ratios. CAR-T cells were normalized with untransduced TransAct-activated PBMCs from each donor to achieve equal T cell counts at each E:T ratio between ENabL-GTT, G.WT, and untransduced (UTD) TransAct-treated PBMC controls. Cytotoxicity (K) of target cells was assessed 18 hours after co-culture by quantifying the decrease in luciferase signal from native CLDN18.2+ target cells NUGC4. ENabL-GTT and G.WT CAR T cells exhibited comparable cytotoxicity to NUGC4 cells. Co-culture with various GFP-expressing target cells at CAR+ cell-to-target (E:T) ratios was also performed on day 9 post-transduction / activation. CAR-T cells were normalized using untransduced TransAct-activated PBMCs from each donor to achieve equal T cell counts at each E:T ratio between ENabL-GTT, G.WT, and untransduced (UTD) TransAct-treated PBMC controls, and the cytotoxicity of target cells was assessed by flow cytometry. Both ENabL-GTT and G.WT CAR T cells efficiently lysed Nalm6 cells engineered to express CLDN18.2 (L), but did not lyse negative control Nalm6 cells engineered to express CLDN18.1 (M) or K562 cells (N). Results are expressed as mean ± SEM on linear scale plots or geometric mean ± geometric SD on logarithmic scale plots for two independent PBMC donors (n=2). Overall, these results indicate that functional CAR-T cells were generated using the TCRab VHHVSV-GI331E eLV vector, demonstrating comparable efficacy.

[0212] Figure 27A graphical representation of the vector used to test the cell specificity and promoter potency of candidate synthetic promoters is shown. The synthetic promoter portion of the vector consists of enhancer DNA elements linked to the core or proximal promoter sequence. Promoter specificity and potency were tested in cultured cell lines and primary cells from various sources by transducing the VSVG broadly tropism-dependent lentiviral vector. Luminescent or fluorescent readings from reporter construct expression were quantified using a luminescent reader or by flow cytometry.

[0213] Figure 28 shows representative flow cytometry plots and normalized luminescence values ​​of Sup-T1 cells transduced with VSVG lentiviral vectors expressing luciferase / GFP dual reporters under the control of pervasive promoters (EF1a), proximal promoters, and different synthetic promoters (Figure 23). Sup-T1 cells were transduced in culture with the same number of functional VSVG lentiviral vectors expressing luciferase / GFP dual reporters under the control of different promoters. Transduction efficiency (percentage of cells expressing GFP) was assessed by flow cytometry (A.), and promoter activity was measured by the luminescence output of the luciferase reporter (B.). This figure shows that the addition of enhancer sequences significantly increased expression from synthetic promoters, with specific synthetic constructs reaching and exceeding expression levels from EF1a in Sup-T1 cells.

[0214] Figure 29 shows representative flow cytometry plots and normalized luminescence values ​​of Jurkat cells transduced with VSVG lentiviral vectors expressing luciferase / GFP dual reporters under the control of pervasive promoters (EF1a), proximal promoters, and different synthetic promoters (Figure 23). Jurkat cells were transduced in culture with VSVG lentiviral vectors of equal functional titers (titer) expressing luciferase / GFP dual reporters under the control of different promoters. Transduction efficiency (percentage of cells expressing GFP) was assessed by flow cytometry (A.), and promoter activity was measured by the luminescence output of the luciferase reporter (B.). These results show that the addition of enhancer sequences significantly increased expression from synthetic promoters, with specific synthetic constructs reaching and exceeding expression levels from EF1a in Jurkat cells.

[0215] Figure 30 shows representative flow cytometry plots and normalized fluorescence values ​​(gMFI) of human primary lymphocytes transduced with VSVG lentiviral vectors expressing luciferase / GFP dual reporters under the control of pervasive promoters (EF1a), proximal promoters, and different synthetic promoters (Figure 23). Activated human primary lymphocytes were transduced in culture with VSVG lentiviral vectors of equal functional titers, expressing luciferase / GFP dual reporters under the control of different promoters. Transduction efficiency was assessed by flow cytometry (the percentage of GFP-expressing CD3+ cells and the gMFI of GFP in CD4+ or CD8+ transduced T cell populations) (A. and C., respectively), and promoter activity was measured by the luminescence output of the luciferase reporter in CD3+ cells (B.). (D.) shows representative flow cytometry histograms of lymphocyte lines (Jurkat and SUPT1) and primary human T cells transduced with VSVG lentiviral vectors that express luciferase / GFP dual reporters under the control of a pervasive promoter (EF1a) and two different synthetic promoters (SYN19 and SYN22). SUP-T1 cells were transduced in culture with VSVG lentiviral vectors of equal functional titers that express luciferase / GFP dual reporters under the control of different promoters. The figure shows that the addition of enhancer sequences significantly increased expression from the synthetic promoters, with the median intensity of reporter expression achieved by a particular synthetic construct within less than twice the median intensity of EF1a in the T cell lines or primary T cells.

[0216] Figure 31 The graph shows a coordinate plot of luminescence values ​​(normalized to cell viability) for each cell type transduced with VSVG lentiviral vectors at pervasive promoters (EF1a), proximal promoters, and different synthetic promoters. Figure 27 The luciferase / GFP dual reporter was expressed under the control of different promoters. Cell lines from different sources were transduced in culture with VSVG lentiviral vectors of equal functional titers, which expressed the luciferase / GFP dual reporter under the control of different promoters. Promoter activity was measured by the luminescence output of the luciferase reporter and normalized to cell viability. To quantify reporter activity in NCI-H929 and THP1 cells, data represent two separate experiments, and the luciferase signal was further normalized for the EF1a condition in each experiment. The results showed that the selected synthetic promoter had limited expression in non-T cell lineages compared to EF1a.

[0217] Figure 32 shows a representative flow cytometry plot of surface BCMA CAR expression on 293T production cells during lentivirus production, and a coordinate plot summarizing the functional titers of the resulting LVGP1x-CD8 lentiviral vectors used to express BFP, BCMA, or CD19 CAR under the control of the ubiquitous promoter (EF1a) or different synthetic promoters (SYN19, SYN21, SYN22). Flow cytometry analysis was performed on the surface expression of the BCMA CAR transgene encoded in the lentiviral transfer vector at the end of the production process in 293T production cell lines transiently transfected with lentiviral transfer and packaging plasmids. Results in A. show that placing the BCMA CAR coding sequence under the control of a T-cell-specific promoter resulted in a more than 10-fold reduction in BCMA CAR surface expression on the production cell surface and, consequently, on the viral envelope of the resulting lentiviral particles. (B.) Titration of the LVGP1x-CD8 lentiviral vector generated in A. on target cells (Sup-T1). The results in B. indicate that restricting the expression of surface-expressed BCMA CAR or CD19 CAR, rather than the expression of the soluble fluorescent reporter protein BFP, during lentiviral particle production resulted in a more than 10-fold increase in the number of transduced competent lentiviral particles per ml.

[0218] Figure 33 This graph shows a coordinate plot of luminescence values ​​from NCI-H929 cells stably expressing luciferase and co-cultured with GFP or CAR-T lymphocytes. Primary T lymphocytes (within a mixed population of peripheral blood mononuclear cells) were transduced with VSVG lentiviral vectors expressing GFP or BCMA CAR, in which the transgenes were controlled by the EF1a or T cell-specific synthetic promoter (SYN19). Transduced cells were expanded and co-cultured with stably luciferase-expressing NCI-H929 cells at a ratio of 1:1 or 1:2 (effectant:target ratio), and luminescence from each condition was measured after 2 days of co-culture. The graph shows a significant decrease (>4-fold) in luminescence from co-cultures of NCI-H929 cells and BCMA CAR-T cells compared to untransduced T cells or GFP-expressing T cells, indicating a loss of NCI-H929 cells in the presence of BCMA CAR-T cells. These results indicate that BCMA CAR-T cells expressing the CAR construct under the control of the T cell synthesis promoter SYN19 are as effective as CAR-T cells expressing the CAR construct under the control of the EF1a promoter.

[0219] Figure 34 shows a coordinate plot illustrating the favorable CAR-T phenotype of BCMA CAR under T-cell-specific SYN23 promoter control relative to the constitutive EF1a promoter. Three independent PBMC donors were activated with anti-CD3 / CD28 and transduced using a VSVG pseudotyped lentiviral vector on day 2 post-activation. CAR expression levels were assessed on day 7 post-activation. Various target cells expressing GFP were then co-cultured at an effector-to-target (E:T) ratio based on CAR+ cells. CAR-T cells were normalized with untransduced PBMCs from each donor to achieve equal T-cell counts at each E:T ratio between EF1a_BCMA CAR, SYN23_BCMA CAR, and untransduced (UTD) PBMC controls. Co-culture was repeated by adding additional BCMAHigh NCI-H929 and BCMANeg K562 target cells at various time points (dashed lines in the coordinate plot). CAR expression was assessed by flow cytometry at various time points during co-culture, showing that CAR+ cells did not expand when co-cultured with BCMANeg K562 cells (A), but CAR+ cells with SYN23_BCMA CAR showed enhanced expansion relative to EF1a_BCMA CAR when co-cultured with BCMAHigh NCI-H929 target cells (B, C). CAR-mediated cytotoxicity was also assessed by flow cytometry at various points during co-culture, showing that under non-critical E:T 1:3 conditions, SYN23_BCMA CAR and EF1a_BCMA CAR had no CAR-mediated cytotoxicity against BCMANeg K562 target cells (D), and were comparable in cytotoxicity against BCMAHigh NCI-H929 target cells (E). Notably, SYN23_BCMA CAR-T cells exhibited enhanced target cell lysis under critical E:T 1:12 conditions (F), while EF1a_BCMA CAR-T cells failed to control target cell growth from the start of co-culture 2. CAR-T activation was also quantitatively assessed by flow cytometry of CD25 expression, with BCMANeg K562 target cells showing no specific activation of CAR-T cells (G); however, EF1a_BCMA CAR-T cells exhibited higher background activation in the absence of BCMA antigen. Conversely, in the presence of BCMAHigh NCI-H929 target cells, Enh023_BCMACAR-T cells showed enhanced BCMA-specific activation in co-cultures 1 and 2 (H, I). Results are expressed as mean ± SEM of linear scale plots or geometric mean ± geometric SD of logarithmic scale plots for three independent PBMC donors (n=3).Overall, these results indicate that CAR expression driven by the SYN23 promoter is associated with lower background activation and more durable CAR expression and function compared to EF1a.

[0220] Figure 35 shows a graph representing the percentage of transduction of unstimulated PBMCs and isolated monocytes using LVGP1X eLV with a scFv exhibiting specific membrane anchoring to CD14. Human PBMCs and isolated primary monocytes were transduced with increasing numbers of LVGP1X-scFVCD14 eLV vectors (physical particles), and the percentage of transduced cells was assessed by flow cytometry 7 days post-transduction. These results demonstrate that monocyte-specific transduction can be achieved by directing LVGP1x eLV to myeloid cells using a membrane-anchored binder targeting CD14.

[0221] Figure 36 shows a graph plot quantified by flow cytometry of the transduction percentage of CD14 target-positive cells (monocytes / macrophages and Jurkat cell lines stably expressing CD14) and CD14 target-negative cells (Jurkat WT cell lines and SUP-T1) using lentiviral vectors pseudotyped with the defective mutant VSVGI331E (G1x) and different CD14 VHH or affinity-matured 3RMB1 CD14 VHH. A. 50,000 Jurkat WT and Jurkat CD14 cells were transduced with 100 μl of unconcentrated CD14 scFv or VHH-targeting lentiviral vectors pseudotyped with G1x and expressing CD14 scFv or different CD14 VHH. Transduction efficiency was assessed by flow cytometry by staining Jurkat cells with anti-VHH antibody 7 days post-transduction. Results are presented as mean ± SD of two different experiments. B. One hundred,000 monocytes isolated from a healthy donor were transduced using vector particles of 250, 500, 1000, and 2000 lentiviral vectors, which were pseudotyped with a CD14 VHH-deficient mutant VSVGI331E (G1x) that did not target or expressed CD14 scFv or a different CD14 VHH targeting defective mutant. The percentage of transduced cells was assessed by flow cytometry 7 days post-transduction. C. One hundred,000 monocytes isolated from a healthy donor were transduced using vector particles of 250 and 1000 lentiviral vectors, which were pseudotyped with a CD14 VHH-deficient mutant VSVGI331E (G1x) that did not target or expressed CD14 scFv or a different affinity maturation of 3RMB1 CD14 VHH targeting defective mutant. Transduction efficiency was assessed by staining macrophages with anti-VHH antibody using flow cytometry 7 days post-transduction. These results show that directing G1x eLV to myeloid cells using a capsule-anchored binder that targets CD14 enables monocyte-specific transduction.

[0222] Figure 37A graph showing the flow cytometry-quantified percentage of transduction of CD14 target-positive cells (monocytes / macrophages, Jurkat CD14 cell lines) and CD14 target-negative cells (stimulated PBMCs, Jurkat WT cell lines, and NCI-H929 cells) using lentiviral vectors pseudotyped with the targeted defective mutant VSVGI331E (G1x) and CD14 VHH or G1x alone. Monocytes / macrophages, CD3 / 28-activated human PBMCs, wild-type or stably CD14-expressing Jurkat lymphocyte lines, and the B-cell lymphocyte line NCI-H929 were transduced from three separate healthy donors using vector particles of 250 or 500 non-targeted eLVs and CD14-targeted eLVs (both pseudotyped with VSVGI331E and expressing the fluorescent reporter GFP). The percentage of transduced cells was assessed by flow cytometry 7 to 14 days post-transduction. Results are expressed as mean ± SD for three donors and two to three separate batches of virus. These results indicate that the VSVGI331E pseudotyped CD14 VHH-targeting eLV vector induced specific transduction in CD14-expressing cells (primary monocytes / macrophages and Jurkat cells stably expressing CD14), while transduction levels in target-negative cells (NCI, Jurkat, and primary PBMCs) were <1%. Importantly, transduction of the VSVGI331E pseudotyped non-targeted (VHH-deficient) eLV vector was undetectable in target-positive cells.

[0223] Figure 38 illustrates the use of six proximal promoters encoding genes derived from macrophage-specific genes (see Figure 38). Figure 27GFP expression levels in different cell types (myeloid, T cells, hepatocytes, B cells, kidneys) transduced with VSVG pseudotyped LV under the control of a reporter gene were assessed as geometric mean fluorescence intensity by flow cytometry. Primary monocytes, THP-1 cells, primary PBMCs, SupT-1 cells, Huh-7 cells, NCI cells, and e293T cells were transduced with VSVG-eGFP transgenes encoding luciferase-eGFP under EF1a, no promoter (empty), or six different proximal promoters. Monocytes were transduced and differentiated into M0-like macrophages in the presence of 50 ng / ml M-CSF for 7 days. PBMCs were stimulated with CD3 / CD28 for 2 days prior to transduction and cultured in the presence of 30 ng / ml human IL-2. A. Percentage of GFP gMFI normalized to EF1a for empty promoter and six proximal promoters. B. FACS plot data of the EF1a and CHIT1 proximal promoters for each cell type tested. These results show that the reporter expression induced by the selected myeloid-specific core promoter in primary macrophages was at a level equal to or higher than that induced by the EF1a promoter.

[0224] Figure 39 illustrates the use of seven synthetic promoters encoding genes derived from macrophage-specific genes (see Figure 39). Figure 27 GFP expression levels in different cell types (myeloid, T cells, hepatocytes, B cells, and kidneys) transduced with VSVG pseudotyped LV under EF1a reporter control were assessed as geometric mean fluorescence intensity by flow cytometry. Primary monocytes, THP-1 cells, primary PBMCs, SupT-1 cells, Huh-7 cells, NCI cells, and e293T cells were transduced with VSVG lentiviruses encoding luciferase-eGFP transgenes under EF1a, no promoter (empty), or six different proximal promoters. Monocytes were transduced and differentiated into M0-like macrophages in the presence of 50 ng / ml M-CSF for 7 days, followed by differentiation into M2-like macrophages in the presence of 50 ng / ml M-CSF, 20 ng / ml IL-4, and 20 ng / ml IL-10 for 2 days. PBMCs were stimulated with CD3 / CD28 for 2 days prior to transduction and cultured in the presence of 30 ng / ml human IL-2. A. Percentage of GFP gMFI normalized to EF1a for the empty promoter, the CHIT1 core promoter, and seven synthetic promoters (including the CHIT1 proximal promoter fused with an enhancer sequence). B. FACS plot data for EF1a and Enh031-CHIT1 synthetic promoters for each tested cell type. These results show that adding an enhancer sequence to the CHIT1 core promoter increases reporter expression levels while preserving expression specificity in myeloid cells.

[0225] Figure 40 shows a coordinate plot of the quantification of human IL-12 secretion in the supernatant of M0 or M2-like macrophages derived from healthy donor primary monocytes and transduced using VSVG LV encoding the h-IL12 transgene under the control of the EF1a, CHIT1 proximal promoter, or Enh031-CHIT1 synthetic promoter. Results in A show the h-IL-12 concentration in the supernatant of M0-like macrophages 7 days post-transduction. Monocytes were transduced and differentiated into M0-like macrophages in the presence of 50 ng / ml M-CSF for 7 days. Results in B show the IL-12 concentration in the supernatant of M2-like macrophages 9 days post-transduction. Monocytes were transduced and differentiated into M0-like macrophages in the presence of 50 ng / ml M-CSF for 7 days, followed by differentiation into M2-like macrophages in the presence of 50 ng / ml M-CSF, 20 ng / ml IL-4, and 20 ng / ml IL-10 for 2 days. Two batches of lentivirus were used for each condition. These results showed that the functional transgene expression level induced by the Enh31-CHIT1 myeloid-specific synthetic promoter was comparable to that induced by the constitutive promoter EF1a.

[0226] Figure 41 A graph showing the flow cytometry quantification of the percentage transduction efficiency of the LVGP1X-VHHCD3 vector in Jurkat cells, where CD3e VHH is anchored to the membrane within different scaffolds, is presented. Jurkat WT cells were transduced with different dilutions of the LVGP1X-VHHCD3 vector, each displaying CD3e VHH in different scaffolds, and the functional titer of each vector type was quantified by the average percentage of transduced cells from at least two linear dilutions. Different scaffolds anchored CD3e VHH to the membrane via either a PDGFR or B7-1 transmembrane domain, which is connected to the VHH via sequences of varying lengths and flexibility (e.g., the CD8a hinge region or the PGFR receptor juxtamembrane region, see X-axis labels). The results show that the scaffold anchoring the VHH to the membrane affects the functional titer of the vector. In the case of CD3e VHH, the anchored scaffold containing both the PDGFR transmembrane domain and the CD8a hinge region provided the architecture for achieving the highest functional titer.

[0227] Figure 42A flow cytometry plot shows the percentage of transduction efficiency of the LVGP1X-VHHCD8a vector in Sup-T1 cells, with CD8a VHH anchored to the membrane within different scaffolds (see Figure 32). Sup-T1 WT cells were transduced with each LVGP1X-VHHCD8a vector at different dilutions, and the functional titer of each vector type was quantified by the percentage of transduced cells averaged from at least two linear dilutions. The results show that scaffolds anchoring VHH to the membrane affect the functional titer of the vector. In the case of CD3e VHH, anchored scaffolds containing the PDGFR transmembrane domain and the CD8a hinge region or the PDGFR juxtamembrane region provide the architecture for achieving the highest functional titer.

[0228] Figure 43 A shows a flow cytometry-quantified graph of the percentage of transduction efficiency of the TCR-targeting vector (VHHTCR eLV) to target cells (Jurkat WT). Jurkat WT cells were transduced with vectors exhibiting TCR VHH, pseudotyped with previously shown to eliminate the broad tropism of wild-type VSVG mutants (K47A, R354), and in which VHH was anchored to the membrane within different scaffolds. TCR1 scaffold: PDGFR transmembrane domain and PDGFR juxtamembrane region; TCR2 scaffold: PDGFR transmembrane domain and IgG4 juxtamembrane region; TCR3 scaffold: PDGFR transmembrane domain and tetrameric coiled helical hinge. The results show that the scaffolds anchoring VHH to the membrane affect the transduction efficiency of the vectors. In the case of TCR VHH, the anchored scaffolds containing the PDGFR transmembrane domain and IgG4 juxtamembrane region provide the architecture that achieves the highest transduction efficiency when linked to the VSVG (K47A, R354) pseudotype. B. A graph showing the enhanced transduction and stimulation capabilities of lentiviruses targeting TCRab when using the CD8a hinge on the TCRab_VHH fusion protein compared to the IgG4 hinge. Stable production cell lines expressing TCRab VHH with different architectures (PDGFR transmembrane domain + CD8a hinge or IgG4 hinge) were used to generate GFP-expressing lentiviruses pseudotyped with a VSVgI331E envelope and targeted by TCR VHH. 1.5 × 10⁵ PBMCs from two independent donors were then transduced with 100 μL of unconcentrated lentivirus. Using the CD8a hinge increased the total number of transduced T cells on day 7 post-transduction compared to the IgG4 hinge. Results are expressed as geometric mean ± geometric SD for two independent PBMC donors and two independent lentiviral formulations (n=4).

[0229] Figure 44 illustrates the quantification of activation, expansion, and transduction of resting CD3 lymphocytes by flow cytometry after exposure to LVGP1X-TCRab VHH or CD8 VHH vectors. Resting hPBMCs were transduced overnight with LVGP1x-TCR VHH, VSVG LV, and LVGP1x-CD8a VHHeLV. The results show that LVGP1X displaying the TCRab VHH binder induced T cell activation (A.), as indicated by an increase in the percentage of T cells expressing the activation marker CD25, and increased T cell expansion (B.) and T cell transduction (C.) compared to resting PBMCs exposed to VSVG LV or LVGP1x-CD8a VHH eLV.

[0230] Figure 45 shows a graph demonstrating the enhanced transduction and stimulation capabilities of TCRab VHH-targeting lentiviruses when using the CD8a hinge on the TCRab_VHH fusion protein compared to the IgG4 hinge. In (A to E), stable production cell lines transduced with a single lentiviral vector expressing TCRab_VHH were used to generate GFP-expressing lentiviruses pseudotyped with a VSVgI331E envelope. 1.5 × 10⁵ PBMCs from two independent donors were then transduced with 100 μL of unconcentrated lentivirus. Anti-CD3 / CD28 (TransAct) was used as a positive control for T cell activation, and the PBMCs were also untreated (NA) to show background levels of T cell expansion and activation. Compared to the IgG4 hinge, the CD8a hinge increased CD69 expression on day 1 post-transduction (A), CD25 and PD1 expression on day 4 post-transduction (B, C), and cell expansion (E), viral titer (F), and total transduced T cells on day 7 post-transduction (G). Results are expressed as mean ± SEM on linear scale plots for two independent PBMC donors and two independent lentiviral formulations (n=4), or geometric mean ± geometric SD on logarithmic scale plots.

[0231] Figure 46 shows a coordinate graph confirming the stimulatory and transduction-mediating capabilities of lentiviral vectors targeting various mutant TCRab_VHH binders. Production cells were transfected with a lentiviral plasmid expressing TCRab_VHH (wild-type or mutant) and TagBFP2, where integration of TCRab_VHH into the lentiviral vector was driven by transient expression of TCRab_VHH from the lentiviral plasmid. The lentiviral vector was pseudotyped using a VSVgI331E envelope. Jurkat cells were transduced with 100 μL of unconcentrated lentivirus. The stimulatory capability of the TCRab_VHH clone was assessed by CD69 expression on Jurkat cells on day 1 post-transduction. Viral titer was assessed by TagBFP2 expression on day 3 post-transduction.

[0232] Figure 47 shows a coordinate graph confirming the increased activation, transduction, and T cell expansion induced by TCRab VHH or CD8a VHH-targeting lentiviruses displaying stimulants or co-stimulants. On day 0, freshly isolated resting human PBMCs were infected via a Ficoll gradient from the erythrocyte sedimentation rate (ESR) amber layer with 2500 viral particles / cells. In one case, the viral particles were pseudotyped CD8a VHH G1x eLV lentiviruses (T8) of the CD8a-targeting VSV-G mutant (I331E), which, in addition to displaying either of the two co-stimulants binding to CD28 (truncated CD80 protein or anti-CD28 nanobodies (VHH), T8K80 or T8K28, respectively), also displayed the stimulating anti-CD3 agonist antibody OKT3 (T8K). In another case, the viral particles were pseudotyped TCRab VHH G1x eLV lentivirus (TT) targeting the VSV-G mutant (I331E). This lentivirus, in addition to exhibiting either of two co-stimulatory agents binding to CD28 (truncated CD80 protein or anti-CD28 nanobodies (VHH, T8K80 or T8K28, respectively), also exhibited the stimulatory anti-CD3 agonist antibody OKT3 (T8K). Flow cytometry was then used to analyze CD3+ T lymphocyte activation at 24 hours post-infection (CD25 surface markers), transduction at 7, 14, and 21 days post-infection (BCMA CAR expression levels on the cell surface, measured by V5 tag staining), and induced proliferation (amplification) at 7, 14, 21, and 28 days post-infection. These results show that although T8 eLV does not activate resting T cells, TCRab VHH targeting eLV, alone or in combination with a co-stimulatory binder (TTK, TT80, or TT28), or CD8 VHH targeting eLV in combination with a co-stimulatory binder, induced a significant increase in T cell activation, expansion, and T cell transduction. These data represent mean ± SEM values ​​from 3 to 12 separate experiments using 3 to 5 different donors.

[0233] Figure 48 shows a coordinate plot of primary T cell phenotypes after transduction with stimulatory / co-stimulatory vectors. In (AF), a single-vector system was used where stable production cell lines transduced with a single lentiviral vector expressing the stimulatory molecule (TCRab_VHH) and various co-stimulatory molecules were used to generate GFP-expressing lentivirus with a VSVgI331E envelope pseudotype. (A) shows the expression of TCRab_VHH on stable production cells. 1.5 × 10⁵ PBMCs from two independent donors were then transduced with 100 μL of unconcentrated lentivirus. Compared to TCRab_VHH alone, the vector carrying the CD28 co-stimulatory molecules CD86 IgV and TGN1412, as well as the 4-1BB co-stimulatory molecule 4B1-4, increased IL2 production (B) and CD69 expression (C) on day 1 post-transduction, increased CD25 expression on day 4 post-transduction (D), and increased T cell expansion on day 7 post-transduction (E). However, overall transduction was reduced in vectors carrying co-stimulatory molecules, likely due to decreased TCRab_VHH expression in these cells. Results are presented as mean ± SEM of linear scale plots or geometric mean ± geometric SD of logarithmic scale plots for two independent PBMC donors (n=2). In (G to L), separate vector systems were used where clonal production cell populations transduced with TCRab_VHH were re-transduced with vectors expressing various co-stimulatory molecules (G to L). Expression of TCRab_VHH and the co-stimulatory molecules CD86 IgV and TGN1412 is shown in (G). These production cells were used to generate GFP-expressing lentivirus pseudotyped with a VSVgI331E envelope. 1.5 × 10⁵ PBMCs from two independent donors were then transduced with two independent formulations of 100 μL of unconcentrated lentivirus. Compared to TCRab_VHH alone, the vector carrying the CD28 co-stimulatory molecules CD86 IgV and TGN1412 increased IL2 production (H), CD25 expression (I), and T cell expansion (J) on day 4 post-transduction. Viral titers remained unchanged due to equivalent TCRab_VHH expression (K). However, the co-stimulatory molecules caused an increase in total transduced cell count (L) due to increased T cell expansion. Results are expressed as mean ± SEM on linear scale plots for two independent PBMC donors and two independent lentiviral formulations (n=4), or geometric mean ± geometric SD on logarithmic scale plots. In all cases, anti-CD3 / CD28 (TransAct) was used as a positive control for T cell activation, and PBMCs were also untreated (NA) to demonstrate background levels of T cell expansion and activation.

[0234] Figure 49 shows a coordinate plot summarizing the flow cytometry quantification of hCD3+ cells expressing NY-ESO TCR after transduction with 10 μl of VHHCD8a (A.) or TCR-targeted LVGP1X eLV (B.) encoding NY-ESO TCR transgenes under the control of EF1a, SYN21, or SYN22 promoters (not shown). Activated human PBMCs were transduced with LVGP1x-VHHCD8a or VHHTCR encoding NY-ESO TCR under the control of EF1a, SYN21, or SYN22. Transduction efficiency in CD3+ T cells was assessed by flow cytometry using PE-conjugated A2 / NY-ESO-1 multimers at days 3, 7, 14, and 21. This result indicates that LVGP1x-VHHCD8a or VHHTCR stably transduces the NY-ESO TCR construct into primary T cells. This result supports... Figure 31 The results presented indicate that the LVGP1X eLV vector expressing NY-ESO under the control of the EF1a promoter has a low functional titer, which may be due to the expression of NY-ESOTCR on the viral envelope.

[0235] Figure 50 shows a graph summarizing the flow cytometry quantification of target and non-target cell numbers in NY-ESO TCR-T cocultures. Activated human PBMCs were transduced with LVGP1x-VHHTCRs of NY-ESO encoded by EF1a or T cell-specific synthetic promoters SYN19 and SYN22. The transduced cells were expanded and co-cultured with NY-ESO peptide-stimulated T2 cells (target cells) or HIV peptide-stimulated T2 cells (non-target cells) at a 1:1 ratio (T cell to target / non-target cell line ratio). The number of target / non-target cells stained with Cell Trace Violet in a fixed volume of culture medium was quantified by flow cytometry and summarized in graphs A and B. The graph shows that the NY-ESO TCR-T cell coculture eradicated target cells (A.) but not non-target cells (B.) compared to the coculture of untransduced T cells. Notably, although the transduction efficiency of the LVGP1X-VHHTCR EF1a NY-ESO vector was poor (see Figure 35), resulting in a lower number of NY-ESO TCR-T cells in the co-culture, the number of target cells was still significantly reduced in these cultures. This result indicates that transduction of primary T cells with LVGP1x-VHH eLV encoding NY-ESO under the control of a T cell-specific promoter leads to functional expression of the TCR construct and specific cytotoxicity of the transduced cells to the target cells.

[0236] Figure 51 shows a coordinate plot of tumor growth quantified by bioluminescence and flow cytometry in tumor-bearing humanized MHCI / II KO NSG mice transduced with human CD3+ T cells via intraperitoneal injection of activated human PBMCs and the TCRab VHH-targeted VSVGI331E pseudotyped eLV vector. The NCI-H929 multiple myeloma cell line, stably expressing luciferase, was intraperitoneally (iv) injected into NSG mice lacking expression of the mouse MHCI and MHCII complex. After tumor transplantation was confirmed, activated human PBMCs were injected intraperitoneally into tumor-bearing animals on day 0, followed one day later by the intraperitoneal injection of the TCRab VHH-targeted VSVGI331E pseudotyped eLV vector expressing BFP or BCMA CAR constructs under the control of the T cell-specific synthetic promoter #23. A. Tumor growth over time measured using in vivo bioluminescence signals from luciferase-expressing NCI-H929 tumor cells. Data represent mean BLI signal ± SD from 6 animals per condition. B. On day 14 post-vector injection, the in vivo transduction efficiency of TCRab VHH VSVGI331EBCMA CAR was assessed by flow cytometry analysis of peripheral blood after immunostaining with CAR-specific antibodies. Data are presented as mean ± SEM from 6 animals for each condition. These results demonstrate that the eLV-G1X-TCRab targeting vector expressing BCMA CAR can efficiently transduce T cells in vivo and that the reprogrammed BCMA CAR-T cells possess potent antitumor activity.

[0237] Figure 52 This image shows a plot of graphs quantifying tumor growth by bioluminescence in tumor-bearing hCD34+ humanized NCG mice injected intravenously with escalating doses of the eLV-G1X-TCRab viral vector expressing either a BCMA CAR construct or a fluorescent reporter (BFP) under the control of a synthetic T-cell promoter #23. Stable luciferase-expressing NCI-H929 multiple myeloma cells were intravenously injected into hCD34+ stable humanized NCG mice. After confirming tumor transplantation, the tumor-bearing animals were intravenously injected with the eLV-G1x-TCRab vector expressing either BFP or a BCMA CAR construct under the control of a T-cell-specific synthetic promoter #23. Tumor growth was measured over time using in vivo bioluminescence signals from luciferase-expressing NCI-H929 tumor cells. Data represent BLI signals for each animal. These results demonstrate that the use of the eLV-G1X-TCRab vector to reprogram BCMA CAR-T cells in vivo resulted in potent antitumor activity, achieving complete and durable tumor clearance in animals treated with 3.25 or 6.5E+10vp, and moderate tumor control in animals treated with 1.3E+10vp.

[0238] Figure 53 shows a coordinate plot of flow cytometry analysis of tissue samples from tumor-bearing hCD34 NCG mice intravenously injected with eLV-G1X-TCRab expressing the BFP reporter or BCMA CAR construct. Flow cytometry analysis of peripheral blood on day 20 post-vector infusion and bone marrow at euthanasia (up to 36 days post-vector infusion) to determine the percentage of CAR+ cells in different immune cell types (mouse CD45+ cells, hCD45+ / CD56+ human NK cells, hCD45+ / CD14+ human myeloid cells, hCD45+ / CD3+ T lymphocytes, hCD45+ / CD3+ / CD4+, and hCD45+ / CD3+ / CD8+ human T cells). Note that NKT cells were below detection levels and CAR expression could not be assessed. Results are expressed as mean ± SEM from 2 to 5 animals per group (depending on tissue) and demonstrate the specific targeting and transduction of the eLV-G1X-TCRab BCMA CAR lentiviral vector to T cells.

[0239] Figure 54 This diagram shows a coordinate plot of tumor growth in tumor-bearing hCD34+ humanized NCG mice that received intravenous injection of TCRab VHH-targeted and / or CD14 VHH VSVGI331E pseudotyped eLV vectors. The NCI-H929 multiple myeloma cell line was subcutaneously injected into NCG cells, and after tumor transplantation was confirmed, tumor-bearing animals were intravenously injected with the vector, CD14 VHH VSVGI331E pseudotyped eLV expressing BFP or human IL12, TCRab VHH VSVGI331E pseudotyped eLV expressing BCMA CAR, or a single injection of the same dose of CD14 VHH VSVGI331E eLV with human IL12 and a combination of both TCRab VHH VSVGI331E pseudotyped eLV and BCMA CAR. Subcutaneous tumor volume was measured over time using calipers. Data represent tumor volume over time for each animal (n=4 or n=8) in each cohort. These results indicate that, following a single injection of the cell type-specific VHH-targeted eLV vector, reprogrammed monocytes / macrophages in vivo exhibit synergistic antitumor activity with BCMA CAR T cells.

[0240] Figure 55 shows the data from... Figure 54Coordinate plots of flow cytometry analysis of tissue samples from tumor-bearing hCD34 NCG mice tested in the study. (A.) Flow cytometry analysis of peripheral blood on day 11 after vector infusion and (C.) tumor tissue at euthanasia to determine the percentage of CAR+ and CD3+ cells, and analysis of the level of secreted human IL12 in plasma (B.). Results are presented as mean ± SEM from 1 to 8 animals per group (depending on tissue and condition). The data indicate that the eLV-G1X-TCRab BCMACAR lentiviral vector and eLV-G1X-CD14 VHH hIL12 effectively reprogram cells in vivo, and (2) the combination of T cell reprogramming (using BCMA CAR) and monocyte / macrophage reprogramming (to secrete human IL12) synergistically enhances the antitumor activity of the individual vectors, at least in part by increasing BCMA CAR-T cell transduction / expansion and increasing tumor T cell infiltration.

[0241] Figure 56 shows data quantifying the recovery of TCRab VHH VSVGI331EeLV, which displays envelope-anchored complement system regulators (RCA). (A) Schematic diagram of RCA constructs, testing their ability to prevent complement-mediated inactivation of VHH-displaying lentiviruses; DAF and factor H are tethered to the lentiviral envelope via VSV-GS anchors or GPI anchors. SP: signal peptide, H: 6xHIS tag, EC: extracellular domain, TM: transmembrane domain, IC: intracellular domain, GPI: phosphatidylinositol anchoring signal. Arrows indicate cleavage sites. (B) Complement inactivation assays were performed in which different lentiviral vectors encoding GFP reporter transgenes were incubated for one hour in serum-free medium, medium supplemented with fresh human serum, or medium supplemented with heat-inactivated human serum. The vector formulations were then incubated with Jurkat cells for 2 hours, and the percentage of transduction was assessed by flow cytometry after 72 hours. Results are expressed as mean ± SEM from three separate experiments and show that LVs exhibiting DAF and factor H are partially protected from complement-mediated inactivation, with mean recoveries increased by up to 24.1% and 16.3%, respectively. In summary, exhibiting RCA on the LV surface protects lentiviral particles from complement-mediated inactivation.

[0242] Figure 57A coordinate plot is shown to quantify the phagocytic activity of macrophages against T cell-targeted TCRab VHH G1x eLV displaying an albumin-binding domain. The albumin-binding domain of Streptococcus G418 was fused with either a CD59-GPI anchor or a VSV-GS anchor, and their ability to prevent macrophage phagocytosis of VHH-displaying lentiviruses was tested. Macrophage phagocytosis assays were performed in which human monocyte-derived macrophages were incubated with a TCRab VHH G1x lentiviral suspension for 2 hours, followed by washing and incubation in cRPMI for 3 days. Chlorpromazine was used as a control to inhibit macrophage activity. gDNA was extracted from macrophages 72 hours post-infection, and vector copy number (VCN) was determined. Results showed that ABD had a protective effect on the eLV envelope displaying membrane-bound hTCR nanobodies (VHH) and I331E VSVG glycoprotein. The data represent the mean ± SEM from three separate experiments using two different PBMC donors, and show that displaying an albumin-binding domain in the lentiviral envelope protects lentiviral particles from macrophage phagocytosis.

[0243] Figure 58 shows the results of culturing under simulated tumor microenvironment conditions and using four different synthetic promoters (e.g., ...). Figure 27 GFP expression levels in macrophages expressing reporter genes under the control of a process described in the figure were assessed by flow cytometry as geometric mean fluorescence intensity. These synthetic promoters are derived from genes specifically upregulated in macrophages within the tumor microenvironment. Human primary monocytes were transduced with a VSVG-dependent lentivirus containing luciferase-eGFP transgenes under the control of EF1a, promoterless (empty condition), or with a proximal CHIT1 promoter (four of which are shown in this figure). Human primary monocytes were transduced and differentiated into M0-like macrophages in the presence of 50 ng / ml M-CSF for 7 days, followed by treatment with TME conditions for 48 hours. For the “cytokine” condition, M0-like macrophages were treated with 20 ng / ml IL-4, 20 ng / ml IL-10, 10 ng / ml TGFβ, and 50 ng / ml M-CSF. For the "hypoxic" condition, M0-like macrophages were treated with 100 μM cobalt chloride (CoCl2). For the "conditioned medium" condition, M0-like macrophages were treated with the supernatant of NUG-C4 cell culture. Results in A show the fold change in GFP gMFI for each TME condition, normalized to reporter expression levels in macrophages before TME treatment. Results in B show flow cytometry data for each tested TME condition for the CHIT1, TAM12-CHIT1, and Enh031-CHIT1 synthetic promoters. Each condition was tested using two batches of lentivirus.

[0244] List of nucleic acid and amino acid sequences

[0245] SEQ ID NO. 1

[0246] QVQLVESGGGLVQAGGSLRLSCALSGRVWGSYAMGWFRQAPGKEREFVAAISRSGGTTYYADAVKGRFTISRDNGKNTVYLQMNSLKPEDTALYYCATHTNQGPAGFGSWGQGTQVTVSS

[0247] SEQ ID NO. 2

[0248] QVQLVESGGGLVQPGGSLRLSCAASGSISSIYVMGWYRQAPGKQRELVADITRTGTINYLKSVMGRFAISRDNAKSTVYLQMNSLKPEDTAVYYCRVVASAAPGRFGSWGQGTQVTVSS

[0249] SEQ ID NO. 3

[0250] QVQLVESGGGLAQPGGSLRLSCAASGSISSIYVMGWYRQAPGKQRELVADITRTGTINYLKSVMGRFAISRDNAKSTVYLQMNSLKPEDTAVYYCNIRLGYSLGDNYWGQGTQVTVSS

[0251] SEQ ID NO. 4

[0252] QLQLVESGGGLVQPGGSLRLSCAASGSISSIYVMGWYRQAPGKQRELVADITRTGTINYLKSVMGRFAISRDNAKSTVYLQMNSLKPEDTAVYYCNIRLGYSLGDNYWGQGTQVTVSS

[0253] SEQ ID NO. 5

[0254] QVQLQESGGGSVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSTINWNGGSAEYAEPVKGRFTISRDNAKNTVYLQMNSLKLEDTAVYYCAKDADLVWYNLSTGQGTQVTVSSAAAYPYDVPDYGSGIRGCRSTSGGGGSGGGGSGGGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0255] SEQ ID NO. 6

[0256] EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYVIGWFRQAPGKGREGVACIRIFDRHTYYADSVKGRFTISSDNSKNTVYLQMNSLRAEDTATYYCAAGSFFGCTRPEGDMDYFGQGTLVQVQSGIRGCRSTSGGGGSGGGGSGGGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0257] SEQ ID NO. 7

[0258] EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKGREGVACIRIFDRHTYYADSVKGRFTISSDNSKNTVYLQMNSLRAEDTATYYCAAGSFWGCTRPEGDMDYFGQGTLVQVQSGASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAKVVVISAILALVVLTIISLIILIMLWQKKPR

[0259] SEQ ID NO. 8

[0260] EVQLVESGGGLVQPGGSLRLSCAASGFTFEDYAIGWFRQAPGKGREGVACIRIFDRHTYYADSVKGRFTISSDNSKNTVYLQMNSLRAEDTATYYCAAGSFFGCTRPEGDMDYFGQGTLVQVQSGASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAKVVVISAILALVVLTIISLIILIMLWQKKPR

[0261] SEQ ID NO. 9.

[0262] EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0263] SEQ ID NO. 10.

[0264] EVQLQQSGPELVKPGASVKMSCKASGYKFTSYVMHWVKQKPGQGLEWIGYINPYNDVTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVHYCARGSYYDYDGFVYWGQGTLVTVSA GGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSATSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0265] SEQ ID NO. 11; Synthetic promoter #17: Enhancer 017 – proximal promoter CD3d

[0266]

[0267] SEQ ID NO. 12; Synthetic promoter #19: Enhancer 019 – proximal promoter CD3d

[0268]

[0269] SEQ ID NO. 13; Synthetic promoter #21: Enhancer 021 – proximal promoter CD3d

[0270]

[0271]

[0272] SEQ ID NO. 14; Synthetic promoter #22: Enhancer 022 – proximal promoter CD3d

[0273]

[0274] SEQ ID NO. 15; Synthetic promoter #23: Enhancer 023 – proximal promoter CD3d

[0275]

[0276] SEQ ID NO. 16; Synthetic promoter #24: Enhancer 024 – proximal promoter CD3d

[0277]

[0278]

[0279] SEQ ID NO. 17; Synthetic promoter #27: Enhancer 027 – proximal promoter CD3d

[0280]

[0281] SEQ ID NO. 18; Synthetic promoter #28: Enhancer 028 – proximal promoter CD3d

[0282]

[0283] SEQ ID NO. 19; Synthetic promoter #29: Enhancer 029 – proximal promoter CD3d

[0284]

[0285]

[0286] SEQ ID NO. 20; Synthetic promoter #30: Enhancer 030 – proximal promoter CD3d

[0287]

[0288] SEQ ID NO. 21 PDGFR short stem

[0289] AVGQDTQEVIVVPHSLPFK

[0290] SEQ ID NO. 22 GAPGAS

[0291] GAPGAS

[0292] SEQ ID NO. 23 GAPG4SAS

[0293] GAPGSGGGGSGGGGSAS

[0294] SEQ ID NO. 24 Tetramer coil-helix

[0295] ASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAG

[0296] SEQ ID NO. 25 IgG4 hinge

[0297] ASESKYGPPCPPCP

[0298] SEQ ID NO. 26 CD8a hinge

[0299] PAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

[0300] SEQ ID NO. 27 PDGFR TM

[0301] VVVISAILALVVLTIISLIILIMLWQKKPR

[0302] SEQ ID NO. 28 B7-1 TM

[0303] WAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV

[0304] SEQ ID NO. 29 IgGk leader sequence OKT3 Vh adapter OKT3 Vl adapter PDGFR Tm

[0305] METDTLLLWVLLLWVPGSTGDQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0306] SEQ ID NO. 30 1G4 Ly TCR alpha chain

[0307] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0308] SEQ ID NO. 31 Y2-1G4 Ly TCR beta chain

[0309] MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0310] SEQ ID NO. 32 LVGP1x Y150A

[0311] MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQAEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSKYWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLNHMGICSCGLYKQPGVPVKWKR*

[0312] SEQ ID NO. 33 LDV including undefined SP [[ID=!!]]

[0313] MWIITALICSFSINPTCLYPHGHEDSPTVRHGISRVLSGDAERNDDEHYHSPPLVLPLQNERTWKPANLSSLKCPEASHLGPDEHRVMEKWLVHRPKSSVLTKVEGSLCHKSRWLTRCEYTWYFSKTVSRKIEPMPPTKQECEEAIKRKEEGLLESLGFPPPACYWARTNDEENVQVDVTDHPMTYDPYSDGVVDNILVGGKCNQRECETVHDSTIWLETQKEKRPSQCEMDVEEQLELVSGIKRVGGSKSKAQRSVFVVGTNYPFMDATGACRLKYCSKSGMLLSNGLWFHITRKISPESNENSKFWLTLSDCSSDKQVGVLGEEYEIGKLQATMEDIMWDLDCFRTLEDLSHHKKVSMLDLFRLSRLTPGTGPAYKLVKGNLMVKEVQYVKAQRDQGELANPLCVAFMTESKNADRCIRYDEYDKEGPYKGQVMNGILINEGMVVFPHERFHLRQWDPEFIIKHEIKQVHHPVLGNYSSQIHDSLHESLIKDHSANLGDVMGNWVQVATSKFSWFFKEIEK

[0314] SEQ ID NO. 34 VHHTCR 2 (IgG4 scaffold) TCR Nb SEQ ID NO 9+SP+Stent

[0315] METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSSASESKYGPPCPPCPAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0316] SEQ ID NO. 35 BCMA CAR J&J, including SP but excluding V5 tags

[0317] MALPVTALLLPLALLLHAARPQVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMSIRPDYWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0318] SEQ ID NO. 36 CD19 CAR FMC63, including SP but excluding V5 label.

[0319] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0320] SEQ ID NO. 37 Y150A H141A F147A LVGP

[0321] MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFALSIPNANQAEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSKYWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLNHMGICSCGLYKQPGVPVKWKR*

[0322] SEQ ID NO. 38 VSVG A51E

[0323] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKEIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0324] SEQ ID NO. 39 VSVG I182E

[0325] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLESMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0326] SEQ ID NO. 40 VSVG I331E

[0327] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0328] SEQ ID NO. 41 The scFV CD14 has no spacer region and no TM structural domain, including SP, VH, VL, and connectors.

[0329] METDTLLLWVLLLWVPGSTGDDVQLQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIK

[0330] SEQ ID NO. 42 Modified gag sequence

[0331] LQSRPEPTAPPEEDPAVDLLKNYPSQKQEPIDKELYPLASLRSLFGSDPSSQ

[0332] SEQ ID NO. 43 Vpx SIV

[0333] MSDPRERIPPGNSGEETIGEAFEWLNRTVEEINREAVNHLPRELIFQVWQRSWEYWHDEQGMSPSYVKYRYLCLIQKALFMHCKKGCRCLGEGHGAGGWRPGPPPPPPPGLA

[0334] SEQ ID NO. 44 TCR1 Nb, including PDGFR™ + PDGFR short stem scaffold

[0335] METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0336] SEQ ID NO. 45 TCR3 Nb, including PDGFR™+CD8 stem support

[0337] METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSSPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIVVVISAILALVVLTIISLIILIMLWQKKPR

[0338] SEQ ID NO. 46 TCR4 Nb, including PDGFR™+ tetramer spiral stem support

[0339] METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSSASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAGVVVISAILALVVLTIISLIILIMLWQKKPR

[0340] SEQ ID NO. 47 VSVG mutant K47A,R354A

[0341] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPASHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0342] SEQ ID NO. 48 scFv TCR1 full-length sequence, including stent and V5 tag.

[0343] MEWSWIFLFLLSGTAGVHSGKPIPNPLLGLDSTEVQLQQSGPELVKPGASVKMSCKASGYKFTSYVMHWVKQKPGQGLEWIGYINPYNDVTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVHYCARGSYYDYDGFVYWGQGTLVTVSAGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSATSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0344] SEQ ID NO. 49 scFv TCR2 full-length sequence, including stent and V5 tag

[0345] MEWSWIFLFLLSGTAGVHSGKPIPNPLLGLDSTEVQLQQSGPELVKPGASVKMSCKASGYKFTSYVMHWVKQKPGQGLEWIGYINPYNDVTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVHYCARGSYYDYDGFVYWGQGTLVTVSAGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSATSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASESKYGPPCPPCPAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0346] SEQ ID NO 50: IgK leader sequence_CD86 IgV_IgG4 hinge_PDGFR TM

[0347] METDTLLLWVLLLWVPGSTGDAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLSASESKYGPPCPPCPVVVISAILALVVLTIISLIILIMLWQKKPR

[0348] SEQ ID 51: IgK leader sequence_TGN1412 scFv_IgG4 hinge_PDGFR TM

[0349] METDTLLLWVLLLWVPGSTGDQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKSASESKYGPPCPPCPVVVISAILALVVLTIISLIILIMLWQKKPR

[0350] SEQ ID 52: IgK leader sequence_TCR-VHH_CD8a hinge_PDGFR TM

[0351] METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSSGASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAKVVVISAILALVVLTIISLIILIMLWQKKPR

[0352] SEQ ID 53: CD8a leader sequence_hu8e5 scFv_CD8a hinge_CD28 TM_CD28 ICD_CD3z ICD (CARsgen CLDN18.2 CAR)

[0353] MALPVTALLLPLALLLHAARPQVQLQESGPGLIKPSQTLSLTCTVSGGSISSGYNWHWIRQPPGKGLEWIGYIHYTGSTNYNPALRSRVTISVDTSKNQFSLKLSSVTAADTAIYYCARIYNGNSFPYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLFNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYSFPYTFGGGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0354] SEQ ID 54: TCR_VHH - B1056

[0355] EVQLVESGGGLVQPGGSLRLSCAASGDVHHINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRADSRIYPYNYWGQGTLVTVSS

[0356] SEQ ID 55: TCR_VHH - B571

[0357] EVQLVESGGGLVQPGGSLRLSCAASGDVSKINILGWYRQAPAKEREMVAHITIGDITDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRALSRIYPYNYWGQGTLVTVSS

[0358] SEQ ID 56:TCR_VHH-B3359

[0359] EVQLVESGGGLVQPGGSLRLSCAASGDVLKINILGWYRQAPAKEREMVAHITIGDAEDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRALSRIYPYNYWGQGTLVTVSS

[0360] SEQ ID 57:TCR_VHH-B162

[0361] EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITSGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0362] SEQ ID 58:TCR_VHH-B197

[0363] EVQLVESGGGLVQPGGSLRLSCAASGDVHVINILGWYRQAPAKEREMVAHITIGDKTDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0364] SEQ ID 59:TCR_VHH-B3710

[0365] EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITYGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCVAYSRIYPYNYWGQGTLVTVSS

[0366] SEQ ID 60:TCR_VHH-B6251

[0367] EVQLVESGGGLVQPGGSLRLSCAASGDVRKINILGWYRQAPAKEREMVAHIHIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCTAYSRIYPYNYWGQGTLVTVSS

[0368] SEQ ID 61:TCR_VHH-B182

[0369] EVQLVESGGGLVQPGGSLRLSCAASLDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0370] SEQ ID 62: TCR_VHH-B341

[0371] EVQLVESGGGLVQPGGSLRLSCAASGDVHRINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0372] SEQ ID 63: TCR_VHH-B291

[0373] EVQLVESGGGLVQPGGSLRLSCAASGDVPKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0374] SEQ ID 64: TCR_VHH-B863

[0375] EVQLVESGGGLVQPGGSLRLSCAASGDVHVINILGWYRQAPAKEREMVAHITIGEATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCSAYSRIYPYNYWGQGTLVTVSS

[0376] SEQ ID 65: TCR_VHH-B2598 ​​​​​​​QVQLQESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSV INSGGGRT KYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYC ALGRDWSQ RGQGTQVTVSS

[0380] SEQ ID NO. 67; Nanobody (VHH) CD14 3RMB96

[0381] QVQLQESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSV INSGGGST RYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYC ALGRDWSQ RGQGTQVIVSS

[0382] SEQ ID NO. 68; Nanobody (VHH) CD14 3RMB138

[0383] QVQLQESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSV INSGGGST RYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYC ALGRDWSQ RGQGTQVIVSS

[0384] SEQ ID NO. 69; Nanobody (VHH) CD14 3RMB187

[0385] QVQLQESGGGLVQPGGSLRLSCAAS GFTFSMYAM TWVRQAPGKGLEWVSA ITSGGGTT RYADSVKGRFTISRDNAKNTLYLQMNSLKADDTAVYYC ALGRDWSQ RGQGTQVTVSS

[0386] SEQ ID NO. 70; Nanobody (VHH) CD14 4EBT28

[0387] QVQLQESGGGLVQAGGSLRLSCAAS GRIFSIRP MGWYRQAPGKEREMVAQ ISSSGIT NYGDSVKGRFTISRGNARNTVYLQMNNLKPEDTAVYYC RLFKTGADS WGQGTQVTVSS

[0388] SEQ ID NO. 71; Nanobody (VHH) CD14 4EBT40

[0389] QVQLQESGGGLVQAGGSLRLSCAAS GRIFSIRP MGWYRQPPGKEREMVAQ ISSNGIT NYGDSVKGRFTISRGSARNTVYLQMNNLKPEDTAVYYC RLFKTGADS WGQGTQVTVSS

[0390] SEQ ID NO. 72; Nanobody (VHH) CD14 4EBT85

[0391] QVQLQESGGGLVQAGGSLRLSCAAS GRIFSIRP MGWYRQPPGKEREMVAQ ISSSGIT NYGDSVKGRFTISRGNARNTVYLQMNNLKPEDTAVYYC RLFKTGADS WGQGTQVTVSS

[0392] SEQ ID NO. 73; Nanobody (VHH) CD14 2EBT32

[0393] QVQLQESGGGLVQAGGSLRLSCVAS GNIRSVGD MGWYRQTLGDQREMVAT IDYGGVA RYADAVKGRFTISRDNAKSAVYLQMNSLNREDTAVYYC NADILYKSDSYARREF WGQGTQVTVSS

[0394] SEQ ID NO. 74; Nanobody (VHH) CD14 2EBT51

[0395] QVQLQESGGGLVQPGGSLRLSCVAS GNIRSVGD MGWYRQTLGDQREMVAT IDYGGVA RYADAVKGRFTISRDNAKSAVYLQMNSLNREDTAVYYC NADILYKSDSYARREF WGQGTQVTVSS

[0396] SEQ ID NO. 75; Nanobody (VHH) CD14 2EBT57

[0397] QVQLQESGGGSVQTGESLRLSCVAS GNIRSVGDMGWYRQTLGDQREMVAT IDYGGVA RYADAVKGRFTISRDNAKSAVYLQMNSLNREDTAVYYC NADILYKSDSYARREF WGQGTQVTVSS

[0398] SEQ ID NO. 76; Nanobody (VHH) CD14 3RMB1

[0399] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSDGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSPYRYLEV WGQGTQVTVSS

[0400] SEQ ID NO. 77; Nanobody (VHH) CD14 3RMB1-2-6

[0401] QVQLQESGGGLVQSGGSLRLSCAVS VSIFSIVD MGWYRQAPGKGRELVAE LSGGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWYPYRFLEV WGQGTQVTVSS

[0402] SEQ ID NO. 78; Nanobody (VHH) CD14 3RMB1-2-2

[0403] QVQLQESGGGLVQSGGSLRLSCAVS SSIFSIVD MGWYRQAPGKGRELVAE LSVGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSLSPYRYLEV WGQGTQVTVSS

[0404] SEQ ID NO. 79; Nanobody (VHH) CD14 3RMB1-2-9

[0405] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSDGSS NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC YADLTGSSWSPYRYLEV WGQGTQVTVSS

[0406] SEQ ID NO. 80; Nanobody (VHH) CD14 3RMB1-2-5

[0407] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSSGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NEDLTGSSWSPYRYLEV WGQGTQVTVSS

[0408] SEQ ID NO. 81; Nanobody (VHH) CD14 3RMB1-2-3

[0409] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSILD MGWYRQAPGKGRELVAE LSNGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSVSPYRYLEV WGQGTQVTDSS

[0410] SEQ ID NO. 82; Nanobody (VHH) CD14 3RMB1-2-19

[0411] QVQLQESGGGLVQSGGSLRLSCAVS RSIFSIVD MGWYRQAPGKGRELVAE LSDGSR NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NDDLTGSSWSPYRYLEV WGQGTQVTVSS

[0412] SEQ ID NO. 83; Nanobody (VHH) CD14 3RMB1-2-7

[0413] QVQLQESGGGLVQSGGSLRLSCAVS GSSFSIVD MGWYRQAPGKGRELVAE LSDGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSFSPYRYLEV WGQGTQVTVSS

[0414] SEQ ID NO. 84; Nanobody (VHH) CD14 3RMB1-2-25

[0415] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSDGSS NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSRYRYLEV WGQGTQVTVSS

[0416] SEQ ID NO. 85; Nanobody (VHH) CD14 3RMB1-2-18

[0417] QVQLQDSGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSVGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSPYRYQEV WGQGTQVTVSS

[0418] SEQ ID NO. 86; Nanobody (VHH) CD14 3RMB1-2-12

[0419] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSYGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGTSWSPYRYLEV WGQGTQVTVSS

[0420] SEQ ID NO. 87; Nanobody (VHH) CD14 3RMB1-2-15

[0421] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVE MGWYRQAPGKGRELVAE LSDGVT NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC TADLTGSSWSPYRYLEV WGQGTQVTVSS

[0422] SEQ ID NO. 88; Nanobody (VHH) CD14 3RMB1-2-16

[0423] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSI VDMGWYRQAPGKGRELVAE LSDGSC NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSLWSPYRYLEV WGQGTQVTVSS

[0424] SEQ ID NO. 89; Nanobody (VHH) CD14 3RMB1-2-20

[0425] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSMVD MGWYRQAPGKGRELVAE LSLGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWEPYRYLEV WGQGTQVTVSS

[0426] SEQ ID NO. 90; Nanobody (VHH) CD14 3RMB1-1-16

[0427] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSILD MGWYRQAPGKGRELVAE LSDGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSEYRYLEV WGQGTQVTVSS

[0428] SEQ ID NO. 91; Nanobody (VHH) CD14 3RMB1-1-28

[0429] QVQLQESGGGLVQSGGSLRLSCAVS GSLFSIV DMGWYRQAPGKGRELVAE LSDGPT NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSLYRYLEH WGQGTQVTVSS

[0430] SEQ ID NO. 92; Nanobody (VHH) CD14 3RMB1-1-9

[0431] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSDGCT NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NIDLTGSSWSPYRYLEV WGQGTQVTVSS

[0432] SEQ ID NO. 92; Nanobody (VHH) CD14 3RMB1-1-14

[0433] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSSGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSPYRYLEV WGQGTQVTVSS

[0434] SEQ ID NO. 93; Nanobody (VHH) CD14 3RMB1-1-27

[0435] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSGGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSPYRYLEV WGQGTQVTVSS

[0436] SEQ ID NO. 94; Nanobody (VHH) CD14 3RMB1-1-13

[0437] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSDGAT NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSYSPYRYLEV WGQGTQVTVSS

[0438] SEQ ID NO. 95; Nanobody (VHH) CD14 3RMB1-1-5

[0439] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSDSST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSPYRYLEV WGQGTQVTVSS

[0440] SEQ ID NO. 96; Nanobody (VHH) CD14 3RMB1-1-6

[0441] QVQLQESGGGLVQSGGSLRLSCSVS GSFFSIVD MGWYRQAPGKGRELVAE LSDGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSPYRYLEV WGQGTQVTVSS

[0442] SEQ ID NO. 97; Nanobody (VHH) CD14 3RMB1-1-24

[0443] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LYDGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSYWSPYRYLEV WGQGTQVTVSS

[0444] SEQ ID NO. 98; Nanobody (VHH) CD14 3RMB1-1-2

[0445] QVQLQESGGGLVQSGGSLRLSCAVS GSSFSIVD MGWYRQAPGKGRELVAE ISDGST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSPYRYLEV WGQGTQVTVSS

[0446] SEQ ID NO. 99; Nanobody (VHH) CD14 3RMB1-1-4

[0447] QVQLQESGGGLVQSGGSLRLSCAVS GSIFSIVD MGWYRQAPGKGRELVAE LSDAST NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC YADLTGSSWSPYRYLEV WGQGTQVTVSS

[0448] SEQ ID NO. 100; Nanobody (VHH) CD14 3RMB1-1-29

[0449] QVQLQESGGGLVQSGGSLRLSCAVS GSLFSIVD MGWYRQAPGKGRELVAE LSDGLT NYADSVLGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSPYRYLEV WGQGTQVTVSS

[0450] SEQ ID NO. 101; Nanobody (VHH) CD14 3RMB32

[0451] QVQLQESGGGLVQPGGSLRLSCAAS GSIFSTNA MSWYRQAPGKERELVAE ITSGGST NYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLTGSSWSPYRYLEV WGQGTQVTVSS

[0452] SEQ ID NO. 102; Nanobody (VHH) CD14 3RMB25

[0453] QVQLQESGGGLVQPGGSLRLSCSAS ESVKSINV MGWYRQAPGKERESVAA ISAEGRT NYTDSVKGRFTISRDKGKDTVYLQMNDLKPEDTAVYYC HADRILYKTDLYYVKNDV WGQGTQVTVSS

[0454] SEQ ID NO. 103; Nanobody (VHH) CD14 3RMB71

[0455] QVQLQESGGGLVQLGGSLRLSCSAS ESVKSINV MGWYRQAPGKERESVAA ISAEGRT NYTDSVKGRFTISRDKGKDTVYLQMNDLKPEDTAVYYC HADRILYKTDLYYVKNDV WGQGTQVTVSS

[0456] SEQ ID NO. 104; Nanobody (VHH) CD14 3RMB142

[0457] QVQLQESGGGLVQAGESLTLSCQPS ERISRIDV LGWYRQAPDKDRELVAL SGKGGRT YYADSVKDRFTISRDTANNRLYLQMNKLEVDDTAIYYC VTEAEGGGYT SGQGTQVTVSS

[0458] SEQ ID NO. 105; Nanobody (VHH) CD14 3RMB62

[0459] QVQLQESGGGLVQAGDSLTISCAAS GAASTFTL MAWYRQAPGKQRKLVGS ITGSGRT AYAQSLKDRFTISRTDAKDTMYLQMNNLKPEDTAVYYC NAISTNGGTMEDY WGQGTQVTVSS

[0460] SEQ ID NO. 106; Nanobody (VHH) CD14 3RMB107

[0461] QVQLQESGGGLVQSGGSLRLSCTVT GRIPARYS LGWYRQLPGKEREFVAA INWNGDNT YYENSVKGRFTISRDNTKNTVDLQMRSLRVEDTAVYYC AASGHSDYIDNGFGT FGQGTQVTVSS

[0462] SEQ ID NO. 107; Nanobody (VHH) CD14 3RMB126

[0463] QVQLQESGGGLVQPGGSLRLSCAAS GRTFSIYA MGWFRQAPGKEHDFVAA ISWSGGNT YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AATTHIPYSDYDADEYDY WGQGTQVTVSS

[0464] SEQ ID NO. 108; Nanobody (VHH) CD14 3RMB76

[0465] QVQLQESGGGSVQAGGSLRLSCTTS RPISSIYA MGWFRQAPGTERESVAA INWSSGNG YYADSVKGRFTISRDNAKKTVYLQMNNLKSEDTAVYYC AAARQATGWSILTAAAHEY WGQGTQVTVSS

[0466] SEQ ID NO. 109; Nanobody (VHH) CD14 3RMB134

[0467] QVQLQESGGGLVQAGGSLRLSCAAS GRTFSSYA MGWFRQAPGKERAFVAA ISWSGGNT DYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AADRRDLYGSRWYAAGWYDY WGQGTQVTVSS

[0468] SEQ ID NO. 110; Nanobody (VHH) CD14 3RMB50

[0469] QVQLQESGGGLVAPGGSLRLSCVAA GSRFGFDYY AIGWFRQALGRQREMVSC ISGSGRGS NYADSVKGRFTISRDNAKNTVYLQMESLKPEDTDVYYC GAVYSPVYSDLALCRLDSIST WGQGTQVTVSS

[0470] SEQ ID NO. 111; Nanobody (VHH) CD14 3RMB18

[0471] QVQLQESGGGLVQSGDSLKLSCHDP GAIIVGQT VAWFRQVPGKEREGIAC ITWDSNWK NYADSVKDRFTISRDNSKKEIYLQMDSLKPEDTAVYYC AGDEYLNYCDSPRAFQRGTYDY WGQGTQVTVSS

[0472] SEQ ID NO. 112; Nanobody (VHH) CD14 3EBT162

[0473] QVQLQESGGGLVQTGGSLRLSCTAS GQTFSDNT MAWWRQAPGKQREMVAI ITRNNKT TYGDSVKGRFTISRDIPKNMVFLQMNNLNSEDTGVYYC NAGFVY WGQGTQVTVSS

[0474] SEQ ID NO. 113; Nanobody (VHH) CD14 2EBT91

[0475] QVQLQESGGGSVQAGGSLRLSCVAS GASSFIA LGWYRQVPGKLRELVAR IVDTGSI NYADHVKGRFTISRDNAKNALYLEMNSLKPEDTAIYYC KMNTKYDVY WGQGTQVTVSS

[0476] SEQ ID NO. 114; Nanobody (VHH) CD14 2EBT9

[0477] QVQLQESGGGSVQPGGSLRLSCSRS GPIYSVDT MGWYRRAPGEQRELVAS ITVGGTT TYADSVKGRFTISRDNTMNTVYLQMNNLKIEDTDVYYC NLNRRLAKSEDY WGRGTQVTVSS

[0478] SEQ ID NO. 115; Nanobody (VHH) CD14 2EBT95

[0479] QVQLQESGGGLVQAGGSLRLSCTVS GASFTFNA MGWYRQAPGKQRELVAR ITKSGTI DYADSVKGRFTISGDSHNNAVYLKMNTLELDDTGVYYC YARLYYMHSWFDV YGKGTQVTVSS

[0480] SEQ ID NO. 116; Nanobody (VHH) CD14 3EBT10

[0481] QVQLQESGGGLVQSGESLRLSCTTS GDIFGVTG VGWYRQAPGKQRELVAA LSNEGYT NYKDPVKGRFTISRDNNKTAVYLQMNDLKPEDTAVYYC KVHCTSSGCQFVGHQSFDY WGQGTQVTVSS

[0482] SEQ ID NO. 117; Nanobody (VHH) CCR2 3RNA74

[0483] QVQLQESGGGLVQTGGSLRLSCAAS GRTFSNYA MGWFRQAPGKGRAFIGG ISRSGGNT HFADSVMGRFTISRDNAKNTVYLQMNSLTPEDTAVYYC AAQYQWTEAVPLGNYNI WGQGTQVTVSS

[0484] SEQ ID NO. 118; Nanobody (VHH) CCR2 2RRNA31

[0485] QVQLQESGGGLVQPGGSLRLSCAAS GSSTSINA MGWYRQAPGKERELVAR ITGSGST SYSDSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC GASRFRVGSQRTEYGY WGQGTQVTVSS

[0486] SEQ ID NO. 119; Nanobody (VHH) CCR2 3RSTM31

[0487] QVQLQESGGGLAQPGGSLRLSCTSS ISIFRLNS MGWYRQTPGKERELVAT ITNGGTV NYADSVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCKSWPFPQEY WGKGTQVTVSS

[0488] SEQ ID NO. 120; Nanobody (VHH) CCR2 3RZK33

[0489] QVQLQESGGGSVQPGGSLRLSCVVS GFTTDNYI IAWYRQAPGKGREGVSS ISRSDGST YYAPSVKGRFSITSDSAKNTVSLQMNSLKPEDTAVYYC NAHDSYGNERDY WGQGTQVTVSS

[0490] SEQ ID NO. 121; Synthetic promoter #11: Enhancer 033 – proximal promoter CD3d

[0491] gggaagatgtgagacaaacatcacaattttgcctaaggtgaatccaacccacaagtagagcacaggccaacagcagctcactagtacacatacttacaccagcagctcactagtacacacacttacaccagacgctcactggtacacactcacaccggcattcatgcttacccacgggctggtcaacaaagaggtgctgacctgagagtagggcacataacctcagccactggggtacacttaccacccccgcccccgtgtagctccctcccctatcctgaaatctcccttagcacactaagtattctaggttaaacagcccagatgttcagggagttcattcgccaggtgggaagtaatgagatgtaaatcggggtcttttaggtaatagctgacaactggggtaggaatgggggttctgagcccagtgctggctgccgggctgtttatctctgagtcacatcagcaccaagccacagcagccacctgccctagctccatctcttacagtcacaacaggatgtggtttgacatttactgggtcctgcatctggggtgcctgtgaaagttgctccctccacccacccaccttcagagcatcatgagaaccacactcaccgcatccggcacccaaccccctcctatgctgctctttctacctgggcccctggttagcatcctggcagtagaaatagaatttacaccgcttccttcctaccctacccctagcccacccccactctgaaaatttcccaccatcaacggcagaaagcagagaagcagacatcttctagttcctcccccactctcctctttccggtacctgtgagtcagctaggggagggcagctctcacccaggctgatagttcggtgacctggctttatctactggatgagttccgctgggag

[0492] [[ID=*3*]]SEQ ID NO. 122; Synthetic promoter #12: Enhancer 034 - proximal promoter CD3d

[0493] Gggaagatgtgagacaaacatcacaattttgcctaaggtgaatccaacccacaagtagagcacaggccaacagcagctcactagtacacatacttacaccagcagctcactagtacacacacttacaccagacgctcactggtacacactcacaccggtgggaagtaatgagatgtaaatcggggtcttttaggtaatagctgacaactggggtaggaatgggggttctgagcccagtgctggctgccgggctgtttatctctgagtcacatcagcaccaagccacagcagccacctgccctagctccatctcttacagtcacaacaggatgtggtttgacatttactgggtcctgcatctggggtgcctgtgaaagttgctccctccacccacccaccttcagagcatcatgagaaccacactcaccgcatccggcacccaaccccctcctatgctgctctttctacctgggcccctggttagcatcctggcagtagaaatagaatttacaccgcttccttcctaccctacccctagcccacccccactctgaaaatttcccaccatcaacggcagaaagcagagaagcagacatcttctagttcctcccccactctcctctttccggtacctgtgagtcagctaggggagggcagctctcacccaggctgatagttcggtgacctggctttatctactggatgagttccgctgggag

[0494] SEQ ID NO. 123; Synthetic promoter #13: Enhancer 035 - proximal promoter CD3d

[0495] ggtgggaagtaatgagatgtaaatcggggtcttttaggtaatagctgacaactggggtaggaatgggggttctgagcccagtgctggctgccgggctgtttatctctgagtcacatcagcaccaagccacagcagccacctgccctagctccatctcttacagtcacaacaggatgtggtttgacatttactgggtcctgcatctggggtgcctgtgaaagttgctccctccacccacccaccttcagagcatcatgagaaccacactcaccgcatccggcacccaaccccctcctatgctgctctttctacctgggcccctggttagcatcctggcagtagaaatagaagggaagatgtgagacaaacatcacaattttgcctaaggtgaatccaacccacaagtagagcacaggccaacagcagctcactagtacacatacttacaccagcagctcactagtacacacacttacaccagacgctcactggtacacactcacacctttacaccgcttccttcctaccctacccctagcccacccccactctgaaaatttcccaccatcaacggcagaaagcagagaagcagacatcttctagttcctcccccactctcctctttccggtacctgtgagtcagctaggggagggcagctctcacccaggctgatagttcggtgacctggctttatctactggatgagttccgctgggag

[0496] SEQ ID NO. 124; Synthetic promoter #14: Enhancer 037 - proximal promoter CD3d

[0497] aaataaacaaggagatagggtgtttattttatggacaagtttcttttgtaacttgtaactcccttgaaagtcagccagagtatgtctcaaaccaaagtcaagatagtgagcaagaagtgttgcacttatgaagggaggtgagtgagcaatgcatgtggtttccaaccgttaatgctagagttatcactttctgttatcaagtggcttcagctatgcaaggaaaccaaacaggggaagttttctcaagcaggttgaaagcaggttccaagaaagccctttaaataaacaaggagatagggtgtttattttatggacaagtttcttttgtaacttgtaactcccttgaaagtcagccagagtatgtctcaaaccaaagtcaagatagtgagcaagaagtgttgcacttatgaagggaggtgagtgagcaatgcatgtggtttccaaccgttaatgctagagttatcactttctgttatcaagtggcttcagctatgcaaggaaaccaaacaggggaagttttctcaagcaggttgaaagcaggttccaagaaagccctttagaaatctagctgctacggcttgcgctatggggccgacggcttctctcaaggggcttcgagatgtggcagtgtttaggttgtgtgtaaatgtggttgcattgtcaatagggacgctaaagttcaggccaccttttccatattctctgccagctccctgctcagagatagagcaatttacaccgcttccttcctaccctacccctagcccacccccactctgaaaatttcccaccatcaacggcagaaagcagagaagcagacatcttctagttcctcccccactctcctctttccggtacctgtgagtcagctaggggagggcagctctcacccaggctgatagttcggtgacctggctttatctactggatgagttccgctgggag

[0498] SEQ ID NO. 125; Synthetic Promoter #15: Proximal Promoter CD5

[0499] Cccacccagacccctgcctcagggacgcctgtcctcagcccagccctcagctgcagccaggccttcagcctccgtaacccccgctcagggtccccaccccctgcagccctgtccctccaggatgcatggccttgtcctgtgtgggggtggccgagagcactgccccagccctgggtaccttgggcaggaagctggcagaggccagggctgccattcaaacaggggcaggtggttttgccaggaggaagttgacagttcaacttcaaacatgggtgacgcaggccccacactgcctgctccccgtcccacccctccctgagcacgccaccccgccctctccctctctgagagcgagatacccggccagacaccctcacctgcggtgcccagctgcccaggctgaggcaagagaaggccagaaaccatgccc

[0500] SEQ ID NO. 126; Synthetic Promoter #16: Proximal Promoter C8a

[0501] cctattcacgggccccagcctcctcgccgggctggaaggcgacaaccgcgaaaaggagggtgactctcctcggcgggggcttcgggtgacatcacatcctccaaatgcgaaatcaggctccgggccggccgaagggcgcaactttcccccctcggcgccccaccggctcccgcgcgcctcccctcgcgcccgagcttcgagccaagcagcgtcctggggagcgcgtc

[0502] SEQ ID NO. 127; Synthetic Promoter #17: Enhancer 023 - NFAT RE – Proximal Promoter CD3d

[0503] ggcattcatgcttacccacgggctggtcaacaaagaggtgctgacctgagagtagggcacataacctcagccactggggtacacttaccacccccgcccccgtgtagctccctcccctatcctgaaatctcccttagcacactaagtattctaggttaaacagcccagatgttcagggagttcattcgccaggtgggaagtaatgagatgtaaatcggggtcttttaggtaatagctgacaactggggtaggaatgggggttctgagcccagtgctggctgccgggctgtttatctctgagtcacatcagcaccaagccacagcagccacctgccctagctccatctcttacagtcacaacaggatgtggtttgacatttactgggtcctgcatctggggtgcctgtgaaagttgctccctccacccacccaccttcagagcatcatgagaaccacactcaccgcatccggcacccaaccccctcctatgctgctctttctacctgggcccctggttagcatcctggcagtagaaatagaaggaaagggaaatagggaaagggaaacagggaaagggaaaagaaatctagctgctacggcttgcgctatggggccgacggcttctctcaaggggcttcgagatgtggcagtgtttaggttgtgtgtaaatgtggttgcattgtcaatagggacgctaaagttcaggccaccttttccatattctctgccagctccctgctcagagatagagcaatttacaccgcttccttcctaccctacccctagcccacccccactctgaaaatttcccaccatcaacggcagaaagcagagaagcagacatcttctagttcctcccccactctcctctttccggtacctgtgagtcagctaggggagggcagctctcacccaggctgatagttcggtgacctggctttatctactggatgagttccgctgggag

[0504] SEQ ID NO. 128; hsa-mir-582-5p:

[0505] AGTAACTGGTTGAACAACTGTAA

[0506] The three repetitive sequences are located at the 3' of the transgene or the 3' of the WPRE.

[0507] SEQ ID NO. 129; hsa-mir-5196-3p

[0508] CTGGGAGGGAGACGAGGATGA

[0509] The three repetitive sequences are located at the 3' end of the transgene.

[0510] SEQ ID NO. 130; Synthetic promoter #1: Proximal promoter CCL13

[0511] Ggcttcccatggtgaatggctggggcgcgtctgtgtccctttctcctctctggctccttgtggcctgaacagccagaaggaagccatgccatgctgtttcagccctcagcttccctcttgcatttcctagaaaagtctttggtgcccagctccagctcagca gattcaggatcccccttcatcatgacttggtcaacgccctgctcaggccaaggtcctctgagagttccaagcttctccactccctataaaaggccggcggaacagccagaggagcagagaggcaaagaaacattgtgaaatctccaactcttaaccttcaac

[0512] SEQ ID NO. 131; Proximal promoter CCL18

[0513] ggggaagatttctcattagatcacgaaaggcctaactgtatataaaagattgaagcatgtgactctcttaaaaggaaaaacaattgtgaaaatccattaaagcattctgctctatcaaaggaggctgtgaccactcatttctgagaaatatctgtcatgtgaaaacataatcacatgattatgcataatcctaagcataatccctgggtgcttccaactcctggtcacccatctggagatacaacttctccataagcccctcaatcaacaagtgaaccactcgtttgtgtaccctaaacctttcaagtaaccataggcaaccctgggctgagaactcacatgacaccaccatagttaaaatgtcttgcatcagcacctccaatgttcccatggtccagtctgcctataaaaaggagagacaacagctcataccccagaaggaggccaggagttgtgagtttccaagccccagctcactctgaccacttctctgcctgcccagcatc

[0514] SEQ ID NO. 132; Synthetic promoter #3: proximal promoter MRC1

[0515] CccaggggaagctccgtcttccggcacaggtaatggcctgcagcttgatctccacccagccccatctgagcaggccgggagctcccaggctgtttcacttcTctccttcctgactcctcaccatcaccatcgccctctctcctccccaccccgccactcctctcccacacgtgtccctttctccccttcctctgcgtctgctcttctcagaagttagcttacgaagcaaagttgttactttgaattcctgtttttccagccaccctcatgtgacaggatgtctcctcagtagaggctttccctaaattcaggagccctttaaaagggagggcttcctctgtagttcttttcagctgggcagctctgggaacttggattaggtggagaggcagttggggggcctcgttgttttgcgtcttagttccgccctcctgtccatcaggagaaggaaaggataaaccctgggcc

[0516] SEQ ID NO. 133; Synthetic promoter #4: proximal promoter LYZ

[0517] catacaaaacaatttcacaaaattgaatgttactcaaaaatcacagctcattttaagctgcacaaaatagtcatttttttctttataattgctcaaattcataatcaaacagaagaaagttcctgtcttggaagtagtgctatgccccaattcttccagagccagtactttaaacaattccatttcattattttcctgtagactaattcttaggacatcagcatatctctcttcaagcattaaaaaaatctctttagagtcagtggatcaatagacagttcctgttttccacacaactgaaagggtggagcccccaaaccacaaggggaagaaggaagttaaaagatgttaaatactggggccagctcaccctggtcagcctagcactctgacctagcagtcaac

[0518] SEQ ID NO. 134; Synthetic promoter #5: Proximal promoter HLA-DR

[0519] Gcaaactctccaactgtcattgcacagacatatgatctgtatttagctctcactttaggtgtttccattgattctattcactaatgtgcttcaggtatatccctgtc tagaagtcagattggggttaaagagtctgtccgtgattgactaacagtcttaaatacttgatttgttgttgttgttgtcctgtttgtttaagaactttacttctttatcc aatgaacggagtatcttgtgtcctggaccctttgcaagaacccttcccctagcaacagatgcgtcatctcaaaatatttttctgattggccaaagagtaattgatttgc attttaatggtcagactctattacaccccacattctcttttcttttattcttgtctgttctgcctcactcccgagctctactgactcccaacagagcgcccaagaagaaa

[0520] SEQ ID NO. 135; Synthetic promoter #6: Enhancer 008 – Proximal promoter CHIT1

[0521] Aggactgagctctgagatccttctgaagaggttgggagaagaggagaatcagcaagagaaactgagaaggagggtcatgggcggtaggcgggaagcaggagagtgtggggtcagaggacagatgtagaaatcagttccaggaagcgagggccgagtgtcagaagctgctgctgagtcactcaagatgaagacagaactcatcactggagtcactcaggcagccttggtgaccttgataaaaatagtttaggtggagtaggtgccgggttggagcacaggcttggttggaatggggttaagggaggatgtgagcagtagaacagggtgcagccaacacagacaactactttggagaagttttgcaaaagggaagtaaaaaatgggcagtggttgatgaggagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0522] SEQ ID NO. 136; Synthetic promoter #7: Enhancer 015 - proximal promoter CHIT1

[0523] Atgtccttcccaggggtgaaagccccctgcctgctgggttcctgccaagactcagctcaaacactaggatgctttggggccccaaggacccagtcacgggactgccccacccgcttcccctctgcggtattaatacgcctcttgctcacttcgggggaaaaacagaaaattgtgcggatgtgggccccagatctgtgggtgtcggttcttctttcagatttgggatcctctgcatgttgggggaggccgcgtctcgctcctcgtttgacctaggtcagaattcaacaggaaggagtttagggtgtccagcaacaacaaccctcaaccatctccagctgagctctcatcccagccccggccctgaccgtaacccgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc <舍

[0524] SEQ ID NO. 137; Synthetic Promoter #8: Enhancer 022 - Proximal Promoter CHIT1

[0525] Note: There seems to be an error in the original text where "舍0001285" is likely a misrepresentation. It should probably be something like "

[0524] " which is preserved as is in the translation. Also, the Chinese part in the original text might be a bit unclear in terms of its correct form and intended meaning. The translation is done based on the best understanding of the provided text.Tagtctcaaaatgcctttcagcataggcaggaaatatgacagaagaaagtcccagtcgaacaaacaataaccgaaaatgctctttgttgctgggccttggcgatcaaagccctgcacatgtttgccagctgtagttcagatggtttctatttcactcagcattaattttttatgaaatctgtggcccaggggcccataagcggcttttagttcttcattctatactgtctggtctaattgccaaggcatggtaagactcagattttaaggtgtcgttaatgtatttaccagccttccggtctacaggcaggctcacctctttgtgtgggattttaatgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0526] SEQ ID NO. 138; Synthetic promoter #9: Enhancer 029 - proximal promoter CHIT1

[0527] agaaagagtgtacttattgaagatgaattgttttcttaatctagtattatcagcctgacagagttattcacaagtagttaagttcctgggaatcacatgagcagcagcaatcccaagaaacctgagtcagtaaaaaaataaattgctgatcttgaagagattcaaagaggaaggcaaaagcattgtcagctttgttcaaactgtatctcaaatttagttgttttttgtttttctatccgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0528] SEQ ID NO. 139; Synthetic promoter #10: Enhancer 031 - proximal promoter CHIT1

[0529] taagaccctaaaagtcacactgtccttagacagttagggaagtagaatggataacatatgcccaaaatacgaatgactaatttccttgcaagatcagctgtctcctgcaagcaacatttgacaattcaccctttcctcgtgatgatgtcttaactaagaagatacaaaaatgcaaggaaatgaaagtgaatatgagatcacttttatgtgctattttaaaaacataaatgcacattgcactgtgtttagggaactatggcttcagaagagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0530] SEQ ID NO. 140; Synthetic Promoter #11: Enhancer 034 - Proximal Promoter CHIT1

[0531] aagctgtggcccagaacaccccgatcccactcagaacactatttataagaatgccccgtatgcagtagtctttacagtttttctttttctcaaatgttctttagcctatgtagatgacatttatgcaattcagaggaagatgcctcaacaggattagtcagcagcaaagtcctctgacgcagcttctgtgatctagttaaaaataggaaagaggatgttttgcataagtcatcaccattgtgcggtattggagaattgaccacttttttggttcccacatcctgctaatttatctaaaaatagatgaaaaccctgtccctgaattcctggacaagaccttttgcttctgtgacctcggggctcaggctcttcttgttctgggctccgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0532] SEQ ID NO. 141; Synthetic promoter #12: TAM1 - proximal promoter CHIT1

[0533] agactctgttccctgtatttgcatccactgcctgcgtgctttctctctcccccatatttctgacatttggattttccccctactaattgacatgcctgtcgcctccttctcctggtgtccatttctggaactgctatttgtatattgcatgatattttatcaggaaaaaatataaaacagatctaatttcatgaaaatgctgttgccataatgctcaggagaacagggggaagagagagattcaagtcgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0534] SEQ ID NO. 142; Synthetic promoter #13: TAM2 - proximal promoter CHIT1

[0535] agaaaagagttcacccaataagtttcccaaatttcaaggagcttttgttttattagatgctgttatcttcatgttagtcacaggaaaaaaaaatgcccacatacacacgcacaactaaatgtagtcagtttgctcagctgctgctgacgtcactgctgcccactaggagtgagttactgtcataacccctcttttattaatcataggctggaggaagcacaaagtgagtgacatcattaggcaaaggaccggctaactcatgctccaatttactcagctttattataacatttctcgagtgtcatatgaaagtgattaccatttcacactttccccatctgagcaatcatccctaaccttcaattcaaattcccagagaaaagcttagcttttcagatggaggtgcgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0536] SEQ ID NO. 143; Synthetic promoter #14: TAM3– proximal promoter CHIT1

[0537] ttctttcagcaaaacctttcggagaagcccaatactaagctcctctggttagagccagccatgagagaaactccaagtacttctgactggttctctctctactcatccaccccttaggtggctgcagaaggaactctgtgcaacccccagagttctcattctcagtgacagggaaatgtaatgattggccctggatgattcagcagatcagatgatacttactcagagcaatttccactcctttgcagtagcatattatcagtattttccagataaataacttggctaaagaaaaatccatttcatttacatctttggcaccttacagcaatagaacttttgtgcaatgattttaatattatatttctacattggctgatagagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0538] SEQ ID NO. 144; Synthetic promoter #15: TAM4 - proximal promoter CHIT1

[0539] gaaatcctcgaaatgagatactgtcccgtgattctgagagtgacccaaaaggatacggaatgaggtactaaaagcaggtgcttggaaacctgagggcagtggtgttcatgcgtttcccggcactttgactttcagcctcctgcaagcctctccttctttgccttcctgaccgttagatacaattaatttggggtttgttttttctgcttcctctttcccctcccaccctatgtttcagttctaaagttagaaggtcctctcatccttttaagaagaacagagacagaaaatggttagtcaccatctaggacagactacaattccagatcattgcaggacgtgcccactcaaatggatgattattgagagctggccacctgaagtgttttacacaggcagtgatctaagggttgaagctatctggcctttttgcttgcttgttatgtatttatggctgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0540] SEQ ID NO. 145; Synthetic promoter #16: TAM5– proximal promoter CHIT1

[0541] ccctagctctggggaaatgaaagccaggctggggttcaaatgagggcagtttcccttcctgtgggctgctgatggaacaaccccatgacgagaaggacccagcctccaagcggccacaccctgtgtgtctctttgtcctgccggcactgaggactcatccatctgcacagctggggcccctgggaggagagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0542] SEQ ID NO. 146; Synthetic promoter #17: TAM6– proximal promoter CHIT1

[0543]

[0544] SEQ ID NO. 147; Synthetic promoter #18: TAM7 - proximal promoter CHIT1

[0545] aaggcggcgccagggagagggcgcagtctctgaatctttccacggggtccatcctagggcccctccaggttcaacggtctcctaacctgtagtcacccacaagagcgtgccctttctgcccgcccttcctagcgttgctccctgcttggctgagcactgcggagtttcacgcctctaaaccccgcctcttcttgcaacctgtgttggcctcatctacccagcaactgtcgacgtcacacgacctgggcctccctgaatgggagatatttactaggcaatcccgcctactgttctgaggtttcccctccaggtgcagcttcagagccaggcgagccaggaaggaccagcgggcgaggtggtgagttgtgaggcgcgcccagagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0546] SEQ ID NO. 148; Synthetic promoter #19: TAM8 - proximal promoter CHIT1

[0547] cttctgcccgacccgaggtacctccaccctgtctaccaggttcagccccgccctctcatcatgtattggcccccaaaacgcggctcttccctcccatcagtttgtctttccactctcattggtcctcaggacgaccgtgactccgcccacctacaccacatttccaccactatccctgacttccaatggccccgccccagccactaaggttcggccttttctgcccagctggccgcctcttccttggtctggtgtcccaggcaccgcccacgggtctagcctcttctcaggagtgctctacagcgccccctaggccaccaagattgtttagctccctgtcgggtcggcccctgactccttattggactcatccatctggctcatccaaggccttgggtctctccagctgactcgacggtgatggggggcaactcggcgggggagctgtggagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0548] SEQ ID NO. 149; Synthetic promoter #20: TAM9–proximal promoter CHIT1

[0549]

[0550] SEQ ID NO. 150; Synthetic promoter #21: TAM10 – proximal promoter CHIT1

[0551] cctagctcttagtgtaatgattatgggaacagggtcaaatagcaaatcacaaattatttcacgactattgtgagacaagagtgagagccaagagtggtggtgcacacctgtaatcccagctcgaaccagggagacagaggttgcagtgagccgagatcgcaccaatggcttaacacaatgggatttggcttttcactcatgtaaatgaccagtgcggttttctctggggcagaatttgaattttctccccaggatgactcagggacacccgcttcctccattgtgctcccatcatgcccttgggcaatggcgtcccgtacttcaaacagcaagaaagaaggagaaacagaatatatctgtgcacttgcaaaatttgctgacacatgtcatttctgtccagaattgttggctataaatagtgttatgaacagtcacacaatgatgcaacaagaccagggaaaaagtcccttggtgggggtagccacttcccagaggttccaatgcttgcactgtaagggtaagtacagaattttttttttttctggtgagcagccagagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc。

[0552] SEQ ID NO. 151; Synthetic promoter #22: TAM11 – proximal promoter CHIT1

[0553] ttattactctatagataaagcagagtctctgaagttaactaacttccccaaagtcacacatcgtggaaccatgaatcttaccaggttctgtgtgactccaaatttgtgctctttcatattgcacaacactgtccagattaattcaagtggagagtgaactttttctggtacatcccttagtctcttgcacagccattgcctttcacacacccagactgtttctttgctttagggactggaaagtggaagtggaaccccctatgttctgtcctgggatatttccggggtgagccaagagtagctgtgtgtcctgctaggtcatggactttcactgaccacagaagttgagaacccttgagcaaactaaattcttatatcttactggtttgggagcaaagcagaagtggcaaaagctgacaaaacccagctggagcaggaatgttgaaatggacacaaataaacataccctgtggacacaatcaatataaatgttctaaatttacttctgctcctatgcttgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0554] SEQ ID NO. 152; Synthetic promoter #23: TAM12 - proximal promoter CHIT1

[0555] Aaaaagtgttaaatcgtccaaaaaggaggaaagaaaggggaaaaaaagtaacaattctaaatctttatgtgcctgataacacggcttcaacacatcgaataaaatgtttacagaactgtaagtagaatcgcagcggaagatttcaacacaattcttttcatatggaaagaggaagaagcacgtaaaaatcacagaactacaagcacgtggagcatgttctctgatcacagtggaactaagcctgaaacctaaaacaaagagataaccagaaacacatatgtttgtaaattaagaaatgcatttctaaataactcctgggtcaaagagcagctcacagtggaaatcagaaagtattttgaactgaaagacgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0556] SEQ ID NO. 153: VSVg-Cl7 I52R, I331E, E355L

[0557] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKARQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTTERLLWDDWA

[0558] SEQ ID NO. 154:VSVg-Cl12 N9Q,S48V,V333I,E353K

[0559] KFTIVFPHQQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKVHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIAAPILSRMVGMISGTTTKRELWDDWA

[0560] SEQ ID NO. 155:VSVg-Cl26 N9G,K47G,I331E,T352S

[0561] KFTIVFPHGQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPGSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTSERELWDDWA

[0562] SEQ ID NO. 156:VSVg-Cl47 H8F,A51P,R329V,T352V

[0563] KFTIVFPFNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKPIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETVYIRVDIAAPILSRMVGMISGTTVERELWDDWA

[0564] SEQ ID NO. 157:VSVg-Cl135 N9Q,K47T,T328S,E355L

[0565] KFTIVFPHQQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPTSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFESRYIRVDIAAPILSRMVGMISGTTTERLLWDDWA

[0566] SEQ ID NO. 158:VSVg-Cl129 N9G,K50S,V333I,T351R

[0567] KFTIVFPHGQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHSAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIAAPILSRMVGMISGTRTERELWDDWA

[0568] SEQ ID NO. 159:VSVg-Cl33 H8F,S48L,R329K

[0569] KFTIVFPFNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKLHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETKYIRVDIAAPILSRMVGMISGTTTERELWDDWA

[0570] SEQ ID NO. 160:VSVg-Cl59 H8Y,K47G

[0571] KFTIVFPYNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPGSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWA

[0572] SEQ ID NO. 161:VSVg-Cl145 N9H,S48R,T352E

[0573] KFTIVFPHHQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKRHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTEERELWDDWA

[0574] SEQ ID NO. 162:VSVg-Cl139 H49F,R329T,T350G

[0575] KFTIVFPHGQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSFKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETTYIRVDIAAPILSRMVGMISGGTTERELWDDWA

[0576] SEQ ID NO. 163:VSVg-Cl58 H8L,M45L,I331E,E355R

[0577] KFTIVFPLNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKLPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTTERRLWDDWA

[0578] SEQ ID NO. 164:VSVg-Cl87 K11E,S48F,Y330L,T352D

[0579] KFTIVFPHNQEGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKFHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRLIRVDIAAPILSRMVGMISGTTDERELWDDWA

[0580] SEQ ID NO. 165:VSVg-Cl386 N9D,A51S,I331T,E355H

[0581] KFTIVFPHDQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKSIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYTRVDIAAPILSRMVGMISGTTTERHLWDDWA

[0582] SEQ ID NO. 166:VSVg-Cl223 A51T,R329W

[0583] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKTIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETWYIRVDIAAPILSRMVGMISGTTTERELWDDWA

[0584] SEQ ID NO. 167:VSVg-Cl363 Q53A,I331E,T352I

[0585] KFTIVFPHNVKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIAADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTIERELWDDWA

[0586] SEQ ID NO. 168:VSVg-Cl549 F6P,Q10K,K50T,V333P,T352R

[0587] KFTIVRPHNKKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHTAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRPDIAAPILSRMVGMISGTTRERELWDDWA

[0588] SEQ ID NO. 169:VSVg-Cl477 S48M,Y330P,T351D

[0589] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKMHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRPIRVDIAAPILSRMVGMISGTDTERELWDDWA

[0590] SEQ ID NO. 170:VSVg-Cl164 N9T,I52T,R329K,T352E

[0591] KFTIVFPHTQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKATQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETKYIRVDIAAPILSRMVGMISGTTEERELWDDWA

[0592] SEQ ID NO. 171:VSVg-Cl167 K47V,D334A,R354D

[0593] KFTIVIPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPVSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVAIAAPILSRMVGMISGTTTEDELWDDWA

[0594] SEQ ID NO. 172:VSVg-Cl436 H8S,H49A,R329H,T352Q

[0595] KFTIVFPSNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSAKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETHYIRVDIAAPILSRMVGMISGTTQERELWDDWA

[0596] SEQ ID NO. 173:VSVg-Cl261 K11A,S48L,I331L,R354K

[0597] KFTIVFPHNQAGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKLHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYLRVDIAAPILSRMVGMISGTTTEKELWDDWA

[0598] SEQ ID NO. 174:VSVg-Cl194 P7W,M45F,V333Y,R354V

[0599] KFTIVFWHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKFPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRYDIAAPILSRMVGMISGTTTEVELWDDWA

[0600] SEQ ID NO. 175:VSVg-Cl1003 N9R,Q53I,I331Q,R354M

[0601] KFTIVFPHRQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIIADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYQRVDIAAPILSRMVGMISGTTTEMELWDDWA

[0602] SEQ ID NO. 176:VSVg-Cl368 H8S,I52V,V333Y,T351S

[0603] KFTIVFPSNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAVQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRYDIAAPILSRMVGMISGTSTERELWDDWA

[0604] SEQ ID NO. 177:VSVg-Cl6915 I331E,E353K

[0605] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTTKRELWDDWA

[0606] SEQ ID NO. 178:VSVg-Cl125 Q10V,K50H,I331M,T352D

[0607] KFTIVFPHNVKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHHAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYMRVDIAAPILSRMVGMISGTTDERELWDDWA

[0608] SEQ ID NO. 179:VSVg-Cl331 N9A,I331E,E353S

[0609] KFTIVFPHAQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTTSRELWDDWA

[0610] SEQ ID NO. 180:VSVg-Cl86 H8G,A51S,E355W

[0611] KFTIVFPGNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKSIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERWLWDDWA

[0612] SEQ ID NO. 181:VSVg-Cl95 H8V,K47W,Y330M,T351D

[0613] KFTIVFPVNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPWSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRMIRVDIAAPILSRMVGMISGTDTERELWDDWA

[0614] SEQ ID NO. 182:VSVg-Cl M N9Q,A51P,R329V,E353K

[0615] KFTIVFPHQQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKPIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETVYIRVDIAAPILSRMVGMISGTTTKRELWDDWA

[0616] SEQ ID NO. 183:DAF-GS

[0617] METDTLLLWVLLLWVPGSTGDDCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAHLQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGHLHECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSGGGAHHHHHHFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0618] SEQ ID NO. 184: DAF - GPI

[0619] METDTLLLWVLLLWVPGSTGDDCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAHLQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGHLHECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSGGGAHHHHHHSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0620] SEQ ID NO. 185: Factor H - GS

[0621] METDTLLLWVLLLWVPGSTGDEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKGGAHHHHHHFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0622] SEQ ID NO. 186: Factor H-GPI

[0623] METDTLLLWVLLLWVPGSTGDEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKGGAHHHHHHSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0624] SEQ ID NO. 187: Kaposica-GS

[0625] METDTLLLWVLLLWVPGSTGDQDNEKCSQKTLIGYRLKMSRDGDIAVGETVELRCRSGYTTYARNITATCLQGGTWSEPTATCNKKSCPNPGEIQNGKVIFHGGQDALKYGANISYVCNEGYFLVGREYVRYCMIGASGQMAWSSSPPFCEKEKCHRPKIENGDFKPDKDYYEYNDAVHFECNEGYTLVGPHSIACAVNNTWTSNMPTCELAGCKFPSVTHGYPIQGFSLTYKHKQSVTFACNDGFVLRGSPTITCNVTEWDPPLPKCVLGGAHHHHHHFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0626] SEQ ID NO. 188:Kaposica-GPI

[0627] METDTLLLWVLLLWVPGSTGDQDNEKCSQKTLIGYRLKMSRDGDIAVGETVELRCRSGYTTYARNITATCLQGGTWSEPTATCNKKSCPNPGEIQNGKVIFHGGQDALKYGANISYVCNEGYFLVGREYVRYCMIGASGQMAWSSSPPFCEKEKCHRPKIENGDFKPDKDYYEYNDAVHFECNEGYTLVGPHSIACAVNNTWTSNMPTCELAGCKFPSVTHGYPIQGFSLTYKHKQSVTFACNDGFVLRGSPTITCNVTEWDPPLPKCVLGGAHHHHHHSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0628] SEQ ID NO. 189:SPICE-GS

[0629] METDTLLLWVLLLWVPGSTGDCCTIPSRPINMTFKNSVETDANANYNIGDTIEYLCLPGYRKQKMGPIYAKCTGTGWTLFNQCIKRRCPSPRDIDNGHLDIGGVDFGSSITYSCNSGYYLIGEYKSYCKLGSTGSMVWNPKAPICESVKCQLPPSISNGRHNGYNDFYTDGSVVTYSCNSGYSLIGNSGVLCSGGEWSNPPTCQIVKCPHPTILNGYLSSGFKRSYSYNDNVDFTCKYGYKLSGSSSSTCSPGNTWQPELPKCVRGGAHHHHHHFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0630] SEQ ID NO. 190:SPICE-GPI

[0631] METDTLLLWVLLLWVPGSTGDCCTIPSRPINMTFKNSVETDANANYNIGDTIEYLCLPGYRKQKMGPIYAKCTGTGWTLFNQCIKRRCPSPRDIDNGHLDIGGVDFGSSITYSCNSGYYLIGEYKSYCKLGSTGSMVWNPKAPICESVKCQLPPSISNGRHNGYNDFYTDGSVVTYSCNSGYSLIGNSGVLCSGGEWSNPPTCQIVKCPHPTILNGYLSSGFKRSYSYNDNVDFTCKYGYKLSGSSSSTCSPGNTWQPELPKCVRGGAHHHHHHSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0632] SEQ ID NO. 191:ABD-GS

[0633] METDTLLLWVLLLWVPGSTGDGHSLQAAQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGAHHHHHHFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0634] SEQ ID NO. 192: ABD - GPI

[0635] METDTLLLWVLLLWVPGSTGDGHSLQAAQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGAHHHHHHNLENGGTSLSEKTVLLLVTPFLAAAWSLHP

[0636] SEQ ID NO. 193: M synthetic promoter #24: Enhancer 030 - proximal promoter CHIT1

[0637] agctagagattctgggggcccttcaaacttctttgggacatgtatgtgtaatttcctagtaaaagagttttgcctgcttcttcttctttctccccctggtgtctgcctgcagtactacaatctccctagtggtgaaatagtgtgcctgcctttgttctccgaggctcacaacatgctattcaactctgcactctgagtcaggcaagacagaaaccagtaccttgggtaacccccacaaaacccaggagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0638] SEQ ID NO. 194 H141A F147A LVGP

[0639] MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFALSIPNANQYEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSKYWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLNHMGICSCGLYKQPGVPVKWKR*

[0640] SEQ ID NO. 195 Y150A F446L LVGP

[0641] MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQYEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSKYWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISILLHLVKIPTHRHIVGKSCPKPHRLNHMGICSCGLYKQPGVPVKWKR*

[0642] SEQ ID NO. 196 LVGP WT

[0643] MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQYEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSKYWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLNHMGICSCGLYKQPGVPVKWKR*

[0644] SEQ ID NO. 197 Synthetic Promoter #25: Proximal Promoter CHIT1

[0645] Gagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0646] SEQ ID NO. 198 hCD80trc273

[0647] MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRE

[0648] SEQ ID NO. 199 CD28 mu9.3 scFv

[0649] EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIELTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKR

[0650] SEQ ID NO. 200 CD28 scaffold (G4S)3 linker, short PDGFR stalk, PDGFR TM and CT truncated

[0651] GGGGSGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0652] 4B1 - 4 in SEQ ID NO. 201 scaffold

[0653] s METDTLLLWVLLLWVPGSTGDQVQLQQPGAELVKPGASVKLSCKASGYTFSSYWMHWVKQRPGQVLEWIGEINPGNGHTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARSFTTARGFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATQSVTPGDRVSLSCRASQTISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQDGHSFPPTFGGGTKLEIKSASESKYGPPCPPCPVVVISAILALVVLTIISLIILIMLWQKKPR

[0654] SEQ ID NO. 202: Indiana vesiculostomatitis virus (San Juan strain) glycoprotein

[0655] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0656] SEQ ID NO. 203: Indiana vesiculostomatitis virus (San Juan strain) glycoprotein, without signal peptide

[0657] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK Example

[0658] Example 1 Production of stable cell lines expressing membrane-anchored single-domain antibodies

[0659] This embodiment describes the generation of a stable production cell line for engineered lentivirus (VHH-eLV) targeting single-domain antibodies (VHH). For this purpose, 293T cells (e293T) stably expressing human CD47 and lacking B2 microglobulin expression were engineered to stably express membrane-anchored VHHs targeting human CD3e (SEQ ID NO. 3), human CD8a (SEQ ID NO. 6), or TCRab (SEQ ID NO. 9).

[0660] Twenty-four hours after inoculation, e293T cells were transduced with a self-inactivated Moroni mouse leukemia virus (SIN gRV)-derived retrovirus or a self-inactivated lentiviral vector (LV) encoding CD3e, CD8a, or TCRab VHH. After infection, the culture medium was changed, and cells were allowed to grow for 3 days. At harvest, cells were stained in PBS with a VHH-specific antibody (Jackson Immunoresearch) and analyzed using a fluorescence-activated cell sorting system (BD FACS Aria). ™Cells were sorted based on VHH expression levels or cloned by limiting dilution. Selected cells were expanded and stored as polyclonal cell lines (CD3eVHH-e293T and CD8aVHH-e293T) or monoclonal cell lines (e.g., e293T B619 clone 38) that stably expressed human CD47 and membrane-anchored CD3e or CD8a or TCRab VHH and lacked MHCI expression.

[0661] As shown in Figure 2A, the e293T cell subset transduced with gRV encoding membrane-anchored CD3e or CD8a nanobodies expressed high levels of CD3e (left) or CD8a (right) membrane-anchored nanobodies. After cell sorting and expansion of cells expressing the highest levels of VHH (P3 gate in Figure 2A), Figure 2B shows that the sorted polyclonal cell lines (CD3eVHH-e293T and CD8aVHH-e293T) expressed stable and homogeneous levels of each VHH. In different iterations of this example, compared with the wild-type cell line HEK 293T, the e293T B619 clone 38 cell line showed increased expression of membrane-anchored TCRab VHH and CD47 and decreased expression of MHCI by flow cytometry. Figure 2C ).

[0662] Example 2 Characterization of cell surface expression of VHH targets in lymphocyte lines and primary peripheral blood mononuclear cells.

[0663] This embodiment describes the characterization of cell surface markers / receptor expression (e.g., CD3, CD4, CD8, and T cell receptors or TCR) on human primary cells (i.e., human peripheral blood mononuclear cells or hPBMCs) or human T cell lines (JurkatWT, clone E6-1; SupT1) to select cell types for specific implementations of testing and titrating VHH-eLV.

[0664] Activated human PBMCs (hPBMCs) from healthy donors (cultured in IL2-containing growth medium supplemented with anti-CD3 and anti-CD28 antibodies), Jurkat WT, and SupT1 were stained with the following antibodies: anti-human CD3 BV510 (BioLegend), anti-human CD4 APC-cyanin 7 (BioLegend), anti-human CD8a PerCP (BioLegend), and anti-human TCRa / b BV421 (BioLegend) and DAPI. CytoFLEX LX was used. ™ (Beckman Coulter) performed flow cytometry analysis.

[0665] As shown in Figure 3A, activated hPBMCs were positive for all four markers (top panel), and there were two main cell populations: CD3+CD4+TCR+ and CD3+CD8+TCR+. Jurkat WT cells were CD3+TCR+ but did not stain for CD8a, and a small subset expressed low levels of the CD4 receptor (middle panel). SupT1 cells were all CD4+CD8+ but did not stain for CD3 or TCR (bottom panel). Internally engineered Jurkat cells that stably expressed hCD8a after lentiviral transduction with a vector encoding hCD8a (see Example 4) were CD3+CD8+TCR+ (Figure 3B).

[0666] In summary, primary human PBMCs, Jurkat WT, SUP-T1, and Jurkat CD8 cells provide different target cell populations that can be used to select and characterize VHH-eLV vectors that target a range of T cell surface markers, including CD3, CD4, CD8, or TCR.

[0667] Example 3 The combination of wild-type vesicular stomatitis virus envelope glycoprotein G and nanobodies targeting CD3e or CD8a failed to redirect the VHH-targeting lentiviral vector to CD3+ or CD8+ T cells.

[0668] Viral entry into cells is an early and crucial step in the transduction of lentiviruses into cells. This occurs through the fusion of the viral envelope with the cell membrane of the target cell. A series of viral proteins have evolved to catalyze this step of viral transduction, one of which is widely used in pseudotyped lentiviral vectors: vesicular stomatitis virus glycoprotein G (VSVG). However, viral envelope glycoproteins also ensure viral targeting; in the case of VSVG, they target a wide range of cell types by binding to cell surface receptors, including LDLR.

[0669] In this embodiment, we tested whether envelope-anchored nanobodies could be combined with VSVG to redirect the broad tropism of VSVG pseudotyped lentiviruses to single-cell types while still benefiting from the membrane fusion properties of VSVG. To this end, CD3e or CD8a envelope-anchored nanobodies were combined with wild-type VSVG protein, and the tropism of the combined vectors was tested on target and non-target cells.

[0670] VSVG pseudotyped CD3e or CD8aVHH-eLV or VSVG eLV were generated by transiently transfecting production cells with a lentiviral transfer plasmid encoding an unstable GFP reporter gene and an envelope construct expressing wild-type VSVG. VHHCD3e, VHHCD8a, or wild-type e293T cells were transiently transfected 24 hours after seeding in culture medium (see Example 1). Transient transfection was performed using PEIpro transfection reagent (58 μg; Invitrogen) and a lentiviral packaging plasmid mixture (12 μg transfer plasmid, 5.5 μg VSVG envelope plasmid, 5.5 μg Gag / Pol packaging plasmid, and 6 μg Rev expression plasmid). The PEIpro / plasmid DNA mixture was added to the cells, and 72 hours post-transfection (cells were cultured at 37°C in 5% CO2), the lentiviral supernatant was harvested, clarified at 500g for 10 minutes, and filtered through a 0.2 mm polyethersulfone (Millipore) filter. Add 5X lentivirus centrator (Origene) to the clarified supernatant and store the mixture at 4°C for 1.5 hours, then centrifuge (3500g, 25 minutes, 4°C). Resuspend the lentivirus-containing precipitate in 150 μL of FBS-free TexMACS medium (Miltenyi).

[0671] To determine functional titers, GFP expression in Jurkat WT or SupT1 cells transduced with serially diluted concentrated viral formulations (final dilutions 100x-1000x-10000x-100000x) was assessed by flow cytometry 72 hours post-infection. Functional titers (transduction units or TU / mL) were calculated for at least two linear dilutions using the following formula: (number of seeded cells × GFP + cells % × dilution factor) / volume (mL).

[0672] For transduction assays of Jurkat and SupT1 cells, growth medium with a functional MOI of 2 was used, and cells were harvested 72 hours post-infection and GFP+ expression was assessed by flow cytometry as described above.

[0673] like Figure 4As shown, the wild-type VSVG pseudotyped eLV vector (VSVG) was transduced in both Jurkat WT and SupT1 cells, consistent with the broad cell tropism of the VSVG envelope protein. However, co-expression of the wild-type VSVG envelope protein with VHHCD3e or VHHCD8 (CD3e VSVG and CD8a VSVG, respectively) failed to redirect the vector's targeting to CD3+ (Jurkat WT) or CD8+ (SupT1) cells, respectively. In fact, the levels achieved by infecting SupT1 cells (which do not express CD3e, see Figure 3) with the CD3e VSVG vector were similar to those achieved by the VSVG vector. Similarly, the levels achieved by infecting Jurkat cells (which do not express CD8, see Figure 3) with the CD8a VSVG eLV were similar to those achieved by the VSVG vector. In summary, these results indicate that envelope-anchored VHHCD3e or VHHCD8a failed to retarget wild-type VSVG pseudotyped eLVs to cells expressing VHH targets, but instead maintained broad tropism associated with wild-type VSVG proteins.

[0674] Example 4 A combination of VHH and Y150A Lassa virus glycoprotein mutants targeting the CD8a receptor or wild-type Raldantec virus envelope proteins can specifically target lentiviral particles to CD8+ T cells.

[0675] The aim of this study was to determine whether the binding of VHH targeting the cell surface to viral envelope glycoproteins other than VSVG could direct the tropism of lentiviral vectors to cell types expressing cell surface proteins targeted by VHH. To this end, the transduction specificity of lentiviral vectors displaying combinations of CD8a-targeting VHH and different wild-type / mutant viral envelope proteins on the envelope to CD8-positive and CD8-negative cell lines was tested. In the results presented in this example, we identified two viral envelope proteins that, in combination with CD8a VHH, induced specific targeting of CD8+ cells.

[0676] To test the specificity of CD8a-directed lentiviral particles, we first established a Jurkat cell line expressing CD8a (Jurkat CD8+). Jurkat WT cells (clone E6-1) were transduced with a lentivirus encoding the human CD8a receptor, expanded, and the population stably expressing hCD8a was sorted and stored as the Jurkat CD8+ polyclonal cell line.

[0677] CD8a VHH eLV was generated by transiently transfecting the VHHCD8a e293T production cell line (see Figure 2) with a transfer plasmid encoding the dsGFP reporter and a viral envelope construct encoding either the Y150A Lassa virus glycoprotein mutant (LVGP1x; SEQ ID NO. 32) or the wild-type LDV envelope protein (LDV; SEQ ID NO. 33). 72 hours post-transfection, the supernatant containing lentivirus was harvested, concentrated, and the vector titrated as outlined in Example 3.

[0678] To test the transduction specificity of LVGP1X- or LDV-VHHCD8a eLV, Jurkat WT and Jurkat CD8+ cells were transduced at an MOI of 2, as described in Example 3. Cells were harvested 72 hours post-infection and GFP+ expression was assessed by flow cytometry.

[0679] like Figure 5 As shown, LVGP1X- or LDV-VHHCD8a eLV specifically transduced CD8a-expressing Jurkat cells (Jurkat CD8+), in contrast to the widely tropism-dependent VSVG pseudotyped eLV, which transduced both Jurkat WT and Jurkat CD8+ T cells. In summary, our results demonstrate that LVGP1X and LDVG pseudotyped lentiviral vectors can specifically target CD8+ T cells via envelope-anchored CD8a-directed VHH.

[0680] Example 5 The combination of VHH and LVGP1x, which target CD3e T cell surface proteins, specifically targets lentiviral particles to CD3+ T cells.

[0681] The aim of this study was to extend the findings of Example 4 to other T cell surface markers and to confirm that the binding of VHH to LVGP1x targeting the cell surface can direct the tropism of lentiviral vectors to target cells. To this end, the transduction specificity of lentiviral vectors exhibiting a combination of CD3e-targeting nanobodies and LVGP1X for CD3e-positive and CD3e-negative cell lines was tested.

[0682] hCD3e VHH display vectors are generated by transiently transfecting e293T production cell lines (similar to those shown in Figure 2) that stably express VHHCD3e H6 (SEQ ID NO. 4) or VHHCD3eG7 (SEQ ID NO. 3) as previously described.

[0683] To test the transduction specificity of LVGP1X-VHHCD3e eLV, SupT1 (which does not express CD3e) and Jurkat WT cells (which express CD3e) were transduced at an MOI of 2, as previously described. Cells were harvested 72 hours post-infection and GFP+ expression was assessed by flow cytometry.

[0684] like Figure 6 As shown, the LVGP1X-VHHCD3e eLV specifically transduced CD3e-expressing Jurkat WT cells, in contrast to the widely tropism-dependent VSVG pseudotyped eLV, which transduced both Jurkat WT and CD3e-negative SupT1 cells. In summary, our results demonstrate that the LVGP1X pseudotyped lentiviral vector can specifically target CD3e T cells via envelope-anchored CD3e-directed VHH.

[0685] Example 6 The combination of a TCR receptor-targeting binder and the envelope protein of the Y150A Lassa virus glycoprotein mutant allows lentiviral particles to specifically target TCR+ cells.

[0686] The aim of this study was to extend VHH-eLV technology to other cell surface markers and to confirm that the binding of VHH targeting the cell surface to selected envelope proteins directs the tropism of lentiviral vectors to VHH target cells. To this end, the transduction specificity of lentiviral vectors exhibiting a combination of TCR-targeting VHH and LVGP1X pr LDV was tested for TCR-positive and TCR-negative cell lines.

[0687] hTCR VHH display vectors were generated by transiently transfecting e293T production cell lines (similar to those shown in Figure 2) that stably express VHH TCR 2 (SEQ ID NO. 34) as previously outlined.

[0688] To test the transduction specificity of VHHTCR2 eLV, SupT1 (which does not express TCR) and Jurkat WT cells (which express TCR) were transduced as in Example 4. Cells were harvested 72 hours post-infection and transgene expression (V5-labeled BCMA CAR construct) was assessed by flow cytometry.

[0689] like Figure 7As shown, the LVGP1X-VHHTCR2 and LDV-VHHTCR2 eLVs specifically transduced TCR-expressing Jurkat WT cells, unlike the widely tropism-dependent VSVG pseudotyped eLV, which equivalently transduced both Jurkat WT and TCR-negative SupT1 cells, although with lower efficiency in this study (likely due to over-dilution of the highly concentrated vectors). In summary, our results demonstrate that the LVGP1X and LDV pseudotyped lentiviral vectors can target surface-expressed TCRs using envelope-anchored TCRVHH.

[0690] Example 7 Lentiviral vectors targeting envelope-anchored CD8a VHH and LVGP1x or LDV envelope proteins induced persistent transduction in CD8+ Jurkat cells.

[0691] The aim of this study was to determine whether LVGP1X- or LDV-VHHCD8 eLV stably transduced target cells. To this end, the transduction persistence of lentiviral vectors displaying a combination of CD8-targeting VHH and LVGP1X- or LDV envelope proteins in CD8-positive Jurkat cell lines was tested.

[0692] LVGP1X- or LDV-VHHCD8a eLV were prepared as described previously and used to transduce Jurkat CD8+ cells. Cells were harvested at 2, 7, and 14 days post-infection and GFP+ expression was assessed by flow cytometry.

[0693] like Figure 8 As shown, transduction of Jurkat CD8+ cells by LVGP1X- or LDV-VHHCD8a eLV persisted for more than 14 days, similar to transduction via eLV with extensive VSVG pseudotypening. These results indicate that LVGP1X- and LDV-CD8a eLV induce stable transduction in target cells.

[0694] Example 8 Lentiviral vectors targeting envelope-anchored CD3e VHH and LVGP1x envelope proteins induced persistent transduction in Jurkat cells.

[0695] The aim of this study was to determine whether LVGP1X-VHHCD3e eLV stably induced persistent transduction in target cells.

[0696] hCD3e VHH eLV was generated as previously described and used to transduce JurkatWT cells at an MOI of 2. Cells were harvested at 2, 7, 14, and 21 days post-infection, and GFP+ expression was assessed by flow cytometry. Furthermore, genomic DNA extracted on day 21 post-transduction was quantified for vector insertion by qPCR. Briefly, genomic DNA (gDNA) from infected cells was extracted and purified using the DNeasy Blood and Tissue Kit (from QIAgen). 50 ng of gDNA was assessed by quantitative PCR using Luna Universal qPCR Master Mixture (New England Biolabs) and primers specific for human albumin and long terminal repeats (LTRs) transcribed from reverse transcriptase. Cell counts and vector copy numbers in samples were quantified using standard curves for albumin (3 to 30,000 cells) and the LTR of transcription (40 to 400,000 viral copies), respectively. Quantitative PCR was performed on a QuantStudio 3 (Life Technologies).

[0697] like Figure 9 As shown, after transduction of Jurkat WT cells with LVGP1X-VHHCD3e eLV, reporter GFP transgene expression persisted for more than 21 days, similar to transduction via extensively VSVG pseudotyped eLV. Furthermore, vector copy number assessment at 21 days indicated that LVGP1X-VHHCD3e eLV integrated into the host genome at an average of 1.6 copies / cell at an MOI of 2. These results demonstrate that LVGP1X-VHHCD3e eLV induces stable and persistent transduction in Jurkat cells through integration into the host genome.

[0698] Example 9 The combination of VHH and LVGP1x envelope proteins, which target the CD8a receptor, allows lentiviral particles to specifically target cell lines that endogenously express CD8a.

[0699] The aim of this study was to extend the findings of Example 4 by using a target cell line that intrinsically expresses CD8a (Sup-T1) instead of a target cell line that exogenously expresses CD8a (Jurkat, which stably expresses CD8a via viral transduction), in order to avoid potential confounding effects associated with transgene overexpression of CD8a in the Jurkat CD8+ cell line. Furthermore, the transduction persistence of LVGP1X-VHHCD8a eLV was evaluated in this example. For this purpose, the transduction specificity of the lentiviral vector demonstrating the combination of a CD8a-targeting nanobody and LVGP1X for CD8a-positive and CD8a-negative cell lines was tested.

[0700] The hCD8a VHH display vector was generated by transiently transfecting an e293T production cell line that stably expresses VHHCD8a (SEQ ID NO. 6) as previously described.

[0701] To test the transduction specificity of LVGP1X-VHHCD8 eLV, SupT1 cells (expressing CD8a) and Jurkat WT cells (not expressing CD8a) were transduced at an MOI of 2. Cells were harvested at 3, 7, 14, and 21 days post-infection, and GFP+ expression was assessed by flow cytometry.

[0702] As shown in Figure 10, LVGP1X-VHHCD8a eLV specifically transduced SupT1 cells endogenously expressing CD8a, but not Jurkat WT cells (A), where CD8a was below detection levels (see Figure 2). Furthermore, LVGP1X-VHHCD8a eLV transduced SupT1 cells for more than 21 days (B), and vector copy number assessment at 21 days indicated that LVGP1X-VHHCD8a eLV integrated into the host genome at an average of 3.1 copies / cell in this experiment (C). These results demonstrate that LVGP1X-VHHCD8a eLV induces stable transduction of target cells through integration into the host genome.

[0703] Example 10 The combination of VHH and LVGP1x or LDV envelope proteins targeting the TCR receptor induced persistent transduction in Jurkat WT cells.

[0704] The aim of this study was to extend previous findings to demonstrate persistent transduction of TCR+ Jurkat WT cells using LVGP1x-VHHTCR and LDV-VHHTCR vectors.

[0705] The TCR VHH display vector was generated by transiently transfecting e293T production cell lines that stably express VHHTCR2 (SEQ ID NO. 34) (similar to those shown in Figure 2) as previously described.

[0706] To test the transduction efficiency of LVGP1X-VHHTCR2 and LDV-VHHTCR2 eLV, Jurkat WT cells were transduced with the vector formulation diluted 1:40 as previously described. Cells were harvested at 4, 7, 12, and 16 days post-infection, and GFP+ expression was assessed by flow cytometry.

[0707] like Figure 11As shown, LVGP1X-VHHTCR2 and LDV-VHHTCR2 eLV efficiently transduced JurkatWT cells expressing TCR, and both types of vectors were observed to exhibit transduction persistence similar to that of the widely tropist VSVG pseudotyped vector.

[0708] Example 11 The combination of VHH and LVGP1x envelope proteins, which target the CD8a receptor, allows lentiviral particles to specifically target primary CD8+ T lymphocytes.

[0709] This example describes the specificity of LVGP1X-VHHCD8a for primary T cells. To this end, the transduction specificity of a lentiviral vector demonstrating a combination of a CD8a-targeting nanobody and LVGP1X for activated hPBMCs containing CD4+ and CD8+ T lymphocytes was tested.

[0710] The hCD8a VHH display vector was generated by transient transfection of the e293T production cell line stably expressing VHHCD8a (SEQ ID NO. 6) as previously described. hPBMCs isolated from healthy donors were stimulated for 2 days with anti-CD3 and anti-CD28 antibodies and transduced with LVGP1x-VHHCD8a eLV or widely tropism-prone VSVG eLV at an MOI of 20. Four days after transduction, half of the cells were collected, washed, and stained with anti-CD4 and anti-CD8 for flow cytometry. The same analysis was performed over time at days 7, 11, 14, and 20 to assess the persistence of GFP expression.

[0711] like Figure 12 As shown, LVGP1X-VHHCD8a eLV specifically transduced primary CD8+ T cells, but not primary CD4+ T lymphocytes, unlike the broadly tropism-dependent VSVG eLV, which non-specifically transduced T lymphocytes. This result indicates that LVGP1x-VHHCD8a eLV can differentiate primary T cell subsets and target the vector only to CD8+ lymphocytes.

[0712] Example 12 The combination of VHH, which targets the TCR, and the LVGP1x envelope protein on the eLV effectively transduced primary T lymphocytes.

[0713] This example describes the transduction efficiency and persistence of LVGP1X-VHHTCR2 eLV in primary T cells. To this end, a lentiviral vector exhibiting the combination of TCR-targeting VHH and LVGP1X was tested on activated hPBMCs.

[0714] A TCR VHH display vector expressing a GFP reporter was generated by transiently transfecting an e293T production cell line stably expressing VHHTCR2 (SEQ ID NO. 34) as previously described, and the vector was titrated on Jurkat WT cells. hPBMCs isolated from healthy donors were transduced at an MOI of 5, stimulated with anti-CD3 and anti-CD28 antibodies for 2 days. Four days post-transduction, half of the cells were collected and stained with anti-CD3, CD4, and CD8 antibodies as previously described, and the percentage of GFP-expressing cells was analyzed by flow cytometry. The same analysis was performed over time at days 7, 11, 14, and 20 to assess the persistence of vector transduction as monitored by GFP expression.

[0715] As shown in Figure 13, LVGP1X-VHHTCR2 persistently transduced CD4+ and CD8+ primary T cells throughout the study duration (20 days), similar to the VSVG vector, but with higher transduction efficiency. In summary, these results demonstrate that lentiviral vectors that bind TCR-targeting VHH to the LVGP1X envelope protein effectively target and stably transduce activated primary T cells.

[0716] Example 13 Transduction of JurkatCD8+ cells with LVGP1X-VHHCD8a or LDV-VHHCD8a eLV encoding BCMA CAR resulted in functional expression of the CAR construct, as demonstrated by activation of transduced cells upon exposure to target cells.

[0717] In this embodiment, CAR-Jurkat cells targeting the BCMA surface antigen were generated using LVGP1X-VHHCD8a or LDV-VHHCD8 eLV to demonstrate the functional delivery of the CAR construct using VHH-targeting eLV. The functional delivery of the CAR construct was evaluated by upregulation of the T cell activation marker CD69 after exposure to target cells expressing the CAR antigen.

[0718] As previously described, LVGP1X-VHHCD8a or LDV-VHHCD8a vectors encoding the BCMA CAR construct (SEQ ID NO. 35) were generated from stably expressing e293T production cells. Jurkat cells were transduced with VHH-eLV at an MOI of 2 and passaged for 7 days before co-culture assays. For co-culture assays, BCMA antigen-negative (K562) and BCMA antigen-positive (NCI-H929) target cells were labeled with CellTrace Violet (Thermo Fisher) to distinguish them from CAR-transduced Jurkat (CAR-Jurkat). CAR-Jurkat cells were co-cultured overnight at a 1:1 effector:target ratio. 18 hours after co-culture, the level of CAR-Jurkat cell activation was assessed by flow cytometry after CD69 antibody staining (BioLegend), and DAPI staining was used to distinguish live cells. The binding domain of the BCMA-CAR used in this study is VHH, and the expression of the CAR construct in live cells was detected by staining with an anti-VHH antibody (Jackson Immunoresearch).

[0719] As shown in Figure 14A, BCMA CAR-Jurkat cells upregulated CD69 expression only in the presence of BCMA+ NCI-H929 target cells, and the degree of upregulation was similar regardless of whether VHHCD8a eLV or widely tropism-dependent VSVG eLV was used to transduce BCMA CAR. Figure 14B shows the quantification of the results in Figure 14A.

[0720] Example 14 Transduction of primary T lymphocytes with the LVGP1X-VHHCD3e vector encoding BCMA CAR resulted in functional expression of the CAR construct, as demonstrated by the activation of transduced cells upon exposure to target cells.

[0721] This example describes the functional delivery of a CAR construct to primary T cells using LVGP1X-VHHCD3e, and the evaluation of the upregulation of the T cell activation marker CD137 after exposure to target cells expressing CAR antigens.

[0722] As previously described, LVGP1X-VHHCD3e eLVs encoding the BCMA CAR construct (SEQ ID NO. 35) were generated from stably expressing e293T production cells. hPBMCs were activated on day 0 with the anti-CD3 / CD28 T cell TransAct reagent (Miltenyi), transduced overnight on day 2 with the eLV encoding the BCMA CAR construct, and the culture medium was changed on day 3. Cytokines (30 ng / mL IL-2; Miltenyi) were supplemented during primary cell culture. Cultures were propagated until day 14 for harvesting for co-culture assays. For co-culture assays, BCMA antigen-negative (K562) and BCMA antigen-positive (NCI-H929) target cells were labeled with CellTrace Violet (Thermo Fisher) to distinguish them from primary human T cells (CAR-T). CAR-T cells were co-cultured overnight at a 1:1 effector:target ratio. Eighteen hours after co-culture, CAR-Jurkat cell activation levels were assessed by flow cytometry following CD137 antibody staining (BioLegend), and 7-AAD staining was used to distinguish live cells. CAR expression in live cells was determined by staining with anti-VHH antibody (Jackson Immunoresearch).

[0723] As shown in Figure 15A, BCMA CAR-T cells upregulated CD137 expression only in the presence of BCMA+ NCI-H929 target cells, and the degree of upregulation was similar regardless of whether CD3e VHH was used to target eLV or to transduce BCMA CAR with widely tropism for VSVG LV. Figure 15B shows the quantification of the results in Figure 15A.

[0724] Example 15 Transduction of primary T lymphocytes with the LVGP1X-VHHCD8a vector encoding BCMA CAR resulted in functional expression of the CAR construct and cytotoxicity of CAR-T cells against BCMA-expressing target cells.

[0725] This example describes the in vitro functionality of BCMA CAR-T cells generated using LVGP1X-VHHCD8a, as measured by killing target cancer cells.

[0726] As previously described, the LVGP1X-VHHCD8a vector encoding a BCMA CAR construct controlled by the T cell-specific promoter SYN19 (SEQ ID NO. 12) was generated from stably expressing CD8aVHH-e293T production cells. hPBMCs were activated in vitro for 48 hours in the presence of anti-CD3 and anti-CD28 antibodies. Two days after activation, cells were transduced and expanded for 7 days in complete IMDM medium supplemented with 30 ng / ml human IL-2 at an MOI of 5. Transduction of PBMCs with the VSVG vector (expressing the BCMA CAR construct under the control of the EF1a promoter) yielded approximately 12% CD3+ CAR+ cells, and transduction of PBMCs with the LVGP1X-VHHCD8a vector yielded approximately 6% CD3+ CAR+ cells, confirming CAR expression. For co-culture assays, BCMA antigen-negative (K562) and BCMA antigen-positive (NCI-H929) target cells were labeled with CellTrace Violet (Thermo Fisher) to distinguish them from CAR-T cells. Forty thousand target cells were co-cultured with CD3+ T cells, with CD3+ T cells adjusted to achieve an effector-to-target ratio of 1:1 or 1:2. Untransduced or GFP-expressing T cells were used as controls to determine background alloreactivity. After five days of co-culture, the cell suspension was stained with anti-CD3 PercP, and the number of target / non-target cells was then quantified by flow cytometry.

[0727] As shown in Figure 16A, compared with untransduced or GFP-transduced controls, the number of NCI-H929 target cells was reduced by >75% when co-cultured with CAR-T cells transduced with LVGP1X-VHHCD8 or VSVGeLV. Conversely, the number of K562 cells remained relatively constant across all groups (Figure 16B), confirming that NCI-H929 depletion is CAR-dependent and BCMA-specific.

[0728] In summary, these results indicate that primary BCMA CAR-T cells obtained via LVGP1X-VHHCD8a eLV transduction are functional and cytotoxic to BCMA+ target cancer cell lines.

[0729] Example 16 Transduction of primary T lymphocytes with the LVGP1X-VHHCD8a vector encoding CD19-directed CAR resulted in functional expression of the CAR construct and cytotoxicity of CAR-T against CD19-expressing target cells.

[0730] The aim of this study is to extend the functional characterization of primary CAR-T cells generated in vitro using LVGP1X-VHHCD8a to other CAR-T constructs.

[0731] As previously described, the LVGP1X-VHHCD8a vector encoding a CD19 CAR construct (SEQ ID NO. 36) under the control of the T cell-specific promoter SYN19 (SEQ ID NO. 12) was generated from stably expressing CD8aVHH-e293T production cells. hPBMCs were activated in vitro for 48 hours in the presence of anti-CD3 and anti-CD28 antibodies. Two days after activation, cells were transduced and expanded for 7 days in complete IMDM medium supplemented with 30 ng / ml human IL-2 at an MOI of 10. Transduction of PBMCs with the VSVG vector (expressing the CD19 CAR construct under the control of the EF1a promoter) yielded approximately 4% CD3+ CAR+ cells, and transduction of PBMCs with LVGP1X-VHHCD8a yielded approximately 12% CD3+ CAR+ cells, as assessed by flow cytometry after staining for CD3 and V5 tags (fused with the CD19 CAR).

[0732] For co-culture assays, target cells from the CD19 antigen-negative myeloid leukemia cell line (K562) and the CD19-expressing B-cell precursor leukemia cell line (Nalm6) were labeled with CellTrace Violet (Thermo Fisher) to distinguish them from CAR-T cells. Target and non-target cells were co-cultured with CD3+ T cells, and CD3+ T cells were regulated to achieve an effector-to-target ratio of 1:1 or 1:2. Untransduced T cells were used as controls to determine non-specific allogeneic reactivity. After three days, the cell suspension was stained with anti-CD3 A700, and the number of target / non-target cells was then quantified by flow cytometry.

[0733] As shown in Figure 17A, compared with the untransduced control, the number of Nalm6 target cells was reduced by >75% when co-cultured with CAR-T cells transduced with LVGP1X-VHHCD8a or VSVG eLV at co-culture ratios of 1:2 and 1:5. Conversely, the number of K562 cells remained relatively constant across all groups (Figure 17B), confirming that NCI-H929 depletion is CAR-dependent and CD19-specific. In summary, these results indicate that primary CD19 CAR-T cells obtained via LVGP1X-VHHCD8a eLV transduction are functional and cytotoxic to CD19+ target cancer cell lines.

[0734] Example 17Transduction of primary T lymphocytes with the LVGP1X-VHHTCR2 vector encoding BCMA CAR resulted in functional expression of the CAR construct and cytotoxicity of CAR-T cells against BCMA-expressing target cells.

[0735] This example describes the in vitro functionality of BCMA CAR-T cells generated using LVGP1X-VHHTCR2, as measured by killing target cancer cells.

[0736] As previously described, the LVGP1X-VHHTCR2 vector encoding a BCMA CAR construct controlled by the T cell-specific promoter SYN19 was generated from stably expressing VHHTCR2-e293T production cells. hPBMCs were activated and transduced at an MOI of 10 as previously described. Transduction of hPBMCs with the LVGP1X-VHHTCR2 vector yielded approximately 80% CD3+ CAR+ cells, confirming CAR expression. For co-culture assays, the BCMA antigen-negative chronic myeloid leukemia (K562) cell line and the BCMA-positive multiple myeloma NCI-H929 cell line were labeled with CellTrace Violet (Thermo Fisher) to distinguish them from CAR-T cells. Target and non-target cells were co-cultured with CD3+ T cells, and CD3+ T cells were regulated to achieve effector-to-target ratios of 1:1, 1:2, or 1:5. Untransduced T cells were used as controls to determine background alloreactivity. After five days of co-culturing, the cell suspension was stained with anti-CD3 PercP, and then the number of target / non-target cells was quantified by flow cytometry.

[0737] As shown in Figure 18A, compared with the untransduced control, the number of NCI-H929 target cells was reduced by >80% to 90% when co-cultured with CAR-T cells transduced with LVGP1X-VHHTCR2 at various effector:target ratios. Conversely, the number of K562 cells remained relatively constant in all groups (Figure 18B), confirming that NCI-H929 depletion is CAR-dependent and BCMA-specific.

[0738] In summary, these results demonstrate that the LVGP1X-VHHTCR2 BCMA CAR eLV vector reprograms T cells into functional BCMA CAR T cells that are cytotoxic to BCMA+ target cancer cell lines.

[0739] Example 18 The LVGP1X-VHHCD8a dsGFP eLV specifically and stably transduced CD8+ human T cells in vivo in a humanized mouse model.

[0740] This example describes the transduction specificity and persistence of LVGP1X-VHHCD8a in humanized mice. Notably, the CD8a VHH targeting LVGP1X eLV does not cross-react with mouse CD8a homologs; therefore, it is necessary to evaluate the in vivo transduction of LVGP1X-VHHCD8a in an immunodeficient mouse model (NOD SCIDγ mice; NSG) that was transplanted with human PBMCs, providing the necessary target for eLV transduction.

[0741] NSG mice were humanized by intraperitoneal injection of 10 million activated hPBMCs (activated for 2 days with anti-CD3 and anti-CD28 antibodies). The following day, 25 million TU (transduction units) or 100 million TU of LVGP1X-VHHCD8a eLVdsGFP was injected intraperitoneally. A control group was included, which received a mediator (TexMACS). The study treatment groups are described in Table 1. As previously described, the LVGP1X-VHHCD8a eLVdsGFP vector encoding GFP under promoter control on EF1a was produced from stably expressing CD8VHH-e293T production cells. In vivo transduction of LVGP1X-VHHCD8a eLVdsGFP was quantified by flow cytometry of the percentage of GFP-expressing cells measured in peripheral blood collected weekly via exsanguination over 27 days. CD3, CD4, CD8, CD19, and CD56 staining was performed on hPBMCs to determine the cell types expressing the GFP transgene and thus to determine the transduction specificity of LVGP1X-VHHCD8aeLV.

[0742] Table 1: Study Treatment Groups

[0743]

[0744] On day 4, GFP expression was observed in both LVGP1X-VHHCD8a eLV dsGFP groups, and GFP expression was specific to CD8+ T cells (Fig. 19A), with GPF+ CD4+ cells ≤0.1% in the high-dose group 3. GFP transgene expression was stable over the 27-day study, ranging from 2% to 3% for a 25 million TU dose and from 3% to 4% for a 100 million TU dose (Fig. 19B). These results indicate that LVGP1X-VHHCD8a eLV can selectively transduce human CD8-positive T cells in vivo, and that transgene expression persists for at least 27 days.

[0745] Example 19 LVGP1X-VHHCD8a, through in vivo reprogramming of T cells using CD19 CAR, resulted in impaired B cell regeneration in a humanized mouse model.

[0746] This example describes the functionality of CD8+ CAR-T cells transduced in vivo using LVGP1X-VHHCD8a eLV encoding a CD19 CAR transgene.

[0747] On day -1, NSG mice were intraperitoneally injected with 10 million activated hPBMCs (as described in Example 19), followed by injection of 100 million TU of LVGP1X-VHHCD8a CD19 CAR eLV on day 2 (day 0). The study treatment groups are described in Table 2: VSVG EF1a dsGFP eLV is a broadly tropophilic VSVG pseudotyped eLV encoding a destabilized GFP reporter under the control of the EF1a promoter; LVGP1X-VHHCD8a CD19 CAR eLV encodes a CD19 CAR construct (SEQ ID NO. 36) under the control of the synthetic T cell-specific promoter SYN19 (SEQ ID NO. 12) and is fused with a V5 tag to facilitate the detection of CAR-expressing cells; VSVG CD19 CAR eLV is a broadly tropophilic VSVG pseudotyped eLV encoding the same CD19 CAR under the control of SYN19. B cells isolated from the donors used for the day-1 injection were intravenously injected one day after vector administration to provide additional targets for in vivo reprogrammed CD19 CAR-T cells. On day 7, half of the animals were collected, and peripheral blood and freshly dissected spleen and bone marrow were subjected to flow cytometry to assess transduction efficiency (% of cells expressing V5) and anti-CD19 CAR activity (% of B cells). The remaining mice were collected on day 14 for the same analysis (data not shown).

[0748] Table 2: Study Treatment Groups

[0749]

[0750] Circulating CAR T cells were detected in both the VSVG and LVGP1X-VHHCD8a eLV CD19 CAR groups on day 7 (Fig. 20A) and day 14 (data not shown). Although VSVG eLV transduced human CD4 and CD8 T cells, CAR-T expression was observed only in the human CD8+ T cell population in the LVGP1X-VHHCD8a eLV CD19 CAR group, confirming the specificity of the LVGP1X-VHHCD8a eLV vector observed in Fig. 19. On day 7 (Fig. 20B) and day 14 (data not shown), CAR expression in groups 2 and 3 was accompanied by depletion of human B cells in peripheral blood, bone marrow, and spleen, with a decrease in the percentage of B cells of approximately 1 to 2 logarithmic orders compared to the control group treated with the VSVG eLV EF1a dsGFP vector. B cells were defined as hCD45+hCD20+ cells. The impaired B-cell regeneration in groups 2 and 3 also confirmed that the T-cell synthesis promoter supports the full expression of CD19 CAR to generate functional CAR-T cells.

[0751] In summary, CD8+ CAR-T cells transduced with the LVGP1X-VHHCD8a eLV CD19 CAR vector are functional and effectively eradicate B cells in vivo.

[0752] Example 20 Delivery of the CD19 CAR construct by CD8a VHH LVGP1X eLV to stable humanized mice resulted in impaired B cell regeneration.

[0753] This example describes the functionality of CD8+ CAR-T cells delivered using CD8a VHH LVGP1x eLV encoding a CD19 CAR transgene in a stable humanized mouse (hCD34 NSG) model, which supports the targeting of CD8a VHH LVGP1x eLV to resting / naive human T cells in the context of an endogenously reconstituted multilineage human immune system using the intended clinical administration route.

[0754] hCD34+ NCG humanized mice were generated by transplanting human CD34+ hematopoietic progenitor cells into chemically ablated NCG mice. At approximately 18 weeks of age, hCD34+ NCG mice with >25% human chimerism were injected with 70E+6 TU of CD8a VHHLVGP1x eLV, which encodes a CD19 CAR or GFP reporter transgene controlled by the T cell-specific synthetic promoter SYN19 (SEQ ID NO 12). Cytokines IL-7 and IL-15 / IL-15Ra were subcutaneously injected four days and one day prior to vector injection to support endogenous T cell expansion and potential T cell transduction. As shown in Figure 20C, significant B cell regeneration impairment was observed in animals injected with CD8a VHHLVGP1x CD19 CAR eLV compared to animals injected with the vector or CD8a VHHLVGP1x GFP eLV. In the CD8a VHH LVGP1x CD19 CAR eLV group, B-cell aplasia persisted until the end of the study on day 53. VCN analysis confirmed the presence of the transgene in the liver, spleen, and bone marrow, with decreasing prevalence in the order of transgene, with levels below 0.3 VCN / cell in the liver and below 0.1 VCN / cell in the spleen. No VCN above background levels was detected in the lungs, kidneys, heart, brain, and ovaries (data not shown). No adverse health effects were reported in any treatment group.

[0755] Conclusion – This study demonstrates that the ENaBL-T8 vector, administered via the intended clinical route, was well-tolerated in a mouse model with endogenous circulating resting T cells, generating sufficient CD19 CAR T cells to achieve the observed elimination of the target B cell population. The transgene was biodistributed only in the liver, spleen, and bone marrow.

[0756] Example 21: In vivo specific delivery of CD19 CAR construct using CD8a or TCRab VHH LVGP1X eLV To T cells and generate functional CD19 CAR T cells that induce rapid B cell regeneration impairment in humanized mouse models. .

[0757] This example describes the in vivo transduction efficiency and specificity of CD8a or TCRab VHH LVGP1X eLV in humanized mice, thereby generating functional CAR-T cells in vivo and eliminating the target cell population.

[0758] NSG mice were humanized by intraperitoneal injection of 10 million activated hPBMCs (activated for 2 days with anti-CD3 and anti-CD28 antibodies). The following day, mice were intraperitoneally injected with 10 million TU (transduction units) of CD8a or TCRabVHH LVGP1x eLV encoding a CD19 CAR transgene, or 50 million TU of TCRab VHH LVGP1x eLV dsGFP. A control group was included, which received 50 million TU of VSVGLV CD19 CAR.

[0759] As shown in Figure 20D, CD19 CAR T cells were detected in animals injected with CD8a or TCRab VHH LVGP1x CD19 CAR eLV or VSVG LV CD19 CAR as early as day 7 post-vector injection. The transduction efficiency of CD3+ T cells with the TCRab VHHLVGP1x CD19 CAR eLV vector was higher than that with the CD8a VHH LVGP1x CD19 CAR eLV vector (9.0%±1.5% and 1.0%±0.15% in peripheral blood on day 7, respectively, at a 10e6 TU dose), and induced greater expansion of the T cell population (10e5 to 10e7 T cells / ml and 10e4 to 10e5 T cells / ml, respectively, on day 7), resulting in a higher total number of circulating CAR T cells (10e5 to 10e6 CAR T cells / ml and 10e2 to 10e4 CAR T cells / ml, respectively). As shown in Figure 20F, the CD8a VHH LVGP1x CD19 CAR eLV vector transduction was specific for CD8+ T cells (1.9% ± 0.3% CD4+CAR+ cells and 22.4% ± 2.2% CD8+CAR+ cells on day 11 at a 10e6 TU dose), while the TCRab VHHLVGP1x CD19 CAR eLV transduced both CD4 and CD8 T cells, with a slight preference for CD4 T cells (14.5% ± 2.3% CD4+CAR+ cells and 7.6% ± 1.4% CD8+CAR+ cells on day 11 at a 10e6 TU dose). In the NKT population, detectable levels of CD19 CAR transduction and expression were observed in both vectors, but not in NK, CD14, or mouse CD45 cells. Although the levels of reprogrammed CD19 CAR T cells varied among the different groups, near-complete or complete B cell regeneration impairment was observed in the blood and spleen of all CD19 CAR groups by day 7 and day 11, respectively.

[0760] This study demonstrates that CD19 CAR T cells generated in vivo from the CD8a or TCRab VHH LVGP1x CD19 CAR eLV vector possess anti-target cytotoxic activity. Compared to vectors targeting CD8, TCRab targeting in the TCRab VHH LVGP1x CD19 CAR eLV resulted in higher levels of transduction and a greater overall expansion of the T cell population. T cell-specific promoter 23 expression of CD19 CAR is specific to both T and NKT cells and leads to potent anti-target cytotoxic activity of the CAR.

[0761] Example 22The combination of a triple mutant of VHH targeting the CD8 receptor and the Lassa virus glycoprotein specifically targets lentiviral particles to cells expressing CD8.

[0762] This embodiment describes cell-specific transduction achieved using a mutant form of eLV exhibiting membrane-anchored VHH and LVGP envelope proteins. For this purpose, several mutants of the Lassa virus envelope glycoprotein were tested, and... Figure 21 The study presents the discovery of a mutant envelope protein that is linked to VHH, which targets CD8a, and can specifically transduce CD8-positive cell lines.

[0763] As previously described, the hCD8a VHH display vector was generated by co-transfecting the VHHCD8a e293T production cell line with a packaged and transferred plasmid and a viral envelope construct expressing either the Y150A Lassa virus glycoprotein mutant (as a positive control) or the Y150A H141A F147A Lassa virus glycoprotein mutant (LVGP3x; SEQ ID NO. 37).

[0764] To test the transduction specificity of LVGP1X- or LVGP3X-VHHCD8a eLV, Jurkat WT, 293T, and Sup-T1 cells were transduced at an MOI of 2, as previously described. Cells were harvested 72 hours post-infection and GFP+ expression was assessed by flow cytometry.

[0765] like Figure 21 As shown, the LVGP3X-VHHCD8a eLV specifically transduced CD8-expressing cells (Sup-T1), similar to the LVGP1X-VHHCD8 vector, and unlike the broadly tropism-dependent VSVG pseudotyped eLV, which transduced both CD8-negative cell lines (Jurkat WT and 293T) and CD8+ SupT1 cells. In summary, these results demonstrate that the LVGP3X pseudotyped lentiviral vector, similar to the LVGP1X eLV, is also targeted by envelope-anchored VHH and efficiently transduced by mutant Lassa virus envelope proteins.

[0766] Example 23: Pseudotype development of Lassa virus glycoproteins using wild-type or H141A / F147A instead of Y150A / F446L The vector for CD8 VHH specifically targets lentiviral particles to CD8+ primary T cells.

[0767] This embodiment describes cell-specific transduction using eLVs that are displayed membrane-anchored CD8 VHH and pseudotyped with WT or H141A / F147A or Y150A / F446L Lassa virus glycoproteins.

[0768] As previously described, vectors displaying CD8a VHH were generated by co-transfecting the CD8a VHH e293T production cell line with a packaged and transferred plasmid and a viral envelope construct expressing wild-type (SEQ ID NO 196), Y150A / F446L (SEQ ID NO 195), or H141A / F147A (SEQ ID NO 194) Lassa virus glycoprotein or the Y150A mutant Lassa virus glycoprotein (used as a positive control). To test the transduction specificity of differentially pseudotyped CD8a VHH eLV, activated PBMCs were transduced at an MOI of 10 as previously described, and the expression of the GFP cargo transgene over time was assessed by flow cytometry.

[0769] like Figure 22 As shown, targeting eLV with wild-type or H141A / F147A Lassa virus glycoprotein pseudotyped CD8 VHH resulted only in transduction of CD8+ primary T cells, although the transduction level was lower compared to the Y150A Lassa virus glycoprotein pseudotyped vector. Conversely, pseudotyped CD8 VHH eLV with Y150A / F446L mutant Lassa virus glycoprotein eliminated the vector's transduction efficiency, while VSVG pseudotyped induced non-specific transduction of both CD8+ and CD8-(CD4+) primary T cells. In summary, these results demonstrate that wild-type and H141A / F147A Lassa virus glycoprotein pseudotyped VHH-displaying eLVs, similar to the Y150A VHH eLV, are also targeted by envelope-anchored VHHs and are efficiently transduced by wild-type and selected mutant Lassa virus envelope proteins.

[0770] Example 24 Cell-specific transduction was achieved by combining VHH-mediated targeting with selected mutants of the VSVG envelope glycoprotein.

[0771] This embodiment describes cell-specific transduction achieved using eLV, which displays membrane-anchored VHH and newly identified VSVG glycoprotein mutants. Based on the crystal structure of the VSVG glycoprotein, we generated specific mutants at key residues located around the binding pocket or in the pathway through which the VSVG protein binds to its native receptor LDLR. These mutations were engineered to prevent (steric hindrance) or block (polarity change) interaction with LDL-R, with the aim of preserving the viral entry activity of VSVG while eliminating the protein's targeting function.

[0772] Figure 23 presents supporting findings of selected VSVG mutants that lack the broad tropism of wild-type VSVG and are linked to VHH, which targets CD8a, and can specifically transduce CD8-positive cell lines.

[0773] As previously described, the hCD8a VHH display vector was generated as follows: the plasmid was packaged and transferred, and expression was performed at... Figure 23A The viral envelope constructs of the VSVG mutants (SEQ ID NO. 38 to 40 and SEQ ID NO. 153 to 182) listed in the protocol were co-transfected into the CD8a VHH e293T production cell line. To test the transduction specificity of the VSVG mutant VHHCD8 eLV, Jurkat WT, CD8-expressing Jurkat cells, and Sup-T1 cells (endogenously expressing CD8a) were transduced at an MOI of 2. Cells were harvested 72 hours post-infection and GFP+ expression was assessed by flow cytometry.

[0774] like Figure 23B and Figure 23C As shown, this includes single mutants A51E, I182E, I331W, I331E, T352W, T352Q or double / triple / quadriple mutants “I52R, I331E, E355L”, “N9G, K47G, I331E, T352S”, “H8F, A51P, R329V, T352V”, “N9Q, K47T, T328S, E355L”, “N9G, K50S, V333I, T351R”, “H8F, S48L, R322,5K”, “H8Y, K47G”, “N9H, S48R, R329T, T352E”, “Q53A”, Multiple VSVG mutant CD8 variants, including “I331E, T352I”, “N9T, I52T, R329K, T352E”, “H8S, H49A, R329H, T352Q”, “K11A, S48L, I331L, R354K”, “N9R, Q53I, I331Q, R354M”, “H8S, I52V, V333Y, T351S”, “I331E, E353K”, “Q10V, K50H, I331M, T352D”, “N9A, I331E, E353S”, and “H8V, K47W, Y330M, T351D”, are included. VHH eLV specifically transduced CD8-expressing cells (Jk-CD8 or SupT1 cells), in contrast to the widely tropism-dependent VSVG pseudotyped eLV, which transduced both CD8-negative cell lines (JurkatWT) and CD8+ cells (Jk-CD8 or SupT1 cells).

[0775] As shown in Figures 24A and 24B, in cell lines endogenously expressing CD8 (Sup T1 or CD8+ primary T cells), using Figure 23BThe identification of pseudotyped CD8 VHH eLVs of VSVG mutants confirmed the target-specific transduction of the A51E, I182E, and I331E mutant VSVG pseudotyped vectors.

[0776] exist Figure 24C Furthermore, the transduction specificity of the I331E mutant VSVG pseudotyped vector was tested using TCRab VHH targeting eLV. These data confirm that I331E pseudotyped eLV retains the targeting specificity of eLVs displaying VHHs other than CD8a VHH, and demonstrate that I331E pseudotyped eLVs are transduction-deficient in the absence of VHH targeting.

[0777] As shown in Figure 25, the TCRab VHH eLV pseudotyped with the VSVG mutant I331E was confirmed to lack LDL-R binding on HEK 293T cells overexpressing LDL-R, while vector binding was observed on HEK 293T cells overexpressing SIRPa (as expected, since CD47 (the ligand of the SIRPa receptor) is displayed on the surface of TCRab VHH eLV).

[0778] In summary, our results show that VSVG mutants designed to eliminate binding to LDLR, particularly those with mutations at position I331, can be used to pseudotype lentiviral vectors, thereby enabling cell-targeted transduction through envelope-anchored target-directed VHH.

[0779] Example 25 Using VSVG, which encodes the CAR construct, to target a pseudotyped defective mutant of TCRab VHH to target eLV transduced primary T lymphocytes resulted in functional expression of the CAR construct and CAR-T cell cytotoxicity against target cells.

[0780] This embodiment describes the use of a targeted defect-type VSVG. I331E The in vitro functionality of CAR-T cells generated by mutant pseudotyped TCRab VHH eLV, as measured by killing target cancer cells.

[0781] As previously described, TCRab VHHG1x eLV vector encoding a BCMA CAR construct (SEQ ID NO 35) controlled by the T cell-specific promoter SYN23 (SEQ ID NO 15) was generated from stably expressing TCRab VHH e293T production cells. Unstimulated hPBMCs were transduced with TCRab VHH G1x eLV encoding the BCMA CAR, and after confirming CAR expression, the cells were co-cultured with the BCMA antigen-negative chronic myeloid leukemia (K562) cell line, the BCMA-positive multiple myeloma NCI-H929 cell line, and the low-BCMA-expressing acute lymphoblastic leukemia lymphocyte line Nalm6 at different ratios of effector to target. As shown in Figures 26A to 26I, compared with the untransduced control, the number of NCI-H929 target cells was reduced by >90% when co-cultured with CAR-T cells transduced with TCRab VHH G1x eLV at various effector:target ratios. Conversely, the number of K562 cells remained relatively constant across all groups, confirming that NCI-H929 depletion is CAR-dependent and BCMA-specific. CAR-T-mediated cytotoxicity was associated with expansion and increased IL2 production, indicating that CAR-T cells generated using the TCRab VHH G1x eLV vector are pluripotent.

[0782] TCRab VHH G1x eLV-mediated delivery of functional CARs is applicable to other constructs. As described above, CAR-T cells targeting sealin 18.2 were generated using the TCRabVHH G1x eLV vector encoding a sealin 18.2 CAR (SEQ ID NO 53) controlled by the T cell-specific promoter SYN23. CAR-T cells were co-cultured at different ratios with sealin 18.2 antigen-negative chronic myeloid leukemia (CML) cell line (K562), sealin 18.2 positive gastric cancer cell line (NUGC4), and Nalm6 acute lymphoblastic leukemia lymphocyte line stably expressing sealin 18.2 or sealin 18.1. As shown in Figures 26K to 26N, the cytotoxic activity of TCRab VHH G1xeLV-transduced sealing protein 18.2 CAR-T cells was detected as early as 18 hours after co-culturing with NUGC4 cells. Moreover, this cytotoxic activity was specific to cells expressing sealing protein 18.2, while no toxicity was observed to K562 cells and Nalm6 cells that stably expressed sealing protein 18.1.

[0783] In summary, these results demonstrate that the TCRab VHH G1x eLV vector effectively delivers CAR constructs to resting T cells and generates CAR-T cells with potent antitumor activity.

[0784] Example 26 Reporter constructs for testing novel cell type-specific synthetic promoters

[0785] The synthesized cell-specific promoter was assembled from genomic sequences upstream of selected cell-specific genes. These genomic sequences included the core and proximal promoters of the selected cell-specific genes, as well as enhancer sequences identified upstream of these genes. The selected sequences were assembled into a synthetic promoter of 1 kb in size and then used... Figure 27 The reporter construct shown was tested. This dual reporter construct allows for qualitative and quantitative assessment of the specificity and potency (expression level) of the upstream promoter. The fluorescent reporter was used to qualitatively assess promoter specificity using flow cytometry, and the luminescent reporter was used to quantitatively assess promoter potency.

[0786] Example 27 The specificity and potency of T-cell synthetic promoters driven by specific combinations of core, proximal, and enhancer sequences.

[0787] This example describes the specificity and potency of 10 synthetic T-cell-specific promoters assembled as described in Example 23. For this purpose, the specificity (using flow cytometry reads of GFP expression in specific cell types) and potency (using luminescent reads from luciferase reporter expression) of multiple candidate synthetic promoters were tested across a range of cell types using the EF1a pervasive promoter as a comparison. Cell type combinations were selected based on predicted vector exposure after systemic administration and included lymphocytes (Jurkat and SupT-1 cell lines and primary T lymphocytes CD4+ and CD8+ from human PBMCs) and non-T lymphocytes from myeloid (THP1) and B-cell (NCI-H929) compartments and derived from the liver (Huh7) and kidney (293T) regions. Figures 28 to 2990. Figure 31 The results obtained on 10 candidates are presented.

[0788] As previously described, VSVG eLVs expressing reporter constructs under the control of EF1a or different synthetic promoters were generated in the e293T production cell line via transient transfection. Activated hPBMCs and cell lines (MOIs of 5 and 2, respectively) were transduced with different VSVG eLVs, and at 7 days post-transduction, cell eGFP expression was evaluated by flow cytometry, cell viability was assessed using Cell-Titer Fluor (Promega), and luciferase expression was evaluated by luminescence using the Steady-Glo assay (Promega). For hPBMCs, flow cytometry staining with anti-CD3 BV510, anti-CD4 APC-Cy7, and anti-CD8 PerCP was used to identify T cell subtypes.

[0789] Figure 28 to Figure 31 As shown, in Sup-T1 (Fig. 28), Jurkat (Fig. 29), and primary T cells (Fig. 30), the luminescence signal from luciferase expression driven by several synthetic promoters (especially SYN22) was equal to or higher than that from EF1a reporter expression. Adding an enhancer sequence to the SYN construct significantly increased luciferase reporter expression compared to the proximal promoter alone. Furthermore, as... Figure 30D As shown, the expression levels of the GFP reporter (measured as the geometric mean of fluorescence signals) were equal in both CD4+ and CD8+ primary T cell types. Conversely, when reporter expression was tested in non-T cell lines, fluorescence in the SYN construct was 4 to 628 times lower than that of EF1a-driven reporter expression, depending on the cell type. Figure 31 ).

[0790] In summary, these results demonstrate that specially constructed T-cell-specific synthetic promoters, optimized in size and fit into lentiviral constructs, effectively restrict reporter transgene expression to cultured T lymphocytes.

[0791] Example 28 Using T-cell synthetic promoters to express CAR transgenes during VHH-directed eLV significantly reduced CAR expression on the surface of producing cell lines during LV manufacturing and increased the functional titer of the vector.

[0792] This example describes the impact of using T-cell-specific synthetic promoters on the fabrication of VHH-targeting lentiviral vectors. We evaluated the yields (using functional titers) of LVGP1x-VHHCD8a vectors encoding BCMA or CD19 CARs controlled by the SYN or EF1a promoters.

[0793] Under the same conditions as previously described, LVGP1x-VHHCD8aSYN19 BCMA / CD19 CAR and LVGP1x-VHHCD8a EF1a BCMA / CD19 CAR eLV were produced from the VHHCD8 e293T production cell line. As shown in Figure 29A, and consistent with Figure 32A, the expression of the transgene (BCMA CAR stained with anti-V5 A647 and assessed by flow cytometry) was significantly reduced on the surface of the production cells (collected after final virus harvest) from the LVGP1x-VHHCD8 SYN19BCMA CAR production process-end cells compared to the process-end cells from LVGP1x-VHHCD8 EF1a BCMA CAR production process-end cells. To assess the functional titer of the vectors, SupT1 cells were transduced with each viral formulation at a series of dilutions (1:50 to 1:3125), and transduction was evaluated 3 days post-transduction after staining with anti-V5 A647 and quantification of the percentage of cells expressing BCMA or CD19 CAR by flow cytometry. The functional titer (transduction units / ml) was calculated based on the minimum of the two dilutions of the vector that produced a 5% to 50% percentage of transduced cells.

[0794] As shown in Figure 32B, the functional titer of LVGP1x-VHHCD8, encoding any CAR construct under the control of the SYN promoter, was 10-fold higher than that of the same construct expressed under the EF1a promoter. In summary, replacing EF1a with a synthetic T-cell-specific promoter to control the expression of the CAR cargo of the LVGP1x-VHHCD8 vector significantly enhanced the functional titer of vector production.

[0795] Example 29 Compared with constitutive promoters, T cell-specific synthetic promoters drive functional CAR expression and high CAR-T cell potency.

[0796] This example describes the evaluation of the efficacy of CAR-T cells expressing CAR constructs under the control of the T cell-specific synthetic promoters selected in Examples 26 to 28. For this purpose, BCMA CAR cytotoxic activity was evaluated in primary T cells transduced with eLV to express BCMA CAR under the control of the EF1a or SYN promoter.

[0797] exist Figure 33In this study, BCMA CAR-T cells, generated by transducing activated human PBMCs with VSVG pseudotyped eLV vectors (SYN19 BCMA CAR expressing the BCMA CAR transgene under the control of synthetic promoter 19 (SYN19); or EF1a BCMA CAR expressing the BCMA CAR transgene under the control of elongation factor 1a (EF1a) promoter; or EF1a GFP expressing the BCMA CAR transgene under the control of EF1a promoter), were cultured in the presence of multiple myeloma BCMA-expressing NCI-H929 cells (stable expression of luciferase reporter) at various effector-to-target ratios. Untransduced or GFP-expressing T cells were used as controls to determine nonspecific allogeneic reactivity. After five days of co-culture, 100 μl (100 µL) of culture was washed, and luciferase expression (correlation with the number of live NCI-H929 target cells) was assessed using the Steady-Glo assay (Promega). Compared to co-culturing untransduced or EF1a-GFP vector-transduced T cells, co-culturing CAR-T cells transduced with SYN19-BCMACAR or EF1a-BCMA CAR at both E:T ratios resulted in a >4-fold decrease in luminescence (correlation with levels in live NCI-H929 target cells). This result indicates enhanced cytotoxicity of SYN19-BCMA CAR-T cells compared to EF1a-BCMA CAR-T cells.

[0798] To further characterize the activity of BCMA CAR-T cells regulated by the T cell synthesis promoter, BCMA CAR-T cells generated from human PBMCs from three different donors were transduced using VSVG pseudotyped eLV vectors (SYN23 BCMA CAR expressing the BCMACAR transgene under the control of the synthesis promoter 23 (SEQ ID NO 15); or EF1a BCMA CAR expressing the BCMA CAR transgene under the control of the elongation factor 1a (EF1a) promoter). These cells were then cultured with target cells (NCI-H929) and non-target cells (K562) at different effector (CAR) to target (cell line) ratios. Fresh target cells were added at different time points to test the durability of CAR-T antitumor activity. The results presented in Figure 34 show that, in contrast to CARs regulated by the EF1a promoter, BCMA CAR-T cells expressing the CAR construct under the control of the SYN23 promoter exhibited (1) enhanced expansion, (2) enhanced antitumor activity in the presence of high levels of target cells (1:12), and (3) lower background activation and increased BCMA-specific activation in the absence of BCMA antigen. In summary, these results indicate that BCMA CAR-T cells expressing the CAR construct under the control of the T cell-specific synthetic promoter #23 possess superior and more durable antitumor activity.

[0799] Example 30 Demonstrates CD14-binding LVGP1x eLV-specific transduction of monocytes.

[0800] Targeting eLV via a combination of envelope-anchored binders and mutant envelope proteins (e.g., LVGP1x, G1x, LDV) is designed to target cell types other than T lymphocytes by switching the envelope-anchored binder. This example describes specific transduction of myeloid cells using LVGP1x eLV, which exhibits a membrane-anchored scFv binder targeting CD14 (a protein expressed on the surface of myeloid cells that acts as part of the LPS receptor complex). Notably, monocytes express multiple viral restriction factors (e.g., SAMHD1), and for efficient transduction of monocytes using lentiviral vectors, it is necessary to package viral protein X (Vpx) within viral particles. Vpx integration is achieved during viral production by adding a Vpx expression plasmid during transient transfection of the production cell line. As shown in Figure 35A, integration of Vpx into VSVG LV or LVGP1x scFV CD14 eLV vector particles (see below for details on vector generation and transduction conditions) significantly increased the transduction of cultured primary monocytes.

[0801] The specificity of the LVGP1x-scFvCD14 eLV vector was evaluated by comparing transduction of human PBMCs with that of primary monocytes isolated from the same donor PBMCs. The GFP-expressing LVGP1x-scFVCD14 scFvCD14 eLV vector was generated by transiently transfecting a stable membrane-anchored anti-CD14 scFv (SEQ ID NO. 41) e293T cell line with a packaging plasmid supplemented with the Vpx expression construct (SEQ ID NO. 43) (using the Gag / pol construct (SEQ ID NO. 42) containing a modified gag sequence to allow Vpx packaging), a transfer plasmid, and a membrane expression plasmid (LVGP1X). The physical titer of the vector suspension was determined using the RT-qPCR Viral Genome Quantification Kit (Takara), and unstimulated hPBMCs (cultured in 30 ng / ml IL-2) or isolated human mononuclear cells (isolated from hPBMCs using the CD14 MACS Isolation Kit from Miltenyi and cultured in 50 ng / ml GM-CSF (130-093-865) + 50 ng / ml IL-4) were transduced at 100, 200, 500, and 1000 vector particles / cells. The percentage of GFP-expressing cells was assessed by flow cytometry 7 days post-transduction.

[0802] As shown in Figure 35B, both CD14-targeting LVGP1 Vpx eLVs efficiently transduced primary monocytes, while transduction in unstimulated PBMCs remained below 2%, even when cells were exposed to 1000 viral particles / cell. In summary, redirecting LVGP1x eLV with an envelope-anchored anti-CD14 binder induced efficient and specific transduction in primary monocytes.

[0803] Example 31: Selecting CD14 VHH to achieve CD14-targeted eLV-mediated targeted transduction of monocytes.

[0804] The use of CD14 scFv-targeted eLV successfully achieved targeting of primary monocytes. To demonstrate that this also applies to VHH-targeted eLV, novel VHHs targeting CD14 or CCR2 were generated via immunization (SEQ ID NOs 66 to 116 and SEQ ID NOs 117 to 120, respectively), and their effectiveness as membrane-anchored binders in targeting and transducing eLV to monocytes was tested. This example describes the selection of CD14 VHHs to achieve optimal transduction of eLV (containing Vpx) to primary monocytes.

[0805] As shown in Figure 36A, several newly generated VHHs (SEQ ID NOs 66 to 76, 101, 102, 104, 106 to 117) specifically targeted and transduced G1x eLVs into CD14-expressing cells (Jurkat cells stably expressing CD14). For this early stage of the screening process, eLVs were generated from e293T cells, and the VHH binder was encoded in the transfer vector. Therefore, the transduction efficiency of the G1x CD14 VHH eLVs generated using this method was evaluated by the expression of VHHs in the transduced cell population.

[0806] From the results in Figure 35A, the most specific subsets of CD14 VHH (SEQ ID NO 70, 72, 76, 107, 111) were selected, and G1x CD14 VHH eLVs were generated from stably expressing e293T cells to test transduction efficiency in cultured primary monocytes. As shown in Figure 35B, G1x CD14 VHH eLVs efficiently transduced monocytes, with the highest level of transduction achieved using G1x eLVs targeting 3RMB1 VHH (SEQ ID NO 76), and transduction was proportional to the number of carrier particles per cell.

[0807] 3RMB1 VHH was selected for further optimization via in vitro maturation. As shown in Figure 36C, the transduction efficiency of mature 3RMB1 (SEQ ID NO 77 to 100) targeting G1x eLV differed from that of the original 3RMB1 VHH targeting vector, with mature 3RMB1-13 VHH (SEQ ID NO 94) achieving the highest level of transduction targeting G1x eLV.

[0808] The specificity of G1x eLV targeted with 3RMB1 VHH was evaluated in other cell types. Figure 37 As shown, 3RMB1 VHH G1x eLV efficiently transduced CD14+ cells (monocytes / macrophages and Jurkat CD14 cell lines), but not CD14-negative cells (stimulated PBMC, Jurkat, or NCI-H929 cell lines). Importantly, transduction of non-targeted (3RMB1 VHH-deficient) G1x eLV was undetectable, highlighting the crucial role of VHH targeting in promoting specific and efficient transduction of target cells.

[0809] These results collectively demonstrate that, using membrane-anchored CD14 VHH, G1x eLV (containing Vpx) can effectively and specifically target CD14+ cells (including primary monocytes).

[0810] Example 32: Monocyte / macrophage synthesis driven by a specific combination of core, proximal, and enhancer sequences promoter specificity and efficacy .

[0811] This example describes the specificity and potency of 11 synthetic myeloid-specific promoters (SEQ ID NOs 130-140 and SEQ ID 197) assembled as described in Example 23. For this purpose, the specificity (using flow cytometry reads of GFP expression in specific cell types) and potency (using luminescent reads from luciferase reporter expression) of multiple candidate synthetic promoters were tested across a range of cell types using the EF1a pervasive promoter as a comparison. Cell type combinations were selected based on predicted vector exposure after systemic administration and included lymphocytes (SupT-1 cell line and human PBMCs) as well as non-T lymphocytes from myeloid cell (THP1) and B cell (NCI-H929) compartments and derived from the liver (Huh7) and kidney (293T) regions. Figures 38-40 present the results obtained on seven candidates.

[0812] As previously described, VSVG eLVs expressing reporter constructs under the control of EF1a or different core promoters or fully synthetic promoters were generated in the e293T production cell line via transient transfection. Primary cells and cell lines were transduced with the purified vector, and the expression level of the reporter eGFP transgene was evaluated in the transduced cells.

[0813] Six proximal promoters derived from macrophage-specific genes were selected for testing. As shown in Figure 38, compared with EF1a promoter-driven reporter vectors (excluding MRC1), the proximal promoters of CCL13, CCL18, CHIT1, HLA-DRA, LYZ, and MRC1 (SEQ ID NO 130, 131, 132, 133, 134, and 197) induced higher eGFP expression in primary monocytes and approximately 5 to 20-fold lower expression in cell lines and activated PBMCs (mainly T cells). These data indicate that HLA-DRA, LYZ, CHIT1, CCL8, and CCL13-induced reporter expression is highly enriched in primary myeloid cells compared with expression controlled by EF1a.

[0814] The CHIT1 sequence was used as the core of the myeloid-specific synthetic promoter tested in Figure 39 and assembled using enhancer sequences (SEQ ID NO 135 to 140) isolated from myeloid-specific genes. As shown in Figure 39, adding enhancer sequences to the CHIT1 core promoter significantly increased eGFP reporter expression compared to the proximal promoter alone or EF1a, while preserving specificity for primary monocytes / macrophages.

[0815] To confirm that the myeloid-derived synthetic promoters tested in Figure 39 induced functional expression levels of transgenes other than the eGFP reporter in monocytes / macrophages, we tested IL12 secretion in macrophages transduced with the VSVG LV vector expressing human IL12 under the control of the EF1a promoter or the myeloid-derived synthetic promoter Enh31 CHIT1 (SEQ ID NO 139). As shown in Figure 40, IL12 secretion levels from macrophages (M0 or M2) transduced with Enh31 CHIT1 were comparable to or higher than those from EF1a, and significantly higher than those from macrophages transduced with IL12 controlled only by the core CHIT1 promoter.

[0816] In summary, these results demonstrate that specially constructed monocyte / macrophage-specific synthetic promoters, optimized in size and suitable for inclusion in lentiviral constructs, effectively restrict reporter transgene expression to cultured monocytes / macrophages.

[0817] Example 33 Optimizing the structure of the VHH envelope anchoring scaffold improves the transduction efficiency of CD8a and CD3e VHH targeting vectors.

[0818] In these embodiments, different hinge regions and transmembrane domains are used to determine the optimal capsule anchoring scaffold for CD3e VHH (SEQ ID NO.3) or CD8 VHH (SEQ ID NO.6) to achieve high-level transduction of target cells.

[0819] VHHCD3e eLV and VHHCD8a eLV vectors were generated by transiently transfecting e293T cells with packaging plasmids encoding Gag / Pol, Rev, and LVGP1x constructs and transfer vectors for expressing VHH-targeting constructs. Each VHH was embedded in a viral envelope anchoring construct consisting of transmembrane domains (PDGFR or B7-1; SEQ ID NO. 27 and SEQ ID NO. 28, respectively) fused to different linker regions with varying flexibility, length, and polymerization potential (see SEQ ID NO. 21 to 26). After harvest, Jurkat WT cells were transduced with the same volume of lentiviral formulation. Three days post-transduction, the functional titer of each vector was assessed by flow cytometry using anti-VHH staining of transduced cells, expressed as transduction units per milliliter.

[0820] like Figure 41 and Figure 42As shown, the transmembrane domain variants PDGFR or B7-1 had little effect on the degree of targeting with CD3e or CD8a VHH, with the PDGFR transmembrane domain variant performing slightly better. Hinge domains had a greater impact. The two best-performing hinge regions in the LVGP1X-VHHCD3e eLV and LVGP1X-VHHCD8a eLV were the PDGFR short stem and the CD8a hinge, exhibiting moderate flexibility and a lower tendency to polymerize compared to shorter, more rigid hinges (e.g., GAPGAS) or polymerized hinges (e.g., tetrameric coiled helices).

[0821] Example 34 Optimizing the structure of the VHH envelope anchoring scaffold improves the transduction efficiency of the TCR VHH targeting vector.

[0822] In this embodiment, different hinge regions and transmembrane domains are used to determine the optimal capsule anchoring scaffold of TCR VHH (SEQ ID NO. 9) to achieve high-level transduction of target cells.

[0823] As previously described, e293T cells stably expressing membrane-anchored TCR VHH generated a VHHTCR eLV vector expressing dsGFP. This membrane-anchored TCR VHH was anchored to the viral envelope via four different scaffolds with varying flexibility, length, and polymerization potential: TCR1: PDGFR transmembrane domain and PDGFR juxtamembrane region (SEQ ID NO. 44); TCR2: PDGFR transmembrane domain and IgG4 juxtamembrane region (SEQ ID NO. 34); TCR3: PDGFR transmembrane domain and CD8a stem (SEQ ID NO. 45); TCR4: PDGFR transmembrane domain and tetrameric coiled helical hinge (SEQ ID NO. 46). Notably, in this study, a VSVG mutant (K47A, R354; SEQ ID NO. 47) (previously shown to eliminate the extensive tropism of wild-type VSVG) was used to pseudotype the VHHTCR eLV. After concentration, Jurkat WT cells were transduced with the same volume of lentiviral formulation, and transduction efficiency (GFP+ cells) was assessed by flow cytometry on day 3.

[0824] As shown in Figure 43A, different hinge sequences within the TCR VHH scaffold affect transduction efficiency. In the case of TCR VHH, the anchored scaffold containing the PDGFR transmembrane domain and the IgG4 juxtamembrane region provides an architecture that achieves the highest transduction efficiency when connected to the VSVG (K47A, R354) pseudotype.

[0825] When targeting the eLV vector with G1x pseudotype, the VHH scaffold architecture was further optimized for TCRab VHH. TCRab VHH G1x eLVs were generated from e293T cells stably expressing TCRab VHH via transient transfection. These TCRab VHHs were housed in a scaffold consisting of the PDGFR transmembrane domain and the IgG4 juxtamembrane region (SEQ ID NO. 34) or a scaffold consisting of the PDGFR transmembrane domain and the CD8a juxtamembrane region (SEQ ID NO. 52). T cell activation, expansion, and transduction were characterized in primary human PBMCs exposed to the same volume of unconcentrated vector. As shown in Figure 43B, compared to the IgG4 hinge, the TCRab VHH G1x eLVs presenting VHH using the PDGFR / CD8a hinge scaffold achieved complete T cell transduction on day 7 post-transduction.

[0826] Example 35: Resting T cells were induced by targeting LVGP1x or G1x eLV with TCRab VHH binder. Activation, amplification, and enhanced transduction

[0827] Upon binding to its homologous antigen, the TCR complex aggregates and triggers a signaling cascade, leading to T cell activation. In this embodiment, we demonstrate that TCRab VHH binders located on a different scaffold on the eLV surface, compared to VSVG LV, induce T cell activation, expansion, and increased transduction.

[0828] LVGP1x-TCRabVHH (SEQ ID NO. 34) expressing dsGFP was generated as previously described. Resting human PBMCs were transduced with 10 μl of LVGP1x-VHHTCR eLV in the presence of low concentrations of rhIL-2 (30 ng / ml), and T cell activation was assessed 24 hours post-transduction by staining with anti-CD25 or anti-CD69 and CD3. As shown in Figure 44, LVGP1x-TCRab VHH eLV induced significant CD25 expression in resting T cells compared to culture medium alone, VSVG LV, and LVGP1x-CD8a VHH eLV. Associated with increased T cell activation, LVGP1x-TCRab VHH successfully transduced resting CD3+ T cells, as measured by GFP expression, and was more effective than VSVG GFP eLV at day 19 (Figure 44C). Compared to VSVG GFP-transduced cells, LVGP1x-TCRab VHH eLV also showed significant activation and transduction of resting T cells within 20 days, which was associated with a substantial increase in GFP-expressing cells (Fig. 44B).

[0829] The activation and expansion of resting T cells observed with TCRab VHH eLV were also regulated by the VHH scaffold in the G1x pseudotyped LV. As shown in Figure 45, TCRab VHH G1x eLV presenting VHH with the PDGFR / CD8a hinge scaffold (SEQ ID NO 34) resulted in higher expression of CD69, CD25, and PD1, as well as increased cell expansion and functional viral titers, compared to the IgG4 hinge (SEQ ID NO 34).

[0830] Similarly, T cell activation was observed with alternative TCRab VHH G1x eLV, which represents an alternative TCRab VHH binder (SEQ ID NO 54 to 65) optimized through in vitro maturation on the original TCRab VHH (SEQ ID NO 9). As shown in Figure 46A, T cell activation was induced in Jurkat cells by exposure to several different TCRab VHH G1x eLVs (each with similar transduction efficiency (Figure 46B)) (assessed by increased CD69 expression).

[0831] In summary, using TCR-specific VHHs in different scaffolds to target T cells with LVGP1x or G1x pseudotyped eLVs induced activation and effective transduction of resting lymphocytes, resulting in a significant expansion of transduced cells.

[0832] Example 36: Displaying co-stimulatory ligands on the surface of VHH-targeted eLV increased the activation and expansion of resting T cells. and transduction

[0833] Resting T lymphocytes exposed to CD8aVHH eLV induced low levels of T cell activation (indicated by CD25 expression) and transduction (indicated by expression of the V5-labeled BCMA CAR transgene) and minimal T cell expansion (Figs. 47A–47C). To test whether the display of co-stimulatory ligands on the surface of the CD8aVHH eLV vector increased T cell activation / expansion and transduction, CD8aVHH G1x eLVs were generated from the CD8aVHH e293T cell line displaying OKT3 alone (a CD3 agonist antibody; SEQ ID NO 29) or in combination with CD28 co-stimulatory receptor activators CD80 or CD28 VHH (SEQ ID NOs. 198 and 199, respectively, and SEQ ID NO. 200 for CD8 scFV scaffolds), as previously described. In the presence of low levels of rhIL-2 (30 ng / ml), resting human PBMCs were transduced with CD8a VHH G1x eLV, CD8a VHH / OKT3 G1x eLV, CD8a VHH / OKT3 / CD80 G1x eLV, or CD8a VHH / OKT3 / CD28 scFv G1x eLV, and T cell activation, expansion, and transduction were assessed by flow cytometry. As shown in Figures 47 A to C, CD8a VHH / OKT3 / G1x eLV, CD8a VHH / OKT3 / CD80 G1x eLV, or CD8a VHH / OKT3 / CD28 scFv G1x eLV induced a significant increase in T cell activation (CD25) and T cell expansion (>10-fold compared to CD8a VHH G1x eLV).

[0834] Similar tests were performed using the TCRab VHH G1x eLV vector, which had already induced T cell activation and expansion, to determine whether the co-stimulatory ligands further increased T cell activation. As shown in Figures 47 D to F, TCRab VHH / displaying CD3 agonist-binding agent OKT3 (TCRab VHH / OKT3scFv G1x eLV), CD28 ligand CD80 (TCRab VHH / CD80 G1x eLV), or CD28 agonist-VHH (TCRab VHH / CD28scFv G1x eLV) increased the activation and transduction of resting T cells, while the increase in T cell expansion levels was minimal. As shown in Figure 48, TCRab VHH G1x eLV with CD28 co-stimulatory molecule CD86 IgV, TGN1412, or 4-1BB co-stimulatory molecule 4B1-4 (SEQ ID NO. 50, 51, and 201, respectively) induced increased IL2 production and CD69 and CD25 expression, indicating increased T cell activation. This is associated with increased amplification and transduction of resting PBMCs compared to the TCRab VHH G1x eLV vector.

[0835] Example 37 Targeting eLV transduction of primary T lymphocytes with CD8a or TCR VHH encoded by engineered TCRs resulted in specific, persistent, and functional expression of the transgene, as demonstrated by the cytotoxicity of TCR-engineered cells.

[0836] Adoptive T-cell therapy using T-cell receptor-engineered T cells (TCR-T cells) has shown promising results in the treatment of certain types of cancer, and in vivo reprogramming of TCR-T cells using VHH-directed eLVs will overcome the challenges associated with currently ex vivo manufactured TCR-T products. To evaluate the ability of VHH-directed eLVs to deliver engineered TCR constructs, we tested CD8a and TCR VHH-targeted LVGP1x eLVs on cultured human PBMCs. These encode clinically validated and widely used engineered TCRs (NY-ESO).

[0837] As previously described, LVGP1X-VHHCD8a and LVGP1X-VHHTCR, which encode TCR transgenes targeting the HLA-A*02:01-binding NY-ESO-1157-165 peptide (i.e., 1G4-LY, TCR α chain SEQ ID NO. 30 and TCR β chain SEQ ID NO. 31), were generated from stably expressed e293T production cells. Human PBMCs (activated with anti-CD3 / CD28) were transduced with 10 μl of concentrated eLV, and transduction efficiency in CD3+ T cells was assessed by flow cytometry using PE-conjugated A2 / NY-ESO-1 multimers at days 3, 7, 14, and 21. On day 17 post-infection, transduced lymphocytes were counted and co-cultured at a 1:1 ratio with BFP-positive HLA-A*02:01-positive T2 lymphoblasts stimulated with either 1 μg / ml control HIV peptide (HIV476-484 amino acid sequence: ILKEPVHGV) or NY-ESO-1157-165 peptide (SLLMWITQC). After 72 hours, cells were collected and their viability and counts were analyzed by flow cytometry.

[0838] As shown in Figures 49A and 49B, targeting activated hPBMCs with LVGP1x eLV using CD8a or TCR VHH encoding NY-ESO resulted in specific and stable transduction into CD8+ T cells or CD3 T cells, respectively. As previously demonstrated with CAR-expressing eLVs, restricting NY-ESO1 transgene expression during viral generation using the T-cell synthesis promoter (SYN21; SEQ ID NO. 12) significantly increased the vector's functional titer compared to EF1a-driven transgene expression. The transduction efficiency of the EF1a NY-ESO construct was approximately one logarithmic order lower than that of the SYN NY-ESO construct.

[0839] After 72 hours, T2 cells stimulated with the control HIV peptide were recovered from co-cultures with untransduced (UT) or NY-ESO-1 transduced cells (Fig. 50B). However, T2 cells stimulated with the NY-ESO-1 peptide were only recovered in the UT group, and there was also some recovery in the EF1a promoter group (where less than 5% of total CD3+ T cells expressed NY-ESO TCR). T2 cells stimulated with the target NY-ESO were not recovered from co-cultures with cells transduced with LVGP1x-VHHTCR SYN NY-ESO (Fig. 50A). These results indicate that T cells transduced with eLV using VHH targeting an engineered TCR construct are functional and highly cytotoxic to target cells.

[0840] Example 38TCRab VHH G1x eLV specifically delivers the BCMA CAR construct to T cells in vivo and generates functional BCMA CAR T cells that eliminate multiple myeloma cell lines transplanted systemically in humanized mouse models.

[0841] This example describes the in vivo transduction efficiency of TCRab VHH G1X eLV in humanized mice, thereby generating functional BCMA CAR-T cells in vivo and eliminating target cancer cell populations.

[0842] Five e^6 NCI-H929 cells were intravenously injected into NSG MHCI / II KO mice, followed by transplantation 7 days later. Then, activated human PBMCs were injected intraperitoneally, and one day later, the TCRab VHH G1X eLV vector encoding a BCMA CAR construct controlled by the T cell-specific synthetic promoter SYN23 was injected intraperitoneally. In vivo reprogrammed BCMA CAR T cells were analyzed weekly in peripheral blood and in bone marrow at euthanasia on day 42 post-vector infusion. The antitumor activity of BCMA CAR-T cells was assessed weekly by bioluminescence imaging of luciferase-expressing NCH-H929 cells, body weight, and clinical observation.

[0843] As shown in Figure 51A, intraperitoneal injection of the ENaBL-GTT vector was well tolerated, and no adverse events were observed during the study. In animals injected with the control TCRab VHH G1X eLV BFP vector, tumor growth exhibited exponential growth starting from day 8, while TCRab VHH G1X eLV BCMA CAR injection led to tumor cell elimination and prevented regrowth until the end of the study on day 42. BCMA CAR T cells were detected only in animals injected with TCRab VHH G1X eLV BCMA CAR (7.9% ± 1.2% CD3+CAR+ cells; Figure 51B). Durability of BCMA CAR T cells was observed throughout the study.

[0844] This study demonstrates that the TCRab VHH G1x eLV BCMA CAR vector reprograms BCMA CAR T cells in vivo, thereby completely eliminating the systemically transplanted multiple myeloma cell line (NCI-H929) in hPBMC humanized mice.

[0845] Example 39 TCRab VHH G1x eLV specifically delivers the BCMA CAR construct to T cells in vivo and generates functional BCMA CAR T cells that eliminate multiple myeloma cell lines transplanted systemically in humanized mouse models.

[0846] This example describes the in vivo T cell transduction specificity and efficacy of TCRab VHH G1X eLV in stable humanized mice carrying systemic multiple myeloma, thereby generating functional BCMA CAR-T cells in vivo and eliminating target cancer cell populations.

[0847] The TCRab VHH G1X eLV vector, encoding a BCMA CAR transgene (or BFP reporter) controlled by the T cell-specific synthetic promoter SYN23, was administered intravenously in a single dose to hCD34+ NCG mice carrying multiple myeloma derived from NCI-H929 cells stably expressing luciferase. Antitumor efficacy was evaluated by monitoring NCI-H929 cell growth (in vivo bioluminescence imaging), animal body weight, clinical status, and survival. Furthermore, flow cytometry was used to analyze the transduction kinetics of the TCRab VHH G1X eLV BCMA CAR and the cell kinetics of transduced cells in peripheral blood. Flow cytometry was also performed on the spleen, bone marrow, and tumor cells to identify the presence and status of CAR T cells in these tissues.

[0848] Although tumor growth was uncontrolled in animals receiving TCRab VHH G1X eLV BFP or the mediator, by day 35 post-tumor injection, mice injected with TCRab VHH G1X eLV BCMA CAR had tumors eradicated in 2 / 8, 8 / 8, and 7 / 8 mice in the low, medium, and high dose groups, respectively. Figure 52 As shown in Figure 53, the transduction of TCRab VHH G1X eLV BCMA CAR is specific for human T cells (both CD4 and CD8 lymphocytes) in peripheral blood (Figure 53A) and bone marrow (Figure 53B), with less than 0.5% of hCD14, hCD20, hCD56 and mouse CD45 cells expressing higher levels of CAR than the background.

[0849] This study demonstrated the effective antitumor activity of (1) TCRab VHH G1X eLV BCMA CAR reprogrammed T cells in vivo and the specificity of the TCRab VHH G1X eLV vector for T lymphocytes in vivo.

[0850] Example 40 The combination of T lymphocyte and monocyte reprogramming synergistically eliminated solid tumors in humanized mice.

[0851] This example describes the in vivo administration of a combination of T cell-specific TCRab VHH G1x eLV BCMA CAR vector and monocyte-specific CD14 VHH G1x eLV expressing human IL12, and the synergistic antitumor activity of this vector combination against NCI-H929 cells subcutaneously implanted to mimic solid tumors.

[0852] Intravenous injection was administered into hCD34+NCG mice with solid tumor-like multiple myeloma carrying 5e6 NCI-H929 cells derived from subcutaneous injection of (1) a monocyte-specific CD14 VHH G1x eLV vector expressing human IL12 or a GFP reporter under the control of a constitutive EF1a promoter; or (2) a TCRab VHH G1X eLV vector encoding a BCMA CAR transgene under the control of a T cell-specific synthetic promoter 23; or (3) the same dose of CD14 VHH G1x eLVhIL12 and TCRab VHH G1x eLV BCMA CAR vector in a single combined injection. Antitumor efficacy was evaluated by monitoring NCI-H929 cell growth (caliper measurement of subcutaneous solid tumor masses) and animal survival. In addition, the levels of BCMA CAR-T cells and human IL12 in peripheral blood transduced cells were analyzed by flow cytometry.

[0853] like Figure 54 As shown, although tumor growth was uncontrolled or poorly controlled in animals injected with TCRab VHH G1x eLV BCMA CAR, CD14 VHH G1xeLV hIL12, or CD14 VHH G1x eLV GFP vector alone, NCI-H929 tumor cells were eliminated in single doses of the combination of TCRab VHH G1x eLV BCMA CAR and CD14 VHH G1x eLV hIL12. As shown in Figure 55A, T lymphocyte transduction was observed only in the TCRab VHH G1x eLV BCMA CAR and the combination of TCRab VHH G1x eLV BCMA CAR and CD14 VHH G1x eLV hIL12 groups, with a significant increase in the percentage of BCMACAR-T cells in the combination group. As shown in Figure 55B, human IL12 was detected only in animals injected with CD14 VHH G1x eLV hIL12. At the end of the study, circulating IL12 levels remained ≥40,000 pg / ml (data not shown). The combination therapy was significantly effective in increasing T-cell infiltration levels within the tumor. As shown in Figure 55B, the combination of BCMA CAR reprogramming of T cells and secretory IL12 reprogramming of monocytes induced a significant increase in T-cell tumor infiltration.

[0854] This study demonstrates that (1) intravenous administration of CD14 VHH G1x eLV hIL12 leads to sustained secretion of high levels of hIL12, indicating persistent and stable transduction of monocytes / macrophages using CD14 VHH G1x eLV hIL12; (2) in vivo reprogramming of multiple cell types can be achieved in the same organism by a single intravenous injection of our VHH targeting vector; and (3) in vivo reprogramming of multiple cell types with enhanced antitumor activity synergistically eliminates tumors that were originally resistant to a single engineered cell type.

[0855] Example 41 : Demonstrating complement activity regulators on the viral envelope of VHH targeting eLV reduced complement-mediated vector particle inactivation.

[0856] The transduction efficiency of systemically injected lentiviral vectors is limited by host viral inactivation mechanisms, including complement-mediated inactivation. This example describes the engineering of the TCRab VHH G1x eLV vector envelope to exhibit exogenous inhibitors or resistance factors that reduce complement inactivation of the viral vector.

[0857] As previously described, transient transfection of e293T production cells stably expressing TCRab VHH and any of the following RCAs was used to generate TCRab VHH targeting G1x eLV, which exhibits complement activity regulators (RCA): (1) DAF (CD55) or (2) factor H, both of which are tethered to the membrane by fusion with the GPI anchor or VSVG transmembrane domain (SEQ ID NO 183 to 186; Fig. 56A).

[0858] Complement-mediated vector particle inactivation was assessed by treating the cells with (1) serum-free (complement-free) medium, (2) medium supplemented with fresh human serum (active complement), or (3) medium supplemented with heat-inactivated human serum (inactivated complement) for one hour. Jurkat cells were then incubated with the lentiviral formulation for 2 hours, and the transduction efficiency of the vector was assessed after 72 hours to quantify the percentage of functional titer recovery of viral particles in the presence of serum. As shown in Figure 56B, TCRab VHH G1x eLVs with DAF-GPI and factor H (GPI or GS) were partially protected from complement-mediated inactivation, with mean recovery rates up to 24.1% and 16.3%, respectively. These results demonstrate the protective effect of RCA against complement-mediated vector inactivation.

[0859] Example 42 Demonstrating an albumin-binding domain on the viral envelope of VHH-targeting eLV reduced complement-mediated vector particle inactivation.

[0860] Albumin-binding domains have previously been used to prolong the pharmacokinetics of intravenously injected therapeutic agents and reduce phagocytosis. This example describes the engineering of the TCRab VHH G1x eLV carrier envelope to exhibit an albumin-binding domain to reduce carrier phagocytosis.

[0861] As previously described, transient transfection of e293T production cells was used to generate TCRab VHH targeting G1x eLV, which displays the albumin-binding domain (ABD) of protein G of Streptococcus strain G148. These cells stably expressed TCRab VHH and ABD, which were tethered to the membrane by fusion with the GPI anchor or VSVG transmembrane domain (SEQ ID NO 192 and 191, respectively).

[0862] The phagocytic activity of the carrier particles was assessed by incubating the carrier formulation with fresh human monocyte-derived macrophages for 2 hours and then quantitatively inserting the carrier 3 days post-exposure. The phagocytic inhibitor chlorpromazine was used as a negative control. Figure 57 As shown, the TCRab VHH G1x eLV vector was phagocytosed by primary macrophages, leading to its integration into the host cell's genomic DNA; this process was inhibited by chlorpromazine. ABD was observed on the envelope of TCRab G1x eLV to reduce the phagocytosis of the lentiviral product, resulting in a significant reduction in the number of viral genomes per macrophage. This result demonstrates the protective effect of ABD on vector phagocytosis.

[0863] Example 43 Synthetic promoters assembled using enhancers of genes preferentially expressed in tumor-associated macrophages compared to tissue-resident macrophages can be induced by signals specific to the tumor microenvironment.

[0864] This example describes the expression of reporter transgenes from synthetic promoters that are induced by signals associated with the tumor microenvironment. Twelve tumor-associated macrophage (TAM)-specific promoters were assembled using enhancers from genes preferentially expressed in TAMs, as described in Example 23 (see SEQ ID NO 141 to 152).

[0865] As previously described, VSVG eLVs expressing GFP / luciferase reporter constructs under the control of EF1a or different synthetic promoters were generated in the e293T production cell line via transient transfection. Primary human monocytes were transduced with different VSVG eLVs and cultured for 7 days, followed by 2 days of culture in media supplemented with the following TME-related signals: (1) IL4, IL10, and TGFb; or (2) CoCl2 (to induce hypoxia signaling); or (3) conditioned medium from the gastric cancer cell line NUGC4. After 2 days of culture, reporter (GFP) expression levels were assessed by flow cytometry.

[0866] As shown in Figure 58, although the expression of reporter from the EF1a promoter decreased when cells were exposed to hypoxia or conditioned medium, the expression driven by the TAM synthesis promoter increased, especially for the TAM12 promoter.

[0867] In summary, these results demonstrate that specially constructed TAM synthetic promoters, with optimized size that can be incorporated into lentiviral constructs, provide a strategy for restricting transgene expression within the tumor microenvironment via TME-related signaling.

Claims

1. A viral vector having a lipid bilayer envelope, the lipid bilayer envelope comprising: (a) a VHH binding domain displayed on the exterior of the envelope, the VHH binding domain being cell type specific; (b) a VSV-G envelope protein displayed on the exterior of the envelope, the VSV-G envelope protein being capable of facilitating infection of the same cell type as in (a), wherein the VSV-G envelope protein comprises an amino acid substitution at position 331 ; and (c) a nucleic acid molecule comprising a promoter capable of being expressed in the same cell type as in (a).

2. The viral vector of claim 1, wherein the promoter is cell type specific.

3. The viral vector of any preceding claim, wherein the VSV-G mutation at position 331 is selected from the group consisting of I331E, I331W, I331M, I331L, I331Q and I331R.

4. The viral vector of any preceding claim, wherein the VSV-G mutation at position 331 is I331E.

5. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position I52.

6. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position E355.

7. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position N9.

8. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position K47.

9. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position T352.

10. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position H8.

11. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position M45.

12. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position K11.

13. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position S408.

14. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position R354.

15. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position Q53.

16. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position E353.

17. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position Q10.

18. The viral vector of any preceding claim, wherein a further VSV-G mutation is provided at position K50.

19. The viral vector of any preceding claim, wherein the mutant VSV-G envelope protein comprises mutations I52R, I331E and E355L.

20. The viral vector of any preceding claim, wherein the mutant VSV-G envelope protein comprises mutations N9G, K47G, I331E and T352S.

21. The viral vector of any preceding claim, wherein the mutant VSV-G envelope protein comprises mutations H8L, M45L, I331E and E355R.

22. The viral vector of any preceding claim, wherein the mutant VSV-G envelope protein comprises mutations Q53A, I331E and T352I.

23. The viral vector of any preceding claim, wherein the mutant VSV-G envelope protein comprises mutations K11A, S408L, I331L and R354K.

24. The viral vector of any preceding claim, wherein the mutant VSV-G envelope protein comprises mutations N9R, Q53I, I331Q and R354M.

25. The viral vector of any preceding claim, wherein the mutant VSV-G envelope protein comprises mutations I331E and E353K.

26. The viral vector of any preceding claim, wherein the mutant VSV-G envelope protein comprises mutations Q10V, K50H, I331M and T352D.

27. The viral vector of any preceding claim, wherein the mutant VSV-G envelope protein comprises mutations N9A, I331E and E353S.

28. The viral vector of any preceding claim, wherein the VHH binding domain and the promoter are specific for use in an immune cell.

29. The viral vector of claim 28, wherein the immune cell is selected from a T cell, a natural killer (NK) cell and / or a macrophage cell.

30. The viral vector of any preceding claim, wherein the viral vector is a retroviral vector, such as a lentiviral vector.

31. The viral vector of any preceding claim, wherein the viral envelope is substantially free of or free of MHC class I.

32. The viral vector of any preceding claim, wherein the viral vector displays high levels of CD47, agonistic CD3 scFv, 41BBL and / or CD80 on the surface of the envelope.

33. The viral vector of claim 32, wherein the viral vector overexpresses CD47.

34. The viral vector of any preceding claim, wherein the VHH binding domain is specific for CD3s, CD8 or T cell receptor (TCR).

35. The viral vector of any one of claims 1 to 33, wherein the VHH binding domain is specific for CD14, CD16, CCR1, CCR2, CXCR4 or CD64.

36. The viral vector of any preceding claim, wherein the nucleic acid molecule encodes a transgene.

37. The viral vector of any preceding claim, wherein the nucleic acid molecule encodes a chimeric antigen receptor (CAR).

38. The viral vector of any preceding claim, wherein the promoter is expressed only in T cells.

39. The viral vector of any one of claims 1 to 37, wherein the promoter is expressed only in monocytes or macrophages.

40. The viral vector of any one of claims 1 to 37, wherein the promoter is expressed only in NK cells.

41. The viral vector of any preceding claim, wherein the promoter is a synthetic promoter comprising a promoter element and an enhancer element.

42. The viral vector of any preceding claim for use in diagnosis, prevention or treatment of a disease.

43. The viral vector for use of claim 42, wherein the disease is cancer.

44. The viral vector for use of claim 42 or claim 43, wherein the viral vector is for use in the treatment of cancer.

45. The viral vector for use of claim 44, wherein the cancer is selected from blood cancer (e.g. multiple myeloma), gastric cancer, lung cancer, colorectal cancer and breast cancer.

46. The viral vector for use of any one of claims 42 to 45, wherein the disease is a human disease.

47. A method of making the viral vector of any one of claims 1 to 41, the method comprising the steps of: (a) engineering a cell to express a cell type specific membrane anchored VHH binding domain; (b) engineering the cell to express a VSV-G envelope protein displayed on the exterior of the envelope, the VSV-G envelope protein being capable of facilitating infection of the same cell type as in (a), wherein the VSV-G envelope protein comprises an amino acid substitution at position 331 ; (c) replicating a nucleic acid vector in the cell, the nucleic acid vector comprising a promoter capable of expression in the same cell type as in (a); and (d) harvesting the viral vector in the form of vesicles from the cell.

48. The method of claim 47, wherein the cell is a 293T derived cell line.

49. The method of claim 47 or claim 48, wherein the cell has been modified to express or overexpress CD47.

50. The method of any one of claims 47 to 49, wherein the cell has been modified to have MHC class I expression knocked out.

51. The method of any one of claims 47 to 50, wherein the nucleic acid vector is a plasmid.

52. A method of modifying a cell, the method comprising exposing the cell to the viral vector of any one of claims 1 to 41.

53. The method of claim 52, wherein the modification is performed in vivo or ex vivo, preferably in vivo.

54. The method of claim 52 or claim 53, wherein the cell to be modified is a T cell.

55. The method of claim 54, wherein the VHH binds the T cell via the T cell receptor (TCR).

56. The method of claim 54, wherein the VHH binds the T cell via CD3s.

57. The method of claim 52 or claim 53, wherein the cell to be modified is a monocyte or macrophage.

58. The method of claim 57, wherein the VHH binds the monocyte or macrophage via one of CD14, CD16, CCR1, CCR2, CXCR4, or CD64.

59. The method of claim 52 or claim 53, wherein the cell to be modified is an NK cell.

60. The method of claim 59, wherein the VHH binds the NK cell via CD16, CXCR4, or CCR1.

61. A virus-like particle (VLP) having a lipid bilayer envelope, the lipid bilayer envelope comprising: (a) a VHH binding domain displayed on the exterior of the envelope, the VHH binding domain being cell type specific; and (b) a VSV-G envelope protein displayed on the exterior of the envelope, the VSV-G envelope protein being capable of facilitating infection of the same cell type as in (a), wherein the VSV-G envelope protein comprises one or more mutations, an amino acid substitution at position 331.

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