Viral vectors and producing cells

Abstract

A viral vector having a lipid bilayer envelope is provided, comprising a cell-type specific antibody-binding domain presented outside the envelope, a viral envelope protein presented outside the envelope capable of promoting infection of the same cell type, and a nucleic acid molecule containing a promoter expressible in the same cell type. Furthermore, a method for producing the viral vector and a method for modifying cells and treating diseases / conditions using this viral vector are also provided.

Description

[Technical Field]

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

[0002] Lentiviruses are a family of retroviruses that infect host cells by inserting their DNA into the host cell's genome. While many such viruses have formed the basis of research using viruses in gene therapy, lentiviruses are unique in 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 in humans to treat CD4 + T cells, CD8 + T cells and / or CD4 +Lentiviruses have been used as vectors for gene transfer to target cells, including transduction into regulatory T cells. Multiple steps are involved in the infection and replication of lentiviruses in host cells. In the first step, the virus attaches to the outer surface of the cell using its surface glycoprotein. For example, lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) target a wide range of cell types due to the specificity of VSVG to receptors (LDLRs) expressed on the surfaces of numerous different cells. Following the targeting and binding of the virus to the target cell, the viral material is then injected into the cytoplasm of the host cell. In the cytoplasm, viral reverse transcriptase reverse transcribes the viral RNA genome to produce the viral DNA genome. The viral DNA is then transported to the nucleus of the host cell, where it is integrated into the host cell genome with the help of the viral enzyme integrase. At this point, the host cell system transcribes the entire viral RNA and expresses structural viral proteins, particularly those that form the viral capsid and envelope. Next, the lentiviral RNA and viral proteins assemble, and the newly formed virions leave the host cell when they have been sufficiently produced from the outer membrane of the infected cell by budding or "blebbing".

[0004] Ex vivo and in vivo methods using lentiviruses for genetic modification are being investigated. In the ex vivo method, cells are extracted from the patient and then cultured. Next, a lentiviral vector carrying the therapeutic transgene is introduced into the culture to infect the cells, resulting in the modification of their genomes. The modified cells are continued to be cultured until they can be transferred to the patient. The cells used in the ex vivo method may be autologous; that is, the cells are taken from the same individual and returned to the same individual. Alternatively, they may be allologous; that is, the cells are taken from one individual and returned to a different individual.

[0005] In contrast, in vivo gene therapy involves injecting a patient with a viral vector, such as a lentivirus, containing a transgene, and the resulting genomic modification.

[0006] Cellular immunotherapy for cancer is a method that utilizes the immune system of cancer patients to induce their own immune system to attack cancer cells, thereby bringing the cancer into remission.

[0007] One known form of cellular immunotherapy is the production 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 that have been engineered to specifically bind to a target antigen. Therefore, when CAR proteins are expressed in T cells that have been genetically modified to express CARs, the T cells target a specific target antigen. The receptor is chimeric in that it combines both antigen-binding function and T cell-activating function into a single receptor.

[0008] Therefore, CAR T-cell therapy treats cancer by using CAR-engineered T cells to define targets as specific antigens presented by cancer cells. In this way, the individual's immune system can be given the ability to effectively target and destroy cancer cells within the individual.

[0009] After CAR T cells are transferred to a subject (or patient), they come into contact with target antigens on the cell surface. The CAR T cells bind to the target, and the T cells are activated. This activation triggers the T cells to proliferate and become cytotoxic. CAR T cells destroy cells through several mechanisms, including causing widespread stimulated cell proliferation, a certain increase in toxicity (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 induced either autologously, i.e., from the patient's own T cells, or allogeneically, i.e., from T cells of another healthy donor. After isolation, the T cells are genetically engineered to express a specific CAR, thereby programming the T cells to target antigens present on the surface of cancer cells, such as tumors. For safety reasons, CAR T cells are typically engineered to be specific to antigens expressed by cancer cells but not on normal cells.

[0011] Inevitably, cell therapies such as CAR T-cell therapy require a complex supply chain of reagents and time-consuming, expensive processing to obtain the pharmaceutical product. In addition to the complexity and difficulty of creating cell-based therapies, there is the difficulty of obtaining the cells that are the basic material for providing therapeutic products. Autologous cells are used because they have a low risk of side effects for patients, but not all patients can provide enough autologous cells (such as T cells) to provide an effective treatment. The use of allogeneic cells (such as T cells) carries a greater risk of side effects.

[0012] Therefore, there is a need for cell-based therapies that have reduced preparation costs and simplified logistics for administration. There is also a need for cell-based therapies that are effective against a wider range of cancer types. [Overview of the project]

[0013] The present invention relates to a viral vector having a lipid bilayer envelope, a) A cell type-specific antibody-binding domain presented on the outside of the envelope, b) The viral envelope protein presented on the outside of the envelope can promote infection of the same cell type as in (a), c) Provides a viral vector comprising a nucleic acid molecule containing a promoter that can be expressed in the same cell type as (a).

[0014] The antibody-binding domain may be derived from scFv. Preferably, the antibody-binding domain is derived from a single-domain antibody (sdAb, i.e., VHH or nanobody®).

[0015] A lipid bilayer (or phospholipid bilayer) is a thin, polar membrane composed of two layers of lipid molecules. These membranes are flat sheets that form a continuous barrier around all cells. The cell membranes of almost all organisms and many viruses are made of lipid bilayers. Therefore, the viral vectors described herein may be based on enveloped viruses.

[0016] A single-domain antibody (sdAb or VHH) is a polypeptide or antibody fragment consisting of a single monomeric variable antibody domain. Like antibodies consisting of associated polypeptide chains, single-domain antibodies can selectively bind to specific antigens. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than typical antibodies (150-160 kDa), which consist of two protein heavy chains and two light chains (typical molecular weight is 12-15 kDa), and even smaller than Fab fragments (approximately 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (approximately 25 kDa, two variable domains, one derived from the light chain and one from the heavy chain).

[0017] The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids, and these are V H This is called the H(VHH) fragment. Cartilaginous fish also have heavy chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which V NAR Single-domain antibodies, known as fragments, can be obtained. An alternative approach to producing single-domain antibodies is to split a dimeric variable domain derived from a common immunoglobulin G (IgG) of human or mouse origin into monomers.

[0018] Nanobodies are typically preferred over other antigen-binding factors, such as scFv, due to their higher solubility, stability, and smaller size. The smaller size of nanobodies allows them to access and remain in conventionally inaccessible regions on therapeutic targets. In addition, nanobodies are less likely to induce undesirable allergic reactions in individuals.

[0019] The VHH and its binding domain can be attached to the lipid bilayer envelope or anchored to a domain via a linker. Therefore, the VHH may 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.

[0020] Advantageously, the linker portion between VHH and its binding domain, as well as the domains that attach or anchor VHH and its binding domain to the lipid bilayer envelope, can modulate transduction efficiency. In particular, the use of a PDGFR / CD8a hinge scaffold for presenting VHH may result in higher transduction efficiency. Furthermore, increased CD69, CD25, and / or PD1 expression, as well as / or increased cell expansion and / or functional viral titer, may be achieved. These advantages can be considered in comparison to the IgG4 linker for the same VHH. While not bound by theory, this technical effect may arise from a linker that provides better physical flexibility, and therefore may improve transduction by improving or optimizing binding to the target and / or achieving an improved distance between two cells.

[0021] The present invention has the advantage that the amount (proportion) of VHH presented on the outside of the envelope can be controlled with respect to the amount (proportion) of viral envelope protein similarly presented on the outside of the envelope. The ability to control the amount or proportion of these two presented parts can optimize their proportion and scaffold (as in the case of the targeted VHH), which means bringing about higher transduction efficiency. Therefore, it is preferable that the antibody domain functioning as the targeting part and the envelope protein are presented separately by the viral vector. That is, fusion / conjugation between the targeting part and the envelope protein should not occur. Similarly, it is preferable that the antibody domain functioning as the targeting part and the envelope protein are expressed separately by the vector-producing cells. That is, fusion / conjugation between the targeting part and the envelope protein should not occur. Without being bound by theory, during the process of virus entry, the envelope protein undergoes very specific conformational changes. Therefore, fusion of VHH to the viral envelope protein is likely to disrupt the virus entry function of the ENV protein. In addition, separating VHH and the envelope protein provides the ability to optimize the VHH scaffold without restrictions related to maintaining the virus entry function of the ENV protein.

[0022] Therefore, the viral vector of the present invention has the advantage of being cell-type specific. In the context of the present invention, "cell-type specific" can mean one specific cell type or a group of related cell types, such as immune cells, lymphocytes, tumour-infiltrating lymphocytes (TIL), monocytes, macrophages, T cells, or NK cells.

[0023] The viral envelope protein can be selected from VSV-G single mutants or double mutants or triple mutants or quadruple mutants. Options include substitution of isoleucine to glutamic acid at position 331 (I331E), or substitution of alanine to glutamic acid at position 51 (A51E), or substitution of isoleucine to glutamic acid at position 182 (I182E). Other options for the viral envelope protein include the wild-type or mutant of the almond virus glycoprotein, the wild-type or mutant of the rudantec virus glycoprotein, the wild-type or mutant of the baculovirus Gp64 glycoprotein, the wild-type or single mutant, double mutant, triple mutant (such as substitution of tyrosine to alanine at position 150 (Y150A), or substitution of histidine to alanine at position 141 (H141A), or substitution of phenylalanine to alanine at position 147 (F147A), etc.) of the Lassa virus glycoprotein, the wild-type or mutant of the influenza HA protein, the wild-type or mutant of the trimetapneumovirus glycoprotein, the wild-type or mutant of the sunshine virus glycoprotein, or the wild-type or mutant of the Newcastle disease virus glycoprotein.

[0024] The amino acid sequence of the non-mutated VSV-G viral envelope protein is shown below as SEQ ID NO: 202 (vesicular stomatitis virus (San Juan strain) glycoprotein). The amino acid sequence of the non-mutated / wild-type VSV-G viral envelope protein without the 16 acids of the amino-terminal signal peptide is shown below as SEQ ID NO: 203. Unless otherwise specified, the mutants of VSV-G described herein are described with reference to the coordinates of SEQ ID NO: 203.

[0025] The VSV-G viral envelope protein can be mutated at I331. The VSV-G viral envelope protein may include the mutant I331E. The VSV-G viral envelope protein may include the mutant I331Q. The VSV-G viral envelope protein may include the mutant I331L. The VSV-G viral envelope protein may include the mutant I331M. The VSV-G viral envelope protein may include the mutant I331W. The VSV-G viral envelope protein may include the mutant I331R. Preferably, the VSV-G viral envelope protein includes the mutant I331E.

[0026] The VSV-G viral envelope protein may contain the A51E variant. The VSV-G viral envelope protein may contain the A51P variant.

[0027] The VSV-G viral envelope protein may contain mutant H8F. The VSV-G viral envelope protein may contain mutant H8L. The VSV-G viral envelope protein may contain mutant H8S. The VSV-G viral envelope protein may contain mutant H8V. The VSV-G viral envelope protein may contain mutant H8Y.

[0028] The VSV-G viral envelope protein may contain the mutant N9A. The VSV-G viral envelope protein may contain the mutant N9G. The VSV-G viral envelope protein may contain the mutant N9H. The VSV-G viral envelope protein may contain the mutant N9Q. The VSV-G viral envelope protein may contain the mutant N9R. The VSV-G viral envelope protein may contain the mutant N9T.

[0029] The VSV-G viral envelope protein may contain the variant Q10V.

[0030] The VSV-G viral envelope protein may contain the K11A variant.

[0031] The VSV-G viral envelope protein may contain the mutant V33Y.

[0032] The VSV-G viral envelope protein may contain the M45L variant.

[0033] The VSV-G viral envelope protein may contain the mutant K47G. The VSV-G viral envelope protein may contain the mutant K47T. The VSV-G viral envelope protein may contain the mutant K47W.

[0034] The VSV-G viral envelope protein may contain the H49A variant.

[0035] The VSV-G viral envelope protein may contain the S48L variant. The VSV-G viral envelope protein may contain the S48R variant.

[0036] The VSV-G viral envelope protein may contain the K50H mutant. The VSV-G viral envelope protein may contain the K50S mutant.

[0037] The VSV-G viral envelope protein may contain the mutant I52R. The VSV-G viral envelope protein may contain the mutant I52T.

[0038] The VSV-G viral envelope protein may contain the mutant Q53A. The VSV-G viral envelope protein may contain the mutant Q53I.

[0039] The VSV-G viral envelope protein may contain the T328S variant.

[0040] The VSV-G viral envelope protein may contain the mutant R329T. The VSV-G viral envelope protein may contain the mutant R329H. The VSV-G viral envelope protein may contain the mutant R329K. The VSV-G viral envelope protein may contain the mutant R329V.

[0041] The VSV-G viral envelope protein may contain the mutant Y330M.

[0042] The VSV-G viral envelope protein may contain the mutant V333Y.

[0043] The VSV-G viral envelope protein may contain the T351D mutant. The VSV-G viral envelope protein may contain the T351R mutant. The VSV-G viral envelope protein may contain the T351S mutant.

[0044] The VSV-G viral envelope protein may contain the T352D mutant. The VSV-G viral envelope protein may contain the T352E mutant. The VSV-G viral envelope protein may contain the T352I mutant. The VSV-G viral envelope protein may contain the T352Q mutant. The VSV-G viral envelope protein may contain the T352S mutant.

[0045] The VSV-G viral envelope protein may contain the mutant E353K. The VSV-G viral envelope protein may contain the mutant E353S.

[0046] The VSV-G viral envelope protein may contain the mutant R354K. The VSV-G viral envelope protein may contain the mutant R354M.

[0047] The VSV-G viral envelope protein may contain the mutant E355L. The VSV-G viral envelope protein may contain the mutant E355R.

[0048] The VSV-G viral envelope protein may contain the variant S408L.

[0049] The VSV-G viral envelope protein may contain the mutant I502V.

[0050] The VSV-G viral envelope protein can be mutated at position I182. The VSV-G viral envelope protein may include the mutant I182E. Amino acid position 182 is an interesting but favorable position for point mutation. This is because the surrounding amino acids at positions 181, 183, and 184 either have side chains pointing away from the CR domain of the LDLR (positions 181 and 183) or are located outside the CR domain binding pocket (position 184), making them unsuitable for mutagenesis.

[0051] The VSV-G viral envelope protein can be mutated at positions I52, I331, and E355. The VSV-G viral envelope protein may contain mutations I52R, I331E, and E355L.

[0052] The VSV-G viral envelope protein can be mutated at positions N9, K47, I331, and T352. The VSV-G viral envelope protein may include mutants N9G, K47G, I331E, and T352S.

[0053] The VSV-G viral envelope protein can be mutated at positions H8, A51, R329, and T352. The VSV-G viral envelope protein may include mutants H8F, A51P, R329V, and T352S.

[0054] The VSV-G viral envelope protein can be mutated at positions N9, K47, T328, and E355. The VSV-G viral envelope protein may contain mutations N9Q, K47T, T328S, and E355L.

[0055] The VSV-G viral envelope protein can be mutated at positions N9, K50, V333, and T351. The VSV-G viral envelope protein may include mutants N9G, K50S, V333Y, and T351R.

[0056] The VSV-G viral envelope protein can be mutated at the H8, S48, and R329 positions. The VSV-G viral envelope protein may contain mutants H8F, S48L, and R329K.

[0057] The VSV-G viral envelope protein can be mutated at the H8 and K47 positions. The VSV-G viral envelope protein may contain the H8Y and K47G mutations.

[0058] The VSV-G viral envelope protein can be mutated at positions N9, S48, R329, and T352. The VSV-G viral envelope protein may include mutants N9H, S48R, R329T, and T352E.

[0059] The VSV-G viral envelope protein can be mutated at positions H8, M45, I331, and E355. The VSV-G viral envelope protein may contain mutations H8L, M45L, I331E, and E355R.

[0060] The VSV-G viral envelope protein can be mutated at positions Q53, I331, and T352. The VSV-G viral envelope protein may contain mutations Q53A, I331E, and T352I.

[0061] The VSV-G viral envelope protein can be mutated at positions N9, I52, R329, and T352. The VSV-G viral envelope protein may include mutants N9T, I52T, R329K, and T352E.

[0062] The VSV-G viral envelope protein can be mutated at positions H8, H49, R329, and T352. The VSV-G viral envelope protein may include mutants H8S, H49A, R329H, and T352Q.

[0063] The VSV-G viral envelope protein can be mutated at positions K11, S408, I331, and R354. The VSV-G viral envelope protein may contain mutants K11A, S408L, I331L, and R354K.

[0064] The VSV-G viral envelope protein can be mutated at positions N9, Q53, I331, and R354. The VSV-G viral envelope protein may contain mutations N9R, Q53I, I331Q, and R354M.

[0065] The VSV-G viral envelope protein can be mutated at positions H8, I502, V33, and T351. The VSV-G viral envelope protein may contain mutants H8S, I502V, V33Y, and T351S.

[0066] The VSV-G viral envelope protein can be mutated at positions I331 and E353. The VSV-G viral envelope protein may contain mutations I331E and E353K.

[0067] The VSV-G viral envelope protein can be mutated at positions Q10, K50, I331, and T352. The VSV-G viral envelope protein may include mutations Q10V, K50H, I331M, and T352D.

[0068] The VSV-G viral envelope protein can be mutated at positions N9, I331, and E353. The VSV-G viral envelope protein may include mutations N9A, I331E, and E353S.

[0069] The VSV-G viral envelope protein can be mutated at positions H8, K47, Y330, and T351. The VSV-G viral envelope protein may include mutants H8V, K47W, Y330M, and T351D.

[0070] The viral envelope protein may be Lassa virus envelope protein. The viral envelope protein may be Lassa virus GP1+2. The viral envelope protein may be Lassa virus GP having the tyrosine-to-alanine substitution Y150A. The viral envelope protein may be Le Dantec virus glycoprotein (LDV).

[0071] The use of antibody domain-targeted lentiviral vectors pseudotyped with the variant Lassa virus glycoprotein Y150A (LVGP1x) advantageously allows for the targeting of the manipulated vector to specific cell types that express the antibody domain target. Similarly, Lassa virus (LVGP3X) glycoproteins H141A F147A or Y150A H141A F147A allow for the specific targeting of the manipulated vector to cells that express the VHH target.

[0072] The antibody domain or VHH may target (bind to) CD8, CD3, or T-cell receptor (TCR), CD14, or CCR2. Preferably, the VHH targets (binds to) the TCR. Therefore, specific targeting of T cells is preferably against cells expressing the TCR, CD3, CD4, or CD8, and most preferably against cells expressing the TCR. Preferably, the antibody-binding domain or VHH binds to the TCR. Preferably, the antibody domain or VHH targets (binds to) CD3, and most preferably, the antibody domain or VHH targets CD3ε (CD3e).

[0073] The antibody domain or VHH may target (bind to) CD14 or CCR2. Preferably, the VHH targets (binds to) CD14. Therefore, specific targeting of myeloid cells is preferably against cells expressing CD14 or CCR2, and most preferably against cells expressing CD14. Preferably, the antibody-binding domain or VHH binds to CD14.

[0074] The use of antibody domain-targeted lentiviral vectors pseudotyped with Rudantec virus glycoprotein (LDV) is advantageous because it allows for the specific targeting of the manipulated vector to cells expressing the antibody domain target. Preferably, the antibody domain or VHH targets (binds to) CD8.

[0075] In contrast, the antibody domain-targeted lentiviral vector of the present invention, which presents membrane-anchored VHH on its surface and is pseudotyped with wild-type vesicular stomatitis virus glycoprotein (VSVG), is insufficient to achieve cell-specific transduction. Therefore, antibody domain-targeted lentiviral vectors pseudotyped with Lassa virus glycoprotein or Rudantec virus glycoprotein (LDV) have advantageous properties compared to antibody domain-targeted lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein.

[0076] However, the use of antibody-domain targeted lentiviral vectors pseudotyped with the A51E, I182E, and I331E mutations in the VSVG protein advantageously allows for the specific targeting of the manipulated vector to cells expressing the antibody domain target. Preferably, the antibody-domain targeted lentiviral vector is pseudotyped with the I331E mutation in the VSVG protein. Although not bound by theory, this is due to the loss of the broad-spectrum targeting function of VSVG. Preferably, the antibody domain or VHH targets (binds to) the TCR. Lentiviral vectors presenting envelope-anchored TCR-targeted VHH pseudotyped with the Lassa virus envelope protein or VSVG protein variants described herein stably transduce primary T-cell lymphocytes. The lentiviral vectors described herein may be pseudotyped with the Lassa virus envelope protein having the Y150A mutation.

[0077] For example, since only a small fraction of the general population is serologically positive for lassa virus, lassa virus is advantageous to use in the context of this invention. In addition, after lassa virus infection, low-titer neutralizing antibodies are produced in the affected population.

[0078] The viral envelope may contain cell-cell fusionogens, which are preferably glycoproteins. In the context of the present invention, cell-cell fusionogens are considered to be proteins that promote membrane fusion between cells, and in particular, cell-cell fusionogens are proteins that promote plasma membrane fusion between different cells. Most preferably, the fusionogens are of viral origin, but may be of nonviral origin.

[0079] The promoter is preferably cell type specific. The promoter may be cell type specific for cells having one or more phenotypes of TCRab, TCRgd, CD3, CD25, CD4, CD8, CD11, CD14, CD16, CCR1, CCR2, CXCR4, CD56, CD20, and / or CD64 [Fc gamma 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 [Fc gamma receptor I (FcγRI)], CD56, CD19, CD20, preferably CD3, CD14, CD56, or CD20.

[0080] The promoter may be cell type specific for cells having the phenotype of CD3, CD25, CD4, CD8, CD11, CD14, CD16, CCR1, CCR2, CXCR4, CD56, CD20, and / or CD64 [Fc gamma receptor I (FcγRI)], CD56, CD19, CD20, preferably CD3, CD14, CD56, or CD20.

[0081] The promoter may be cell type specific to cells expressing CD3, CD25, CD4, CD8, CD11, CD14, CD16, CCR1, CCR2, CXCR4, CD56, CD20, and / or CD64 [Fc gamma receptor I (FcγRI)], CD56, CD19, CD20, preferably CD3, CD11, CD14, CD56, or CD20.

[0082] The present invention also has the advantage that, by using a target cell-specific promoter (and therefore implicitly inactive in the cells producing the viral vector particles), the producing cells do not need to bear the transcriptional and translational load when 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 is expressed in the target cells. This has the advantage that by using a cell-specific promoter in a producing cell where the cell-specific promoter is inactive, the titer of the vector produced by this producing cell is higher. As just one example, advantageously, the number of transdependent lentiviral particles per milliliter (mL) of product is increased by restricting the expression of surface-expressed proteins and / or protein complexes during the production of viral vector particles. Preferably, this increase is 5 times or more, or 10 times or more. Most preferably, this increase is 5 times or more.

[0083] A nucleic acid molecule may contain an open reading frame that can be expressed from a promoter. The promoter is a region of the gene's nucleotide sequence from which gene transcription begins. The promoter may be a synthetic promoter comprising promoter elements and enhancer elements, preferably the promoter elements and enhancer elements are not usually associated with each other.

[0084] In the context of this disclosure, an enhancer is a region of nucleotide sequence that modulates the activity of a promoter. Preferably, the presence of an enhancer and / or interaction with the promoter makes the activity of the synthetic promoter specific to a particular cell type or subset of cell types. The open reading frame may encode one or more 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, metabolites, and switch receptors. In some embodiments, the viral genome may also encode regulatory RNA elements, including interfering RNA, short hairpin RNA, microRNA, and / or microRNA binding sites.

[0085] A nucleic acid molecule may contain both a promoter element and an enhancer element on the same nucleic acid molecule.

[0086] The addition of enhancer sequences offers the advantage of increasing expression from the synthetic promoter. Therefore, the synthetic construct can reach and / or exceed the expression levels obtained from the ubiquitous promoter EF1a. In non-T cell lineages, the T cell-specific synthetic promoter may have limited expression compared to expression from the ubiquitous promoter EF1a (human elongation factor 1α) or PGK (mammalian phosphoglycerate kinase-derived promoter).

[0087] Preferably, the promoter is expressed only in T cells.

[0088] Preferably, the promoter is expressed only in monocytes, macrophages, or tumor-associated myeloid cells.

[0089] Preferably, the promoter is expressed only in natural killer (NK) cells.

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

[0091] The synthetic promoter may include one, two, or more enhancer elements, preferably two enhancer elements.

[0092] The synthetic promoter may contain one or more cell-specific microRNA elements. Preferably, the microRNA elements may have sequences selected from SEQ ID NOs. 128 or 129.

[0093] The promoter element for specific expression in T cells may include a sequence selected from either SEQ ID NOs: 11-20 or 121-127.

[0094] Preferably, the viral vector encodes a payload under the control of a T cell-specific promoter containing or having one of the sequences of SEQ ID NOs: 12-15.

[0095] Preferably, the viral vector encodes the payload under the control of a promoter containing or having the sequence of SEQ ID NO: 15.

[0096] The promoter element for specific expression in bone marrow cells may include a sequence selected from any one of SEQ ID NOs: 130-152 and SEQ ID NO: 193. The promoter may have a sequence selected from any one of SEQ ID NOs: 130-152, 193, and SEQ ID NO: 197.

[0097] Preferably, the viral vector encodes the payload under the control of a promoter containing or having the sequence of SEQ ID NO: 138, 139, or 193.

[0098] The promoter element for specific expression in tumor-associated myeloid cells may include a sequence selected from any one of SEQ ID NOs: 141-152. The promoter may have a sequence selected from any one of SEQ ID NOs: 141-152 and SEQ ID NO: 193.

[0099] Preferably, the viral vector encodes the payload under the control of a promoter containing or having sequence number 143, 146, or 152.

[0100] The viral vector may be a retroviral vector, preferably a lentiviral vector.

[0101] The bioavailability of viral vectors can be increased by reducing phagocytosis, extending the vector's half-life, and / or protecting it from serum components. Viral vector envelopes may present protein domains that shield the vector or protein modifications that function as self-markers. The presented protein domains and modifications may include albumin-binding domains derived from G418, albumin-binding domains derived from Zag, and / or hypersialization of lentiviral glycoproteins.

[0102] Viral vectors may display CD47 on the surface of their envelope. Preferably, viral vectors display a level of CD47 on the surface of their envelope at a level at least 1.2 times higher than the level of CD47 on the envelope of vectors produced using cells that do not overexpress CD47. Preferably, viral vectors display a level of CD47 on the surface of their envelope at a level at least 1.2 times higher than the level of CD47 on the envelope of vectors produced from cells that do not further express CD47 or have not been modified to express higher levels of CD47. CD47 expression on the envelope has the advantages of reducing vector phagocytosis, reducing off-target transduction, and increasing the amount of vector available for on-target transduction.

[0103] The viral vector optionally presents high levels of CD47, agonist CD3 scFv, 41BBL, and / or CD80 on the envelope surface. Optionally, anti-4-1BB scFv / VHH, anti-CD40 scFv / VHH, anti-CD28 scFv / VHH, CD86, CD86 immunoglobulin variable-like domain, anti-CD40 scFv / VHH, anti-OX40 scFv / VHH, anti-ICOS scFv / VHH, IL2, and / or IL7 are presented.

[0104] Viral vectors can be shielded from complement system-mediated degradation or inactivation. The viral vector envelope may present complement system modulators, or protein domains or modifications that prevent recognition by the complement system. Possible complement system modulators include C1 protease inhibitors, C3 or C5 convertase inhibitors, C3b or C4b inactivators, membrane invasion complex disruptors, C8 or C9C1-INH binding factors, H factors, disintegration promoters, DAF (CD55), MCP (CD46), CR1 (CD35), CD59, CFHR5, CR2, C4BP, kaposica (KSHV ORF4), EMICE, MOPICE, VCP, HVS-CCPH, and / or SPICE. Possible protein domains and modifications include albumin-binding domains derived from G418, albumin-binding domains derived from Zag, and / or hypersialization of lentiviral glycoproteins.

[0105] Preferably, the viral vector envelope is substantially free of MHC class I. Most preferably, the viral envelope is MHC class I-deficient.

[0106] A viral vector envelope that is substantially free of MHC class I has the advantage of reducing the immunogenicity of the vector and reducing complement-mediated inactivation.

[0107] The viral vector may present a T cell activator on the surface of its envelope. The T cell activator may be an agonist-binding factor of CD3 or TCR. The agonist-binding factor of CD3 or TCR has the advantage of inducing T cell activation, proliferation, and enhancing transduction of resting T cells. The VHH binding factor is particularly preferably TCRab VHH. Optionally, the T cell activator is combined with co-stimulatory ligand expression to achieve further advantages. The co-stimulatory ligand may be, for example, OKT3 via scFv. The co-stimulatory ligand may be, for example, CD28 via scFV. The co-stimulatory ligand may be CD80. Preferably, the co-stimulatory ligand is 4B1-4.

[0108] Viral vectors may present binding factors or proteins on the surface of their envelope that induce T cell costimulatory signals. Preferably, the binding factors or proteins induce costimulation through at least one of OX40 or 41BB, CD28, ICOS, HVEM, CD27, DR3, GITR, CD30, SLQM, CD2, 2B4, TIM1, TIM2, TNFRSF15, CD40L, or CD226. Binding factors or proteins that induce T cell costimulation have the advantage of inducing T cell proliferation, memory, and survival in activated T cells.

[0109] The antibody-binding domain may be specific to T cells.

[0110] The antibody-binding domain may be specific to CD3ε. VHH may contain any of the amino acid sequences of SEQ ID NOs: 1 to 4. Preferably, VHH contains the amino acid sequence of SEQ ID NO: 3.

[0111] The antibody-binding domain may be specific to the CD8a or b subunit. VHH may contain any of the amino acid sequences of SEQ ID NOs. 5 to 8. Preferably, VHH contains the amino acid sequence of SEQ ID NO. 7.

[0112] The antibody-binding domain may have any of the amino acid sequences of SEQ ID NOs: 1-8, 9, 10, 29, 34, 41, 44-46, 48, 49, or 54-65.

[0113] The antibody-binding domain may be specific to the T cell receptor (TCR), preferably the constant region of the TCR. Advantageously, the viral vectors described herein efficiently transduce T cells expressing endogenous levels of TCR, preferably primary T cells. The antibody-binding domain specific to the T cell receptor (TCR) may contain any of the amino acid sequences of SEQ ID NOs: 9-10 or 54-65. The antibody-binding domain specific to the T cell receptor (TCR) may be the VHH of SEQ ID NOs: 9, 56, or 62. Preferably, the VHH of the T cell receptor (TCR) is the VHH of SEQ ID NO: 9.

[0114] The antibody-binding domain may be specific to monocytes or macrophages.

[0115] The antibody-binding domain may be specific to CD14, CD16, CCR1, CCR2, CXCR4, or CD64, preferably CD14 or CCR2.

[0116] The antibody-binding domain may be specific to CD14 or CCR2, preferably CD14. Advantageously, the viral vectors described herein efficiently transduce monocytes / macrophages, preferably primary monocytes / macrophages, expressing endogenous levels of CD14 or CCR2. The antibody-binding domain specific to CD14 or CCR2 may contain any of the amino acid sequences of SEQ ID NOs. 66-120. The antibody-binding domain specific to CD14 may be the VHH of SEQ ID NOs. 70, 72, 76, 107, or 111. Preferably, the CD14 VHH is the VHH of SEQ ID NO. 76.

[0117] The antibody-binding domain may be specific to natural killer (NK) cells.

[0118] The antibody-binding domain may be specific to CD56, NKp46, or NKG2C.

[0119] The antibody-binding domain may be specific to B cells.

[0120] The antibody-binding domain may be specific to CD19, MS4A1, FCRL1, or CNR2.

[0121] The antibody-binding domain may be specific to dendritic cells.

[0122] The antibody-binding domain may be specific to CD19, MS4A1, FCRL1, or CNR2.

[0123] In all cases, the antibody-binding domain is preferably in the form of a VHH or a VHH-binding domain.

[0124] The present invention also provides a viral vector according to any of the preceding claims for use in therapeutic purposes.

[0125] In certain embodiments, the viral vectors described herein also include a CD8 or TCRab-specific VHH binding domain, and CD3, CD28, CD86, 41BB, CD40, OX40, IL2 receptor, or IL7 receptor-specific VHH, scFv, or (ir) ligand-binding domains presented outside the envelope. The use of these viral vectors results in the advantageous property of transducing resting T cells.

[0126] 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 a viral vector allows for the genetic modification of target cells to express a nucleic acid sequence encoded internally. Preferably, this is the expression of a specific protein, such as a chimeric antigen receptor (CAR).

[0127] Preferably, the viral vector of the present invention may be administered by systemic or local injection, thereby delivering the "payload" gene contained in the vector to target cells in the individual or patient. In this way, the target cells are genetically modified in vivo rather than in vitro.

[0128] In vivo administration of viral vectors has the advantage of not requiring the collection or donation of target cells from the patient or another individual for ex vivo modification. Therefore, the production of modified cells that are active in cell therapy is greatly simplified, as is the production and administration of pharmaceutically effective substances. The following are examples of tasks that become unnecessary when ex vivo processing and re-administration are required: cell collection (apheresis), cell processing (e.g., centrifugation and cryopreservation), transport, proliferation, manufacturing, product packaging and cryopreservation, distribution to the treatment site, processing, and preparation for transfer to an individual, and further administration or re-administration of cells to an individual.

[0129] The viral vector of the present invention can be used for ex vivo modification of autologous or allogeneic target cells, and can be administered to an individual or a patient. The use of this viral vector yields advantageous properties, for example, in T cells, it transduces a subpopulation of T cells and reduces the possibility of transduction of transformed cells in circulating peripheral blood cells collected from a patient. The subpopulation of T cells may be a resting population of T cells.

[0130] The present invention also provides a viral vector for use in the diagnosis, prevention, or treatment of a disease, preferably cancer.

[0131] The cancer may be blood cancer. The cancer may be stomach cancer. The cancer may be lung cancer. The cancer may be colorectal cancer. The cancer may be breast cancer.

[0132] The cancer may be multiple myeloma.

[0133] The present invention further provides viral vectors for use in therapy. The therapy may be cancer therapy. Optionally, the therapy may be for autoimmune diseases. The cancer therapy may be for cancers selected from bladder cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0134] Cancers include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenal cortical carcinoma, astrocytoma, childhood (brain cancer), atypical teratoma / rhabdoid tumor, childhood, central nervous system (brain cancer), basal cell carcinoma of the skin (see skin cancer), bile duct cancer, bladder cancer, bone cancer (Ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoma), brain tumor, breast cancer, bronchial tumor (lung cancer), Burkitt lymphoma, central nervous system cancer (atypical teratoma / rhabdoid tumor, childhood (brain cancer), cervical cancer, childhood extracranial germ cell tumor, childhood hemangiomas (soft tissue sarcoma), bile duct cancer, chordoma (bone cancer), and chronic lymphocytic leukemia. Chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma (brain cancer), cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome), ductal carcinoma in situ (DCIS), endometrial cancer (uterine cancer), ependymoma, childhood brain cancer, nasal neuroblastoma (head and neck cancer), Ewing's sarcoma (bone cancer), extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer (intraocular melanoma), fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal neuroendocrine tumor, gastrointestinal stromal tumorGerm cell tumors (soft tissue sarcomas), 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 cancers, hepatocellular carcinoma (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer (head and neck cancer), intraocular melanoma (eye), islet cell tumor, pancreatic neuroendocrine tumor, Kaposi's sarcoma (soft tissue sarcoma), renal (renal cell) carcinoma, Langerhans histiocytosis, laryngeal cancer (head and neck cancer) ), leukemia, lip and oral cancer (head and neck cancer), liver cancer, lung cancer (non-small cell, small cell, pleuropulmonary blastoma, inflammatory myofibroblastic tumor of the lung, and tracheobronchial tumor), lymphoma, medulloblastoma and other CNS embryonic tumors, childhood (brain cancer), melanoma, Merkel cell carcinoma (skin cancer), mesothelioma, metastatic squamous cell carcinoma of the neck (head and neck cancer), midline cancer, oral cancer (head and neck cancer), multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytocytosis Mycosis fungoides (lymphoma), myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, myeloid leukemia, chronic (CML), myeloid leukemia, acute (AML), myeloproliferative neoplasms, chronic, nasal cavity and paranasal sinus cancer (head and neck cancer), nasopharyngeal cancer (head and neck cancer), neuroblastoma, neuroendocrine tumors (gastrointestinal tract), non-Hodgkin lymphoma, non-small cell lung cancer, esophageal cancer, oral cancer, lip and oral cancer, and oropharyngeal cancer (head and neck cancer), oropharyngeal Head cancer, osteosarcoma (bone cancer), undifferentiated pleomorphic sarcoma of bone, ovarian cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic neuroendocrine tumor (island cell tumor), papilloma (childhood larynx), paraganglioma, sinus and nasal cavity cancer (head and neck cancer), parathyroid carcinoma, penile cancer, pharyngeal cancer (head and neck cancer), pheochromocytoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma (lung cancer), pregnancy and breast cancer, primary central nervous system cancer.(CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Pneumonitis Myofibroblastoma (Lung Cancer), Rectal Cancer, Renal Cell (Kidney) Cancer, Retinoblastoma, Rhabdomyosarcoma, Childhood (Soft Tissue Sarcoma), Salivary Gland Cancer (Head and Neck Cancer), Sarcoma (Childhood Rhabdomyosarcoma (Soft Tissue Sarcoma), Sézary Syndrome (Lymphoma), Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma of the Skin, Metastatic Squamous Neck Cancer of Unknown Primary Origin ( The following may be selected: head and neck cancer, gastric cancer, T-cell lymphoma, skin cancer (mycosis fungoides and Sézary syndrome), testicular cancer, throat cancer (head and neck cancer, thymoma and thymic cancer), thyroid cancer, tracheobronchial tumors (lung cancer), transitional cell carcinoma of the renal pelvis and ureter (kidney (renal cell) carcinoma), transitional cell carcinoma of the ureter and renal pelvis (kidney (renal cell) carcinoma), uterine cancer, uterine sarcoma, vaginal cancer, hemangioma (soft tissue sarcoma), vulvar cancer, and Wilms' tumor.

[0135] Treatment may be 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 (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, septicemia, multi-organ injury syndromes following trauma or hemorrhage, eye disorders, for example, but not limited to allergic conjunctivitis, vernal keratoconjunctivitis, uveitis, and thyroid eye disease, eosinophilic granuloma, asthma, chronic bronchitis, allergic rhinitis, adult respiratory distress syndrome (ARDS), severe acute respiratory syndrome. Lung or respiratory conditions including, but not limited to, pulmonary inflammatory diseases (e.g., chronic obstructive pulmonary disease), silicosis, pulmonary sarcoidosis, pleurisy, alveolitis, vasculitis, pneumonia, bronchiectasis, hereditary emphysema, and pulmonary oxygen toxicity, such as ischemia-reperfusion injury of the myocardium, brain, or limbs, fibrosis including, but not limited to, cystic fibrosis, keloid formation or scarring, atherosclerosis, systemic lupus erythematosus (SLE), lupus nephritis, autoimmune thyroiditis, multiple sclerosis, several forms of diabetes mellitus, and autoimmune diseases including, but not limited to, Raynaud's syndrome, and graft-versus-host disease.Tissue or organ transplant rejection disorders, including but not limited to disease (GvHD) and allograft rejection; inflammatory bowel disease, including but not limited to chronic or acute glomerulonephritis, 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; central or peripheral nervous system inflammatory conditions, including but not limited to meningitis (e.g., acute purulent meningitis), encephalitis, and brain or spinal cord injury due to minor trauma; Sjögren's syndrome; diseases with leukocytosis; alcoholic hepatitis; bacterial pneumonia; community-acquired pneumonia (CAP); and Pneumocystis carinii pneumonia. These include pneumonia (PCP), antigen-antibody complex-mediated disorders, hypovolemic shock, type 1 diabetes, acute and delayed-type hypersensitivity reactions, diseases due to leukocyte abnormalities and metastases, burns, granulocyte transfusion-associated syndrome, cytokine-induced toxicity, stroke, pancreatitis, myocardial infarction, respiratory syncytial virus (RSV) infection, and spinal cord injury.

[0136] In the context of the present invention, a chimeric antigen receptor (CAR) for targeting an epitope presented by one, more, or any of the cancer cells of the above-mentioned cancers may be used. The CAR may be a B-cell maturation antigen (BCMA) CAR. The CAR may be a CD19 CAR. The CAR may be a claudin 18.2 CAR, and optionally, the sequence encoding the claudin 18.2 CAR is derived from SEQ ID NO: 53. The CAR may be a HER2 CAR. The CAR may be a mesothelin CAR.

[0137] In the context of the present invention, a modified TCR for targeting an epitope presented by one, more, or any of the cancer cells of the above-mentioned cancers may be used. The TCR may be specific to a cancer-testicular antigen. The cancer-testicular antigen may be NY-ESO, MAGEA4, or PRAME. The TCR may be specific to a viral antigen. The viral antigen may be derived from HPV. The TCR may be specific to WT1.

[0138] The modified TCR may be a “domain-exchanged” TCR. Exogenous human TCRαβ transduced into human CD3+ T lymphocytes may mispair with endogenous TCRαβ chains, potentially leading to toxicity from the recognition of autoantigens by the mispaired TCR. To reduce this risk of mispairing between exogenous and endogenous TCR chains, the constant domain regions of the α chain (Calpha, Cα) and β chain (Cbeta, Cβ) are “exchanged” between these two chains. Using this “domain-exchanged” approach, the α receptor consists of a variable α (Vα) and Cβ domain (denoted as VaCb), while the β receptor consists of a variable β (Vβ) and Cα domain (denoted as VbCa). The two domain-exchanged receptors can still form heterodimers together, but can no longer pair with any wild-type TCRα or β chain. Thus, mispairing of TCR chains is prevented.

[0139] The present invention relates to a method for producing a viral vector, (a) A step of manipulating cells to express a cell-type specific antibody-binding domain (preferably a membrane-anchored type), (b) A step of manipulating cells to express a viral envelope protein that can promote infection of the same cell type as in (a), (c) A step of replicating a nucleic acid vector containing a promoter that can be expressed in the same cell type as (a) within a cell, (d) A method further comprising the step of recovering a viral vector in the form of vesicles released from cells is provided.

[0140] The present invention also provides cells modified to produce the viral vector particles disclosed herein. In this context, cells modified to produce the viral vector particles disclosed herein are referred to as “producing cells”.

[0141] A vesicle is a structure containing fluid or cytoplasm surrounded by a lipid bilayer. Vesicles released from a cell may also be blebs. In cell biology, a bleb is a spherical, "vesicle-like" bulge of the cell plasma membrane. Preving, or zeiosis, refers to the phenomenon of bleb formation. Preving is a method by which viral particles of enveloped viruses are released from the infected cells that produce them.

[0142] The nucleic acid vector may also be a plasmid.

[0143] This method may include a step of manipulating the producing cells to knock down the expression of MHC class I. Preferably, this method includes a step of manipulating the producing cells to knock out the expression of MHC class I.

[0144] Most preferably, the method includes a step of modifying the expression of MHC class I by introducing a change in the sequence of the nuclear genome. Thus, the viral vector envelope can be manipulated to lack MHC class I.

[0145] Deletion of MHC class I on the envelope of the producing cell results in a vector lacking MHC class I on its surface, which then favorably reduces the immunogenicity of the associated vector and diminishes complement-mediated inactivation.

[0146] Editing proteins in the form of nucleases, base editors, and prime editors can be packaged within vectors to mediate the introduction of alterations in the nuclear genome sequence.

[0147] In this context, "mediate" is defined as causing all the first steps or substrates of a physiological pathway or process.

[0148] The endonuclease may be an RNA-directed DNA-specific endonuclease. Preferably, the endonuclease is a CRISPR-Cas ribonucleoprotein complex.

[0149] The endonuclease may be a TALEN. The endonuclease may be a zinc finger nuclease.

[0150] The producing cell line may be a 293T cell line or a cell line derived therefrom.

[0151] The producing cells may be engineered to express or overexpress a cell surface protein or an intracellular protein. In some embodiments, this protein is CD47. Advantageously, the expression of CD47 on the envelope reduces the phagocytosis of the vector produced by the producing cells, reduces off-target transduction, and increases the amount of vector available for on-target transduction.

[0152] In some embodiments, the producing cells may express a protein that will be packaged into budding virus particles. In some embodiments, this protein may be Vpx. Advantageously, by delivering Vpx to transduced cells in vivo, the efficiency of target cell reprogramming can be increased by inhibiting the cell's antiviral defenses with Vpx. In other embodiments, this protein may be a gene-editing protein comprising Cas9, Cas12, Cpf1, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a base editor, or a prime editor. The gene-editing protein may be modified to enhance packaging within the virus particle. Advantageously, the delivery of gene-editing proteins using the vectors described herein provides a mechanism for cell-specific gene editing in vivo.

[0153] In any case, it should be understood that the producing cells can be modified in an appropriate manner to enable the production of the viral vector according to the present invention, as outlined above and below.

[0154] The present invention further provides a method for modifying cells, comprising the step of exposing cells to a viral vector described herein.

[0155] The cells to be modified may present an antigen on their outer surface that can be bound by an antibody-binding domain (preferably a VHH-binding domain) presented on the envelope of the viral vector.

[0156] Cell modification is preferably carried out in vivo. Advantageously, the present invention enables this through a combination of viral vector features that produce highly specific and effective agents.

[0157] In some embodiments, vectors targeting different cell types may be administered together or at short intervals to enable in vivo reprogramming of different cell types. The different cell-specific vectors may carry the same cargo or transgene, or each may carry a different cargo or transgene. Advantageously, this provides a method for reprogramming different cell types with potentially synergistic and non-overlapping disease-treating mechanisms to provide a more effective disease treatment. The cells to be modified may be T cells. Modified T cells are used in various therapies, such as CAR-T cell therapy, but 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 means that an effective method for overcoming previous limitations and producing modified T cells in vivo is provided.

[0158] The cells to be modified are preferably immune effector cells.

[0159] The cells to be modified may also be T cells.

[0160] The antigen is preferably one that is highly conserved in T cells and expressed on the cell membrane.

[0161] The antigen may also be a T cell receptor (TCR).

[0162] The antigen may be of the CD3ε phenotype.

[0163] The cells to be modified may be monocytes or macrophages. The cells to be modified may be B cells. The cells to be modified may be dendritic cells (DCs).

[0164] The antigen is preferably highly conserved in monocytes and / or their differentiated forms and expressed on the cell membrane.

[0165] The antigen can be selected from CD14, CD16, CCR1, CCR2, CXCR4, and CD64. Preferably, the antigen is selected from CD16, CXCR4, and CCR1.

[0166] The cell to be modified may be a natural killer (NK) cell.

[0167] Preferably, the antigen is highly conserved in NK cells and is expressed on the cell membrane.

[0168] The viral vector is 10 6 sup, 10 7 sup, 10 8 sup, 10 9 sup, 10 10 sup, or 10 11 sup of vector particles, preferably 10 8 sup of vector particles and can be administered.

[0169] The viral vector may be administered at a frequency of less than 2 weeks. That is, the second or supplemental administration of the viral vector is performed less than 2 weeks after the first administration of the viral vector. Preferably, the second or supplemental administration of the viral vector is performed less than 1 week after the first administration of the viral vector.

[0170] Administer the first course (one or more doses) of the viral vector, and then, after a period selected from 6 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, or 6 years, a second or supplemental administration of the viral vector may be performed.

[0171] In another embodiment, the present invention is a virus-like particle having a lipid bilayer envelope, (a) a VHH binding domain presented on the outside of the envelope that is cell-type specific, (b) Provided are virus-like particles comprising an envelope protein, which is presented outside the envelope and is capable of promoting infection of the same cell type as in (a), the envelope protein being one of those described elsewhere in this specification.

[0172] Virus-like particles are suitable for the delivery of substances contained within them to certain types of cells, as described elsewhere in this specification.

[0173] Therefore, the present invention provides highly specific viral vectors or virus-like particles, methods for using them in therapy, methods for producing them, and methods for modifying cells both ex vivo and in vivo using them. [Brief explanation of the drawing]

[0174] Next, the present invention will be illustrated with reference to the following figures. [Figure 1] This is a diagram illustrating one example of a vector according to the present invention. [Figure 2-1] This is a flow cytometry scatter plot of a 293T packaging monoclonal cell line used to generate a lentiviral vector in a specific embodiment. Parental 293T cells overexpressing surface hCD47 (center panel) and parental 293T cells lacking MHCI (e293T) were transduced with gamma retroviruses (gRVs) encoding CD3e or CD8a membrane-anchored VHHs, gated (P3), and stable expression cells were isolated by fluorescence-activated cell sorting (A). The resulting polyclonal cell lines (B) were then expanded and frozen for long-term storage and use. [Figure 2-2](C) Flow cytometry scatter plot of the Operation 293T packaging monoclonal cell line used to generate the Operation 293T lentiviral vector in a specific embodiment. In another embodiment, parent e293T was transduced with a lentivirus (LV) encoding TCRab membrane-anchored VHH, and the monoclonal cell line was isolated by limiting dilution cloning. e293T B619 clone 38 was a monoclonal-producing cell line induced by such method, and the expression of B2M, CD47, and membrane-anchored nanobodies (VHH) was confirmed by flow cytometry compared to a reference wild-type cell line HEK293T, which is known to be B2M-positive, CD47 at baseline levels, and VHH-negative. [Figure 3] This is a flow cytometry scatter plot of Jurkat wild-type cells (WT), Sup-T1 cells, a Jurkat cell line stably expressing hCD8a (Jurkat CD8+), and primary human PBCM stained with CD3, CD4, CD8, and TCR antibodies. The results show that, in contrast to Sup-T1 cells expressing surface CD3 and CD8, Jurkat WT cells do not express CD8 but express TCR, while Jurkat cells stably expressing CD8 exhibit surface expression of CD3, CD4, TCR, and transgenic CD8a. [Figure 4]This graph shows the quantitative transduction rate, determined by GFP reporter expression, in Jurkat WT and Sup-T1 cells transduced with different types of wild-type vesicular stomatitis virus (VSVG) and pseudotyped lentivirus, as measured by flow cytometry. Jurkat WT and Sup-T1 cells were transduced with VSVG pseudotyped lentiviruses expressing a GFP reporter and displaying either CD8a VHH (CD8 VSVG), CD3e VHH (CD3 VSVG), or no VHH (VSVG) on their envelope. Three days after transduction, the cells were analyzed for GFP reporter expression by flow cytometry. As shown in this figure, transduction by VSVG wild-type pseudotyped vectors displaying either CD3e or CD8a membrane-anchored nanobodies is equivalent between Jurkat WT (no CD8 expression) and Sup-T1 (no CD3 expression). These results demonstrate that presenting membrane-anchored VHH on the surface of a lentiviral vector pseudotyped with wild-type vesicular stomatitis virus glycoprotein is insufficient to achieve cell-specific transduction. [Figure 5] For comparison, the graph shows the quantitative transduction rate, determined by GFP reporter expression, in Jurkat WT cells and Jurkat CD8+ cells transduced with different types of manipulated lentiviruses, as measured by flow cytometry. Jurkat cells (Jurkat WT) or Jurkat cells stably expressing human CD8a (Jurkat CD8+) were transduced with GFP-expressing lentiviral vectors that were pseudotyped with mutant Lassa virus glycoprotein Y150A (LVGP1x) or wild-type Rudantec virus glycoprotein (LDV) and presented membrane-anchored CD8aVHH on the viral envelope. These results demonstrate that, in contrast to wild-type VSVG lentiviral particles with broad tropism, the CD8a VHH-targeting lentiviral particles LVGP1x-CD8 and LDV-CD8 specifically target the manipulated vectors to Jurkat cells (Jurkat CD8+) expressing the VHH target. [Figure 6]This graph shows the transduction rates of targeted vectors (LVGP1x-CD3) and non-targeted vectors (VSVG) to target cells (Jurkat WT) and non-target cells (Sup-T1), quantified by flow cytometry. Sup-T1 cells and Jurkat WT cells were transduced using either a VSVG lentiviral vector with broad tropism or a CD3 VHH-presenting vector pseudotyped with LVGP1x glycoprotein. The results indicate that, in contrast to the VSVG vector, the CD3e VHH-presenting LVGP Y150A lentiviral particle transduced only CD3-expressing cells. This demonstrates that target-specific transduction can also be achieved by targeting a pseudotyped LVGP1x vector with a different VHH than CD8a. [Figure 7] This graph shows the transduction efficiency ratios of targeted vectors (LVGP1x-TCR2 and LDV-VHHTCR2) and non-targeted vectors (VSVG) to target cells (Jurkat WT) and non-target cells (Sup-T1), quantified by flow cytometry. Sup-T1 cells and Jurkat WT cells were transduced using either the broad-tropism VSVG lentiviral vector (VSVG) or TCR VHH-presenting vectors pseudotyped with LVGP1x or LDV (LVGP1x-TCR2 and LDV-VHHTCR2). The results show that, in contrast to the VSVG vector, TCR VHH-targeted LVGP1x or LDV lentiviral particles transduce only TCR-expressing cells (Jurkat WT). This demonstrates that target-specific transduction can be achieved by targeting LVGP1x and LDV pseudotyped vectors that have nanobodies for different T cell markers. [Figure 8]This graph shows the transduction efficiency ratios of targeted vectors (LVGP1x-CD8a and LDV-CD8a) and non-targeted vectors (VSVG) to target cells, quantified over time by flow cytometry. Jurkat CD8+ cells, which stably express human CD8a, were transduced with VSVG, LVGP1x-CD8a, or LDV-CD8a GFP expression vectors, and GFP expression was quantified by flow cytometry over 14 days of culture. These results demonstrate that CD8a VHH targeted lentiviral particles pseudotyped with LVGP1x and LDV, similar to VSVG lentiviral particles, stably transduce target cells. [Figure 9] This graph shows the transduction efficiency ratios of targeted vectors (LVGP1x-CD3) and non-targeted vectors (VSVG) to target cells, quantified over time by flow cytometry, and also quantifies viral genome insertion by quantitative PCR. Jurkat WT cells in culture were transduced with either a VSVG vector with broad tropism, or a target-specific vector pseudotyped with LVGP1x combined with one 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 evaluated on day 21 by quantitative PCR amplification of the inserted vector sequence. These results indicate that transduction using the LVGP1x-CD3e VHH targeted vector was stable for 21 days (A) and resulted in viral genome insertion into host DNA (B). [Figure 10]This graph shows the transduction efficiency ratios of targeted vector (LVGP1x-CD8) and non-targeted vector (VSVG) to target cells (Sup-T1) and non-target cells (Jurkat WT) quantified by flow cytometry on day 3 (A) and over time (B), and also quantified viral genome insertion by quantitative PCR (C). Jurkat WT and Sup-T1 cells in culture were transduced with a VSVG vector with broad tropism or a target-specific vector pseudotyped with LVGP1x that displays CD8a VHH. GFP transgene expression was monitored over time using flow cytometry, and genomic vector insertion was evaluated on day 21 by quantitative PCR amplification of the inserted vector sequence. These results demonstrate that transduction using the LVGP1x-CD8 VHH-targeted vector is specific to target cells expressing CD8a (A), stable for 21 days (B), and results in insertion of the viral genome into host DNA (C). [Figure 11] This graph shows the transduction efficiency ratios of targeted vectors (LVGP1x-TCR and LDV-TCR) and non-targeted vectors (VSVG) to the target (Jurkat WT) over time, quantified by flow cytometry. Jurkat WT cells in culture were transduced with either a VSVG vector with broad tropism or a target-specific vector pseudotyped with LVGP or LDV presenting TCR-targeted VHH. GFP transgene expression was monitored over time using flow cytometry. The results indicate that transduction using LVGP or LDV TCR VHH-targeted vectors is stable for 16 days. [Figure 12]This graph shows the quantitative ratio of transduction efficiency of human primary CD4+ and CD8+ T cells using targeted vectors (LVGP1x-CD8) and non-targeted vectors (VSVG), as measured by flow cytometry. Human PBMCs were activated in culture and transduced with either the LVGP1x-CD8 vector or the VSVG vector. GFP expression was quantified by flow cytometry 7 days after transduction. These results demonstrate that, in contrast to the VSVG pseudotype vector, the LVGP1x-CD8 vector specifically transduces primary T cells expressing endogenous levels of CD8a VHH target, i.e., CD8a. [Figure 13] The graphs show the ratio of transduction efficiency for targeted vectors (LVGP1x-TCR) and untargeted vectors (VSVG) for human primary CD4+ and CD8+ T cells using flow cytometry. Human PBMCs were activated in culture and transduced with either LVGP1x-TCR or VSVG vectors. GFP expression was quantified by flow cytometry 4 days (A) and more than 20 days (B) after transduction. These results demonstrate that the LVGP1x-TCR vector efficiently transduces primary T cells expressing endogenous levels of TCR, and that LVGP1x-TCR transduction is stable under the same conditions and more efficient than transduction using the VSVG pseudotyped vector. [Figure 14]This is a flow cytometry plot of BCMA CAR-transduced Jurkat-CD8+ cells in co-culture with BCMA-expressing or non-expressing cells, or with transduced cells alone. Jurkat-CD8 cells were transduced with LVGP1x-CD8 or LDV-CD8, or a VSVG lentiviral vector with broad tropism encoding BCMA CAR. Transduced cells were cultured alone (untargeted), 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 evaluated by flow cytometry (A), and a graph summarizing the data is shown in (B). These results indicate that co-culture of BCMA CAR-expressing Jurkat-CD8 cells with NCI-H929 cells results in increased CD69 surface expression compared to co-culture with T cells alone or with untargeted cells. These results demonstrate that transduction of Jurkat-CD8 with the LVGP1x-CD8 vector or LDV-CD8 vector encoding the BCMA CAR results in functional expression of the CAR construct and signaling consistent with T cell activation comparable to that of cells transduced with the broad-tropism VSVG vector. [Figure 15]This is a flow cytometry plot of primary T cells transduced with BCMA CAR in co-culture with BCMA-expressing or non-expressing cells, or with T cells alone. Activated primary human T cells were transduced with a targeted LVGP1x-CD3 vector encoding BCMA CAR or a VSVG lentiviral vector with broad tropism. Transduced cells were cultured alone (untargeted), 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 evaluated by flow cytometry (A), and a graph summarizing the data is presented in (B). This figure shows that co-culture of BCMA CAR-expressing T cells with NCI-H929 cells results in increased CD137 surface expression compared to conditions in which BCMA CAR-T cells were cultured alone or with non-targeted cells. These results demonstrate that transduction of primary T cells with the LVGP1x-CD3 vector encoding the BCMA CAR leads to functional expression of the CAR construct and signaling consistent with T cell activation, comparable to that of cells transduced with the VSVG vector, which exhibits broad tropism. [Figure 16]This graph summarizes the results of quantifying the number of target and non-target cells in BCMA CAR-T co-cultures using flow cytometry. Activated human PBMCs were transduced with LVGP1x-CD8 vectors encoding BCMA CARs or VSVG lentiviral vectors with broad tropism, under the control of a T cell-specific synthetic promoter (SYN17; for LVGP1x-CD8 vectors) or a constitutive promoter (EF1a; for VSVG). Transduced cells were expanded and co-cultured with NCI-H929 (BCMA-expressing cells) or K562 non-target cells (non-BCMA-expressing cells) in a 1:1 or 1:2 ratio (ratio of effector T cells to target / non-target cell lines). The number of target / non-target cells stained with Cell Trace Violet was quantified by flow cytometry in 50 μL of culture medium and summarized in graphs A and B. This figure shows that co-culture of CD19 CAR-T cells significantly reduces the number of target cells (NCI-H929) but not non-target cells (K562) compared to co-culture with VSVG-GFP transduced T cells or non-transduced T cells. This result demonstrates that transduction of primary T cells with LVGP1x-CD8 encoding the BCMA CAR results in functional expression of the CAR construct and induces specific cytotoxicity of transduced cells against target cells comparable to that of T cells transduced with the broad-tropism VSVG vector. [Figure 17]This graph summarizes the results of quantifying the number of target (Nalm6) and non-target (K562) cells in co-culture of CD19 CAR-T cells by flow cytometry. Activated human PBMCs were transduced with LVGP1x-CD8 vectors encoding CD19 CARs or VSVG lentiviral vectors with broad tropism, under the control of a T cell-specific synthetic promoter (SYN17; for LVGP1x-CD8 vectors) or a constitutive promoter (EF1a; for VSVG). Transduced cells were expanded and co-cultured with Nalm6 (CD19-expressing cells) or K562 non-target cells (non-CD19-expressing cells) in a ratio of 1:2 or 1:5 (ratio of effector T cells to target / non-target cell lines). The number of target or non-target cells stained with Cell Trace Violet was quantified by flow cytometry in 50 μL of culture medium and summarized in graphs A and B, respectively. This figure shows that co-culture of CD19 CAR-T cells significantly reduces the number of target cells (Nalm6) but not non-target cells (K562) compared to co-culture with non-transduced T cells. This result demonstrates that functional transduction using the LVGP1x-CD8 vector is applicable beyond BCMA CARs, and that transduction of primary T cells with LVGP1x-CD8 encoding the CD19 CAR results in functional expression of the CAR construct and produces target-cell-specific cytotoxicity of transduced cells comparable to that of T cells transduced with the broad-tropic VSVG vector. [Figure 18]This graph summarizes the results of quantifying the number of target and non-target cells in co-culture of BCMA CAR-T cells by flow cytometry. Activated human PBMCs were transduced with LVGP1x-TCR encoding BCMA CAR under the control of 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 (non-BCMA-expressing cells) in a ratio of 1:1, 1:2, or 1:5 (ratio of effector T cells to target / non-target cell lines). The number of target / non-target cells stained with Cell Trace Violet was quantified by flow cytometry in fixed-volume culture medium and summarized in graphs A and B. This figure shows that co-culture of BCMA CAR-T cells significantly reduces the number of target cells (NCI-H929) but not the number of non-target cells (K562) compared to co-culture with non-transduced T cells. These results demonstrate that transduction of primary T cells by LVGP1x-TCR encoding a BCMA CAR under the control of a T cell-specific promoter leads to the functional expression of the CAR construct and to target cell-specific cytotoxicity of the transduced cells. [Figure 19]This figure shows flow cytometry plots of human CD8 T cells or CD4 T cells in the peripheral blood of humanized NSG mice injected with a targeted LVGP1x-CD8 vector, and graphs showing the persistence of CD8+ transduced cells over 27 days. Activated human PBMCs were intraperitoneally injected into immunodeficient NSG mice on day 0, and a viral vector encoding GFP under the control of a ubiquitous EF1a promoter was intraperitoneally injected on day 1. Transduction of human T cells was evaluated by flow cytometry for GFP and cell-specific markers in the peripheral blood of mice on days 4, 7, 14, 21, and 27. This figure shows that injection of LVGP1x-CD8, which is a 25x10⁶ or 100x10⁶ transducing unit (TU), results in specific transduction of CD8+ human T cells (A), and that transgene expression and transduced CD8 T cells persist for 27 days (B). These results demonstrate the target-specific and stable transduction of human CD8 T cells in vivo using the CD8 VHH-targeting vector LVGP1x-CD8. [Figure 20-1](A, B) are graphs summarizing the results of quantification of transduced human CD8 T cells and CD4 T cells, as well as the levels of CD20 B cells, in humanized NSG mice injected intraperitoneally with activated human PBMCs and viral vectors, as measured by flow cytometry. Activated human PBMCs were injected intraperitoneally into immunodeficient NSG mice on day 0, and on day 1, VSVG vectors encoding GFP or CD19 CAR under the control of the EF1a promoter (VSVG GFP and VSVG Ef1a CD19 CAR) or LVGP1x-CD8 vectors encoding CD19 CAR under the control of a synthetic T cell-specific promoter (LVGP1x-CD8 SYN CD19 CAR) were injected intraperitoneally. CD19 CAR expression in human T cells was evaluated in peripheral blood on day 7 after viral injection by flow cytometry (A). B cell dysplasia was assessed in dissociated spleen, bone marrow, or peripheral blood using flow cytometry for the B cell marker CD20 on day 7 after virus injection (B). This figure shows that injection of LVGP1x-CD8, a 100e6 transduction unit, resulted in specific transduction of CD8+ human T cells, while injection of the same dose of VSVG vector resulted in broad transduction of both CD4 and CD8 cell populations. In addition, the proportion of B cells in the spleen, bone marrow, and peripheral blood was significantly reduced in mice injected with the CD19 CAR-coding vector compared to animals injected with the GFP-coding vector. These results demonstrate in vivo target-specific transduction of human CD8 T cells using LVGP1x-CD8 encoding the CAR construct, resulting in target cell deletion at levels similar to those observed in mice injected with the VSVG vector, which has broad tropism encoding the CAR. (C) is a graph showing the results of flow cytometry quantification of the percentage of circulating human B cells in hCD34+ humanized NCG mice intravenously injected with the eLV-LVGP1X-CD8a viral vector. Normal hCD34+ stable humanized NCG mice were intravenously injected with the eLV-LVGP1X-CD8a vector expressing GFP or a CD19 CAR construct under the control of T cell-specific synthesis promoter #19.The percentage of CD20+ (B cells) in peripheral blood was assessed by flow cytometry. This data represents the mean ± SEM from 4–6 animals per condition. These results demonstrate potent and sustained anti-target cell activity of reprogrammed CD19 CAR-T cells reprogrammed in vivo by a single injection of an eLV-LVGP1X-CD8a targeted vector expressing BCMA CAR under the control of T cell-specific promoter #19. [Figure 20-2](D~F) are graphs summarizing the results of flow cytometry quantification of human CD20+ (B cells), CD3+, CD4+, and CD8+ (T lymphocytes) cells in the peripheral blood of humanized NSG mice injected with targeted LVGP1x-CD8-CD19 CAR(10E+06 TU), LVGP1x-TCRab-CD19 CAR(10E+06 TU), or LVGP1x-TCRab-GFP(50E+06 TU) vectors, or VSVG LV vectors with broad tropism expressing CD19-CAR(50E+06 TU). Activated human PBMCs were intraperitoneally injected into immunodeficient NSG mice on day 0, and on day 1, viral vectors encoding the CD19 CAR construct under the control of the T cell-specific synthesis promoter SYN23, or encoding GFP under the control of the ubiquitous EF1a promoter, were intraperitoneally injected. The levels of human B cells and T cell transduction were evaluated by flow cytometry in the peripheral blood of mice on day 7. (D.) CD19 CAR T cells were detected as early as day 7 after vector administration in animals injected with the CD19 CAR vector. The LVGP1x-TCRab-CD19 CAR vector efficiently transduced both CD4+ T cells and CD8+ T cells, while the LVGP1x-CD8-CD19 CAR vector transduction was specific to CD8+ T cells. The transduction efficiency of LVGP1X-TCRab was significantly higher than that of VSVG LV, which has broad tropism, and the same level of T cell transduction was achieved with 1 / 5 of the dose. (E.) B cell dysplasia was completed on day 7 after vector injection in the LVGP1x-TCRab-CD19 CAR group and was almost completed for LVGP1x-CD8. These results demonstrate potent anti-targeting activity of CD19 CAR-T cells reprogrammed in vivo using the LVGP1X T cell targeting vector. (F.) Flow cytometry analysis of peripheral blood for the percentage of CAR+ cells in different immune cell types (mouse CD45+ cells, hCD45+ / CD56+ human NK cells, hCD45+ / CD3+ / CD56+ human NKT cells, hCD45+ / CD14+ human bone marrow cells, hCD45+ / CD3+ / CD4+ and CD8+ human T cells) 11 days after vector injection.The results represent the mean ± SEM from 8 animals. Note: CD19CAR B cell expression was not evaluated due to B cell dysplasia. [Figure 21] This graph shows the results of quantitative analysis by flow cytometry of the transduction rates between CD8a-target-negative cell lines (Jurkat WT and 293T) and CD8a-positive cell lines (Sup-T1) using VSVG lentiviral vectors with broad tropism, and CD8a-targeted lentiviral vectors pseudotyped with Y150A (LVGP1X) and Y150A H141A F147A Lassa virus (LVGP3X) glycoproteins. Jurkat WT and 293T cells, which are CD8a-negative cell lines, and Sup-T1 cells, which are CD8a-positive cell lines, were transduced with VSVG and LVGP1x-CD8 or LVGP3x-CD8 lentiviral vectors, and GFP expression was quantified by flow cytometry three days after transduction. These results demonstrate that the LVGP1 vector x and LVGP3x-CD8 vector specifically transduce target cells compared to the VSVG lentiviral vector, which exhibits broad tropism. This shows that by combining mutations in the LVGP envelope glycoprotein that target residues crucial for Lassa target binding with membrane-anchored nanobodies, transduction into specific cell types can be targeted. [Figure 22]This graph shows the in vitro transduction rate of human PBMCs transduced with GFP-expressing CD8-targeted lentiviral vectors pseudotyped with WT Lassa GP protein or Lassa GP variants (Lassa Y150A; Lassa H141A, F147A; Lassa Y150A, F446L), quantified by flow cytometry. The vectors were used for in trio transduction at MOI 10 (G, Lassa Y150A; Lassa H141A, F147A; Lassa Y150A, F446L) or MOI 2 (WT), based on titrations performed on SupT1. The results represent the mean ± SEM of PBMC transduction from three different human donors. These results demonstrate that while mutant F446L eliminates the ability of Lassa GP protein to fuse, Lassa WT, Lassa Y150A, or Lassa H141A / F147A pseudotypes enable specific and sustained transduction of CD8+ cells by CD8-targeted vectors. [Figure 23A] This document presents data supporting the selection and testing of VSVG variants to identify point mutations that eliminate VSVG binding to LDLR and promote target-specific transduction of VHH-targeted eLVs. (A) A table of mutation sites in the original Indiana strain bullous stomatitis virus glycoprotein (VSV-G, accession number P03522 on UniProtKB). Twenty-four positions identified on the available crystal structure of VSV-G were mutated to sterically inhibit binding of VSV-G to its native 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). [Figure 23B]This shows data supporting the selection and testing of VSVG variants to identify point mutations that eliminate VSVG binding to LDLR and promote target-specific transduction of VHH-targeted eLVs. (B) Flow cytometry analysis of wild-type Jurkat cells (CD8-negative) transduced with wild-type or VSV-G mutant envelope protein alone (gray) or with GFP-expressing lentivirus pseudotyped with CD8 protein-targeting VHH (blue), or Jurkat CD8+ (stable transduction of CD8α / β heterodimer) transduced with wild-type or VSV-G mutant envelope protein and CD8 protein-targeting VHHH (orange, shown as 8G_Jk-CD8 at the end of the list in the figure). The graph shows the percentage of GFP+ normalized to 100% for wild-type VSV-G. [Figure 23C] This shows data supporting the selection and testing of VSVG variants to identify point mutations that eliminate VSVG binding to LDLR and promote target-specific transduction of VHH-targeted eLVs. (C) is a graph of the results of flow cytometry quantification of the transduction rate between CD8a-target-negative cell lines (Jurkat WT) and CD8a-positive cell lines (Sup-T1) using a VSVG lentiviral vector (WT) with broad tropism and a CD8a-targeted lentiviral vector pseudotyped with a VSVG variant engineered to eliminate VSVG protein tropism. [Figure 24-1]This graph shows the results of flow cytometry analysis of the transduction rates of primary CD4+ and CD8+ human T lymphocytes using CD8a target-negative and positive cell lines, as well as CD8a VHH-targeted vectors pseudotyped with different VSVG variants engineered to eliminate VSVG protein tropism. (A) CD8a target-negative cell line (Jurkat WT) versus CD8a-positive cell line (Sup-T1), or (B) activated primary CD4+ and CD8+ human T lymphocytes were transduced using a VSVG lentiviral vector with broad tropism (WT) and CD8a-targeted lentiviral vectors pseudotyped with VSVG variants. GFP expression was quantified by flow cytometry three days after transduction. These results indicate that the three CD8 lentiviral vectors pseudotyped with mutant VSVG glycoproteins specifically transduce target cells compared to the VSVG lentiviral vector with broad tropism. This demonstrates that mutations in the VSVG protein A51E, I182E, and I331E eliminate VSVG's broad-tropic targeting function, supporting VHH-mediated targeting for transduction of marker-positive cell types. [Figure 24-2](C) is a graph showing the results of quantitative analysis by flow cytometry of the transduction rates of primary CD4+ and CD8+ human T lymphocytes using CD8a target-negative and positive cell lines, as well as CD8a VHH-targeted vectors pseudotyped with different VSVG mutants engineered to eliminate VSVG protein tropism. CD3 / 28-activated human CD4 / CD8 primary human T cells, isolated primary monocytes, and T cell lymphoblastic cell lines (Jurkat and SupT1) were transduced using VSV-GI331E-flash-typed untargeted G1x eLV, G1x CD8 eLV, or G1x TCRab eLV, increasing the number of vector particles. The percentage of transduced cells was evaluated by flow cytometry 7 days post-transduction. These results indicate that fauxtyped CD8a VHH or TCRab VHH-targeted eLV vectors using VSV-GI331E (ENaBL-T8 and ENaBL-TT, respectively) induce specific transduction of CD8a-expressing cells (CD8+ T cells, SupT1) and TCRab-expressing cells (CD4+ T cells, CD8+ T cells, Jurkat), respectively, with transduction levels of target-negative cells being <1%. Importantly, in target-positive cells, there was no detectable transduction of the untargeted (VHH-deficient) ENaBL vector pseudotyped with VSV-GI331E. The results represent the mean ± SEM of two separate studies using two individual human PBMC donors. [Figure 25-1]This figure shows the binding of TCRab VHH VSVGI331E eLV vector particles to a Retrogenix cell microarray consisting of HEK293 T cells expressing surface human proteins, in order to evaluate the potential off-target binding of the vector. TCRab VHH VSV-GI331E eLV, which encodes BCMA CAR, was seeded at three different physical MOIs (6000, 8000, and 11000 vector particles per cell, A, B, and C) on immobilized HEK293 microarrays expressing six different targets (SIRPa, CD19, CD8A+CD8B2 heterodimer, CD4, LDLR, and EGFR), and the binding of the vector to selected targets was detected using a fluorescently labeled anti-VHH antibody. When this array was probed with a vehicle (PBS, D.), no signal was detected, and the expression of each target was confirmed with a transfection control (E.). The Retrogenix cell microarray did not contain HEK293 cells expressing the TCRab complex (the TCRab complex requires the simultaneous expression of multiple proteins for proper expression on the membrane). SIPRa, the CD47 receptor on the surface of the vector, was used as a positive control. The results showed that the TCRab VHH VSV-GI331E eLV bound to HEK293 cells expressing SIPRa, while binding to CD19, CD8A+CD8B2 heterodimer, CD4, and EGFR was not observed. A signal slightly above background was detected for LDLR, which was not reproduced in a full library screening, further supporting the lack of targeting ability of the VSV-GI331E variant. [Figure 25-2]Figures (F-G) show the binding of TCRab VHH VSVGI331E eLV vector particles to a Retrogenix cell microarray consisting of HEK293T cells expressing surface human proteins, in order to evaluate the potential off-target binding of the vector. Based on these results in A-E and the signal intensity compared to the background, 6000 vector particles per cell were selected for a complete library screening. TCRab VHH VSV-GI331E eLV binding was tested on a complete cell microarray (Retrogenix cell microarray technology) consisting of HEK293 cells individually expressing 6105 full-length human plasma membrane proteins, human secretory proteins tethered to the cell surface, and an additional 400 human surface protein heterodimers. As shown in Figures 25F and 25G, the results of two replicates of complete library screening and confirmatory screening indicate that TCRab VHH VSV-GI331E eLV specifically binds to SIRPa (isoforms 1, 2, and 4) and has very weak / weak binding to CD7, VSIG8, SIRPG, EPHB2, and EPHA7. In conclusion, data obtained from Retrogenix cell microarrays demonstrate that the TCRab VHH VSV-GI331E eLV vector has minimal off-target binding. [Figure 25-3]Figures (F-G) show the binding of TCRab VHH VSVGI331E eLV vector particles to a Retrogenix cell microarray consisting of HEK293T cells expressing surface human proteins, in order to evaluate the potential off-target binding of the vector. Based on these results in A-E and the signal intensity compared to the background, 6000 vector particles per cell were selected for a complete library screening. TCRab VHH VSV-GI331E eLV binding was tested on a complete cell microarray (Retrogenix cell microarray technology) consisting of HEK293 cells individually expressing 6105 full-length human plasma membrane proteins, human secretory proteins tethered to the cell surface, and an additional 400 human surface protein heterodimers. As shown in Figures 25F and 25G, the results of two replicates of complete library screening and confirmatory screening indicate that TCRab VHH VSV-GI331E eLV specifically binds to SIRPa (isoforms 1, 2, and 4) and has very weak / weak binding to CD7, VSIG8, SIRPG, EPHB2, and EPHA7. In conclusion, data obtained from Retrogenix cell microarrays demonstrate that the TCRab VHH VSV-GI331E eLV vector has minimal off-target binding. [Figure 26-1]This graph demonstrates the in vitro production of functional BCMA CAR-T cells using TCRab VHH VSV-GI331E eLV transduction of dormant human PBMCs. (A) 1 x 10⁶ unstimulated PBMCs from two independent donors were transduced with a BCMA CAR-expressing lentiviral vector targeted by TCRab_VHH and pseudotyped with VSV-GI331E at a fixed infection multiplicity of 0.4 transduction units per cell. CAR expression was evaluated by flow cytometry using BCMA-Fc, and CAR+ cells were identified on days 7 and 11 after transduction. (B) T cell expansion and proliferation were also evaluated by flow cytometry, and cell activation and cell expansion and proliferation were compared with PBMCs treated with anti-CD3 / CD28 (UTD TransAct) or untreated PBMCs (UTD inactivated) as positive and negative controls, respectively. The number of CAR-T cells is shown in (C), and compared to the initial number of PBMCs, approximately 10 times more CAR+ T cells were obtained by day 11 posttransduction. On day 11 posttransduction, co-culture with various GFP-expressing target cells was performed using effector-to-target (E:T) ratios based on CAR+ cells. To achieve equivalent T cell numbers under each E:T ratio between BCMA_CAR, BCMA_CAR_2, and untransduced (UTD) TransAct-treated PBMC controls, CAR-T cells were normalized with untransduced PBMCs from each donor, and the cytotoxicity of target cells was evaluated by flow cytometry. Two experiments were conducted separately: one using BCMAHigh NCI-H929 target cells and BCMANeg K562 cells (D-F), and the other using BCMALow Nalm6 target cells and BCMANeg K562 cells (G-H). BCMA_CAR and BCMA_CAR_2 T cells did not exhibit background cytotoxicity in K562 cells under unloaded E:T ratio conditions (D), while effectively lysing BCMAHigh NCI-H929 cells (E). BCMA CAR_2 demonstrated more effective control of target cell proliferation under more challenging conditions with BCMAHigh NCI-H929 cells at low E:T ratios (F), or in co-culture with BCMALow Nalm6 cells (H).(I) IL2 production was also evaluated by titration of HEK_Blue_IL2 cells in the supernatant from the co-culture on day 1 after co-culture, demonstrating clear IL2 production in co-culture with BCMAHigh NCI-H929 target cells. Results are shown for two independent PBMC donors (n=2), representing mean ± SEM on a linear scale graph or geometric mean ± geometric SD on a logarithmic scale graph. Overall, these results demonstrate the production of pluripotent CAR-T cells by transduction of resting human PBMCs using the TCRab VHH VSV-GI331E eLV vector. [Figure 26-2]This graph demonstrates the in vitro production of functional BCMA CAR-T cells using transduction of resting human PBMCs with TCRab VHH VSV-GI331E eLV. (K~N) is a graph demonstrating the production of functional CLDN18.2CAR-T cells using the TCRab VHH VSV-GI331E eLV vector. 1x10⁶ unstimulated PBMCs from two independent donors were transduced with a CLDN18.2CAR-expressing lentiviral vector containing the TCRab_VHH targeting molecule and then pseudotyped with VSVgI331E(ENabL-GTT). Alternatively, 1x10⁶ PBMCs were stimulated with TransAct, and after 24 hours, transduced with a CLDN18.2CAR-expressing lentiviral vector pseudotyped with VSVg(G.WT). Next, co-cultures were performed with various target cells expressing luciferase using various effector-to-target (E:T) ratios based on CAR+ cells. To achieve equivalent T cell numbers under each E:T ratio between ENabL-GTT, G.WT, and untransduced (UTD) TransAct-treated PBMC controls, CAR-T cells were normalized with untransduced TransAct-activated PBMCs from each donor. The cytotoxicity of target cells was evaluated 18 hours after co-culture by quantifying the reduction of luciferase signaling from endogenous CLDN18.2+ target cells, NUGC4(K). ENaBL-GTT and G.WT CAR T cells demonstrated equivalent cytotoxicity to NUGC4 cells. Furthermore, on day 9 after transduction / activation, co-cultures were performed with various target cells expressing GFP using effector-to-target (E:T) ratios based on CAR+ cells. To achieve comparable T cell counts under each E:T ratio among ENabL-GTT, G.WT, and untransduced (UTD) TransAct-treated PBMC controls, CAR-T cells were normalized with untransduced TransAct-activated PBMCs from each donor, and the cytotoxicity of target cells was evaluated by flow cytometry. Both ENaBL-GTT and G.WT CAR T cells effectively lysed Nalm6 cells (L) engineered to express CLDN18.2, but did not lyse the negative control, Nalm6 cells (M) or K562 cells (N) engineered to express CLDN18.1.The results for two independent PBMC donors (n=2) represent mean ± SEM on a linear scale graph, or geometric mean ± geometric SD on a logarithmic scale graph. Overall, these results demonstrate comparable functional CAR-T cell production using the TCRab VHH VSV-GI331E eLV vector. [Figure 27] This figure shows the graphic representation of the vector used to test the cell specificity and promoter expression ability of candidate synthetic promoters. The synthetic promoter portion of the vector consists of an enhancer DNA element combined with a core promoter sequence or a proximal promoter sequence. Promoter specificity and expression ability are tested in cultured cell lines and primary cells of different origins by transduction of a VSVG lentiviral vector with broad tropism. The luminescence or fluorescence readout from the expression of the reporter construct is quantified using a luminescence reader or by flow cytometry. [Figure 28] These are representative flow cytometry plots and normalized luminescence graphs for Sup-T1 cells transduced with VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of a ubiquitous promoter (EF1a), a proximal promoter, and different synthetic promoters (Figure 23). Sup-T1 cells were transduced in culture using the same number of functional VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of different promoters. Transduction efficiency (percentage of cells expressing GFP) was evaluated by flow cytometry (A), and promoter activity was measured by luminescence output from the luciferase reporter (B). This figure shows that the addition of enhancer sequences significantly increased expression from the synthetic promoter, and that specific synthetic constructs reached and exceeded the expression level from EF1a in Sup-T1 cells. [Figure 29]These are representative flow cytometry plots and normalized luminescence graphs for Jurkat cells transduced with VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of a ubiquitous promoter (EF1a), a proximal promoter, and different synthetic promoters (Figure 23). Jurkat cells were transduced in culture using equal functional titers (tites) of VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of different promoters. Transduction efficiency (percentage of cells expressing GFP) was evaluated by flow cytometry (A), and promoter activity was measured by luminescence output from the luciferase reporter (B). These results indicate that the addition of enhancer sequences significantly increases expression from the synthetic promoter, and that specific synthetic constructs reach and exceed the expression levels from EF1a in Jurkat cells. [Figure 30-1] This figure shows representative flow cytometry plots, normalized emission values, and geometric mean fluorescence intensity (gMFI) for human primary lymphocytes transduced with VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of a ubiquitous promoter (EF1a), a proximal promoter, and different synthetic promoters (Figure 23). Activated human primary lymphocytes were transduced in culture using VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of different promoters, with equal functional titers. Transduction efficiency (percentage of CD3+ cells expressing GFP) and gMFI of GFP in CD4+ transduced T cell populations or CD8+ transduced T cell populations were evaluated by flow cytometry (A and C, respectively), and promoter activity was measured by the emission output from the luciferase reporter in CD3+ cells (B). [Figure 30-2]This figure shows representative flow cytometry plots, normalized emission values, and geometric mean fluorescence intensity (gMFI) for human primary lymphocytes transduced with VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of a ubiquitous promoter (EF1a), a proximal promoter, and different synthetic promoters (Figure 23). (D.) shows representative flow cytometry plots for lymphocyte cell lines (Jurkat and SUPT1) and primary human T cells transduced with VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of a ubiquitous promoter (EF1a) and two different synthetic promoters (SYN19 and SYN22). SUP-T1 cells were transduced in culture using VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of different promoters at equal functional titers. This figure shows that the addition of enhancer sequences significantly increases expression from the synthetic promoter, and that specific synthetic constructs reach a median reporter expression intensity of less than twice that of EF1a in T cell lines or primary T cells. [Figure 31] This graph shows the luminescence values ​​(normalized for cell viability) for each cell type transduced with VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of a ubiquitous promoter (EF1a), a proximal promoter, and different synthetic promoters (Figure 27). Cell lines of different origins were transduced in culture using equal functional titers of VSVG lentiviral vectors expressing a luciferase / GFP dual reporter under the control of different promoters. Promoter activity was measured by luminescence output from the luciferase reporter and normalized for cell viability. For quantification of reporter activity in NCI-H929 and THP1 cells, data represented two separate experiments, and the luciferase signal was further normalized for each experiment against the EF1a condition. These results indicate that the selected synthetic promoter has limited expression in non-T cell lineages compared to EF1a. [Figure 32]This graph summarizes representative flow cytometry plots of surface BCMA CAR expression on 293T-producing cells during lentivirus production, and the functional titers of LVGP1x-CD8 lentiviral vectors produced for the expression of BFP, BCMA, or CD19 CAR under the control of a ubiquitous promoter (EF1a) or different synthetic promoters (SYN19, SYN21, SYN22). 293T-producing cell lines transiently transfected with lentiviral transfection plasmids and packaging plasmids were analyzed by flow cytometry at the end of the production process for the surface expression of the BCMA CAR transgene encoded in the lentiviral transfection vector. (A) Results show that placing the BCMA CAR coding sequence under the control of a T cell-specific promoter reduces surface expression of BCMA CAR on the surface of producing cells, and therefore on the viral envelope of the resulting lentiviral particles, by more than 10-fold. (B) The LVGP1x-CD8 lentiviral vector produced in A was titrated on target cells (Sup-T1). The results in B demonstrate that restricting the expression of surface-expressed BCMA CAR or CD19 CAR, rather than the expression of soluble fluorescent reporter protein BFP, during lentiviral particle production increases the number of transduction-capable lentiviral particles per mL by more than 10 times. [Figure 33]This graph shows the luminescence values ​​from NCI-H929 cells that stably express luciferase and are co-cultured with GFP lymphocytes or CAR-T lymphocytes. Primary T lymphocytes (within a mixed population of peripheral blood mononuclear cells) were transduced with GFP or BCMA CAR-expressing VSVG lentiviral vectors whose transgene is EF1a or controlled by the T cell-specific synthetic promoter (SYN19). Transduced cells were grown and co-cultured with NCI-H929 cells that stably express luciferase in a 1:1 or 1:2 (effector:target ratio), and luminescence under each condition was measured 2 days after co-culture. This figure shows a significant decrease (>4 times) 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 the loss of NCI-H929 cells in the presence of BCMA CAR-T cells. These results demonstrate 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. [Figure 34]This graph demonstrates the favorable CAR-T phenotype of BCMA CAR under the control of the T cell-specific SYN23 promoter compared to the constitutive EF1a promoter. Three independent donor PBMCs were activated with anti-CD3 / CD28 and transduced with the VSVG pseudotyped lentiviral vector on day 2 postactivation. CAR expression levels were assessed on day 7 postactivation. Co-culture with various GFP-expressing target cells was then performed using effector-to-target (E:T) ratios based on CAR+ cells. CAR-T cells were normalized with untransduced PBMCs from each donor to achieve comparable T cell counts under each E:T ratio between BCMA_CAR, BCMA_CAR_2, and untransduced (UTD) TransAct-treated PBMC controls. Repeated co-cultures were performed at various time points by adding additional BCMAHigh NCI-H929 and BCMANeg K562 target cells (dotted lines in the graph). CAR expression was evaluated by flow cytometry at various time points during co-culture, demonstrating the lack of expansion and proliferation of CAR+ cells in co-culture with BCMANeg K562 cells (A), while expansion and proliferation of CAR+ cells using SYN23_BCMA CAR was enhanced compared to EF1a_BCMA CAR in co-culture with BCMAHigh NCI-H929 target cells (B, C). CAR-mediated cytotoxicity was also evaluated by flow cytometry at various time points during co-culture, demonstrating the absence of CAR-mediated cytotoxicity in BCMANeg K562 target cells (D), and that the cytotoxicity of BCMAHigh NCI-H929 target cells was equivalent between SYN23_BCMA CAR and EF1a_BCMA CAR under unloaded E:T1:3 conditions (E). In particular, SYN23_BCMA CAR-T cells exhibited enhanced target cell lysis under challenging E:T1:12 conditions (F), and EF1a_BCMA CAR-T cells failed to control target cell proliferation initiated in co-culture 2. CAR-T activation was also assessed by flow cytometry quantification of CD25 expression and was shown to be unrelated to specific activation of CAR-T cells by BCMANeg K562 target cells (G).However, EF1a_BCMA CAR-T cells demonstrated higher background activation in the absence of BCMA antigen. Conversely, in the presence of BCMAHigh NCI-H929 target cells, Enh023_BCMA CAR-T cells demonstrated increased BCMA-specific activation in co-cultures 1 and 2 (H, I). Results are shown for three independent PBMC donors (n=3), representing mean ± SEM on a linear scale graph or geometric mean ± geometric SD on a logarithmic scale graph. Overall, these results indicate lower background activation, as well as more sustained CAR expression and function, with SYN23 promoter-driven CAR expression, compared to EF1a. [Figure 35] This graph shows the results of quantitative analysis by flow cytometry of the transduction rate of unstimulated PBMCs and isolated monocytes using LVGP1xeLV, which presents a CD14-specific membrane-anchored scFv. Human PBMCs and isolated primary monocytes were transduced with an increasing number of LVGP1X-scFVCD14 eLV vectors (physical particles), and the proportion of transduced cells was evaluated by flow cytometry on day 7 after transduction. These results indicate that specific transduction of monocytes is possible by directing LVGP1x eLV to bone marrow cells using an envelope-anchored binding factor that targets CD14. [Figure 36]This graph shows the results of quantitative analysis by flow cytometry of the transduction rate 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 targeting-deficient mutant VSVGI331E(G1x) and different CD14 VHH or affinity-matured 3RMB1 CD14 VHH. A. 50,000 Jurkat WT and Jurkat Cd14 cells were transduced using 100 μL of unenriched CD14 scFv-targeted lentiviral vector or VHH-targeted lentiviral vector. These lentiviral vectors were pseudotyped with G1x and expressed either CD14 scFv or a different CD14 VHH. Transduction efficiency was evaluated by flow cytometry on day 7 post-transduction by staining Jurkat cells with anti-VHH antibody. The results represent the mean ± SD of two different experiments. B. 100,000 monocytes isolated from one healthy donor were transduced with 250, 500, 1,000, and 2,000 vector particles of a lentiviral vector pseudotyped with the targeting-deficient mutant VSVGI331E(G1x). This lentiviral vector either lacked targeting or expressed either CD14 scFv or a different CD14 VHH. The percentage of transduced cells was assessed by flow cytometry 7 days post-transduction. C. 100,000 monocytes isolated from one healthy donor were transduced with 250 and 1,000 vector particles of a lentiviral vector pseudotyped with the targeting-deficient mutant VSVGI331E(G1x). This lentiviral vector either lacked targeting or expressed either CD14 scFv or a different affinity-matured 3RMB1 CD14 VHH. Transduction efficiency was evaluated by flow cytometry on day 7 posttransduction by staining macrophages with anti-VHH antibody. These results indicate that specific transduction of monocytes is possible by directing G1x eLV to bone marrow cells using an envelope-anchored binding factor that targets CD14. [Figure 37]This graph shows the results of quantitative analysis by flow cytometry of the transduction rates of CD14-target-positive cells (monocytes / macrophages, and Jurkat CD14 cells) and CD14-target-negative cells (stimulated PBMCs, Jurkat WT cell lines, and NCI-H929 cells) using lentiviral vectors pseudotyped with the targeting-deficient mutant VSVGI331E(G1x) and CD14 VHH or G1x alone. Monocytes / macrophages, CD3 / 28-activated human PBMCs, wild-type Jurkat lymphoblast cell lines, or Jurkat lymphoblast cell lines and B-cell lymphoblast cell lines NCI-H929 that stably express CD14, isolated from three separate healthy donors, were transduced with 250 or 500 vector particles of untargeted eLVs and CD14-targeted eLVs. Both of these eLVs were pseudotyped with VSVGI331E and express the fluorescent reporter GFP. The percentage of transduced cells was assessed by flow cytometry 7–14 days post-transduction. The results represent the mean ± SD for three donors and 2–3 separate batches of virus. These results indicate that pseudotyped CD14 VHH-targeted eLV vectors with VSVGI331E induce specific transduction of CD14-expressing cells (primary monocytes / macrophages and Jurkat cells that stably express CD14), with transduction levels in target-negative cells being <1% (NCI, Jurkat, and primary PBMCs). Importantly, there was no detectable transduction of the untargeted (VHH-deficient) eLV vector pseudotyped with VSVGI331E in target-positive cells. [Figure 38A]This figure shows the levels of GFP expression, as assessed by flow cytometry as geometric mean fluorescence intensity, in different cell types (bone marrow, T cells, hepatocytes, B cells, kidney) transduced with a VSVG pseudotype LV encoding a reporter under the control of six proximal promoters derived from macrophage-specific genes (see Figure 27). Primary monocytes, THP-1, primary PBMCs, SupT-1, Huh-7, NCI, and e293 T cells were transduced with the broad-tropism VSVG lentivirus. This lentivirus encodes a luciferase-eGFP transgene under the control of EF1a, no promoter (empty), or six different proximal promoters. Monocytes were transduced and differentiated in M0-like macrophages for 7 days in the presence of 50 ng / mL M-CSF. 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. The proportion of GFP gMFI normalized to EF1a for empty promoters and six proximal promoters. [Figure 38B]This figure shows the levels of GFP expression, as assessed by flow cytometry as geometric mean fluorescence intensity, in different cell types (bone marrow, T cells, hepatocytes, B cells, kidney) transduced with a VSVG pseudotype LV encoding a reporter under the control of six proximal promoters derived from macrophage-specific genes (see Figure 27). Primary monocytes, THP-1, primary PBMCs, SupT-1, Huh-7, NCI, and e293 T cells were transduced with the broad-tropism VSVG lentivirus. This lentivirus encodes a luciferase-eGFP transgene under the control of EF1a, no promoter (empty), or six different proximal promoters. Monocytes were transduced and differentiated in M0-like macrophages for 7 days in the presence of 50 ng / mL M-CSF. PBMCs were stimulated with CD3 / CD28 for 2 days prior to transduction and cultured in the presence of 30 ng / mL human IL-2. These are FACS plot data for each test cell type for the B.EF1a and CHIT1 proximal promoters. These results indicate that reporter expression induced by the selected bone marrow-specific core promoter induces specific expression in primary macrophages at levels equivalent to or exceeding those of the EF1a promoter. [Figure 39A]This figure shows the levels of GFP expression, as assessed by flow cytometry as geometric mean fluorescence intensity, in different cell types (bone marrow, T cells, hepatocytes, B cells, kidney) transduced with a VSVG pseudotype LV encoding a reporter under the control of seven synthetic promoters derived from macrophage-specific genes (see Figure 27). Primary monocytes, THP-1, primary PBMCs, SupT-1, Huh-7, NCI, and e293 T cells were transduced with the broad-tropism VSVG lentivirus. This lentivirus encodes a luciferase-eGFP transgene under the control of EF1a, no promoter (empty), or six different proximal promoters. Monocytes were transduced and differentiated in M0-like macrophages for 7 days in the presence of 50 ng / mL M-CSF, and then differentiated in M2-like macrophages for 2 days in the presence of 50 ng / mL M-CSF, 20 ng / mL IL-4, and 20 ng / mL IL-10. PBMCs were stimulated with CD3 / CD28 for 2 days before transduction and cultured in the presence of 30 ng / mL human IL-2. A. Percentage of GFP gMFI normalized to EF1a for seven synthetic promoters incorporating an empty promoter, a CHIT1 core promoter, and a CHIT1 proximal promoter fused to an enhancer sequence. [Figure 39B]This figure shows the levels of GFP expression, as assessed by flow cytometry as geometric mean fluorescence intensity, in different cell types (bone marrow, T cells, hepatocytes, B cells, kidney) transduced with a VSVG pseudotype LV encoding a reporter under the control of seven synthetic promoters derived from macrophage-specific genes (see Figure 27). Primary monocytes, THP-1, primary PBMCs, SupT-1, Huh-7, NCI, and e293 T cells were transduced with the broad-tropism VSVG lentivirus. This lentivirus encodes a luciferase-eGFP transgene under the control of EF1a, no promoter (empty), or six different proximal promoters. Monocytes were transduced and differentiated in M0-like macrophages for 7 days in the presence of 50 ng / mL M-CSF, and then differentiated in M2-like macrophages for 2 days in the presence of 50 ng / mL M-CSF, 20 ng / mL IL-4, and 20 ng / mL IL-10. PBMCs were stimulated with CD3 / CD28 for 2 days before transduction and cultured in the presence of 30 ng / mL human IL-2. FACS plot data for each test cell type are shown for B.EF1a and Enh031-CHIT1 synthetic promoters. These results indicate that the addition of enhancer sequences to the CHIT1 core promoter increases reporter expression levels while maintaining expression specificity in bone marrow cells. [Figure 40]This graph shows the quantitative results of human IL-12 secretion in the supernatant of M0-like macrophages or M2-like macrophages transduced from primary monocytes derived from healthy donors and transduced with VSVG LV encoding the h-IL-12 transgene under the control of EF1a, CHIT1 proximal promoter, or Enh031-CHIT1 synthetic promoter. Result A shows the h-IL-12 concentration in the supernatant of M0-like macrophages 7 days after transduction. Monocytes were transduced and differentiated in M0-like macrophages for 7 days in the presence of 50 ng / mL of M-CSF. Result B shows the IL-12 concentration in the supernatant of M2-like macrophages 9 days after transduction. Monocytes were transduced and differentiated in M0-like macrophages for 7 days in the presence of 50 ng / mL M-CSF, and then differentiated in M2-like macrophages for 2 days in the presence of 50 ng / mL M-CSF, 20 ng / mL IL-4, and 20 ng / mL IL-10. The experiments were performed using two batches of lentivirus per condition. These results demonstrate that the Enh31-CHIT1 bone marrow-specific synthetic promoter induces functional transgene expression levels equivalent to those induced by the constitutive promoter EF1a. [Figure 41] This graph shows the results of flow cytometry analysis of the transduction efficiency ratios of LVGP1X-VHHCD3 vectors to Jurkat cells, where CD3e VHH is anchored to the envelope within different scaffolds. Jurkat WT cells were transduced with different dilutions of LVGP1X-VHHCD3 vectors, each presenting CD3e VHH in different scaffolds. The functional titer of each vector type was quantified by the average percentage of transduced cells from at least two serial dilutions. The different scaffolds anchor CD3e VHH to the envelope via transmembrane domains of PDGFR or B7-1 linked to VHH through sequences of varying lengths and flexibility (e.g., CD8a hinge region or PGFR receptor proximal membrane region, see X-axis labeling). This result indicates that the scaffold that anchors VHH to the membrane influences the functional titer of the vector. In the case of CD3e VHH, an anchor scaffold containing the PDGFR transmembrane domain and the CD8a hinge region provided a structure that achieved the highest functional titer. [Figure 42] This graph shows the results of flow cytometry quantification of the transduction efficiency ratio of LVGP1X-VHHCD8a vectors to Sup-T1 cells, where CD8a VHH is anchored on envelopes within different scaffolds (see Figure 32). Sup-T1 WT cells were transduced with different dilutions of each LVGP1X-VHHCD8a vector, and the functional titer of each vector type was quantified by the average percentage of transduced cells from at least two serial dilutions. This result indicates that the scaffold that anchors the VHH to the membrane influences the functional titer of the vector. In the case of CD3e VHH, anchor scaffolds containing the PDGFR transmembrane domain and the CD8a hinge region or PDGFR proximal membrane region provided the structure that achieved the highest functional titer. [Figure 43]A. is a graph showing the results of quantifying the transduction efficiency ratio of target cells (Jurkat WT) by TCR targeting (VHHTCR eLV) using flow cytometry. Jurkat WT cells were transduced with a TCR VHH-presenting vector pseudotyped with VSVG mutants (K47A, R354) that were previously shown to eliminate the broad tropism of wild-type VSVG. In this vector, VHH is anchored to envelopes within different scaffolds: TCR1 scaffold: PDGFR transmembrane domain and proximal membrane region of PDGFR; TCR2 scaffold: PDGFR transmembrane domain and proximal membrane region of IgG4; TCR3 scaffold: PDGFR transmembrane domain and tetrameric coiled-coil hinge. These results indicate that the scaffold that anchors VHH to the membrane affects the transduction efficiency of the vector. In the case of TCR VHH, an anchor scaffold containing the PDGFR transmembrane domain and the proximal membrane region of IgG4, combined with the VSVG(K47A, R354) pseudotype, provided a structure that achieved the highest transduction efficiency. B is a graph demonstrating the increased transduction and stimulating ability of TCRab-targeted lentivirus using the CD8a hinge compared to the IgG4 hinge on the TCRab_VHH fusion protein. Using stable-producing cell lines that stably express TCRab VHH in different structures (PDGFR transmembrane domain + CD8a hinge or IgG4 hinge), pseudotyped with the VSVgI331E envelope and GFP-expressing lentivirus targeted by TCR VHH were produced. Subsequently, 1.5 x 10⁵ PBMCs from two independent donors were transduced with 100 μL of unenriched lentivirus. Compared to the IgG4 hinge, the use of the CD8a hinge resulted in higher total transduced T cells at day 7 posttransduction. These results represent the geometric mean ± geometric SD of two independent PBMC donors and two independent lentiviral preparations (n=4). [Figure 44]This graph shows the results of quantifying the activation, proliferation, 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 VHH eLV. These results indicate that LVGP1X, which presents the TCRab VHH binding factor, induces increased T cell activation (A.), T cell proliferation (B.), and T cell transduction (C.) compared to resting PBMCs exposed to VSVG LV or LVGP1X-CD8a VHH eLV, as indicated by the increased percentage of T cells expressing the activation marker CD25. [Figure 45] This graph demonstrates the increased transduction and stimulating ability of TCRab-targeted lentiviruses using the CD8a hinge compared to the IgG4 hinge on the TCRab_VHH fusion protein. (A-E) Stable-producing cell lines transduced with a single lentiviral vector expressing TCRab_VHH were used to produce GFP-expressing lentiviruses pseudotyped with a VSVgI331E envelope. Subsequently, 1.5 x 10⁵ PBMCs from two independent donors were transduced with 100 μL of unenriched lentivirus. Anti-CD3 / CD28 (TransAct) was used as a positive control for T cell activation. Untreated PBMCs were also included (NA) to demonstrate the background levels of T cell proliferation and activation. The use of the CD8a hinge compared to the IgG4 hinge resulted in higher CD69 expression on post-transduction day 1 (A), CD25 and PD1 expression on post-transduction day 4 (B, C), and increased cell expansion and proliferation (E), viral titer (F), and total transduced T cells (G) on post-transduction day 7. Results are shown for two independent PBMC donors and two independent lentiviral preparations (n=4), with linear scale graphs representing mean ± SEM or logarithmic mean ± geometric SD. [Figure 46]This graph demonstrates the stimulating and transduction-mediated capabilities of lentiviral vectors targeted with various mutant TCRab_VHH binding factors. Producing cells were transfected with lentiviral plasmids expressing both TCRab_VHH (wild-type or mutant) and TagBFP2. Here, the 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 with a VSVgI331E envelope. Jurkat cells were transduced with 100 μL of unenriched lentivirus. The stimulating ability of the TCRab_VHH clone was evaluated by CD69 expression on Jurkat cells 1 day post-transduction. Viral titer was evaluated by TagBFP2 expression 3 days post-transduction. [Figure 47]This graph demonstrates increased activation, transduction, and T cell expansion induced by TCRab VHH or CD8a VHH-targeted lentiviruses presenting stimulating or co-stimulating factors. Resting human PBMCs, newly isolated from buffy coat by Ficoll gradient, were infected with 2500 viral particles per cell on day 0. In one example, the viral particles were CD8a VHH G1x eLV lentivirus (T8) pseudotyped with the CD8a-targeted VSV-G variant (I331E), presenting one of two CD28-binding co-stimulating factors (cleaved CD80 protein or anti-CD28 nanobody (VHH), T8K80, or T8K28, respectively) in addition to the stimulating anti-CD3 agonist antibody OKT3 (T8K). In another example, the viral particles were TCRab VHH G1x eLV lentivirus (TT) pseudotyped with the TCRab-targeted VSV-G variant (I331E), which presented the stimulating anti-CD3 agonist antibody OKT3 (T8K) in addition to one of two CD28-binding costimulators (cleaved CD80 protein or anti-CD28 nanobody (VHH), T8K80, or T8K28, respectively). CD3+ T lymphocytes were then analyzed by flow cytometry for activation (CD25 surface marker) at 24 hours post-infection, for transduction (level of BCMA CAR expression on the cell surface, measured by V5 tag staining) at 7, 14, 21, and 21 days post-infection, and for induced proliferation (expanding proliferation) at 7, 14, 21, and 28 days post-infection. These results indicate that while T8 eLVs do not activate resting T cells, TCRab VHH-targeted eLVs, alone or in combination with costimulatory factors (TTK, TT80, or TT28), or CD8 VHH-targeted eLVs in combination with costimulatory factors, induce a significant increase in T cell activation, proliferation, and T cell transduction. These data represent the mean ± SEM of 3 to 12 separate experiments using 3 to 5 different donors. [Figure 48-1]This graph shows the phenotype of primary T cells after transduction with a stimulating vector / co-stimulating vector. Single-vector systems were used in (A-F). Here, stable-producing cell lines transductioned with a single lentiviral vector expressing a stimulating molecule (TCRab_VHH) and various co-stimulating molecules were used to produce GFP-expressing lentiviruses pseudotyped with a VSVgI331E envelope. The expression of TCRab_VHH on stable-producing cells is shown in (A). Next, 1.5 x 10⁵ PBMCs from two independent donors were transductioned with 100 μL of unenriched lentivirus. Vectors containing the CD28 co-stimulatory molecules CD86 IgV and TGN1412, and the 4-1BB co-stimulatory molecule 4B1-4, resulted in increased IL2 production (B) and CD69 expression (C) on post-transduction day 1, increased CD25 expression (D) on post-transduction day 4, and increased T cell expansion and proliferation (E) on post-transduction day 7, compared to TCRab_VHH alone. However, in vectors containing the co-stimulatory molecules, overall transduction was reduced, likely due to decreased TCRab_VHH expression in these cells. Results are shown for two independent PBMC donors (n=2), representing mean ± SEM on a linear scale graph or geometric mean ± geometric SD on a logarithmic scale graph. [Figure 48-2]This graph shows the phenotype of primary T cells after transduction with a stimulating vector / co-stimulating vector. (G~L) shows the use of different vector systems. This allowed for the retransduction of a population of clone-producing cells transduced with TCRab_VHH using vectors expressing various co-stimulatory molecules (G~L). Expression of TCRab_VHH and the co-stimulatory molecules CD86 IgV and TGN1412 is shown in (G). Using these producing cells, pseudotyped GFP-expressing lentiviruses with a VSVgI331E envelope were produced. Subsequently, 1.5 x 10⁵ PBMCs from two independent donors were transduced with two independent preparations consisting of 100 μL of unenriched lentivirus. Vectors containing the CD28 costimulatory molecules CD86 IgV and TGN1412 resulted in increased IL2 production (H), increased CD25 expression on day 4 posttransduction (I), and increased T cell expansion and proliferation on day 7 posttransduction (J) compared to TCRab_VHH alone. Viral titer was not altered by equivalent TCRab_VHH expression (K). However, as a result of increased T cell expansion and proliferation, the costimulatory molecules resulted in an increase in the overall number of transduced cells (L). Results are shown for two independent PBMC donors and two independent lentiviral preparations (n=4), with linear scale graphs representing mean ± SEM or logarithmic scale graphs representing geometric mean ± geometric SD. In all cases, anti-CD3 / CD28 (TransAct) was used as a positive control for T cell activation. PBMCs were also left untreated (NA) to demonstrate background levels of T cell expansion and proliferation and activation. [Figure 49]This graph summarizes the results of quantification by flow cytometry of hCD3+ cells expressing NY-ESO TCR after transduction with VHH Cd8a 10uL (A.) encoding the NY-ESO TCR transgene or TCR-targeted LVGP1x eLV (B.) under the control of EF1a, SYN21, or SYN22 promoters (not shown). Activated human PBMCs were transduced with LVGP1x-VHHCD8a or VHHTCR encoding the NY-ESO TCR under the control of EF1a, SYN21, or SYN22. Transduction efficiency in CD3+ T cells was evaluated on days 3, 7, 14, and 21 by flow cytometry using PE-bound A2 / NY-ESO-1 multimers. These results demonstrate that LVGP1x-VHHCD8a or VHHTCR stably transduce primary T cells using the NY-ESO TCR construct. As supported by the results presented in Figure 31, these results indicate that the LVGP1x eLV vector expressing NY-ESO under the control of the EF1a promoter has a low functional titer, potentially due to the expression of the NY-ESO TCR on the viral envelope. [Figure 50]This graph summarizes the results of quantifying the number of target and non-target cells in co-culture of NY-ESO TCR-T cells by flow cytometry. Activated human PBMCs were transduced with LVGP1x-VHHTCR, which encodes NY-ESO, under the control of EF1a or T cell-specific synthetic promoters SYN19 and SYN22. Transduced cells were expanded and co-cultured with T2 cells pulsed with NY-ESO peptide (target cells) or HIV peptide (non-target cells) in a 1:1 ratio (ratio of T cells to target cell lines / non-target cell lines). The number of target / non-target cells stained with Cell Trace Violet was quantified by flow cytometry in fixed-volume culture medium and summarized in graphs A and B. This figure shows that co-culture of NY-ESO TCR-T cells eradicated target cells (A) but not non-target cells (B) compared to co-culture with untransduced T cells. Notably, despite the low transduction efficiency of the LVGP1X-VHHTCR EF1a NY-ESO vector (see Figure 35), and therefore the low number of NY-ESO TCR-T cells in these co-cultures, the number of target cells in these cultures was still significantly reduced. This result demonstrates that transduction of primary T cells by LVGP1x-VHH eLV encoding NY-CAR under the control of a T cell-specific promoter leads to the functional expression of the TCR construct and to target-cell-specific cytotoxicity of transduced cells. [Figure 51]This graph shows the results of quantifying tumor growth, measured by bioluminescence, and transduced human CD3+ T cells by flow cytometry in tumor-bearing humanized MHCI / II KO NSG mice that were intraperitoneally injected with activated human PBMCs and a TCRab VHH-targeted VSVGI331E pseudotype eLV vector. The NCI-H929 multiple myeloma cell line, which stably expresses luciferase, was (iv) systemically injected into NSG mice lacking expression of mouse MHCI and MHCII complexes. After confirming tumor engraftment, activated human PBMCs were injected intraperitoneally into tumor-bearing animals on day 0, and a TCRab VHH-targeted VSVGI331E pseudotype eLV vector expressing BFP or BCMA CAR constructs under the control of T cell-specific synthesis promoter #23 was injected intraperitoneally on day 1. A. Tumor growth over time, measured using in vivo bioluminescence signals from luciferase-expressing NCI-H929 tumor cells. The data represent the mean BLI signal ± SD from 6 animals per condition. B. In vivo transduction efficiency of TCRab VHH VSVGI331E BCMA CAR, evaluated by flow cytometry analysis of peripheral blood after immunostaining with CAR-specific antibody 14 days after vector injection. The data represent the mean ± SEM from 6 animals per condition. These results demonstrate efficient in vivo transduction of T cells using an eLV-G1X-TCRab-targeted vector expressing BCMA CAR, and the potent antitumor activity of BCMA CAR-T cells reprogrammed in vivo. [Figure 52]This graph shows tumor growth quantified by bioluminescence in tumor-bearing hCD34+ humanized NCG mice, after intravenous injection of an eLV-G1X-TCRab viral vector expressing a BCMA CAR construct or a fluorescent reporter (BFP) under the control of synthetic T cell promoter #23. NCI-H929 multiple myeloma cells stably expressing luciferase were (iv) systemically injected into stable hCD34+ humanized NCG mice. After confirming tumor engraftment, the tumor-bearing animals were intravenously injected with an eLV-G1x-TCRab vector expressing BFP or a BCMA CAR construct under the control of T cell-specific synthetic promoter #23. Tumor growth was measured over time using in vivo bioluminescence signals from luciferase-expressing NCI-H929 tumor cells. The data represent the BLI signal for each animal. These results demonstrate the potent antitumor activity of BCMA CAR-T cells reprogrammed in vivo using the eLV-G1X-TCRab vector, with complete and sustained tumor clearance observed in animals treated with 3.25 or 6.5E+10 vp, and moderate tumor control observed in animals treated with 1.3E+10 vp. [Figure 53] This graph shows the flow cytometry analysis of tissue samples from tumor-bearing hCD34 NCG mice that were intravenously injected with eLV-G1X-TCRab expressing either a BFP reporter or a BCMA CAR construct. The proportion of CAR+ cells within different immune cell types (mouse CD45+ cells, hCD45+ / CD56+ human NK cells, hCD45+ / CD14+ human bone marrow cells, hCD45+ / CD3+ T lymphocytes, hCD45+ / CD3+ / CD4+ human T cells, and hCD45+ / CD3+ / CD8+ human T cells) was analyzed by flow cytometry in (A) peripheral blood 20 days after vector injection and (B) bone marrow at the time of euthanasia (up to 36 days after vector injection). Note that NKT cells were below detection level, and therefore CAR expression could not be evaluated. The results, representing the mean ± SEM from 2 to 5 animals per group depending on the tissue, demonstrate the specific targeting and transduction of T cells by the eLV-G1X-TCRab BCMA CAR lentiviral vector. [Figure 54] This graph shows tumor growth in tumor-bearing hCD34+ humanized NCG mice after intravenous injection of TCRab VHH-targeted and / or CD14 VHH VSVGI331E pseudotype eLV vectors. NCI-H929 multiple myeloma cell line was subcutaneously injected into NCG mice, and after confirming tumor engraftment, tumor-bearing animals were intravenously injected in a single dose with either CD14 VHH VSVGI331E pseudotype eLV expressing vehicle, BFP, or human IL12, TCRab VHH VSVGI331E pseudotype eLV expressing BCMA CAR, or a combination of the same dose of CD14 VHH VSVGI331E eLV with human IL12 and TCRab VHH VSVGI331E pseudotype eLV with BCMA CAR. Subcutaneous tumor volume was measured over time using a caliper. The data represent tumor volume over time in each animal (n=4 or n=8) for each cohort. These results demonstrate synergistic antitumor activity between in vivo reprogrammed monocytes / macrophages and BCMA CAR T cells after a single injection of cell type-specific VHH-targeted eLV vectors. [Figure 55] Figure 54 shows a graph of flow cytometry analysis results for tissue samples derived from tumor-carrying hCD34 NCG mice tested. The proportion of CAR+ cells and CD3+ cells was analyzed by flow cytometry in (A) peripheral blood 11 days after vector injection and (C) tumor tissue at the time of euthanasia, and (B) the level of secreted human IL-12 in plasma. The results represent the mean ± SEM from 1 to 8 animals per group, depending on the tissue and conditions. The data demonstrate that (1) the eLV-G1X-TCRab BCMA CAR lentiviral vector and eLV-G1X-CD14 VHH hIL12 effectively reprogram cells in vivo, and (2) the combination of T cell reprogramming (with BCMA CAR) and monocytes / macrophages (for secreting human IL12) synergistically enhances the antitumor activity of the individual vectors by increasing the transduction / growth of BCMA CAR-T cells and increasing tumor T cell infiltration. [Figure 56]This figure shows data quantifying the recovery of TCRab VHH VSVGI331E eLV, which presents an envelope-anchored complement regulatory factor (RCA). (A) Schematic diagram of an RCA construct tested for its ability to prevent complement-mediated inactivation of VHH-presenting lentiviruses. DAF and H factors were tethered to the lentiviral envelope via a VSV-GS anchor or a GPI anchor. SP: signal peptide, H: 6xHIS-Tag, EC: extracellular domain, TM: transmembrane domain, IC: intracellular domain, GPI: phosphatidylinositol anchor signal. Arrows indicate cleavage sites. (B) A complement inactivation assay was performed. Different lentiviral vectors encoding GFP reporter transgenes were incubated for 1 hour in serum-free medium supplemented with fresh human serum or in complement medium containing heat-inactivated human serum. The vector preparations were then incubated with Jurkat cells for 2 hours, and the transduction rate was evaluated by flow cytometry after 72 hours. The results, representing the mean ± SEM from three separate experiments, show that LVs presenting DAF and factor H are partially shielded from complement-mediated inactivation, resulting in increased mean recovery rates of up to 24.1% and 16.3%, respectively. In conclusion, by presenting RCA on the surface of LVs, lentiviral particles are shielded from complement-mediated inactivation. [Figure 57]This graph shows the quantitative results of macrophage phagocytosis of T-cell-targeted TCRab VHH G1x eLV presenting an albumin-binding domain. The albumin-binding domain of Streptococcus G418 was fused to a CD59-GPI anchor or a VSV-GS anchor to test the ability of macrophages to prevent phagocytosis of VHH-presenting lentiviruses. A macrophage phagocytosis assay was performed. Human monocyte-derived macrophages were incubated with a TCRab VHH G1x lentivirus suspension for 2 hours, then washed and incubated in cRPMI for 3 days. Chlorpromazine was used as a control for macrophage activity inhibition. 72 hours post-infection, gDNA was extracted from macrophages and the vector copy number (VCN) was determined. The results demonstrate the protective effect of ABD on the envelope of eLV presenting membrane hTCR nanobodies (VHH) and I331E VSVG glycoprotein. The data, representing the mean ± SEM from three separate experiments using two different PBMC donors, demonstrate that presenting an albumin-binding domain within the lentiviral envelope shields lentiviral particles from phagocytosis by macrophages. [Figure 58]This figure shows the level of GFP expression, as evaluated by flow cytometry as geometric mean fluorescence intensity, in macrophages that expressed a reporter under the control of four different synthetic promoters (constructed as shown in Figure 27) derived from genes specifically upregulated in macrophages of the tumor microenvironment, cultured under conditions mimicking the tumor microenvironment. Human primary monocytes were transduced with VSVG lentivirus, which has broad tropism including the luciferase-eGFP transgene, under the control of EF1a, no promoter (empty condition), or 12 TAM enhancers with the CHIT1 proximal promoter (four of which are shown in this figure). Transduced human primary monocytes were differentiated in M0-like macrophages for 7 days in the presence of 50 ng / mL M-CSF, and then treated under 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 "hypoxia" condition, M0-like macrophages were treated with 100 μM cobalt chloride (CoCl2). For the "conditional medium" condition, M0-like macrophages were treated with the supernatant of NUG-C4 cell culture. Results A show the multiplier change in GFP gMFI for each TME condition, normalized to the reporter expression level in macrophages before TME treatment. Results B show flow cytometry plot data for the synthetic promoters of CHIT1, TAM12-CHIT1, and Enh031-CHIT1 for each test TME condition. Experiments were performed using two batches of lentivirus per condition. [Modes for carrying out the invention]

[0175] List of nucleic acid sequences and amino acid sequences Sequence ID 1 QVQLVESGGGLVQAGGSLRLSCALSGRVWGSYAMGWFRQAPGKEREFVAAISRSGGTTYYADAVKGRFTISRDNGKNTVYLQMNSLKPEDTALYYCATHTNQGPAGFGSWGQGTQVTVSS

[0176] Sequence ID 2 QVQLVESGGGLVQPGGSLRLSCAASGSISSIYVMGWYRQAPGKQRELVADITRTGTINYLKSVMGRFAISRDNAKSTVYLQMNSLKPEDTAVYYCRVVASAAPGRFGSWGQGTQVTVSS

[0177] Sequence ID 3 QVQLVESGGGLAQPGGSLRLSCAASGSISSIYVMGWYRQAPGKQRELVADITRTGTINYLKSVMGRFAISRDNAKSTVYLQMNSLKPEDTAVYYCNIRLGYSLGDNYWGQGTQVTVSS

[0178] Sequence ID 4 QLQLVESGGGLVQPGGSLRLSCAASGSISSIYVMGWYRQAPGKQRELVADITRTGTINYLKSVMGRFAISRDNAKSTVYLQMNSLKPEDTAVYYCNIRLGYSLGDNYWGQGTQVTVSS

[0179] Sequence ID 5 QVQLQESGGGSVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSTINWNGGSAEYAEPVKGRFTISRDNAKNTVYLQMNSLKLEDTAVYYCAKDADLV WYNLSTGQGTQVTVSSAAAYPYDVPDYGSGIRGCRSTSGGGGSGGGGSGGGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0180] Sequence ID 6 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYVIGWFRQAPGKGREGVACIRIFDRHTYYADSVKGRFTISSDNSKNTVYLQMNSLRAEDTATYYCAAGSFFGCTRPEGDMDYFGQGTLVQVQSGIRGCRSTSGGGGSGGGGSGGGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0181] Sequence ID 7 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKGREGVACIRIFDRHTYYADSVKGRFTISSDNSKNTVYLQMNSLRAEDTATYYCAAGSFWGCTRPEGDMDYFGQGTLVQVQSGASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAKVVVISAILALVVLTIISLIILIMLWQKKPR

[0182] Sequence ID 8 EVQLVESGGGLVQPGGSLRLSCAASGFTFEDYAIGWFRQAPGKGREGVACIRIFDRHTYYADSVKGRFTISSDNSKNTVYLQMNSLRAEDTATYYCAAGSFFGCTRPEGDMDYFGQGTLVQVQSGASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAKVVVISAILALVVLTIISLIILIMLWQKKPR

[0183] Sequence ID 9 EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0184] Sequence ID 10 EVQLQQSGPELVKPGASVKMSCKASGYKFTSYVMHWVKQKPGQGLEWIGYINPYNDVTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVHYCARGSYYDYDGFVYWGQGTLVTVSA GGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSATSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0185] Sequence ID 11; Synthetic promoter #17: Enhancer 017 - Proximal promoter CD3d

[0186] [Table 1]

[0187] Sequence ID 12; Synthetic promoter #19: Enhancer 019 - Proximal promoter CD3d

[0188] [Table 2]

[0189] Sequence ID 13; Synthetic promoter #21: Enhancer 021 - Proximal promoter CD3d

[0190] [Table 3]

[0191] Sequence ID 14; Synthetic promoter #22: Enhancer 022 - Proximal promoter CD3d

[0192] [Table 4]

[0193] Sequence ID 15; Synthetic promoter #23: Enhancer 023 - Proximal promoter CD3d

[0194] [Table 5]

[0195] Sequence ID 16; Synthetic promoter #24: Enhancer 024 - Proximal promoter CD3d

[0196] [Table 6]

[0197] Sequence ID 17; Synthetic promoter #27: Enhancer 027 - Proximal promoter CD3d

[0198] [Table 7]

[0199] Sequence ID 18; Synthetic promoter #28: Enhancer 028 - Proximal promoter CD3d

[0200] [Table 8]

[0201] Sequence ID 19; Synthetic promoter #29: Enhancer 029 - Proximal promoter CD3d

[0202] [Table 9]

[0203] Sequence ID 20; Synthetic promoter #30: Enhancer 030 - Proximal promoter CD3d

[0204] [Table 10]

[0205] Sequence ID 21 PDGFR short stem AVGQDTQEVIVVPHSLPFK

[0206] Sequence ID 22 GAPGAS GAPGAS

[0207] Sequence ID 23 GAPG4SAS GAPGSGGGGSGGGGSAS

[0208] Sequence ID No. 24: Tetramer Coiled Coil ASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAG

[0209] Sequence ID 25 IgG4 Hinge ASESKYGPPCPPCP

[0210] Sequence ID 26 CD8a Hinge PAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

[0211] Sequence ID 27 PDGFR TM VVVISAILALVVLTIISLIILIMLWQKKPR

[0212] Sequence ID 28 B7-1 TM WAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV

[0213] Sequence ID 29 IgGk leader OKT3 Vh linker OKT3 Vl linker PDGFR Tm METDTLLLWVLLLWVPGSTGDQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSP AIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0214] Sequence ID 30 1G4 LY TCRα chain METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQN PDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0215] Sequence ID 31 Y2-1G4 LY TCRβ chain MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISH TQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0216] Sequence ID 32 LVGP1x Y150A MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSDA HKKNLYDHALMSIISTFHLSIPNFNQAEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLS QRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSKY WYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLNHMGICSCGLYKQPGVPVKWKR*

[0217] Sequence ID 33: LDV containing an undefined SP MWIITALICSFSINPTCLYPHGHEDSPTVRHGISRVLSGDAERNDDEHYHSPPLVLPLQNERTWKPANLSSLKCPEASHLGPDEHRVMEKWLVHRPKSSVLTKVEGSLCHKSRWLTRCEYTWYFSKTVSR KIEPMPPTKQECEEAIKRKEEGLLESLGFPPPACYWARTNDEENVQVDVTDHPMTYDPYSDGVVDNILVGGKCNQRECETVHDSTIWLETQKEKRPSQCEMDVEEQLELVSGIKRVGGSKSKAQRSVFVVG TNYPFMDATGACRLKYCSKSGMLLSNGLWFHITRKISPESNENSKFWLTLSDCSSDKQVGVLGEEYEIGKLQATMEDIMWDLDCFRTLEDLSHHKKVSMLDLFRLSRLTPGTGPAYKLVKGNLMVKEVQYV KAQRDQGELANPLCVAFMTESKNADRCIRYDEYDKEGPYKGQVMNGILINEGMVVFPHERFHLRQWDPEFIIKHEIKQVHHPVLGNYSSQIHDSLHESLIKDHSANLGDVMGNWVQVATSKFSWFFKEIEK

[0218] Sequence ID 34 VHHTCR2 (IgG4 scaffold) TCR Nb Sequence ID 9 + SP + scaffold METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSSASESKYGPPCPPCPAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0219] BCMA CAR J&J containing sequence number 35 SP, but without V5 tag. MALPVTALLLPLALLLHAARPQVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMSIRPDYWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0220] CD19 CAR FMC63 that contains SEQ ID NO: 36 SP and does not contain the V5 tag MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0221] Sequence number 37 Y150A H141A F147A LVGP

[0222]

Table 11

[0223] Sequence number 38 VSVG A51E

[0224]

Table 12

[0225] Sequence number 39 VSVG I182E

[0226]

Table 13

[0227] Sequence ID 40 VSVG I331E

[0228] [Table 14]

[0229] Sequence ID 41 scFV CD14, no spacer, no TM domain, includes SP, VH, VL, and linker.

[0230] [Table 15]

[0231] Sequence ID 42 modified gag sequence LQSRPEPTAPPEEDPAVDLLKNYPSQKQEPIDKELYPLASLRSLFGSDPSSQ

[0232] Sequence ID 43 Vpx SIV MSDPRERIPPGNSGEETIGEAFEWLNRTVEEINREAVNHLPRELIFQVWQRSWEYWHDEQGMSPSYVKYRYLCLIQKALFMHCKKGCRCLGEGHGAGGWRPGPPPPPPPGLA

[0233] Sequence ID 44 PDGFR TM + PDGFR Short-Pedestal Scaffolding TCR1 Nb METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0234] Sequence ID 45 PDGFR TM+CD8 TCR3 Nb containing stem scaffold METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSSPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIVVVISAILALVVLTIISLIILIMLWQKKPR

[0235] TCR4 Nb containing SEQ ID NO: 46 PDGFR TM + tetramer coil scaffold METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSSASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAGVVVISAILALVVLTIISLIILIMLWQKKPR

[0236] SEQ ID NO: 47 VSVG mutants K47A, R354A MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSSDLNWHNDLIGTALQVKMPASHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPP QSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEM ADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVE IGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0237] Sequence ID 48: Complete scFv TCR1 array including scaffolding and V5 tags MEWSWIFLFLLSGTAGVHSGKPIPNPLLGLDSTEVQLQQSGPELVKPGASVKMSCKASGYKFTSYVMHWVKQKPGQGLEWIGYINPYNDVTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVHYCARGSYYDYDGFVYWGQGTLVTVSAGGGGSGGG GSGGGGSQIVLTQSPAIMSASPGEKVTMTCSATSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0238] Sequence ID 49: Complete scFv TCR2 array including scaffolding and V5 tags. MEWSWIFLFLLSGTAGVHSGKPIPNPLLGLDSTEVQLQQSGPELVKPGASVKMSCKASGYKFTSYVMHWVKQKPGQGLEWIGYINPYNDVTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVHYCARGSYYDYDGFVYWGQGTLVTVSAGGGGSGGGGSGGGS QIVLTQSPAIMSASPGEKVTMTCSATSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASESKYGPPCPPCPAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0239] Sequence ID 50: IgK Leader_CD86 IgV_IgG4 Hinge_PDGFR TM METDTLLLWVLLLWVPGSTGDAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLSASESKYGPPCPPCPVVVISAILALVVLTIISLIILIMLWQKKPR

[0240] Sequence ID 51: IgK Leader_TGN1412 scFv_IgG4 Hinge_PDGFR TM METDTLLLWVLLLWVPGSTGDQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSGGGGGSGGGGSGGG GSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKSASESKYGPPCPPCPVVVISAILALVVLTIISLIILIMLWQKKPR

[0241] Sequence ID 52: IgK Leader_TCR-VHH_CD8a Hinge_PDGFR TM METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPE DTAVYFCRAYSRIYPYNYWGQGTLVTVSSGASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAKVVVISAILALVVLTIISLIILIMLWQKKPR

[0242] Sequence ID 53: CD8a Leader_hu8e5 scFv_CD8a Hinge_CD28 TM_CD28 ICD_CD3z ICD(CARsgen CLDN18.2CAR) MALPVTALLLPLALLLHAARPQVQLQESGPGLIKPSQTLSLTCTVSGGSISSGYNWHWIRQPPGKGLEWIGYIHYTGSTNYNPALRSRVTISVDTSKNQFSLKLSSVTAADTAIYYCARIYNGNSFYWGQGTTVTVSSGGGGSGGGGSDGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLFNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFLTISSLQAEDVAVYY CQNAYSFPYTFGGGTKLEIKRTTTPAPPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLHSDYMNMTPRRPPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0243] sequence number 54:TCR_VHH-B1056 EVQLVESGGGLVQPGGSLRLSCAASGDVHHINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRADSRIYPYNYWGQGTLVTVSS

[0244] sequence number 55:TCR_VHH-B571 EVQLVESGGGLVQPGGSLRLSCAASGDVSKINILGWYRQAPAKEREMVAHITIGDITDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRALSRIYPYNYWGQGTLVTVSS

[0245] sequence number 56:TCR_VHH-B3359 EVQLVESGGGLVQPGGSLRLSCAASGDVLKINILGWYRQAPAKEREMVAHITIGDAEDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRALSRIYPYNYWGQGTLVTVSS

[0246] Sequence ID 57: TCR_VHH-B162 EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITSGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0247] Sequence ID 58: TCR_VHH-B197 EVQLVESGGGLVQPGGSLRLSCAASGDVHVINILGWYRQAPAKEREMVAHITIGDKTDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0248] Sequence ID 59: TCR_VHH-B3710 EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITYGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCVAYSRIYPYNYWGQGTLVTVSS

[0249] Sequence ID 60: TCR_VHH-B6251 EVQLVESGGGLVQPGGSLRLSCAASGDVRKINILGWYRQAPAKEREMVAHIHIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCTAYSRIYPYNYWGQGTLVTVSS

[0250] Sequence ID 61: TCR_VHH-B182 EVQLVESGGGLVQPGGSLRLSCAASLDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0251] Sequence ID 62: TCR_VHH-B341 EVQLVESGGGLVQPGGSLRLSCAASGDVHRINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0252] Sequence ID 63: TCR_VHH-B291 EVQLVESGGGLVQPGGSLRLSCAASGDVPKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

[0253] Sequence ID 64: TCR_VHH-B863 EVQLVESGGGLVQPGGSLRLSCAASGDVHVINILGWYRQAPAKEREMVAHITIGEATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCSAYSRIYPYNYWGQGTLVTVSS

[0254] Sequence ID 65: TCR_VHH-B2598 EVQLVESGGGLVQPGGSLRLSCAASGDVHTINILGWYRQAPAKEREMVAHITIGDAPDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRLYPYNYWGQGTLVTVSS

[0255] Sequence ID 66; Nanobody (VHH) CD14 3RMB85

[0256] [Table 16]

[0257] Sequence ID 67; Nanobody (VHH) CD14 3RMB96

[0258] [Table 17]

[0259] Sequence ID 68; Nanobody (VHH) CD14 3RMB138

[0260] [Table 18]

[0261] Sequence ID 69 Nanobody (VHH) CD14 3RMB187

[0262] [Table 19]

[0263] Sequence ID 70; Nanobody (VHH) CD14 4EBT28

[0264] [Table 20]

[0265] Sequence ID 71; Nanobody (VHH) CD14 4EBT40

[0266] [Table 21]

[0267] Sequence ID 72; Nanobody (VHH) CD14 4EBT85

[0268] [Table 22]

[0269] Sequence ID 73; Nanobody (VHH) CD14 2EBT32

[0270] [Table 23]

[0271] Sequence ID 74; Nanobody (VHH) CD14 2EBT51

[0272] [Table 24]

[0273] Sequence ID 75; Nanobody (VHH) CD14 2EBT57

[0274] [Table 25]

[0275] Sequence ID 76; Nanobody (VHH) CD14 3RMB1

[0276] [Table 26]

[0277] Sequence ID 77; Nanobody (VHH) CD14 3RMB1-2-6

[0278] [Table 27]

[0279] Sequence ID 78; Nanobody (VHH) CD14 3RMB1-2-2

[0280] [Table 28]

[0281] Sequence ID 79; Nanobody (VHH) CD14 3RMB1-2-9

[0282] [Table 29]

[0283] Sequence ID 80; Nanobody (VHH) CD14 3RMB1-2-5

[0284] [Table 30]

[0285] Sequence ID 81; Nanobody (VHH) CD14 3RMB1-2-3

[0286] [Table 31]

[0287] Sequence ID 82; Nanobody (VHH) CD14 3RMB1-2-19

[0288] [Table 32]

[0289] Sequence ID 83; Nanobody (VHH) CD14 3RMB1-2-7

[0290] [Table 33]

[0291] Sequence ID 84; Nanobody (VHH) CD14 3RMB1-2-25

[0292] [Table 34]

[0293] Sequence ID 85; Nanobody (VHH) CD14 3RMB1-2-18

[0294] [Table 35]

[0295] Sequence ID 86; Nanobody (VHH)CD14 3RMB1-2-12

[0296] [Table 36]

[0297] Sequence ID 87; Nanobody (VHH) CD14 3RMB1-2-15

[0298] [Table 37]

[0299] Sequence ID 88; Nanobody (VHH) CD14 3RMB1-2-16

[0300] [Table 38]

[0301] Sequence ID 89; Nanobody (VHH) CD14 3RMB1-2-20

[0302] [Table 39]

[0303] Sequence ID 90; Nanobody (VHH) CD14 3RMB1-1-16

[0304] [Table 40]

[0305] Sequence ID 91; Nanobody (VHH) CD14 3RMB1-1-28

[0306] [Table 41]

[0307] Sequence ID 92; Nanobody (VHH) CD14 3RMB1-1-9

[0308] [Table 42]

[0309] Sequence ID 93; Nanobody (VHH) CD14 3RMB1-1-27

[0310] [Table 43]

[0311] Sequence ID 94; Nanobody (VHH) CD14 3RMB1-1-13

[0312] [Table 44]

[0313] Sequence ID 95; Nanobody (VHH) CD14 3RMB1-1-5

[0314] [Table 45]

[0315] Sequence ID 96; Nanobody (VHH) CD14 3RMB1-1-6

[0316] [Table 46]

[0317] Sequence ID 97; Nanobody (VHH) CD14 3RMB1-1-24

[0318] [Table 47]

[0319] Sequence ID 98; Nanobody (VHH) CD14 3RMB1-1-2

[0320] [Table 48]

[0321] Sequence ID 99; Nanobody (VHH) CD14 3RMB1-1-4

[0322] [Table 49]

[0323] Sequence ID 100; Nanobody (VHH) CD14 3RMB1-1-29

[0324] [Table 50]

[0325] Sequence ID 101; Nanobody (VHH) CD14 3RMB32

[0326] [Table 51]

[0327] Sequence ID 102; Nanobody (VHH) CD14 3RMB25

[0328] [Table 52]

[0329] Sequence ID 103; Nanobody (VHH) CD14 3RMB71

[0330] [Table 53]

[0331] Sequence ID 104; Nanobody (VHH) CD14 3RMB142

[0332] [Table 54]

[0333] Sequence ID 105; Nanobody (VHH) CD14 3RMB62

[0334] [Table 55]

[0335] Sequence ID 106; Nanobody (VHH) CD14 3RMB107

[0336] [Table 56]

[0337] Sequence ID 107; Nanobody (VHH) CD14 3RMB126

[0338] [Table 57]

[0339] Sequence ID 108; Nanobody (VHH) CD14 3RMB76

[0340] [Table 58]

[0341] Sequence ID 109; Nanobody (VHH) CD14 3RMB134

[0342] [Table 59]

[0343] Sequence ID 110; Nanobody (VHH) CD14 3RMB50

[0344] [Table 60]

[0345] Sequence ID 111; Nanobody (VHH) CD14 3RMB18

[0346] [Table 61]

[0347] Sequence ID 112; Nanobody (VHH) CD14 3EBT162

[0348] [Table 62]

[0349] Sequence ID 113; Nanobody (VHH) CD14 2EBT91

[0350] [Table 63]

[0351] Sequence ID 114; Nanobody (VHH) CD14 2EBT9

[0352] [Table 64]

[0353] Sequence ID 115; Nanobody (VHH) CD14 2EBT95

[0354] [Table 65]

[0355] Sequence ID 116; Nanobody (VHH) CD14 3EBT10

[0356] [Table 66]

[0357] Sequence ID 117; Nanobody (VHH)CCR2 3RNA74

[0358] [Table 67]

[0359] Sequence ID 118; Nanobody (VHH)CCR2 2RRNA31

[0360] [Table 68]

[0361] Sequence ID 119; Nanobody (VHH) CCR2 3RSTM31

[0362] [Table 69]

[0363] Sequence ID 120; Nanobody (VHH) CCR2 3RZK33

[0364] [Table 70]

[0365] Accession number 121; Synthetic promoter #11: Enhancer 033 - proximal promoter CD3d gggaagatgtgagacaaacatcacaattttgcctaaggtgaatccaacccacaagtagagcacaggccaacagcagctcactagtacacatacttacaccagcagctcactagtacacacacttacaccagacgctcactggtacacactcacaccggcattcatgcttacccacgggctggtcaacaaagaggtgctgacctgagagtagggcacataacctcagccactggggtacacttaccacccccgcccccgtgtagctccctcccctatcctgaaatctcccttagcacactaagtattctaggttaaacagcccagatgttcagggagttcattcgccaggtgggaagtaatgagatgtaaatcggggtcttttaggtaatagctgacaactggggtaggaatgggggttctgagcccagtgctggctgccgggctgtttatctctgagtcacatcagcaccaagccacagcagccacctgccctagctccatctcttacagtcacaacaggatgtggtttgacatttactgggtcctgcatctggggtgcctgtgaaagttgctccctccacccacccaccttcagagcatcatgagaaccacactcaccgcatccggcacccaaccccctcctatgctgctctttctacctgggcccctggttagcatcctggcagtagaaatagaatttacaccgcttccttcctaccctacccctagcccacccccactctgaaaatttcccaccatcaacggcagaaagcagagaagcagacatcttctagttcctcccccactctcctctttccggtacctgtgagtcagctaggggagggcagctctcacccaggctgatagttcggtgacctggctttatctactggatgagttccgctgggag

[0366] Accession number 122; Synthetic promoter #12: Enhancer 034 - proximal promoter CD3d Gggaagatgtgagacaaacatcacaattttgcctaaggtgaatccaacccacaagtagagcacaggccaacagcagctcactagtacacatacttacaccagcagctcactagtacacacacttacaccagacgctcactggtacacactcacaccggtgggaagtaatgagatgtaaatcggggtcttttaggtaatagctgacaactggggtaggaatgggggttctgagcccagtgctggctgccgggctgtttatctctgagtcacatcagcaccaagccacagcagccacctgccctagctccatctcttacagtcacaacaggatgtggtttgacatttactgggtcctgcatctggggtgcctgtgaaagttgctccctccacccacccaccttcagagcatcatgagaaccacactcaccgcatccggcacccaaccccctcctatgctgctctttctacctgggcccctggttagcatcctggcagtagaaatagaatttacaccgcttccttcctaccctacccctagcccacccccactctgaaaatttcccaccatcaacggcagaaagcagagaagcagacatcttctagttcctcccccactctcctctttccggtacctgtgagtcagctaggggagggcagctctcacccaggctgatagttcggtgacctggctttatctactggatgagttccgctgggag

[0367] Accession number 123; Synthetic promoter #13: Enhancer 035 - proximal promoter CD3d ggtgggaagtaatgagatgtaaatcggggtcttttaggtaatagctgacaactggggtaggaatgggggttctgagcccagtgctggctgccgggctgtttatctctgagtcacatcagcaccaagccacagcagccacctgccctagctccatctcttacagtcacaacaggatgtggtttgacatttactgggtcctgcatctggggtgcctgtgaaagttgctccctccacccacccaccttcagagcatcatgagaaccacactcaccgcatccggcacccaaccccctcctatgctgctctttctacctgggcccctggttagcatcctggcagtagaaatagaagggaagatgtgagacaaacatcacaattttgcctaaggtgaatccaacccacaagtagagcacaggccaacagcagctcactagtacacatacttacaccagcagctcactagtacacacacttacaccagacgctcactggtacacactcacacctttacaccgcttccttcctaccctacccctagcccacccccactctgaaaatttcccaccatcaacggcagaaagcagagaagcagacatcttctagttcctcccccactctcctctttccggtacctgtgagtcagctaggggagggcagctctcacccaggctgatagttcggtgacctggctttatctactggatgagttccgctgggag

[0368] SEQ ID NO: 124; Synthetic Promoter #14: Enhancer 037 - proximal promoter CD3d aaataaacaaggagatagggtgtttattttatggacaagtttcttttgtaacttgtaactcccttgaaagtcagccagagtatgtctcaaaccaaagtcaagatagtgagcaagaagtgttgcacttatgaagggaggtgagtgagcaatgcatgtggtttccaaccgttaatgctagagttatcactttctgttatcaagtggcttcagctatgcaaggaaaccaaacaggggaagttttctcaagcaggttgaaagcaggttccaagaaagccctttaaataaacaaggagatagggtgtttattttatggacaagtttcttttgtaacttgtaactcccttgaaagtcagccagagtatgtctcaaaccaaagtcaagatagtgagcaagaagtgttgcacttatgaagggaggtgagtgagcaatgcatgtggtttccaaccgttaatgctagagttatcactttctgttatcaagtggcttcagctatgcaaggaaaccaaacaggggaagttttctcaagcaggttgaaagcaggttccaagaaagccctttagaaatctagctgctacggcttgcgctatggggccgacggcttctctcaaggggcttcgagatgtggcagtgtttaggttgtgtgtaaatgtggttgcattgtcaatagggacgctaaagttcaggccaccttttccatattctctgccagctccctgctcagagatagagcaatttacaccgcttccttcctaccctacccctagcccacccccactctgaaaatttcccaccatcaacggcagaaagcagagaagcagacatcttctagttcctcccccactctcctctttccggtacctgtgagtcagctaggggagggcagctctcacccaggctgatagttcggtgacctggctttatctactggatgagttccgctgggag

[0369] Sequence ID 125; Synthetic promoter #15: Proximal promoter CD5 Cccacccagacccctgcctcagggacgcctgtcctcagcccagccctcagctgcagccaggccttcagcctccgtaacccccgctcagggtccccaccccctgcagc cctgtccctccaggatgcatggccttgtcctgtgtgggggtggccgagagcactgccccagccctgggtaccttgggcaggaagctggcagaggccagggctgccatt caaacaggggcaggtggttttgccaggaggaagttgacagttcaacttcaaacatgggtgacgcaggccccacactgcctgctccccgtcccacccctccctgagca cgccacccccgccctctccctctctgagagcgagatacccggccagacaccctcacctgcggtgcccagctgcccaggctgaggcaagagaaggccagaaaccatgccc

[0370] Sequence ID 126; Synthetic promoter #16: Proximal promoter C8a cctattcacgggccccagcctcctcgccgggctggaaggcgacaaccgcgaaaaggagggtgactctcctcggcgggggcttcgggtgacatcacatcctccaaatgcgaaat caggctccgggccggccgaagggcgcaactttcccccctcggcgccccaccggctcccgcgcgcctcccctcgcgcccgagcttcgagccaagcagcgtcctggggagcgcgtc

[0371] Sequence ID 127; Synthetic promoter #17: Enhancer 023-NFAT RE-Proximal promoter CD3d ggcattcatgcttacccacgggctggtcaacaaagaggtgctgacctgagagtagggcacataacctcagccactggggtacacttaccacccccgcccccgtgtagctccctcccctatcctgaaatctcccttagcacactaagtattctaggttaaacagcccagatgttcagggagttcattcgccaggtgggaagtaatgagatgtaaatcggggtcttttaggtaatagctgacaactggggtaggaatgggggttctgagcccagtgctggctgccgggctgtttatctctgagtcacatcagcaccaagccacagcagccacctgccctagctccatctcttacagtcacaacaggatgtggtttgacatttactgggtcctgcatctggggtgcctgtgaaagttgctccctccacccacccaccttcagagcatcatgagaaccacactcaccgcatccggcacccaaccccctcctatgctgctctttctacctgggcccctggttagcatcctggcagtagaaatagaaggaaagggaaatagggaaagggaaacagggaaagggaaaagaaatctagctgctacggcttgcgctatggggccgacggcttctctcaaggggcttcgagatgtggcagtgtttaggttgtgtgtaaatgtggttgcattgtcaatagggacgctaaagttcaggccaccttttccatattctctgccagctccctgctcagagatagagcaatttacaccgcttccttcctaccctacccctagcccacccccactctgaaaatttcccaccatcaacggcagaaagcagagaagcagacatcttctagttcctcccccactctcctctttccggtacctgtgagtcagctaggggagggcagctctcacccaggctgatagttcggtgacctggctttatctactggatgagttccgctgggag

[0372] Sequence ID 128; hsa-mir-582-5p: AGTAACTGGTTGAACAACTGTAA A triple repeat sequence located at the 3' end of the transgene or the 3' end of the WPRE.

[0373] Sequence ID 129; hsa-mir-5196-3p CTGGGAGGGAGACGAGGATGA A triple repeat sequence located at the 3' end of the transgene.

[0374] Sequence ID 130; Synthetic promoter #1: Proximal promoter CCL13 Ggcttcccatggtgaatggctggggcgcgtctgtgtccctttctcctctctggctccttgtggcctgaacagccagaaggaagccatgccatgctgtttcagccctcagcttccctcttgcatttcctagaaaagtctttggtgcccagctccagctcagca gattcaggatcccccttcatcatgacttggtcaacgccctgctcaggccaaggtcctctgagagttccaagcttctccactccctataaaaggccggcggaacagccagaggagcagagaggcaaagaaacattgtgaaatctccaactcttaaccttcaac

[0375] Sequence ID 131; Proximal promoter CCL18 ggggaagatttctcattagatcacgaaaggcctaactgtatataaaagattgaagcatgtgactctcttaaaaggaaaaacaattgtgaaaatccattaaagcattctgctctatcaaaggaggctgtgaccactcatttctgagaaatatctgtcatgtgaaaacataatcacatgattatgcataatcctaagcataatccctgggtgcttccaactcctggtcacccatctggagatacaacttctccataagcccctcaatcaacaagtgaaccactcgtttgtgtaccctaaacctttcaagtaaccataggcaaccctgggctgagaactcacatgacaccaccatagttaaaatgtcttgcatcagcacctccaatgttcccatggtccagtctgcctataaaaaggagagacaacagctcataccccagaaggaggccaggagttgtgagtttccaagccccagctcactctgaccacttctctgcctgcccagcatc

[0376] SEQ ID NO: 132; Synthetic Promoter #3: proximal promoter MRC1 CccaggggaagctccgtcttccggcacaggtaatggcctgcagcttgatctccacccagccccatctgagcaggccgggagctcccaggctgtttcacttcTctccttcctgactcctcaccatcaccatcgccctctctcctccccaccccgccactcctctcccacacgtgtccctttctccccttcctctgcgtctgctcttctcagaagttagcttacgaagcaaagttgttactttgaattcctgtttttccagccaccctcatgtgacaggatgtctcctcagtagaggctttccctaaattcaggagccctttaaaagggagggcttcctctgtagttcttttcagctgggcagctctgggaacttggattaggtggagaggcagttggggggcctcgttgttttgcgtcttagttccgccctcctgtccatcaggagaaggaaaggataaaccctgggcc

[0377] SEQ ID NO: 133; Synthetic Promoter #4: proximal promoter LYZ catacaaaacaatttcacaaaattgaatgttactcaaaaatcacagctcattttaagctgcacaaaatagtcatttttttctttataattgctcaaattcataatcaaacagaagaaagttcctgtcttggaagtagtgctatgccccaattcttccagagccagtactttaaacaattccatttcattattttcctgtagactaattcttaggacatcagcatatctctcttcaagcattaaaaaaatctctttagagtcagtggatcaatagacagttcctgttttccacacaactgaaagggtggagcccccaaaccacaaggggaagaaggaagttaaaagatgttaaatactggggccagctcaccctggtcagcctagcactctgacctagcagtcaac

[0378] Sequence ID 134; Synthetic promoter #5: Proximal promoter HLA-DR Gcaaactctccaactgtcattgcacagacatatgatctgtatttagctctcactttaggtgtttccattgattctattctcactaatgtgcttcaggtatatccctgtc tagaagtcagattggggttaaagagtctgtccgtgattgactaacagtcttaaatacttgatttgttgttgttgttgtcctgtttgtttaagaactttacttctttatcc aatgaacggagtatcttgtgtcctggaccctttgcaagaacccttcccctagcaacagatgcgtcatctcaaaatatttttctgattggccaaagagtaattgatttgc attttaatggtcagactctattacaccccacattctcttttcttttattcttgtctgttctgcctcactcccgagctctctgactcccaacagagcgcccaagaagaaa

[0379] Sequence ID 135; Synthetic promoter #6: Enhancer 008 - Proximal promoter CHIT1 Aggactgagctctgagatccttctgaagaggttgggagaagaggagaatcagcaagagaaactgagaaggagggtcatgggcggtaggcgggaagcaggagagtgtggggtcagaggacagatgtagaaatcagttccaggaagcgagggccgagtgtcagaagctgctgctgagtcactcaagatgaagacagaactcatcactggagtcactcaggcagccttggtgaccttgataaaaatagtttaggtggagtaggtgccgggttggagcacaggcttggttggaatggggttaagggaggatgtgagcagtagaacagggtgcagccaacacagacaactactttggagaagttttgcaaaagggaagtaaaaaatgggcagtggttgatgaggagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0380] SEQ ID NO: 136; Synthetic Promoter #7: Enhancer 015 - proximal promoter CHIT1 Atgtccttcccaggggtgaaagccccctgcctgctgggttcctgccaagactcagctcaaacactaggatgctttggggccccaaggacccagtcacgggactgccccacccgcttcccctctgcggtattaatacgcctcttgctcacttcgggggaaaaacagaaaattgtgcggatgtgggccccagatctgtgggtgtcggttcttctttcagatttgggatcctctgcatgttgggggaggccgcgtctcgctcctcgtttgacctaggtcagaattcaacaggaaggagtttagggtgtccagcaacaacaaccctcaaccatctccagctgagctctcatcccagccccggccctgaccgtaacccgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0381] SEQ ID NO: 137; Synthetic Promoter #8: Enhancer 022 - proximal promoter CHIT1 Tagtctcaaaatgcctttcagcataggcaggaaatatgacagaagaaagtcccagtcgaacaaacaataaccgaaaatgctctttgttgctgggccttggcgatcaaagccctgcacatgtttgccagctgtagttcagatggtttctatttcactcagcattaattttttatgaaatctgtggcccaggggcccataagcggcttttagttcttcattctatactgtctggtctaattgccaaggcatggtaagactcagattttaaggtgtcgttaatgtatttaccagccttccggtctacaggcaggctcacctctttgtgtgggattttaatgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0382] SEQ ID NO: 138; Synthetic Promoter #9: Enhancer 029 - proximal promoter CHIT1 agaaagagtgtacttattgaagatgaattgttttcttaatctagtattatcagcctgacagagttattcacaagtagttaagttcctgggaatcacatgagcagcagcaatcccaagaaacctgagtcagtaaaaaaataaattgctgatcttgaagagattcaaagaggaaggcaaaagcattgtcagctttgttcaaactgtatctcaaatttagttgttttttgtttttctatccgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0383] SEQ ID NO: 139; Synthetic Promoter #10: Enhancer 031 - proximal promoter CHIT1 taagaccctaaaagtcacactgtccttagacagttagggaagtagaatggataacatatgcccaaaatacgaatgactaatttccttgcaagatcagctgtctcctgcaagcaacatttgacaattcaccctttcctcgtgatgatgtcttaactaagaagatacaaaaatgcaaggaaatgaaagtgaatatgagatcacttttatgtgctattttaaaaacataaatgcacattgcactgtgtttagggaactatggcttcagaagagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0384] Accession No. 140; Synthetic Promoter #11: Enhancer 034 - proximal promoter CHIT1 aagctgtggcccagaacaccccgatcccactcagaacactatttataagaatgccccgtatgcagtagtctttacagtttttctttttctcaaatgttctttagcctatgtagatgacatttatgcaattcagaggaagatgcctcaacaggattagtcagcagcaaagtcctctgacgcagcttctgtgatctagttaaaaataggaaagaggatgttttgcataagtcatcaccattgtgcggtattggagaattgaccacttttttggttcccacatcctgctaatttatctaaaaatagatgaaaaccctgtccctgaattcctggacaagaccttttgcttctgtgacctcggggctcaggctcttcttgttctgggctccgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0385] SEQ ID NO: 141; Synthetic Promoter #12: TAM1 - proximal promoter CHIT1 agactctgttccctgtatttgcatccactgcctgcgtgctttctctctcccccatatttctgacatttggattttccccctactaattgacatgcctgtcgcctccttctcctggtgtccatttctggaactgctatttgtatattgcatgatattttatcaggaaaaaatataaaacagatctaatttcatgaaaatgctgttgccataatgctcaggagaacagggggaagagagagattcaagtcgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0386] Accession No. 142; Synthetic Promoter #13: TAM2 - proximal promoter CHIT1 agaaaagagttcacccaataagtttcccaaatttcaaggagcttttgttttattagatgctgttatcttcatgttagtcacaggaaaaaaaaatgcccacatacacacgcacaactaaatgtagtcagtttgctcagctgctgctgacgtcactgctgcccactaggagtgagttactgtcataacccctcttttattaatcataggctggaggaagcacaaagtgagtgacatcattaggcaaaggaccggctaactcatgctccaatttactcagctttattataacatttctcgagtgtcatatgaaagtgattaccatttcacactttccccatctgagcaatcatccctaaccttcaattcaaattcccagagaaaagcttagcttttcagatggaggtgcgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0387] Sequence number 143; Synthetic promoter #14: TAM3 - proximal promoter CHIT1 ttctttcagcaaaacctttcggagaagcccaatactaagctcctctggttagagccagccatgagagaaactccaagtacttctgactggttctctctctactcatccaccccttaggtggctgcagaaggaactctgtgcaacccccagagttctcattctcagtgacagggaaatgtaatgattggccctggatgattcagcagatcagatgatacttactcagagcaatttccactcctttgcagtagcatattatcagtattttccagataaataacttggctaaagaaaaatccatttcatttacatctttggcaccttacagcaatagaacttttgtgcaatgattttaatattatatttctacattggctgatagagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0388] SEQ ID NO: 144; Synthetic Promoter #15: TAM4 - proximal promoter CHIT1 gaaatcctcgaaatgagatactgtcccgtgattctgagagtgacccaaaaggatacggaatgaggtactaaaagcaggtgcttggaaacctgagggcagtggtgttcatgcgtttcccggcactttgactttcagcctcctgcaagcctctccttctttgccttcctgaccgttagatacaattaatttggggtttgttttttctgcttcctctttcccctcccaccctatgtttcagttctaaagttagaaggtcctctcatccttttaagaagaacagagacagaaaatggttagtcaccatctaggacagactacaattccagatcattgcaggacgtgcccactcaaatggatgattattgagagctggccacctgaagtgttttacacaggcagtgatctaagggttgaagctatctggcctttttgcttgcttgttatgtatttatggctgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0389] SEQ ID NO: 145; Synthetic Promoter #16: TAM5 - proximal promoter CHIT1 ccctagctctggggaaatgaaagccaggctggggttcaaatgagggcagtttcccttcctgtgggctgctgatggaacaaccccatgacgagaaggacccagcctccaagcggccacaccctgtgtgtctctttgtcctgccggcactgaggactcatccatctgcacagctggggcccctgggaggagagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0390] SEQ ID NO: 146; Synthetic Promoter #17: TAM6 - proximal promoter CHIT1

[0391] Array number 147; Synthetic promoter #18: TAM7 - proximal promoter CHIT1 aaggcggcgccagggagagggcgcagtctctgaatctttccacggggtccatcctagggcccctccaggttcaacggtctcctaacctgtagtcacccacaagagcgtgccctttctgcccgcccttcctagcgttgctccctgcttggctgagcactgcggagtttcacgcctctaaaccccgcctcttcttgcaacctgtgttggcctcatctacccagcaactgtcgacgtcacacgacctgggcctccctgaatgggagatatttactaggcaatcccgcctactgttctgaggtttcccctccaggtgcagcttcagagccaggcgagccaggaaggaccagcgggcgaggtggtgagttgtgaggcgcgcccagagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0392] Array number 148; Synthetic promoter #19: TAM8 - proximal promoter CHIT1 cttctgcccgacccgaggtacctccaccctgtctaccaggttcagccccgccctctcatcatgtattggcccccaaaacgcggctcttccctcccatcagtttgtctttccactctcattggtcctcaggacgaccgtgactccgcccacctacaccacatttccaccactatccctgacttccaatggccccgccccagccactaaggttcggccttttctgcccagctggccgcctcttccttggtctggtgtcccaggcaccgcccacgggtctagcctcttctcaggagtgctctacagcgccccctaggccaccaagattgtttagctccctgtcgggtcggcccctgactccttattggactcatccatctggctcatccaaggccttgggtctctccagctgactcgacggtgatggggggcaactcggcgggggagctgtggagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0393] SEQ ID NO: 149; Synthetic Promoter #20: TAM9 - proximal promoter CHIT1

[0394] Sequence ID 150; Synthetic promoter #21: TAM10-proximal promoter CHIT1 cctagctcttagtgtaatgattatgggaacagggtcaaatagcaaatcacaaattatttcacgactattgtgagacaagagtgagagccaagagtggtggtgcacacctgtaatcccagctcgaaccagggagacagaggttgcagtgagccgagatcgcaccaatggcttaacacaatgggatttggcttttcactcatgtaaatgaccagtgcggttttctctggggcagaatttgaattttctccccaggatgactcagggacacccgcttcctccattgtgctcccatcatgcccttgggcaatggcgtcccgtacttcaaacagcaagaaagaaggagaaacagaatatatctgtgcacttgcaaaatttgctgacacatgtcatttctgtccagaattgttggctataaatagtgttatgaacagtcacacaatgatgcaacaagaccagggaaaaagtcccttggtgggggtagccacttcccagaggttccaatgcttgcactgtaagggtaagtacagaattttttttttttctggtgagcagccagagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc。

[0395] Sequence ID 151; Synthetic promoter #22: TAM11-proximal promoter CHIT1 ttattactctatagataaagcagagtctctgaagttaactaacttccccaaagtcacacatcgtggaaccatgaatcttaccaggttctgtgtgactccaaatttgtgctctttcatattgcacaacactgtccagattaattcaagtggagagtgaactttttctggtacatcccttagtctcttgcacagccattgcctttcacacacccagactgtttctttgctttagggactggaaagtggaagtggaaccccctatgttctgtcctgggatatttccggggtgagccaagagtagctgtgtgtcctgctaggtcatggactttcactgaccacagaagttgagaacccttgagcaaactaaattcttatatcttactggtttgggagcaaagcagaagtggcaaaagctgacaaaacccagctggagcaggaatgttgaaatggacacaaataaacataccctgtggacacaatcaatataaatgttctaaatttacttctgctcctatgcttgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0396] Accession number 152; Synthetic promoter #23: TAM12 - proximal promoter CHIT1 Aaaaagtgttaaatcgtccaaaaaggaggaaagaaaggggaaaaaaagtaacaattctaaatctttatgtgcctgataacacggcttcaacacatcgaataaaatgtttacagaactgtaagtagaatcgcagcggaagatttcaacacaattcttttcatatggaaagaggaagaagcacgtaaaaatcacagaactacaagcacgtggagcatgttctctgatcacagtggaactaagcctgaaacctaaaacaaagagataaccagaaacacatatgtttgtaaattaagaaatgcatttctaaataactcctgggtcaaagagcagctcacagtggaaatcagaaagtattttgaactgaaagacgagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0397] Accession number 153: VSVg - Cl7 I52R, I331E, E355L KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKARQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTTERLLWDDWA

[0398] Sequence ID 154: VSVg-Cl12 N9Q, S48V, V333I, E353K KFTIVFPHQQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKVHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIAAPILSRMVGMISGTTTKRELWDDWA

[0399] Sequence ID 155: VSVg-Cl26 N9G, K47G, I331E, T352S KFTIVFPHGQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPGSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTSERELWDDWA

[0400] Sequence ID 156: VSVg-Cl47 H8F, A51P, R329V, T352V KFTIVFPFNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKPIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETVYIRVDIAAPILSRMVGMISGTTVERELWDDWA

[0401] Sequence ID 157: VSVg-Cl135 N9Q, K47T, T328S, E355L KFTIVFPHQQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPTSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFESRYIRVDIAAPILSRMVGMISGTTTERLLWDDWA

[0402] Sequence ID 158: VSVg-Cl129 N9G, K50S, V333I, T351R KFTIVFPHGQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHSAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIAAPILSRMVGMISGTRTERELWDDWA

[0403] Sequence ID 159: VSVg-Cl33 H8F, S48L, R329K KFTIVFPFNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKLHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETKYIRVDIAAPILSRMVGMISGTTTERELWDDWA

[0404] Sequence ID 160: VSVg-Cl59 H8Y, K47G KFTIVFPYNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPGSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWA

[0405] Sequence ID 161: VSVg-Cl145 N9H, S48R, T352E KFTIVFPHHQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKRHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTEERELWDDWA

[0406] Sequence ID 162: VSVg-Cl139 H49F, R329T, T350G KFTIVFPHGQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSFKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETTYIRVDIAAPILSRMVGMISGGTTERELWDDWA

[0407] Sequence ID 163: VSVg-Cl58 H8L, M45L, I331E, E355R KFTIVFPLNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKLPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTTERRLWDDWA

[0408] Sequence ID 164: VSVg-Cl87 K11E, S48F, Y330L, T352D KFTIVFPHNQEGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKFHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRLIRVDIAAPILSRMVGMISGTTDERELWDDWA

[0409] Sequence ID 165: VSVg-Cl386 N9D, A51S, I331T, E355H KFTIVFPHDQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKSIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYTRVDIAAPILSRMVGMISGTTTERHLWDDWA

[0410] Sequence ID 166: VSVg-Cl223 A51T, R329W KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKTIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETWYIRVDIAAPILSRMVGMISGTTTERELWDDWA

[0411] Sequence ID 167: VSVg-Cl363 Q53A, I331E, T352I KFTIVFPHNVKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIAADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTIERELWDDWA

[0412] Sequence ID 168: VSVg-Cl549 F6P, Q10K, K50T, V333P, T352R KFTIVRPHNKKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHTAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRPDIAAPILSRMVGMISGTTRERELWDDWA

[0413] Sequence ID 169: VSVg-Cl477 S48M, Y330P, T351D KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKMHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRPIRVDIAAPILSRMVGMISGTDTERELWDDWA

[0414] Sequence ID 170: VSVg-Cl164 N9T, I52T, R329K, T352E KFTIVFPHTQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKATQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETKYIRVDIAAPILSRMVGMISGTTEERELWDDWA

[0415] Sequence ID 171: VSVg-Cl167 K47V, D334A, R354D KFTIVIPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPVSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVAIAAPILSRMVGMISGTTTEDELWDDWA

[0416] Sequence ID 172: VSVg-Cl436 H8S, H49A, R329H, T352Q KFTIVFPSNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSAKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETHYIRVDIAAPILSRMVGMISGTTQERELWDDWA

[0417] Sequence ID 173: VSVg-Cl261 K11A, S48L, I331L, R354K KFTIVFPHNQAGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKLHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYLRVDIAAPILSRMVGMISGTTTEKELWDDWA

[0418] Sequence ID 174: VSVg-Cl194 P7W, M45F, V333Y, R354V KFTIVFWHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKFPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRYDIAAPILSRMVGMISGTTTEVELWDDWA

[0419] Sequence ID 175: VSVg-Cl1003 N9R, Q53I, I331Q, R354M KFTIVFPHRQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIIADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYQRVDIAAPILSRMVGMISGTTTEMELWDDWA

[0420] Sequence ID 176: VSVg-Cl368 H8S, I52V, V333Y, T351S KFTIVFPSNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAVQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRYDIAAPILSRMVGMISGTSTERELWDDWA

[0421] Sequence ID 177: VSVg-Cl6915 I331E, E353K KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTTKRELWDDWA

[0422] Sequence ID 178: VSVg-Cl125 Q10V, K50H, I331M, T352D KFTIVFPHNVKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHHAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYMRVDIAAPILSRMVGMISGTTDERELWDDWA

[0423] Sequence ID 179: VSVg-Cl331 N9A, I331E, E353S KFTIVFPHAQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYERVDIAAPILSRMVGMISGTTTSRELWDDWA

[0424] Sequence ID 180: VSVg-Cl86 H8G, A51S, E355W KFTIVFPGNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKSIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERWLWDDWA

[0425] Sequence ID 181: VSVg-Cl95 H8V, K47W, Y330M, T351D KFTIVFPVNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPWSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRMIRVDIAAPILSRMVGMISGTDTERELWDDWA

[0426] Sequence ID 182: VSVg-Cl M N9Q, A51P, R329V, E353K KFTIVFPHQQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKPIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSN LISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETVYIRVDIAAPILSRMVGMISGTTTKRELWDDWA

[0427] Sequence ID 183: DAF-GS METDTLLLWVLLLWVPGSTGDDCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAHLQIDN GIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGHLHECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSGGGAHHHHHHFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTTIEMNRLGK

[0428] sequence number 184:DAF-GPI METDTLLLWVLLLWVPGSTGDDCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSS FCLISGSSVQWSDPLPECREIYCPAHLQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGHLHECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTPNAQATRSPVSRTTKHFHETPNKGSGTTSGGGAHHHHHHGSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0429] SEQ ID NO:185:H-GS factor METDTLLLWVLLLWVPGSTGDEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSG YKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKGGAHHHHHHFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFILIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0430] Sequence ID 186: H-GPI factor METDTLLLWVLLLWVPGSTGDEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVT APENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKGGAHHHHHHSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0431] Sequence ID 187: Kaposica-GS METDTLLLWVLLLWVPGSTGDQDNEKCSQKTLIGYRLKMSRDGDIAVGETVELRCRSGYTTYARNITATCLQGGTWSEPTATCNKKSCPNPGEIQNGKVIFHGGQDALKYGANISYVCNEGYFLVGREYVRYCMIGASGQMAWSSSPPFCEKEKCHRPKIENGDFKPDKDYYEYNDAVHFECNEG YTLVGPHSIACAVNNTWTSNMPTCELAGCKFPSVTHGYPIQGFSLTYKHKQSVTFACNDGFVLRGSPTITCNVTEWDPPLPKCVLGGAHHHHHHFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0432] Sequence ID 188: Kaposica-GPI METDTLLLWVLLLWVPGSTGDQDNEKCSQKTLIGYRLKMSRDGDIAVGETVELRCRSGYTTYARNITATCLQGGTWSEPTATCNKKSCPNPGEIQNGKVIFHGGQDALKYGANISYVCNEGYFLVGREYVRYCMIGASGQMAWSSSPPFCEKEK CHRPKIENGDFKPDKDYYEYNDAVHFECNEGYTLVGPHSIACAVNNTWTSNMPTCELAGCKFPSVTHGYPIQGFSLTYKHKQSVTFACNDGFVLRGSPTITCNVTEWDPPLPKCVLGGAHHHHHHSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0433] Sequence ID 189: SPICE-GS METDTLLLWVLLLWVPGSTGDCCTIPSRPINMTFKNSVETDANANYNIGDTIEYLCLPGYRKQKMGPIYAKCTGTGWTLFNQCIKRRCPSPRDIDNGHLDIGGVDFGSSITYSCNSGYYLIGEYKSYCKLGSTGSMVWNKPAPICESVKCQLPPSISNGRHNGYNDFYTDGSVVTYSCNSGY SLIGNSGVLCSGGEWSNPPTCQIVKCPHPTILNGYLSSGFKRSYSYNDNVDFTCKYGYKLSGSSSSTCSPGNTWQPELPKCVRGGAHHHHHFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0434] sequence number 190:SPICE-GPI METDTLLLWVLLLWVPGSTGDCCTIPSRPINMTFKNSVETDANANYNIGDTIEYLCLPGYRKQKMGPIYAKCTGTGWTLFNQCIKRRCPSPRIDNGHLDIGGVDFGSSITYSCNSGYYLIGEYKSYCKLGSTGSMVWNPKAPICESVKCQLPPSISNGRHNGYNDFYTDGSVVTYSCNSGYSLIGNSGVLCSGGEWSNPTCQIVKCPHPTILNGYLSSGFKRSYSYNDNVDFTCKYGYKLSGSSSSTCSGPNTWQPELPKCVRGGAHHHHHHSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0435] sequence number 191:ABD-GS METDTLLLWVLLLWVPGSTGDGHSLQAAQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGAHHHHHFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0436] Sequence ID 192: ABD-GPI METDTLLLWVLLLWVPGSTGDGHSLQAAQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGAHHHHHHNLENGGTSLSEKTVLLLVTPFLAAAWSLHP

[0437] SEQ ID NO: 193: M Synthetic Promoter #24: Enhancer 030 - Proximal Promoter CHIT1 agctagagattctgggggcccttcaaacttctttgggacatgtatgtgtaatttcctagtaaaagagttttgcctgcttcttcttctttctccccctggtgtctgcctgcagtactacaatctccctagtggtgaaatagtgtgcctgcctttgttctccgaggctcacaac atgctattcaactctgcactctgagtcaggcaagacagaaaccagtaccttgggtaaccccacaaaacccaggagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctatta agtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttg cccaattcatgctgcttgacatcttaccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0438] Sequence ID 194 H141A F147A LVGP

[0439] [Table 71]

[0440] Sequence ID 195 Y150A F446L LVGP MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSD AHKKNLYDHALMSIISTFHLSIPNFNQYEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLL SQRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSK YWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISILLHLVKIPTHRHIVGKSCPKPHRLNHMGICSCGLYKQPGVPVKWKR *

[0441] Sequence ID 196 LVGP WT MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSD AHKKNLYDHALMSIISTFHLSIPNFNQYEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLL SQRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSK YWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLNHMGICSCGLYKQPGVPVKWKR *

[0442] Sequence ID No. 197 Synthetic Promoter #25: Proximal Promoter CHIT1 Gagaaacagtttgagagagggcagtggagaagcaagagagctggtctggaacccagctgcctgactcgtgagtcatttctcttcctccaaagccctattaagtctggggctgcttatggtcctcaccaactacacaaagaaagtcagggtcatgctcatccatcaggaggttcagggccacttcccgctttcctctgtttggcaacactaagagtcaccctctgattgttccagaaaagcaagaattgctaaaggccattgtgtcaagtttgcccaattcatgctgcttgacatcttacccctccccaccctgctcccatcccccccacccagtgcccctgattgcaacactcctggctggggtgggacagggtggccagataaaagcagagcaggacctggaaagctggtttgtatgggctgcagcctgccgctgagctgcatc

[0443] SEQ ID NO: 198 hCD80trc273 MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRE

[0444] SEQ ID NO: 199 CD28 mu9.3 scFv EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTSVTVSSGGG GSGGGGSGGGGSDIELTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKR

[0445] Sequence ID 200 CD28 Scaffold (G4S) 3 Linker, Short PDGFR Stem, PDGFR™ and CT Cutting Type GGGGSGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0446] Sequence ID 201 4B1-4 on scaffolding METDTLLLWVLLLWVPGSTGDQVQLQQPGAELVKPGASVKLSCKASGYTFSSYWMHWVKQRPGQVLEWIGEINPGNGHTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARSFTTARGFAYWGQGTLVTVSAGGGGGSGGGGSGGG GSDIVMTQSPATQSVTPGDRVSLSCRASQTISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQDGHSFPPTFGGGTKLEIKSASESKYGPPCPPCPVVVISAILALVVLTIISLIILIMLWQKKPR

[0447] Sequence ID No. 202: Vesicular stomatitis virus (San Juan strain) glycoprotein MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPP QSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEM ADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVE IGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0448] SEQ ID NO: 203: Signal peptide-free vesicular stomatitis virus (San Juan strain) glycoprotein KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0449] Sequence ID 204; Nanobody (VHH) CD14 3RMB1-1-14

[0450] [Table 72] [Examples]

[0451] Example 1: Production of a stable cell line expressing a membrane-anchored single-domain antibody. This example describes the generation of a stable production cell line for the production of single-domain antibody (VHH) targeted lentivirus (VHH-eLV). For this purpose, 293T cells (e293T) that stably express human CD47 and lack B2 microglobulin expression were engineered to stably express membrane-anchored VHH targeting human CD3e (SEQ ID NO: 3), human CD8a (SEQ ID NO: 6), or TCRab (SEQ ID NO: 9).

[0452] Cell 293T was transduced 24 hours after seeding with a retrovirus derived from self-inactivated Moloney mouse leukemia virus (SIN gRV) or a self-inactivated lentiviral vector (LV) encoding either CD3e, CD8a, or TCRab VHH. After infection, the culture medium was changed and the cells were grown for 3 days. Upon harvesting, the cells were stained in PBS with a VHH-specific antibody (Jackson Immunoresearch) and sorted based on VHH expression levels using a fluorescently labeled cell sorter (BD FACS Aria®) or cloned by limiting dilution. The selected cells were grown and banked as polyclonal cell lines (CD3eVHH-e293T and CD8aVHH-e293T) or monoclonal cell lines (e.g., e293T B619 clone 38). These cell lines stably express human CD47 and membrane-anchored CD3e or CD8a, or TCRab VHH, and lack MHCI expression.

[0453] As shown in Figure 2A, a subpopulation of e293T cells transduced with membrane-anchored CD3e or CD8a nanobodies encoding gRV expresses either high levels of CD3e (left panel) or CD8a (right panel) membrane-anchored nanobodies. After cell selection (P3 gate in Figure 2A) and expansion of cells expressing the highest levels of VHH, Figure 2B shows that the selected polyclonal cell lines (CD3eVHH-e293T and CD8aVHH-e293T) express each VHH at stable and uniform levels. In different replicates of this example, increased expression of membrane-anchored TCRab VHH and CD47, and decreased MHCI expression were confirmed by flow cytometry in 38 e293T B619 clone cells compared to the wild-type cell line HEK293T (Figure 2C).

[0454] Example 2: Characterization of lymphocyte cell lines and primary peripheral blood mononuclear cells for cell surface expression of VHH targets This example describes the characterization of cell surface marker / receptor expression (e.g., CD3, CD4, CD8, and T cell receptor, or TCR) in human primary cells (i.e., human peripheral blood mononuclear cells or hPBMCs) or human T cell lines (Jurkat WT, clone E6-1; SupT1) to select cell types for testing and titration of specific embodiments of VHH-eLV.

[0455] Activated human PBMCs (hPBMCs) derived from healthy donors (cultured in IL2-containing growth medium supplemented with anti-CD3 and anti-CD28 antibodies), Jurkat WT, and SupT1 were stained with anti-human CD3 BV510 (BioLegend), anti-human CD4 APC-cyanine 7 (BioLegend), anti-human CD8a PerCP (BioLegend), anti-human TCR / b BV421 (BioLegend), and DAPI. Flow cytometry analysis was performed using CytoFLEX LX® (Beckman Coulter).

[0456] As shown in Figure 3A, activated hPBMCs stain positively for all four markers (top panel) and have two major cell populations: CD3+ CD4+ TCR+ and CD3+ CD8+ TCR+. Jurkat WT cells are CD3+ TCR+ but do not stain for CD8a, and a small subpopulation expresses low levels of the CD4 receptor (center panel). SupT1 cells are both CD4+ CD8+ but do not stain for CD3 or TCR (bottom panel). Jurkat cells engineered in-house to stably express hCD8a after lentiviral transduction with a vector encoding hCD8a (see Example 4) are CD3+ CD8+ TCR+ (Figure 3B).

[0457] In conclusion, primary human PBMCs, Jurkat WT, SUP-T1, and Jurkat CD8 cells provide distinct targeting cell populations that may be useful for selecting and characterizing VHH-eLV vectors that target a range of T cell surface markers, including CD3, CD4, CD8, or TCR.

[0458] Example 3: The combination of wild-type vesicular stomatitis virus envelope glycoprotein G and a nanobody targeting either CD3e or CD8a fails to redirect the VHH-targeted lentiviral vector to CD3+ or CD8+ T cells. Viral cell entry is an early and crucial step in the process of lentiviral cell transduction. This occurs through the fusion of the viral envelope with the target cell membrane. A range of viral proteins have evolved to catalyze this step in viral transduction, one of which is vesicular stomatitis virus glycoprotein G (VSVG), widely used to pseudotype lentiviral vectors. However, viral envelope glycoproteins also ensure viral targeting, which in the case of VSVG targets a wide range of cell types through binding to cell surface receptors, including LDLRs.

[0459] In this example, we investigated whether the broad tropism of VSVG pseudotyped lentiviruses can be redirected to single-cell types by using envelope-anchored nanobodies in combination with VSVG, while still benefiting from the membrane fusion properties of VSVG. For this purpose, CD3e or CD8a envelope-anchored nanobodies were used in combination with wild-type VSVG protein. The tropism of these combined vectors was then tested against both target and non-target cells.

[0460] VSVG pseudotype CD3e or CD8aVHH-eLV, or VSVG eLV, was generated by transient transfection of producing cells using a lentiviral transfection plasmid encoding an unstable GFP reporter gene and an envelope construct expressing wild-type VSVG. VHHCD3e, VHHCD8a, or wild-type e293T cells (see Example 1) were transiently transfected 24 hours after seeding in culture medium. Transient transfection was performed using PEIpro transfection reagent (58 ug, Invitrogen) and a mixture of lentiviral packaging plasmids (12 ug of transfection plasmid, 5.5 ug of VSVG envelope plasmid, 5.5 ug of Gag / Pol packaging plasmid, and 6 ug of Rev expression plasmid). PEIpro / plasmid DNA mixture was added to cells, and 72 hours after transfection (cells were cultured at 37°C in 5% CO2), the supernatant containing the lentivirus was collected, clarified at 500g for 10 minutes, and filtered through a 0.2mm polyethersulfone filter (Millipore). 5X Lenticoncentrator (Origene) was added to the clarified supernatant, and the mixture was stored at 4°C for 1.5 hours before being centrifuged (3500g, 25 minutes, 4°C). The lentivirus-containing pellet was resuspended in 150 μL of FBS-free TexMACS medium (Miltenyi).

[0461] To determine functional titer, Jurkat WT cells or SupT1 cells transduced with series dilutions of concentrated virus preparations (final dilutions of 100x, 1000x, 10000x, and 100000x) were evaluated for GFP expression by flow cytometry 72 hours post-infection. Functional titer (transduction units or TU / mL) is calculated for at least two serial dilutions using the following formula: (number of seeded cells x GFP + percentage of cells x dilution factor) / volume in milliliters (mL).

[0462] For transduction assays of Jurkat and SupT1 cells, two functional MOIs were used in growth medium. Cells were harvested 72 hours post-infection, and GFP+ expression was evaluated by flow cytometry as described above.

[0463] As shown in Figure 4, the wild-type VSVG pseudotype eLV vector (VSVG) transduces both Jurkat WT and SupT1 cells, consistent with the broad cellular tropism of the VSVG envelope protein. However, co-expression of the wild-type VSVG envelope protein with either VHHCD3e or VHHCD8 (CD3e VSVG and CD8a VSVG, respectively) failed to redirect the vector's targeting to CD3+ (Jurkat WT) cells or CD8+ (SupT1) cells, respectively. In fact, the CD3e VSVG vector infected SupT1 cells (which do not express CD3e, see Figure 3) to a similar level as the VSVG vector. Similarly, the CD8a VSVG eLV infected Jurkat cells (which do not express CD8, see Figure 3) to a similar level as the VSVG vector. In summary, these results demonstrate that envelope-anchored VHHCD3e or VHHCD8a failed to retarget wild-type VSVG pseudotype eLVs to cells expressing VHH targets, but instead maintained targeting with broad tropism associated with wild-type VSVG proteins.

[0464] Example 4: The combination of VHH targeting the CD8a receptor and the Y150A Lassa virus glycoprotein variant or wild-type Rudantec virus envelope protein specifically targets lentiviral particles to CD8+ T cells. The objective of this study was to determine whether combinations of cell surface-targeting VHHs and viral envelope glycoproteins other than VSVG could direct the lentiviral vector's tropism to cell types expressing the cell surface proteins targeted by the VHHs. For this purpose, lentiviral vectors presenting combinations of CD8a-targeting VHHs and different wild-type / mutant viral envelope proteins on the envelope were tested for transduction specificity to CD8-positive and CD8-negative cell lines. In the results presented in this example, the inventors identified two viral envelope proteins that, in combination with CD8aVHHs, induce specific targeting of CD8+ cells.

[0465] To test the specificity of CD8a-targeting lentiviral particles, the inventors first established a CD8a-expressing Jurkat cell line (Jurkat CD8+). Jurkat WT cells (clone E6-1) were transduced with a lentivirus encoding the human CD8a receptor, expanded, and a population that stably expressed hCD8a was selected and banked as a Jurkat CD8+ polyclonal cell line.

[0466] CD8a VHH eLV was generated by transient transfection of a VHHCD8a e293T-producing cell line (see Figure 2) using a transduction plasmid encoding a dsGFP reporter and a viral envelope construct encoding either the Y150A Lassa virus glycoprotein variant (LVGP1x, SEQ ID NO: 32) or the wild-type Rudantec virus envelope protein (LDV, SEQ ID NO: 33). 72 hours post-transfection, the supernatant containing the lentivirus was collected, concentrated, and the vector titer was measured as outlined in Example 3.

[0467] 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 in Example 3. Cells were harvested 72 hours post-infection, and GFP+ expression was evaluated by flow cytometry.

[0468] As shown in Figure 5, LVGP1X- or LDV-VHHCD8a eLV exhibited broad tropism and specifically transduced CD8a-expressing Jurkat cells (Jurkat CD8+) in contrast to VSVG pseudotyped eLV, which transduced both Jurkat WT cells 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.

[0469] Example 5: The combination of VHH, which targets CD3e T cell surface protein, and LVGP1x specifically targets lentiviral particles to CD3+ T cells. The objective of the study was to extend the findings of Example 4 to other T cell surface markers and to confirm that the combination of cell surface-targeted VHH and LVGP1x can direct lentiviral vector tropism to target cells. For this purpose, lentiviral vectors presenting a combination of CD3e-targeted nanobodies and LVGP1X were tested for transduction specificity in CD3e-positive and CD3e-negative cell lines.

[0470] The hCD3e VHH-presenting vector was generated by transient transfection of an e293T-producing cell line (similar to the one shown in Figure 2) that stably expresses VHHCD3e H6 (SEQ ID NO: 4) or VHHCD3e G7 (SEQ ID NO: 3), as previously described.

[0471] To test the transduction specificity of LVGP1X-VHHCD3e eLVs, SupT1 (CD3e-nonexpressing) and Jurkat WT cells (CD3e-expressing) were transduced at MOI 2 as previously described. Cells were harvested 72 hours post-infection and GFP+ expression was evaluated by flow cytometry.

[0472] As shown in Figure 6, the LVGP1X-VHHCD3e eLV exhibited broad tropism and specifically transduced CD3e-expressing Jurkat WT cells, in contrast to the VSVG pseudotyped eLV which transduced both Jurkat WT cells and CD3e-negative SupT1 cells. In summary, our results demonstrate that the LVGP1X pseudotyped lentiviral vector can specifically target CD3eT cells via envelope-anchored CD3e-directed VHH.

[0473] Example 6: The combination of a binding factor targeting the TCR receptor and the Y150A Lassa virus glycoprotein mutant viral envelope protein specifically targets lentiviral particles to TCR+ cells. The objective of the study was to extend VHH-eLV technology to other cell surface markers and to confirm that the combination of cell surface-targeted VHH and selected envelope proteins directs lentiviral vector tropism to VHH-targeted cells. For this purpose, lentiviral vectors presenting combinations of TCR-targeted VHH and LVGP1X or (pr)LDV were tested for transduction specificity to TCR-positive and TCR-negative cell lines.

[0474] The hTCR VHH presentation vector was generated by transient transfection of an e293T-producing cell line (similar to the one shown in Figure 2) that stably expresses VHH TCR 2 (SEQ ID NO: 34), as previously outlined.

[0475] To test the transduction specificity of VHHTCR2 eLV, SupT1 (non-TCR expressing) and Jurkat WT cells (TCR expressing) were transduced as in Example 4. 72 hours post-infection, the cells were harvested, and transgene expression (V5-tagged BCMA CAR construct) was evaluated by flow cytometry.

[0476] As shown in Figure 7, LVGP1X-VHHTCR2 and LDV-VHHTCR2 eLVs exhibited broad tropism and specifically transduced TCR-expressing Jurkat WT cells, in contrast to VSVG pseudotyped eLVs which equally transduced both Jurkat WT cells and TCR-negative SupT1 cells, although the efficiency in this test was low (likely due to excessive dilution of the highly enriched vector). In summary, our results demonstrate that LVGP1X and LDV pseudotyped lentiviral vectors can target surface-expressed TCRs through the use of envelope-anchored TCR VHH.

[0477] Example 7: Lentiviral vectors targeted with envelope-anchored CD8aVHH and LVGP1x or LDV envelope proteins induce sustained transduction of CD8+ Jurkat cells. The purpose of this study was to determine whether LVGP1X- or LDV-VHHCD8 eLV reliably transduces target cells. For this purpose, lentiviral vectors presenting combinations of CD8-targeted VHH and LVGP1X- or LDV envelope proteins were tested for the persistence of transduction into CD8-positive Jurkat cell lines.

[0478] LVGP1X- or LDV-VHHCD8a eLV were prepared as before and used to transduce Jurkat CD8+ cells. Cells were harvested on days 2, 7, and 14 post-infection, and GFP+ expression was evaluated by flow cytometry.

[0479] As shown in Figure 8, transduction of Jurkat CD8+ cells with LVGP1X- or LDV-VHHCD8a eLV persists for 14 days, similar to transduction with VSVG pseudotype eLV, which exhibits broad tropism. This result demonstrates that LVGP1X- and LDV-CD8a eLV induce stable transduction of target cells.

[0480] Example 8: Lentiviral vectors targeted with envelope-anchored CD3e VHH and LVGP1x envelope proteins induce sustained transduction of Jurkat cells. The purpose of this study is to determine whether LVGP1X-VHHCD3e eLV reliably induces sustained transduction of target cells.

[0481] hCD3e VHH eLV was generated as before and used to transduce Jurkat WT cells at an MOI of 2. Cells were harvested on days 2, 7, 14, and 21 post-infection, and GFP+ expression was evaluated by flow cytometry. In addition, vector insertion was quantified by qPCR on genomic DNA extracted on day 21 post-transduction. Briefly, genomic DNA (gDNA) was extracted from infected cells and purified using the DNeasy Blood and Tissue Kit (QIAgen). 50 ng of gDNA was evaluated by quantitative PCR using the Luna Universal qPCR master mix (New England Biolabs) with primers specific to human albumin and reverse-transcribed long terminal repeats (LTRs). The number of cells and vector copies in the test samples were quantified using standard curves for albumin (3-30,000 cells) and transcribed LTRs (40-400,000 viral copies), respectively. Quantitative PCR was performed using QuantStudio3 (Life Technologies).

[0482] As shown in Figure 9, reporter GFP transgene expression persisted for 21 days after transduction of Jurkat WT cells with LVGP1X-VHHCD3e eLV, which is similar to transduction with VSVG pseudotype eLV exhibiting broad tropism. In addition, vector copy number evaluation on day 21 demonstrated that LVGP1X-VHHCD3e eLV were integrated into the host genome at an average of 1.6 copies per cell with an MOI of 2. These results demonstrate that LVGP1X-VHHCD3e eLVs induce stable and sustained transduction of Jurkat cells through integration into the host genome.

[0483] Example 9: The combination of VHH targeting the CD8a receptor and LVGP1x envelope protein specifically targets lentiviral particles in cell lines that endogenously express CD8a. The objective of this study is to expand upon the findings of Example 4 by using a target cell line (Sup-T1) that endogenously expresses CD8a (instead of exogenously expressing CD8a in Jurkat CD8+ cell lines) to avoid potential confounding effects associated with transgenic overexpression of CD8a in the Jurkat CD8+ cell line. In addition, the persistence of transduction of LVGP1X-VHHCD8a eLV was evaluated in this example. For this purpose, lentiviral vectors presenting a combination of CD8a-targeted nanobodies and LVGP1X were tested for transduction specificity to CD8a-positive and CD8a-negative cell lines.

[0484] As before, the hCD8a VHH presentation vector was generated by transient transfection of an e293T-producing cell line that stably expresses VHHCD8a (SEQ ID NO: 6).

[0485] To test the transduction specificity of LVGP1X-VHHCD8 eLV, SupT1 (CD8a-expressing) and Jurkat WT cells (CD8a-non-expressing) were transduced at a MOI of 2. Cells were harvested on days 3, 7, 14, and 21 post-infection, and GFP+ expression was evaluated by flow cytometry.

[0486] As shown in Figure 10, LVGP1X-VHHCD8a eLV specifically transduced SupT1 cells that endogenously express CD8a, but did not transduce Jurkat WT cells (A) in which CD8a levels were below detection (see Figure 2). Furthermore, transduction of SupT1 by LVGP1X-VHHCD8a eLV persisted for 21 days (B), and vector copy number evaluation on day 21 demonstrated that LVGP1X-VHHCD8a eLV was integrated into the host genome at an average of 3.1 copies per cell in this experiment (C). These results demonstrate that LVGP1X-VHHCD8a eLVs induce stable transduction of target cells through integration into the host genome.

[0487] Example 10: Combining VHH targeting the TCR receptor with LVGP1x or LDV envelope protein induces sustained transduction in Jurkat WT cells. The objective of this study is to expand upon previous findings to demonstrate sustained transduction of TCR+Jurkat WT cells using LVGP1x-VHHTCR and LDV-VHHTCR vectors.

[0488] The TCR VHH presentation vector was generated by transient transfection of an e293T-producing cell line (similar to the one shown in Figure 2) that stably expresses VHHTCR2 (SEQ ID NO: 34), as previously described.

[0489] To test the transduction efficiency of LVGP1X-VHHTCR2 and LDV-VHHTCR2 eLV, Jurkat WT cells were transduced using a 1:40 dilution of the vector preparation, as previously done. Cells were harvested on days 4, 7, 12, and 16 post-infection, and GFP+ expression was evaluated by flow cytometry.

[0490] As shown in Figure 11, LVGP1X-VHHTCR2 and LDV-VHHTCR2 eLV efficiently transduced TCR-expressing Jurkat WT cells, and both vectors exhibited transduction persistence similar to that of the VSVG pseudotype vector, which possesses broad tropism.

[0491] Example 11: The combination of VHH targeting the CD8a receptor and LVGP1x envelope protein specifically targets lentiviral particles to primary CD8+ T lymphocytes. This example describes the specificity of LVGP1X-VHHCD8a to primary T cells. For this purpose, lentiviral vectors presenting a combination of CD8a-targeting nanobodies and LVGP1X were tested for specificity of transduction into activated hPBMCs, including both CD4+ T lymphocytes and CD8+ T lymphocytes.

[0492] The hCD8a VHH presentation vector was generated by transient transfection of an e293T-producing cell line stably expressing VHHCD8a (SEQ ID NO: 6), as previously described. hPBMCs isolated from healthy donors were stimulated with anti-CD3 and anti-CD28 antibodies for 2 days and transduced at a MOI of 20 using LVGP1x-VHHCD8a eLV or VSVG eLV with broad tropism. Four days posttransduction, half of the cells were harvested, washed, and stained with anti-CD4 and anti-CD8 for flow cytometry. The same analysis was performed sequentially on days 7, 11, 14, and 20 to evaluate the persistence of GFP expression.

[0493] As shown in Figure 12, LVGP1X-VHHCD8a eLV specifically transduced primary CD8+ T cells and did not transduce primary CD4+ T lymphocytes, in contrast to VSVG eLV, which exhibited broad tropism and nonspecific transduction of T lymphocytes. This result demonstrates that LVGP1x-VHHCD8a eLV can distinguish between subpopulations of primary T cells and target the vector only to CD8+ lymphocytes.

[0494] Example 12: The combination of TCR-targeting VHH and LVGP1x envelope protein on eLV effectively transduces primary T lymphocytes. This example describes the transduction efficiency and persistence of LVGP1X-VHHTCR2 eLV into primary T cells. For this purpose, lentiviral vectors presenting a combination of TCR-targeted VHH and LVGP1X were tested against activated hPBMCs.

[0495] A TCR VHH presentation vector expressing a GFP reporter was generated by transient transfection of an e293T-producing 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 stimulated with anti-CD3 and anti-CD28 antibodies for 2 days were transduced at a MOI of 5. On day 4 posttransduction, half of the cells were collected and stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, as previously described, before flow cytometry analysis of the percentage of GFP-expressing cells. The same analysis was performed sequentially on days 7, 11, 14, and 20 to evaluate the persistence of vector transduction monitored by GFP expression.

[0496] As shown in Figure 13, LVGP1X-VHHTCR2, like the VSVG vector, sustainably transduces both CD4+ and CD8+ primary T cells over the test period (20 days), but exhibits higher transduction efficiency. In conclusion, these results demonstrate that a lentiviral vector combining TCR-targeted VHH and the LVGP1X envelope protein effectively targets and stably transduces activated primary T cells.

[0497] Example 13: Transduction of Jurkat CD8+ cells with LVGP1X-VHHCD8a or LDV-VHHCD8a eLV encoding the BCMA CAR results in functional expression of the CAR construct, as demonstrated by activation of the transduced cells after exposure to target cells. In this example, to demonstrate the functional delivery of CAR constructs using VHH-targeted eLVs, CAR-Jurkat cells targeting BCMA surface antigens were generated using LVGP1X-VHHCD8a or LDV-VHHCD8 eLVs. Functional delivery of CAR constructs was evaluated by the increase in the expression of the T cell activation marker CD69 after exposure to target cells expressing the CAR antigen.

[0498] LVGP1X-VHHCD8a vectors or LDV-VHHCD8a vectors encoding the BCMA CAR construct (SEQ ID NO: 35) were generated from stably expressing e293T-producing cells, as previously described. Jurkat cells were transduced with VHH-eLV at an MOI of 2 and passaged for 7 days before use in the co-culture assay. For the co-culture assay, BCMA antigen-negative (K562) target cells and BCMA antigen-positive (NCI-H929) target cells were labeled with CellTrace Violet (Thermo Fisher) to distinguish them from CAR-transduced Jurkat cells (CAR-Jurkat). CAR-Jurkat cells were co-cultured overnight in a 1:1 effector:target ratio. The level of CAR-Jurkat cell activation was evaluated 18 hours after co-culture by flow cytometry after CD69 antibody staining (BioLegend), and viable cells were distinguished using DAPI staining. The binding factor domain of BCMA-CAR used in this study is VHH, and the expression of the CAR construct in living cells was detected by staining with an anti-VHH antibody (Jackson Immunoresearch).

[0499] As shown in Figure 14A, BCMA CAR-Jurkat cells showed increased CD69 expression only in the presence of BCMA+NCI-H929 target cells, and the degree of this increase was similar regardless of whether VHHCD8a eLV or VSVG eLV with broad tropism was used to transduce BCMA CAR. Figure 14B shows a quantification of the results in Figure 14A.

[0500] Example 14: Transduction of primary T lymphocytes with the LVGP1X-VHHCD3e vector encoding the BCMA CAR results in functional expression of the CAR construct, as demonstrated by activation of the transduced cells after exposure to target cells. This example describes the functional delivery of a CAR construct using LVGP1X-VHHCD3e to primary T cells, and its evaluation by the increased expression of the T cell activation marker CD137 after exposure to target cells expressing the CAR antigen.

[0501] LVGP1X-VHHCD3e eLVs encoding the BCMA CAR construct (SEQ ID NO: 35) were generated from stably expressing e293T-producing cells, as previously described. hPBMCs were activated with anti-CD3 / CD28 T Cell TransAct reagent (Miltenyi) on day 0, transduced overnight with the BCMA CAR construct-encoding eLV on day 2, and the culture medium was changed on day 3. Cytokine supplementation (30 ng / mL IL-2, Miltenyi) was performed during primary cell culture. The cultures were grown until harvest on day 14 for co-culture assay. For co-culture assay, 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 in a 1:1 effector:target ratio. The level of CAR-Jurkat cell activation was evaluated 18 hours after co-culture by flow cytometry after CD137 antibody staining (BioLegend), and viable cells were distinguished using 7-AAD staining. CAR expression in viable cells was determined by staining with anti-VHH antibody (Jackson Immunoresearch).

[0502] As shown in Figure 15A, BCMA CAR-T cells showed increased CD137 expression only in the presence of BCMA+NCI-H929 target cells, and the degree of this increase was similar regardless of whether CD3e VHH-targeted eLVs or VSVG LVs with broad tropism were used to transduce BCMA CARs. Figure 15B shows a quantification of the results in Figure 15A.

[0503] Example 15: Transduction of primary T lymphocytes with the LVGP1X-VHHCD8a vector encoding BCMA CAR results in functional expression of the CAR construct and cytotoxicity of CAR-T against BCMA-expressing target cells. This example describes the in vitro function of BCMA CAR-T cells generated using LVGP1X-VHHCD8a, as measured by the death of target cancer cells.

[0504] The LVGP1X-VHHCD8a vector, encoding the BCMA CAR construct under the control of the T cell-specific promoter SYN19 (SEQ ID NO: 12), was generated from stably expressing CD8aVHH-e293T-producing cells, as previously described. hPBMCs were activated in vitro for 48 hours in the presence of anti-CD3 and anti-CD28 antibodies. Two days after activation, the cells were transduced at a MOI of 5 in complete IMDM medium supplemented with 30 ng / mL human IL-2 and expanded for 7 days. Transduction of PBMCs resulted in approximately 12% CD3+ CAR+ cells with the VSVG vector (expressing the BCMA CAR construct under the control of the ubiquitous EF1a promoter) and 6% with the LVGP1X-VHHCD8a vector, thereby confirming CAR expression. For the co-culture assay, BCMA antigen-negative (K562) target cells and BCMA antigen-positive (NCI-H929) target cells were labeled with CellTrace Violet (Thermo Fisher) to distinguish them from CAR-T cells. 40,000 target cells were co-cultured with CD3+ T cells adjusted to obtain an effector-to-target ratio of 1:1 or 1:2. Untransduced T cells or GFP-expressing T cells were used as controls to determine background alloreactivity. After 5 days of co-culture, the target cell / non-target cell count was quantified by flow cytometry after staining the cell suspension with anti-CD3 PercP.

[0505] As shown in Figure 16A, in co-culture with CAR-T cells transduced with LVGP1X-VHHCD8 or VSVG eLV, the number of NCI-H929 target cells decreased by >75% compared to untransduced or GFP-transduced controls. In contrast, the number of K562 cells remained relatively constant across all groups (Figure 16B), confirming that the deletion of NCI-H929 was CAR-dependent and BCMA-specific.

[0506] In summary, these results demonstrate that primary BCMA CAR-T cells transduced from LVGP1X-VHHCD8a eLV are functional and cytotoxic against BCMA+ targeted cancer cell lines.

[0507] Example 16: Transduction of primary T lymphocytes with the LVGP1X-VHHCD8a vector encoding a CD19-directed CAR results in functional expression of the CAR construct and cytotoxicity of CAR-T cells against CD19-expressing target cells. The objective 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.

[0508] The LVGP1X-VHHCD8a vector, encoding the 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-producing cells, as previously described. hPBMCs were activated in vitro for 48 hours in the presence of anti-CD3 and anti-CD28 antibodies. Two days after activation, the cells were transduced at a MOI of 10 in complete IMDM medium supplemented with 30 ng / mL of human IL-2 and expanded for 7 days. After staining the cells for CD3 and V5 tags (fused to the CD19 CAR), evaluation by flow cytometry showed that transduction of PBMCs yielded approximately 4% CD3+ CAR+ cells with the VSVG vector (expressing the CD19 CAR construct under the control of the ubiquitous EF1a promoter) and 12% with LVGP1X-VHHCD8a.

[0509] For the co-culture assay, target cells from CD19 antigen-negative myeloid leukemia cell lines (K562) and CD19-expressing B-cell precursor leukemia cell lines (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 adjusted to obtain an effector-to-target ratio of 1:1 or 1:2. Untransduced T cells were used as a control for nonspecific alloreactivity. After 3 days, the target / non-target cell count was quantified by flow cytometry after staining the cell suspension with anti-CD3 A700.

[0510] As shown in Figure 17A, in co-culture with CAR-T cells transduced with LVGP1X-VHHCD8a or VSVG eLV at co-culture ratios of 1:2 and 1:5, the number of Nalm6 target cells decreased by >75% compared to the untransduced control. In contrast, the number of K562 cells remained relatively constant across all groups (Figure 17B), confirming that the deletion of NCI-H929 was CAR-dependent and CD19-specific. In summary, these results demonstrate that primary CD19 CAR-T cells transduced with LVGP1X-VHHCD8a eLV are functional and cytotoxic against CD19+ target cancer cell lines.

[0511] Example 17: Transduction of primary T lymphocytes with the LVGP1X-VHHTCR2 vector encoding BCMA CAR results in functional expression of the CAR construct and cytotoxicity of CAR-T cells against BCMA-expressing target cells. This example describes the in vitro function of BCMA CAR-T cells generated using LVGP1X-VHHTCR2, as measured by the death of target cancer cells.

[0512] The LVGP1X-VHHTCR2 vector encoding the BCMA CAR construct was generated from stably expressing VHHTCR2-e293T-producing cells under the control of the T cell-specific promoter SYN19, as previously described. hPBMCs were activated and transduced at a MOI of 10, as previously described. Transduction of hPBMCs resulted in approximately 80% CD3+ CAR+ cells using the LVGP1X-VHHTCR2 vector, thereby confirming CAR expression. For co-culture assays, BCMA antigen-negative chronic myeloid leukemia (leukemia) cell lines (K562) and multiple myeloma BCMA-positive NCI-H929 cell lines 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 adjusted to obtain effector-to-target ratios of 1:1, 1:2, or 1:5. Untransduced T cells were used as a control to determine background alloreactivity. After 5 days of co-culture, the target cell / non-target cell count was quantified by flow cytometry after staining the cell suspension with anti-CD3 PercP.

[0513] As shown in Figure 18A, in co-culture with CAR-T cells transduced with LVGP1X-VHHTCR2, the number of NCI-H929 target cells decreased by >80–90% compared to untransduced controls across effector:target ratios. In contrast, the number of K562 cells remained relatively constant across all groups (Figure 18B), confirming that the deletion of NCI-H929 was CAR-dependent and BCMA-specific.

[0514] In summary, these results demonstrate that the LVGP1X-VHHTCR2 BCMA CAR eLV vector reprograms T cells into BCMA CAR T cells that are functional against BCMA+ targeted cancer cell lines and are cytotoxic.

[0515] Example 18: LVGP1X-VHHCD8a dsGFP eLV specifically and stably transduces CD8+ human T cells in vivo in a humanized mouse model. This example describes the transduction specificity and persistence of LVGP1X-VHHCD8a in humanized mice. Notably, 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 immunodeficient mouse models (NOD SCID gamma mice, NSG) that engraft PBMCs providing the target necessary for eLV transduction.

[0516] 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 eLV dsGFP were injected intraperitoneally. A control group injected with a vehicle (TexMACS) was also included. The test treatment groups are listed in Table 1. The LVGP1X-VHHCD8a eLV dsGFP vector encoding GFP under the control of the EF1a ubiquitous promoter was produced from stably expressing CD8VHH-e293T-producing cells, as previously described. In vivo transduction by LVGP1X-VHHCD8a eLV dsGFP was quantified by flow cytometry of the percentage of GFP-expressing cells in peripheral blood collected weekly for 27 days. hPBMCs were stained for CD3, CD4, CD8, CD19, and CD56 to determine the cell type expressing the GFP transgene, and therefore the transduction specificity of LVGP1X-VHHCD8a eLV.

[0517] [Table 73]

[0518] On day 4, GFP expression was observed in both LVGP1X-VHHCD8a eLV dsGFP groups and was specific to CD8+ T cells (Figure 19A), with GPF+ CD4+ cells ≤0.1% in the high-dose group 3. GFP transgene expression remained stable throughout the 27-day trial, ranging from 2–3% at a 25 million TU dose and 3–4% at a 100 million TU dose (Figure 19B). These results demonstrate that LVGP1X-VHHCD8a eLV can selectively transduce human CD8-positive T cells in vivo, and that transgene expression persists for at least 27 days.

[0519] Example 19: In vivo reprogramming of T cells with LVGP1X-VHHCD8a using CD19 CARs results in B cell dysplasia in a humanized mouse model. This example describes the function of CD8+ CAR-T cells transduced in vivo using LVGP1X-VHHCD8a eLV, which encodes the CD19 CAR transgene.

[0520] On day minus 1, 10 million activated hPBMCs were intraperitoneally injected into NSG mice (as described in Example 19), and on the following day (day 0), 100 million TU of LVGP1X-VHHCD8a CD19 CAR eLV were intraperitoneally injected. The test treatment groups are listed in Table 2. VSVG EF1a dsGFP eLV is a broad-tropism VSVG pseudotype eLV encoding an unstable GFP reporter under the control of the EF1a promoter, LVGP1X-VHHCD8a CD19 CAR eLV encoding the CD19 CAR construct (SEQ ID NO: 36) under the control of the synthetic T cell-specific promoter SYN19 (SEQ ID NO: 12), fused with a V5 tag to facilitate detection of CAR-expressing cells, and VSVG CD19 CAR eLV is a broad-tropism VSVG pseudotype eLV encoding the same CD19 CAR under the control of SYN19. B cells isolated from donors used for injection on day minus 1 were intravenously injected one day after vector administration to provide further targets for CD19 CAR-T cells reprogrammed in vivo. Half of the animals were collected on day 7, and flow cytometry was performed on peripheral blood and newly dissected spleen and bone marrow to evaluate transduction efficiency (V5-expressing cells %) and anti-CD19 CAR activity (percentage of B cells). The remaining mice were collected on day 14 for the same analysis (data not shown).

[0521] [Table 74]

[0522] Circulating CAR T cells were detected in both the VSVG and LVGP1X-VHHCD8a eLV CD19 CAR groups on day 7 (Figure 20A) and day 14 (data not shown). While VSVG eLV transduced both 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, supporting the specificity of the LVGP1X-VHHCD8a eLV vector observed in Figure 19. CAR expression in groups 2 and 3 was accompanied by deletion of human B cells in peripheral blood, bone marrow, and spleen on day 7 (Figure 20B) and day 14 (data not shown), with a B cell percentage approximately 1–2 log decrease compared to the control group treated with the VSVG eLV EF1a dsGFP vector. B cells were defined as hCD45+ hCD20+ cells. The B cell dysplasia in groups 2 and 3 also confirms that the T cell synthesis promoter allows for sufficient CD19 CAR expression to generate functional CAR-T cells.

[0523] In conclusion, CD8+ CAR-T cells transduced in vivo with the LVGP1X-VHHCD8a eLV CD19 CAR vector are functional and effectively eradicate B cells in vivo.

[0524] Example 20: In vivo delivery of the CD19 CAR construct CD8a VHH LVGP1x eLV in stably humanized mice results in B cell dysplasia. This example describes the functionality of CD8+ CAR-T cells delivered using CD8a VHH LVGP1x eLV encoding a CD19 CAR transgene in a stably humanized mouse (hCD34 NSG) model. This model enables the targeting of CD8a VHH LVGP1x eLV to resting / naive human T cells in an endogenously reconstituted multiseries human immune system setting using the intended clinical administration route.

[0525] hCD34+ NCG humanized mice were generated by transplanting human CD34+ hematopoietic progenitor cells into chemically pre-treated NCG mice. At approximately 18 weeks of age, hCD34+ NCG mice with a human blood chimera rate >25% were injected with 70E+6 TU of CD8a VHH LVGP1x eLV encoding a CD19 CAR or GFP reporter transgene under the control of the T cell-specific synthesis promoter SYN19 (SEQ ID NO: 12). To promote endogenous T cell expansion and potentially T cell transduction, cytokines IL-7 and IL-15 / IL-15Ra were subcutaneously injected 4 and 1 days prior to vector injection. As shown in Figure 20C, significant B cell dysplasia was observed in animals injected with CD8a VHH LVGP1x CD19 CAR eLV compared to animals injected with the vehicle or CD8a VHH LVGP1x GFP eLV. B-cell dysplasia persisted in the CD8a VHH LVGP1x CD19 CAR eLV group until the end of the study on day 53. VCN analysis revealed the presence of the transgene in the liver, spleen, and bone marrow, with levels decreasing in prevalence being <0.3 VCNs per cell in the liver and <0.1 VCNs per cell in the spleen. No VCNs exceeding background levels were detected in the lungs, kidneys, heart, brain, and ovaries (data not shown). No adverse health effects were reported in any of the treatment groups.

[0526] Conclusion – This study demonstrates that injection of the ENaBL-T8 vector using the intended clinical administration route was well tolerated in a mouse model with endogenously circulating resting T cells and generated sufficient CD19 CAR T cells to observe the elimination of the target B cell population. The in vivo distribution of the transgene was limited to the liver, spleen, and bone marrow.

[0527] Example 21: CD8a or TCRab VHH LVGP1x eLV specifically delivers CD19 CAR constructs to T cells in vivo, generating functional CD19 CAR T cells that induce rapid B cell dysplasia in a humanized mouse model. This example describes the in vivo transduction efficiency and specificity of CD8a or TCRab VHH LVGP1x eLV in humanized mice, resulting in the in vivo production of functional CAR-T cells and the elimination of target cell populations.

[0528] 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, 10 million TU (transduction units) of CD8a or TCRab VHH LVGP1x eLV encoding the CD19 CAR transgene, or 50 million TU of TCRab VHH LVGP1x eLV dsGFP were injected intraperitoneally. A control group was also included, which received 50 million TU of VSVG LV CD19 CAR.

[0529] As shown in Figure 20D, CD19 CAR T cells were detected as early as day 7 after vector injection in animals injected with CD8a or TCRab VHH LVGP1x CD19 CAR eLV or VSVG LV CD19 CAR. The transduction efficiency of CD3+ T cells with the TCRab VHH LVGP1x CD19 CAR eLV vector was higher compared to the CD8a VHH LVGP1x CD19 CAR eLV vector (9.0% ± 1.5 and 1.0% ± 0.15, respectively, in peripheral blood at day 7 at a 10e6 TU dose), induced a higher expansion and proliferation of the T cell population (10e5–10e7 T cells / mL vs. 10e4–10e5 T cells / mL, respectively, at day 7), and was therefore higher than the total number of circulating CAR T cells (10e5–10e6 CAR T cells / mL vs. 10e2–10e4 CAR T cells / mL, respectively). As shown in Figure 20F, transduction of the CD8a VHH LVGP1x CD19 CAR eLV vector was specific to CD8+ T cells (1.9% + 0.3 CD4+ CAR+ cells vs. 22.4% + 2.2 CD8+ CAR+ cells at a 10e6 TU dose on day 11), while TCRab VHH LVGP1x 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 vs. 7.6% + 1.4 CD8+ CAR+ cells at a 10e6 TU dose on day 11). Detectable levels of CD19 CAR transduction and expression were observed for both vectors in the NKT population, but not in NK, CD14, or mouse CD45 cells. Despite varying levels of CD19 CAR T cells reprogrammed in vivo across different groups, near-complete or complete B-cell dysplasia was observed in all CD19 CAR groups by day 7 in the blood and by day 11 in the spleen.

[0530] This study demonstrates that CD19 CAR T cells generated in vivo using CD8a or TCRab VHH LVGP1x CD19 CAR eLV vectors possess anti-targeted cytotoxic activity. TCRab targeting of TCRab VHH LVGP1x CD19 CAR eLV results in higher levels of transduction and overall greater expansion and proliferation of the T cell population compared to CD8-targeted vectors. Expression of CD19 CAR by T cell-specific synthetic promoter 23 is specific to T cells and NKT cells, resulting in potent anti-targeted cytotoxic activity of the CAR.

[0531] Example 22: The combination of VHH targeting the CD8 receptor and a triple mutant of Lassa virus glycoprotein specifically targets lentiviral particles to CD8-expressing cells. This example describes cell-specific transduction achieved using eLV presenting membrane-anchored VHH and LVGP envelope protein variants. For this purpose, several variants of Lassa virus envelope glycoprotein were tested, and Figure 21 presents findings regarding one such variant envelope protein that specifically transduces CD8-positive cell lines in combination with CD8a-targeted VHH.

[0532] The hCD8a VHH presentation vector was generated as previously described by co-transfection of a VHHCD8a e293T-producing cell line with a packaging and transduction plasmid and a viral envelope construct expressing either the Y150A Lassa virus glycoprotein variant (as a positive control) or the Y150A H141A F147A Lassa virus glycoprotein variant (LVGP3x, SEQ ID NO: 37).

[0533] To test the transduction specificity of LVGP1X- or LVGP3X-VHHCD8a eLVs, Jurkat WT, 293T, and Sup-T1 cells were transduced at a MOI of 2 as before. Cells were harvested 72 hours post-infection and evaluated for GFP+ expression by flow cytometry.

[0534] As shown in Figure 21, the LVGP3X-VHHCD8a eLV, like the LVGP1X-VHHCD8 vector, specifically transduced CD8-expressing cells (Sup-T1), in contrast to the VSVG pseudotype eLV, which exhibited broad tropism by transducing both CD8-negative cell lines (Jurkat WT and 293T) and CD8+SupT1 cells. In summary, these results indicate that, similar to the LVGP1X eLV, the LVGP3X pseudotype lentiviral vector is targeted by envelope-anchored VHH, and that effective transduction is mediated by mutant Lassa envelope protein.

[0535] Example 23: Pseudotyping of a CD8 VHH presentation vector containing wild-type or H141A / F147A but lacking the Y150A / F446L Lassa virus glycoprotein specifically targets lentiviral particles to CD8+ primary T cells. This example describes cell-specific transduction achieved using eLVs that present membrane-anchored CD8 VHH and are pseudotyped with either WT or H141A / F147A or Y150A / F446L Lassa virus glycoprotein.

[0536] CD8a VHH presentation vectors were generated as previously described by co-transfection of a CD8a VHH e293T-producing cell line with a packaging and transduction plasmid, as well as 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 Y150A mutant Lassa virus glycoprotein (used as a positive control). To test the transduction specificity of differentially pseudotyped CD8a VHH eLVs, activated PBMCs were transduced at a MOI of 10 as previously described, and the expression of the GFP cargo transgene was evaluated over time by flow cytometry.

[0537] As shown in Figure 22, CD8 VHH-targeted eLVs pseudotyped with wild-type or H141A / F147A Lassa virus glycoprotein transduced only CD8+ primary T cells, but the level of transduction was lower compared to vectors pseudotyped with Y150A Lassa virus glycoprotein. In contrast, pseudotyped CD8 VHH eLVs using the Y150A / F446L mutant Lassa virus glycoprotein eliminated the transduction efficiency of the vector, while pseudotyped VSVG induced nonspecific transduction to both CD8+ and CD8-(CD4+) primary T cells. In summary, these results indicate that wild-type and H141A / F147A Lassa virus glycoprotein pseudotyped VHH-presenting eLVs, like Y150A VHH eLVs, are targeted by envelope-anchored VHH, and that effective transduction is mediated by both wild-type and selected mutant Lassa virus envelope proteins.

[0538] Example 24: Cell-specific transduction is achieved by combining VHH-mediated targeting with selected mutants of the VSVG envelope glycoprotein. This example describes cell-specific transduction achieved using newly identified mutants of membrane-anchored VHH-presenting eLV and VSVG glycoproteins. Based on the crystal structure of VSVG glycoprotein, the inventors generated specific mutants in key residues located around the binding pocket or in the pathway by which the VSVG protein binds to its native receptor, LDL-R. These mutations were engineered to either prevent (steric hindrance) or block (polarity change) interaction with LDL-R, in order to preserve the viral entry activity of VSVG while eliminating the protein's targeting function.

[0539] Figure 23 presents findings that support the selection of VSVG mutants that lack the broad tropism-mediated targeting of wild-type VSVG and specifically transduce CD8-positive cell lines when combined with CD8a-targeted VHH.

[0540] The hCD8a VHH presentation vector was generated as previously described by co-transfection of a CD8a VHH e293T-producing cell line with packaging and transduction plasmids, as well as viral envelope constructs expressing VSVG variants listed in Figure 23A (SEQ ID NOs. 38-40 and 153-182). To test the transduction specificity of VSVG variant VHHCD8 eLVs, Jurkat WT, CD8-expressing Jurkat cells, and Sup-T1 cells (endogenously expressing CD8a) were transduced at a MOI of 2. Cells were harvested 72 hours post-infection and GFP+ expression was evaluated by flow cytometry.

[0541] As shown in Figures 23B and 23C, single mutants A51E, I182E, I331W, I331E, T352W, T352Q, or double / triple / quadruplicate 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 Several VSVG variant CD8 including "Q53A, 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". In contrast to VSVG pseudotype eLVs, which exhibit broad tropism by transducing both CD8-negative cell lines (Jurkat WT) and CD8-positive cells (Jk-CD8 or SupT1 cells), VHH eLV specifically transduced CD8-expressing cells (Jk-CD8 or SupT1 cells).

[0542] As shown in Figures 24A and 24B, in cell lines endogenously expressing CD8 (Sup T1 or CD8+ primary T cells), CD8 VHH eLV pseudotyped with the VSVG variants identified in Figure 23B confirmed target-specific transduction of the A51E, I182E, and I331E mutant VSVG pseudotype vectors.

[0543] In Figure 24C, the transduction specificity of the I331E mutant VSVG pseudotype vector was also tested using a TCRab VHH-targeted eLV. These data confirm that I331E pseudotypes retain targeting specificity for eLVs presenting VHHs other than CD8a VHH, and demonstrate that without VHH targeting, the I331E pseudotyped eLV is transduction-deficient.

[0544] As shown in Figure 25, the lack of LDL-R binding in TCRab VHH eLV pseudotyped with the VSVG mutant I331E was confirmed on HEK 293T cells overexpressing LDL-R, while vector binding was observed on HEK 293T cells overexpressing SIRPa (as expected, since CD47, a ligand for the SIRPa receptor, is presented on the surface of TCRab VHH eLV).

[0545] In summary, our results demonstrate that VSVG variants manipulated to eliminate binding to LDLR, particularly VSVG variants containing a mutation at position I331, can be used to pseudotype lentiviral vectors to achieve cell-targeted transduction through the use of envelope-anchored, target-directed VHHs.

[0546] Example 25 Transduction of primary T lymphocytes in TCRab VHH-targeted eLV pseudotyped with a VSVG-targeted knockout mutant encoding a CAR construct results in functional expression of the CAR construct and CAR-T cytotoxicity against target-expressing cells. This example measures target-deficient VSVG, which is measured by the death of target cancer cells. I331EWe describe the in vitro function of CAR-T cells generated using pseudotyped TCRab VHH eLV with a mutant.

[0547] A TCRab VHH G1x eLV vector encoding the BCMA CAR construct (SEQ ID NO: 35) under the control of the T cell-specific promoter SYN23 (SEQ ID NO: 15) was generated from stably expressing TCRab VHH e293T-producing cells, as previously described. Unstimulated hPBMCs were transduced with the BCMA CAR-encoding TCRab VHH G1x eLV, and after confirming CAR expression, the cells were co-cultured with BCMA antigen-negative chronic myeloid leukemia (leukemia) cell line (K562), multiple myeloma BCMA-positive NCI-H929 cell line, and Nalm6, an acute lymphoblastic leukemia lymphocyte cell line expressing low levels of BCMA, at different effector-to-target ratios. As shown in Figures 26A to 26I, in co-culture with CAR-T cells transduced with TCRab VHH G1x eLV, the number of NCI-H929 target cells decreased by >90% compared to untransduced controls across effector:target ratios. In contrast, the number of K562 cells remained relatively constant across all groups, confirming that NCI-H929 deletion was CAR-dependent and BCMA-specific. CAR-T-mediated cytotoxicity correlated with increased proliferation and IL2 production, demonstrating the pluripotency of CAR-T cells generated using the TCRab VHH G1x eLVs vector.

[0548] The TCRab VHH G1x eLV-mediated delivery of functional CARs is applicable to other constructs. CAR-T cells targeting claudin 18.2 were generated as described above using the TCRab VHH G1x eLV vector encoding the claudin 18.2 CAR (SEQ ID NO: 53) under the control of the T cell-specific promoter SYN23. The CAR-T cells were co-cultured in different ratios with claudin 18.2 antigen-negative chronic myeloid leukemia (leukemia) cell line (K562), claudin 18.2-positive gastric cancer cell line (NUGC4), and acute lymphoblastic leukemia lymphocyte cell line Nalm6 that stably expresses either claudin 18.2 or claudin 18.1. As shown in Figures 26K to 26N, the cytotoxic activity of TCRab VHH G1x eLV transduced claudin 18.2 CAR-T cells was detected as early as 18 hours after co-culture with NUGC4 cells, was specific to claudin 18.2-expressing cells, and no toxicity was observed against K562 cells or Nalm6 cells that stably express claudin 18.1.

[0549] In summary, these results demonstrate that the TCRab VHH G1x eLV vector effectively delivers CAR constructs to resting T cells, producing CAR-T cells with potent antitumor activity.

[0550] Example 26: Reporter construct for testing novel cell type-specific synthetic promoters Synthetic cell-specific promoters are constructed from the upstream genomic sequences of selected cell-specific genes. These genomic sequences include the core and proximal promoters of the selected cell-specific genes, as well as enhancer sequences identified upstream of these genes. The selected sequences are constructed as synthetic promoters less than 1 kb in size and tested using the reporter construct shown in Figure 27. This dual reporter construct allows for qualitative and quantitative evaluation of the specificity and expression capacity (expression level) of the upstream promoter. A fluorescent reporter is used for qualitative evaluation of promoter specificity using flow cytometry, and a luminescent reporter is used for quantitative evaluation of promoter expression capacity.

[0551] Example 27: Specificity and expression ability of a T cell synthesis promoter driven by a specific combination of core sequence, proximal sequence, and enhancer sequence. This example describes the specificity and expression potential of 10 synthetic T cell-specific promoters constructed as described in Example 23. For this purpose, several candidate synthetic promoters were tested for specificity in a range of cell types (using flow cytometry readout of GFP expression in specific cell types) and expression potential (using luminescence readout from luciferase reporter expression) using the EF1a ubiquitous promoter as a comparator. The cell type panel was selected based on predicted vector exposure after systemic administration and included lymphocytes (Jurkat and SupT-1 cell lines, as well as primary T lymphocytes CD4+ and CD8+ from human PBMCs), non-T lymphocytes from bone marrow (THP1) and B cell (NCI-H929) compartments, and cells from the liver (Huh7) and kidney (293T). Figures 28–31 present the results obtained for the 10 candidates.

[0552] VSVG eLVs expressing the reporter construct under the control of EF1a or a different synthetic promoter were generated in e293T-producing cell lines by transient transfection, as previously described. Activated hPBMCs and cell lines were transduced with different VSVG eLVs (MOI of 5 and MOI of 2, respectively), and on day 7 posttransduction, cells were evaluated for eGFP expression by flow cytometry, viability using Cell-Titer Fluor (Promega), and luciferase expression by luminescence using Steady-Glo assay (Promega). For flow cytometry of hPBMCs, cells were stained with anti-CD3 BV510, anti-CD4 APC-Cy7, and anti-CD8 PerCP to identify T cell subtypes.

[0553] As shown in Figures 28–31, the luminescence signals from some of the synthetic promoter-driven luciferase expressions (particularly SYN22) were equivalent to or greater than those of EF1a reporter expression in Sup-T1 cells (Figure 28), Jurkat cells (Figure 29), and primary T cells (Figure 30). Addition of enhancer sequences in the SYN construct significantly increased luciferase reporter expression compared to the proximal promoter alone. Furthermore, as shown in Figure 30D, the GFP reporter expression levels (measured as the geometric mean of the fluorescence signal) were equivalent in CD4+ primary T cells and CD8+ primary T cells. Conversely, when reporter expression was tested in non-T cell lines, the luminescence signal was 4–628 times lower with the SYN construct compared to EF1a-driven reporter expression, depending on the cell type (Figure 31).

[0554] In summary, these results demonstrate that a target-construction type T cell-specific synthetic promoter, sized for integration into lentiviral constructs, effectively restricts reporter transgene expression in cultured T lymphocytes.

[0555] Example 28: Using a T cell synthesis promoter to express CAR transgenes using VHH-oriented eLV significantly reduces CAR expression on the surface of the producing cell line during LV production and increases the functional titer (titer) of the vector. This example describes the effect of using a T cell-specific synthetic promoter on the production of VHH-targeted lentiviral vectors. The inventors evaluated the production yield of LVGP1x-VHHCD8a vectors encoding BCMA or CD19 CAR under the control of a SYN promoter or an EF1a promoter (evaluated using functional titer).

[0556] LVGP1x-VHHCD8a SYN19 BCMA / CD19 CAR and LVGP1x-VHHCD8a EF1a BCMA / CD19 CAR eLV were produced from VHHCD8 e293T-producing cell lines under the same conditions as previously described. As shown in Figure 29A, and consistent with Figure 32A, the expression of the transgene (BCMA CAR, stained with anti-V5 A647 and evaluated by flow cytometry) was significantly reduced on the surface of process-terminate cells from LVGP1x-VHHCD8 SYN19 BCMA CAR production (productive cells collected after final virus recovery) compared with process-terminate cells from LVGP1x-VHHCD8 EF1a BCMA CAR production. To evaluate the functional titer of the vectors, SupT1 cells were transduced with each viral product at a range of dilutions (1:50 to 1:3125). Transduction was assessed on day 3 post-transduction based on the quantification of the percentage of BCMA or CD19 CAR-expressing cells by anti-V5 A647 staining and flow cytometry. Functional titer (transduction units / mL) was calculated from the minimum value of two vector dilutions representing 5-50% transduced cell percentages.

[0557] As shown in Figure 32B, the functional titer of LVGP1x-VHHCD8 encoding any of the CAR constructs under the control of the SYN promoter was 10-fold higher than that of the same construct expressed under the control of the EF1a promoter. In conclusion, replacing EF1a with a synthetic T cell-specific promoter to control the expression of the CAR cargo in the LVGP1x-VHHCD8 vector significantly enhances the functional titer of vector production.

[0558] Example 29: T cell-specific synthetic promoters drive functional CAR expression and enhance the activity of CAR-T cells compared to constitutive promoters. This example describes the evaluation of the efficacy of CAR-T cells expressing CAR constructs under the control of T cell-specific synthetic promoters selected in Examples 26-28. For this purpose, BCMA CAR cytotoxic activity was evaluated in primary T cells transduced with eLV for BCMA CAR expression under the control of EF1a or SYN promoters.

[0559] Figure 33 shows that BCMA CAR-T cells (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) produced by transducing activated human PBMCs with a VSVG pseudotype eLV vector were cultured in the presence of multiple myeloma BCMA-expressing NCI-H929 cells (which stably express a luciferase reporter) at various effector-to-target ratios. Non-transduced T cells or GFP-expressing T cells were used as controls for nonspecific alloreactivity. After 5 days of co-culture, 100 μL (100 microliters) of the culture was washed, and luciferase expression (correlated to the number of surviving NCI-H929 target cells) was evaluated using the Steady-Glo assay (Promega). Compared to co-cultures with untransduced T cells or EF1a-GFP vector-transduced T cells, co-cultures with SYN19-BCMA CAR or EF1a-BCMA CAR-transduced CAR-T cells showed a >4-fold decrease in luminescence (correlated to the level of surviving NCI-H929 target cells) at all E:T ratios. This result demonstrates that the cytotoxicity of SYN19-BCMA CAR-T cells is enhanced compared to that of EF1a-BCMA CAR-T cells.

[0560] To further characterize the activity of BCMA CAR-T cells regulated by the T cell synthesis promoter, BCMA CAR-T cells produced from human PBMCs from three different donors (either BCMA CAR-T cells expressing the BCMA CAR transgene under the control of the VSVG pseudotype eLV vector (SYN23 BCMA CAR, which expresses the BCMA CAR transgene under the control of the synthesis promoter 23 and SEQ ID NO: 15, or EF1a BCMA CAR, which expresses the BCMA CAR transgene under the control of the elongation factor 1a (EF1a) promoter) were co-cultured with target cells (NCI-H929) and non-target cells (K562) in different effector (CAR) to target (cell line) ratios. Fresh target cells were added at various time points to test the persistence of CAR-T antitumor activity. The results presented in Figure 34 show that BCMA CAR-T cells expressing the CAR construct under the control of the SYN23 promoter demonstrated (1) enhanced proliferation, (2) enhanced antitumor activity in the presence of high levels of target cells (1:12), and (3) reduced background activation and increased BCMA-specific activation in the absence of BCMA antigen, in contrast to CARs regulated by the EF1a promoter. In summary, these results demonstrate that BCMA CAR-T cells expressing the CAR construct under the control of the T cell-specific synthetic promoter #23 possess superior and more sustained antitumor activity.

[0561] Example 30: CD14-binding factor presenting LVGP1x eLV specifically transduces monocytes. Targeting of eLVs via combinations of envelope-anchored binding factors and mutant envelope proteins (e.g., LVGP1x, G1x, LDV) has been engineered to target cell types beyond T lymphocytes by switching the envelope-anchored binding factors. This example describes the specific transduction of myeloid cells using LVGP1x eLV presenting a membrane-anchored scFv binding factor that targets CD14 (a protein expressed on the surface of myeloid cells that functions as part of the LPS receptor complex). Notably, monocytes express multiple viral restriction factors (e.g., SAMHD1), and packaging viral protein X (Vpx) within the viral particle is necessary to achieve effective transduction of monocytes using lentiviral vectors. Vpx uptake is achieved during virus production by adding a Vpx expression plasmid during transient transfection of a producing cell line. As shown in Figure 35A, the incorporation of Vpx into VSVG LV or LVGP1x scFV CD14 eLV vector particles (see below for details on vector production and transduction conditions) significantly increases the transduction of primary monocytes during culture.

[0562] The specificity of the LVGP1x-scFvCD14 eLV vector was evaluated by comparing the transduction of human PBMCs with the transduction of primary monocytes isolated from the same donor PBMCs. The GFP-expressing LVGP1x-scFVCD14 scFvCD14 eLV vector was produced by transient transfection of an e293T cell line stably expressing a membrane-anchored anti-CD14 scFv (sequence number 41) containing a transduction plasmid supplemented with a Vpx expression construct (sequence number 43) and an envelope expression plasmid (LVGP1X), packaged (using a Gag / pol construct containing a modified gag sequence, sequence number 42, to enable Vpx packaging), and envelope expression plasmid (LVGP1X). The physical titer of the vector suspension was determined using the RT-qPCR viral genome quantification kit (Takara). Unstimulated hPBMCs (cultured in 30 ng / mL IL-2) or isolated human monocytes (isolated from hPBMCs using the Miltenyi CD14 MACS isolation kit and cultured in 50 ng / mL GM-CSF (130-093-865) + 50 ng / mL IL-4) were transduced with 100, 200, 500, and 1000 vector particles per cell. On day 7 after transduction, the percentage of cells expressing GFP was evaluated by flow cytometry.

[0563] As shown in Figure 35B, CD14-targeted LVGP1 Vpx eLVs transduced primary monocytes with high efficiency, while transduction of unstimulated PBMCs remained below 2%, even when cells were exposed to 1000 viral particles per cell. In conclusion, retargeting LVGP1x eLV with an envelope-anchored anti-CD14 binding factor induces effective and specific transduction of primary monocytes.

[0564] Example 31: Selection of CD14 VHH for targeted monocyte transduction using CD14-targeted eLV Targeting of primary monocytes was successfully achieved using eLVs targeted with CD14 scFv. To demonstrate its applicability to VHH-targeted eLVs, de novo VHHs targeting CD14 or CCR2 (sequence numbers 66-116 and 117-120, respectively) were immunized and tested as membrane-anchored binding factors targeting eLV transduction into monocytes. This example describes the selection of CD14 VHHs for optimal transduction of primary monocytes by eLVs (including Vpx).

[0565] As shown in Figure 36A, some of the newly generated VHHs (SEQ ID NOs. 66-76, 101, 102, 104, 106-117) specifically targeted CD14-expressing cells (Jurkat cells that stably express CD14) for G1x eLV transduction. In this early stage of the screening process, eLVs were produced from e293T cells, and the VHH-binding factor was encoded in the transduction vector. Therefore, the transduction efficiency of G1x CD14 VHH eLVs produced in this manner was evaluated by VHH expression in the transduction cell population.

[0566] A subset of the most specific CD14 VHH (SEQ ID NOs. 70, 72, 76, 107, 111) was selected from the results in Figure 35A, and G1x CD14 VHH eLV was produced from stably expressing e293T cells to test the transduction efficiency of primary monocytes in culture. As shown in Figure 35B, G1x CD14 VHH eLV efficiently transduced monocytes, and G1x eLV targeted with 3RMB1 VHH (SEQ ID NOs. 76) achieved the highest level of transduction, with transduction proportional to the number of vector particles per cell.

[0567] 3RMB1 VHH was selected for further optimization through in vitro maturation. As shown in Figure 36C, the transduction efficiency of mature 3RMB1 (SEQ ID NOs. 77-100)-targeted G1x eLV varied compared to the original 3RMB1 VHH-targeted vector, with mature 3RMB1-13 VHH (SEQ ID NOs. 94)-targeted G1x eLV achieving the highest level of transduction.

[0568] The specificity of 3RMB1 VHH-targeted G1x eLV was evaluated in further cell types. As shown in Figure 37, 3RMB1 VHH G1x eLV efficiently transduced CD14+ cells (monocytes / macrophages and Jurkat CD14 cell lines) but not CD14-negative cells (stimulated PBMCs, Jurkat, or NCI-H929 cell lines). Importantly, there was no detectable transduction of untargeted (3RMB1 VHH-deficient) G1x eLV, highlighting the crucial role of targeted VHH in promoting specific and effective transduction of target cells.

[0569] In summary, these results demonstrate that G1x eLV (incorporating Vpx) can effectively and specifically target CD14+ cells (including primary monocytes) using membrane-anchored CD14 VHH.

[0570] Example 32: Specificity and expression ability of a monocyte / macrophage cell synthesis promoter driven by a specific combination of core sequence, proximal sequence, and enhancer sequence. This example describes the specificity and expression potential of 11 synthetic bone marrow-specific promoters (SEQ ID NOs: 130-140 and SEQ ID NO: 197) constructed as described in Example 23. For this purpose, several candidate synthetic promoters were tested for specificity in a range of cell types (using flow cytometry readout of GFP expression in specific cell types) and expression potential (using luminescence readout from luciferase reporter expression) using the EF1a ubiquitous promoter as a comparator. The cell type panel was 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 bone marrow (THP1) and B cell (NCI-H929) compartments, and cells from the liver (Huh7) and kidney (293T). Figures 38-40 present the results obtained for seven candidate promoters.

[0571] VSVG eLVs expressing the reporter construct under the control of EF1a or a different core promoter or a fully synthetic promoter were generated in e293T-producing cell lines by transient transfection, as previously described. Primary cells and cell lines were transduced with purified vectors, and the expression levels of the reporter eGFP transgene were evaluated in transduced cells.

[0572] Six proximal promoters derived from macrophage-specific genes were selected for testing. As shown in Figure 38, the proximal promoters of CCL13, CCL18, CHIT1, HLA-DRA, LYZ, and MRC1 (SEQ ID NOs. 130, 131, 132, 133, 134, and 197) induced higher eGFP expression in primary monocytes compared to EF1a promoter-driven reporter vectors (excluding MRC1), and approximately 5–20-fold lower expression in cell lines and activated PBMCs (primarily T cells). These data demonstrate that HLA-DRA, LYZ, CHIT1, CCL8, and CCL13 induce highly enriched reporter expression in primary myeloid cells compared to EF1a-regulated expression.

[0573] The CHIT1 sequence was used as the core of the bone marrow-specific synthetic promoter tested in Figure 39, and the promoter was constructed using enhancer sequences (SEQ ID NOs. 135-140) isolated from bone marrow-specific genes. As shown in Figure 39, the addition of enhancer sequences to the CHIT1 core promoter significantly increased eGFP reporter expression compared to the proximal promoter alone or EF1a, while maintaining specificity for primary monocytes / macrophages.

[0574] To confirm that the bone marrow synthesis promoters tested in Figure 39 induce functional levels of expression of transgenes other than the eGFP reporter in monocytes / macrophages, the inventors tested the secretion of IL12 from macrophages transduced with a VSVG LV vector expressing human IL12 under the control of the EF1a promoter or the bone marrow-specific synthesis promoter Enh31 CHIT1 (SEQ ID NO: 139). As shown in Figure 40, the level of IL12 secretion from Enh31 CHIT1 transduced macrophages (M0 or M2) was comparable to or higher than that of EF1a, and significantly higher than that of macrophages transduced with IL12 under the control of the core CHIT1 promoter alone.

[0575] In summary, these results demonstrate that a target-construction monocyte / macrophage cell-specific synthetic promoter, sized for integration into lentiviral constructs, effectively restricts reporter transgene expression in cultured monocytes / macrophages.

[0576] Example 33: Optimizing the structure of the VHH envelope anchor scaffold increases the transduction efficiency of CD8a and CD3e VHH targeted vectors. In these examples, different hinge regions and transmembrane domains were used to determine the optimal envelope anchor scaffold for CD3e VHH (SEQ ID NO: 3) or CD8 VHH (SEQ ID NO: 6), achieving high levels of transduction of target cells.

[0577] VHHCD3e and VHHCD8a eLV vector expression was produced by transient transfection of e293T cells using packaging plasmids encoding Gag / Pol, Rev, and LVGP1x constructs, as well as transfection vectors for the expression of VHH-targeted constructs. Each VHH was incorporated into a viral envelope anchor construct consisting of a transmembrane domain (PDGFR or B7-1, SEQ ID NO: 27 and SEQ ID NO: 28, respectively) and fused to linker regions with varying flexibility, length, and multimerization ability (see SEQ ID NOs: 21-26). After recovery, Jurkat WT cells were transduced with the same volume of lentiviral preparations. The functional titer of each vector was evaluated on day 3 posttransduction by anti-VHH staining and flow cytometry of transduced cells, and the value was expressed as transduced units per milliliter.

[0578] As shown in Figures 41 and 42, the transmembrane domain variants PDGFR or B7-1 did not significantly affect the degree of targeting by CD3e or CD8a VHH, with PDGFR transmembrane showing slightly better performance. Hinge domains had a greater impact. The two hinge regions that showed the best performance in both LVGP1X-VHHCD3e eLV and LVGP1X-VHHCD8a eLV were the PDGFR short-stemmed and CD8a hinges, which have moderate flexibility and a low tendency to polymerize compared to stiffer, shorter hinges (e.g., GAPGAS) or multimerizing hinges (e.g., tetramerized coils).

[0579] Example 34: Optimizing the structure of the VHH envelope anchor scaffold increases the transduction efficiency of the TCR VHH targeted vector. In this example, the optimal envelope anchor scaffold for TCR VHH (SEQ ID NO: 9) was determined using different hinge regions and transmembrane domains, achieving a high level of transduction of target cells.

[0580] VHHTCR eLV vectors expressing dsGFP were produced from e293T cells stably expressing membrane-anchored TCR VHH anchored to the viral envelope via four different scaffolds with varying flexibility, length, and multimerization capabilities, as previously described: TCR1: PDGFR transmembrane domain and proximal membrane region of PDGFR (SEQ ID NO: 44), TCR2: PDGFR transmembrane domain and proximal membrane region of IgG4 (SEQ ID NO: 34), TCR3: PDGFR transmembrane domain and CD8a stem (SEQ ID NO: 45), and TCR4: PDGFR transmembrane domain and tetrameric coiled-coil hinge (SEQ ID NO: 46). Notably, VHHTCR eLVs were pseudotyped in this study using VSVG variants (K47A, R354, SEQ ID NO: 47) that have been previously shown to eliminate the broad tropism of wild-type VSVG. After concentration, Jurkat WT cells were transduced with the same volume of lentiviral preparation, and the transduction efficiency (GFP+ cells) was evaluated by flow cytometry on day 3.

[0581] As shown in Figure 43A, different hinge sequences within the TCR VHH scaffold affected transduction efficiency. In the case of TCR VHH, an anchor scaffold containing the PDGFR transmembrane domain and the membrane-proximal region of IgG4, combined with the VSVG(K47A, R354) pseudotype, provided a structure that achieved the highest transduction efficiency.

[0582] The structure of the VHH scaffold was further optimized for TCRab VHH when the targeted eLV vector was pseudotyped with G1x. The TCRab VHH G1x eLV produced e293 T cells stably expressing TCRab VHH on scaffolds composed of the PDGFR transmembrane domain and the proximal membrane region of the IgG4 scaffold (SEQ ID NO: 34) or the PDGFR transmembrane domain and the proximal membrane region of CD8a (SEQ ID NO: 52) via transient transfection. Primary human PBMCs exposed to the same volume of unenriched vector were characterized for T cell activation, proliferation, and transduction. As shown in Figure 43B, the TCRab VHH G1x eLV presenting VHH using the PDGFR / CD8a hinge scaffold yielded total transduced T cells on day 7 post-transduction compared to the IgG4 hinge.

[0583] Example 35: Targeting LVGP1x or G1x eLV to T cells using TCRab VHH binding factor induces enhanced activation, proliferation, and transduction of resting T cells. When the TCR complex binds to its corresponding antigen, the TCR complex aggregates, triggering a signaling cascade and leading to T cell activation. In this embodiment, we demonstrate that TCRab VHH binding factors on the surface of eLV in different scaffolds induce increased T cell activation, proliferation, and transduction compared to VSVG LV.

[0584] 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-concentration 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 expression of both CD25 in resting T cells compared to medium alone, VSVG LV, and LVGP1x-CD8a VHH eLV. Correlating with increased T cell activation, LVGP1x-TCRab VHH successfully transduced resting CD3+ T cells more efficiently than VSVG GFP eLV on day 19, as measured by GFP expression (Figure 44C). Activation and transduction of resting T cells by LVGP1x-TCRab VHH eLV also correlated with significant expansion and proliferation of GFP-expressing cells over 20 days compared to VSVG GFP-transduced cells (Figure 44B).

[0585] The activation and proliferation of resting T cells observed using TCRab VHH eLV were regulated by the VHH scaffold in G1x pseudotype LV. As shown in Figure 45, TCRab VHH G1x eLV using the PDGFR / CD8a hinge scaffold (SEQ ID NO: 52) to present VHH resulted in higher CD69, CD25, and PD1 expression, as well as increased cell proliferation and functional viral titer, compared to the IgG4 hinge (SEQ ID NO: 34).

[0586] Similarly, T cell activation was observed with substituted TCRab VHH G1x eLVs presenting substituted TCRab VHH binding factors (SEQ ID NOs. 54-65) optimized by in vitro maturation on the original TCRab VHH (SEQ ID NO: 9). As shown in Figure 46A, T cell activation (assessed by increased CD69 expression) was induced in Jurkat cells by exposure to several different TCRab VHH G1x eLVs, each with similar transduction efficiencies (Figure 46B).

[0587] In conclusion, LVGP1x or G1x pseudotype eLV targeted to T cells using TCR-specific VHH in different scaffolds induces activation of resting lymphocytes and effective transduction, resulting in significant expansion and proliferation of transduced cells.

[0588] Example 36: Presenting a co-stimulatory ligand on the surface of VHH-targeted eLV increases the activation, proliferation, and transduction of resting T cells. Exposure of resting T lymphocytes to CD8aVHH eLV induces low levels of T cell activation (indicated by CD25 expression) and transduction (indicated by expression of V5-tagged BCMA CAR transgenes) and minimal T cell expansion and proliferation (Figures 47A-47C). To test whether the presentation of costimulatory ligands on the surface of the Cd8a VHH eLV vector increases T cell activation / expansion and transduction, CD8a VHH G1x eLV was produced from CD8a VHH e293 T cell lines presenting OKT3 (CD3 agonist antibody, SEQ ID NO: 29) alone or in combination with CD28 costimulatory receptor activators, i.e., CD80 or CD28 VHH (SEQ ID NOs: 198 and 199, respectively, and SEQ ID NO: 200 for the CD8 scFV scaffold), as previously described. Residual 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 in the presence of low levels of rhIL-2 (30 ng / mL), and T cell activation, proliferation, and transduction were observed by flow cytometry. As shown in Figures 47A to 47C, CD8a VHH / OKT3 / G1x eLV, CD8a VHH / OKT3 / CD80 G1x eLV, or CD8a VHH / OKT3 / CD28 scFv G1x eLV significantly increased T cell activation (CD25) and T cell proliferation (>10 times compared to CD8a VHH G1x eLV).

[0589] To determine whether the co-stimulatory ligand further increases T cell activation, similar tests were performed using the TCRab VHH G1x eLV vector, which had already induced T cell activation and proliferation. As shown in Figures 47D to 47F, TCRab VHH / presenting the CD3 agonist binding factor OKT3 (TCRab VHH / OKT3scFv G1x eLV), the CD28 ligand CD80 (TCRab VHH / CD80 G1x eLV), or the CD28 agonist VHH (TCRab VHH / CD28scFv G1x eLV) increased the activation and transduction of resting T cells with minimal increases in T cell proliferation levels. As shown in Figure 48, TCRab VHH G1x eLV presenting the CD28 costimulatory molecule CD86 IgV or TGN1412, or the 4-1BB costimulatory molecule 4B1-4 (SEQ ID NOs. 50, 51, and 201, respectively) induced increased IL2 production and CD69 and CD25 expression. This resulted in increased T cell activation associated with increased expansion and transduction of resting PBMCs compared to the TCRab VHH G1x eLV vector.

[0590] Example 37: Transduction of primary T lymphocytes using CD8a or a TCR VHH-targeted eLV encoding a manipulated TCR results in specific, sustained, and functional expression of the transgene, as demonstrated by the cytotoxicity of the TCR-manipulated cells. Adoptive T-cell therapy using T-cell receptor-modified 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 would overcome the challenges associated with current ex vivo-produced TCRT-T cell products. To evaluate the ability of VHH-directed eLVs to deliver modified TCR constructs, we tested TCR VHH-targeted LVGP1x eLVs encoding CD8a and a clinically validated and widely used modified TCR (NY-ESO) on human PBMCs in culture.

[0591] LVGP1X-VHHCD8a and LVGP1X-VHHTCR (i.e., 1G4-LY, TCRα chain, SEQ ID NO: 30 and TCRβ chain, SEQ ID NO: 31), encoding TCR transgenes for the HLA-A*02:01-bound NY-ESO-1157-165 peptide, were generated from stably expressing e293T-producing cells, as previously described. Human PBMCs (activated with anti-CD3 / CD28) were transduced with 10 μL of enriched eLV, and the transduction efficiency in CD3+ T cells was evaluated on days 3, 7, 14, and 21 by flow cytometry using PE-bound A2 / NY-ESO-1 polymers. On day 17 post-infection, transduced lymphocytes were counted and co-cultured in a 1:1 ratio with lymphoblastic BFP-positive HLA-A*02:01-positive T2 cells pulsed with 1 μg / mL of control HIV peptide (HIV476-484 amino acid sequence: ILKEPVHGV) or NY-ESO-1157-165 peptide (SLLMWITQC). After 72 hours, cells were collected and analyzed for viability and cell count by flow cytometry.

[0592] As shown in Figures 49A and 49B, transduction of activated hPBMCs with CD8a or TCR VHH-targeted LVGP1x eLV encoding NY-ESO resulted in specific and stable transduction of CD8+ T cells or CD3+ T cells, respectively. As previously demonstrated for CARs expressing eLV, restricting the expression of the NY-ESO1 transgene during virus production using a T cell synthesis promoter (SYN21, SEQ ID NO: 12) significantly increased the functional titer of the vector compared to EF1a-driven transgene expression. The transduction efficiency of the EF1a NY-ESO construct was approximately 1 log lower than that of the SYN NY-ESO construct.

[0593] After 72 hours, T2 cells pulsed with the control HIV peptide were recovered from co-culture with either non-transduced (UT) cells or NY-ESO-1 transduced cells (Figure 50B). However, T2 cells pulsed with the NY-ESO-1 peptide were recovered only in the UT group, and to some extent in the EF1a promoter group (which represented less than 5% of total CD3+ T cells expressing the NY-ESO TCR). Targeted NY-ESO pulsed T2 cells were not recovered from co-culture with LVGP1x-VHHTCR SYN NY-ESO transduced cells (Figure 50A). These results demonstrate that T cells transduced with VHH-targeted eLV encoding the manipulated TCR construct are functional and highly cytotoxic to target cells.

[0594] Example 38: TCRab VHH G1x eLV specifically delivers BCMA CAR constructs to T cells in vivo, generating functional BCMA CAR T cells and eliminating systemically engrafted multiple myeloma cell lines in a humanized mouse model. This example describes the in vivo transduction efficiency of TCRab VHH G1x eLV in humanized mice, resulting in the in vivo production of functional BCMA CAR-T cells and the elimination of a target cancer cell population.

[0595] In NSG MHCI / II KO mice, 5e6 NCI-H929 cells were intravenously injected and allowed to engraft for 7 days. Activated human PBMCs were then intraperitoneally injected, followed one day by intraperitoneal injection of TCRab VHH G1x eLV, which encodes a BCMA CAR construct under the control of the T cell-specific synthesis promoter SYN23 vector. BCMA CAR T cells reprogrammed in vivo were analyzed weekly in peripheral blood and bone marrow at euthanasia 42 days after vector injection. The antitumor activity of BCMA CAR-T cells was evaluated weekly by bioluminescence imaging, body weight, and clinical observation of luciferase-expressing NCH-H929 cells.

[0596] As shown in Figure 51A, intraperitoneal injection of the ENaBL-GTT vector was well tolerated without any adverse events observed during the study. Tumor growth began exponentially from day 8 in animals injected with the control TCRab VHH G1x eLV BFP vector, but TCRab VHH G1x eLV BCMA CAR injection resulted in the elimination of tumor cells 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). The persistence of BCMA CAR T cells was observed throughout this study.

[0597] The study demonstrates that the TCRab VHH G1x eLV BCMA CAR vector reprograms BCMA CAR T cells in vivo, resulting in the complete elimination of systemically engrafted multiple myeloma cell line (NCI-H929) in hPBMC humanized mice.

[0598] Example 39: TCRab VHH G1x eLV specifically delivers BCMA CAR constructs to T cells in vivo, generating functional BCMA CAR T cells and eliminating systemically engrafted multiple myeloma cell lines in a humanized mouse model. This example describes the in vivo T-cell transduction specificity and effects of TCRab VHH G1x eLV in stably humanized mice carrying systemic multiple myeloma tumors, as well as its ability to induce in vivo production of functional BCMA CAR-T cells and elimination of the target cancer cell population.

[0599] The TCRab VHH G1X eLV vector encoding a BCMA CAR transgene (or BFP reporter) under the control of the T cell-specific synthesis promoter SYN23 was administered as a single intravenous infusion to hCD34+ NCG mice carrying multiple myeloma tumors via injection of 5e6 NCI-H929, which stably expresses luciferase. The antitumor effect was evaluated by monitoring NCI-H929 cell proliferation (in vivo bioluminescence imaging), animal body weight, clinical status, and survival. In addition, the transduction dynamics of TCRab VHH G1X eLV BCMA CAR and the cellular dynamics of transduced cells were analyzed by flow cytometry of peripheral blood. Flow cytometry of the spleen, bone marrow, and tumors was also performed to identify the presence and status of CAR T cells in these tissues.

[0600] Tumor growth was not controlled in animals treated with TCRab VHH G1X eLV BFP or vehicle, but mice injected with TCRab VHH G1x eLV BCMA CAR had tumors eradicated in 2 / 8, 8 / 8, and 7 / 8 mice in the low-dose, medium-dose, and high-dose groups by 35 days post-tumor injection (Figure 52). As shown in Figure 53, transduction of TCRab VHH G1X eLV BCMA CAR was specific to 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 CAR levels above background.

[0601] This study demonstrates (1) the effective antitumor activity of T cells reprogrammed with TCRab VHH G1X eLV BCMA CAR in vivo, and (2) the specificity of the TCRab VHH G1X eLV vector to T lymphocytes in vivo.

[0602] Example 40: The combination of T lymphocyte and monocyte reprogramming works synergistically to eliminate solid tumors in humanized mice. This example describes the in vivo administration of a T cell-specific TCRab VHH G1x eLV BCMA CAR vector in combination with a human IL12-expressing monocyte-specific CD14 VHH G1x eLV, and the synergistic antitumor activity of this vector combination against NCI-H929 cells subcutaneously transplanted to simulate solid tumors.

[0603] hCD34+ NCG mice harboring multiple myeloma tumors in solid tumor form, induced by subcutaneous injection of 5e6 NCI-H929, were intravenously injected with either (1) a monocyte-specific CD14 VHH G1x eLV vector expressing human IL12 or a GFP reporter under the control of the constitutive EF1a promoter, (2) a TCRab VHH G1X eLV vector encoding a BCMA CAR transgene under the control of the T cell-specific synthetic promoter 23, or (3) the same dose of CD14 VHH G1x eLV hIL12 and TCRab VHH G1X eLV BCMA CAR vector in a single combined injection. Antitumor effects were evaluated by monitoring NCI-H929 cell proliferation (calipass measurement of subcutaneous solid tumor volume) and animal survival. In addition, levels of BCMA CAR-T cells and human IL12 in peripheral blood transdextrose cells were analyzed by flow cytometry.

[0604] As shown in Figure 54, tumor growth was not controlled or was poorly controlled in animals injected with TCRab VHH G1x eLV BCMA CAR, CD14 VHH G1x eLV hIL12, or CD14 VHH G1x eLV GFP vector alone. In contrast, NCI-H929 tumor cells were eliminated by a single combined administration 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 group and the combined group of TCRab VHH G1x eLV BCMA CAR and CD14 VHH G1x eLV hIL12, and the proportion of BCMA CAR-T cells was significantly increased in the combined group. As shown in Figure 55B, human IL12 was detected only in animals injected with CD14 VHH G1x eLV hIL12. Circulating IL12 levels remained ≥40000 pg / mL at the end of the study (data not shown). The effect of this combination treatment was significant in the level of T cell infiltration within the tumor. As shown in Figure 55B, the combination of T cell reprogramming with BCMA CAR and monocyte reprogramming with secretible IL12 induced a significant increase in T cell tumor infiltration.

[0605] This study demonstrates that (1) intravenous administration of CD14 VHH G1x eLV hIL12 results in sustained secretion of high levels of hIL12, suggesting sustained 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-targeted vector; and (3) in vivo reprogramming of multiple cell types with enhanced antitumor activity synergistically eliminates tumors that would otherwise be resistant to single-operated cell types.

[0606] Example 41: Presenting a complement activity regulator on the viral envelope of a VHH-targeted eLV reduces complement-mediated inactivation of the vector particle. The transduction efficiency of a lentiviral vector injected systemically is limited by host virus inactivation mechanisms, including complement-mediated inactivation. This example describes manipulating the TCRab VHH G1x eLV vector envelope to present an exogenous inhibitor or resistance factor that reduces complement-mediated inactivation of the viral vector.

[0607] TCRab VHH-targeted G1x eLVs presenting complement activity regulators (RCAs) were produced, as previously described, by transient transfection of e293T-producing cells stably expressing TCRab VHH and either (1) DAF(CD55) or (2) factor H, both RCAs being anchored to the envelope via fusion to the transmembrane domain of GPI anchors or VSVG (SEQ ID NOs. 183-186, Figure 56A).

[0608] Complement-mediated inactivation of vector particles was evaluated for 1 hour using (1) serum-free (complement-free) medium, (2) medium supplemented with fresh human serum (active complement), or (3) medium complement containing heat-inactivated human serum (inactivated complement). Jurkat cells were then incubated with lentivirus preparations for 2 hours, and the transduction efficiency of the vector was evaluated after 72 hours to quantify the percentage of functional titer recovery of viral particles incubated in the presence of serum. As shown in Figure 56B, TCRab VHH G1x eLV presenting DAF-GPI and factor H (GPI or GS) was partially protected against complement-mediated inactivation, with mean recovery rates increased by up to 24.1% and 16.3%, respectively. This result demonstrates the protective effect of RCA against vector complement-mediated inactivation.

[0609] Example 42: Presenting an albumin-binding domain on the viral envelope of a VHH-targeted eLV reduces complement-mediated inactivation of the vector particle. Albumin-binding domains have long been used to prolong the pharmacokinetics of intravenously injected therapeutic agents and reduce phagocytosis. This example describes the manipulation of a TCRab VHH G1x eLV vector envelope to present an albumin-binding domain in order to reduce vector phagocytosis.

[0610] TCRab VHH-targeted G1x eLVs, which display the albumin-binding domain to protein G(ABD) of Streptococcus strain G148, were produced using transient transfection of e293T-producing cells stably expressing TCRab VHH and ABD, which are anchored to the envelope via fusion to a GPI anchor or the transmembrane domain of VSVG (SEQ ID NOs. 192 and 191, respectively), as previously described.

[0611] The phagocytosis of vector particles was evaluated by incubating vector preparations with fresh human monocyte-derived macrophages for 2 hours and quantifying vector insertion on day 3 after exposure. Chlorpromazine, a phagocytosis inhibitor, was used as a negative control. As shown in Figure 57, the TCRab VHH G1x eLV vector was phagocytosed by primary macrophages, resulting in the integration of the vector into host cell genomic DNA, which is inhibited by chloropromazine. By presenting ABD on the envelope of the TCRab G1x eLV, the phagocytosis of the lentiviral article was reduced, resulting in a significant decrease in the number of viral genomes per macrophage. This result demonstrates the protective effect of ABD against vector phagocytosis.

[0612] Example 43: A synthetic promoter constructed using an enhancer of a gene preferentially expressed in tumor-associated macrophages compared to tissue-resident macrophages can be induced by a signal specific to the tumor microenvironment. This example describes the expression of reporter transgenes from synthetic promoters that can be induced by signals related to the tumor microenvironment. Twelve tumor-associated macrophage (TAM) specific promoters were constructed using enhancers derived from genes preferentially expressed in TAMs, as described in Example 23 (see Sequence IDs 141-152).

[0613] VSVG eLVs expressing a GFP / luciferase reporter construct under the control of EF1a or a different synthetic promoter were generated in e293T-producing cell lines by transient transfection, as previously described. Primary human monocytes were transduced with different VSVG eLVs and cultured for 7 days, followed by 2 days in medium supplemented with TME-related signals. These TME-related signals were either (1) IL4, IL10, and TGFb, or (2) CoCl2 (to induce hypoxic signaling), or (3) a condition medium derived from the gastric cancer cell line NUGC4. After 2 days of culture, the level of reporter (GFP) expression was evaluated by flow cytometry.

[0614] As shown in Figure 58, reporter expression from the EF1a promoter decreases when cells are exposed to hypoxia or the conditional medium, while expression driven by the TAM synthesis promoter increases, particularly for the TAM 12 promoter.

[0615] In summary, these results demonstrate that a target-construction TAM synthesis promoter, sized for integration into lentiviral constructs, can be induced by TME-related signals, thus providing a strategy for restricting transgene expression to the tumor microenvironment.

Claims

1. A viral vector having a lipid bilayer envelope, (a) A cell-type specific VHH-binding domain presented on the outside of the envelope, (b) A VSV-G envelope protein presented on the outside of the envelope, which can promote infection of the same cell type as in (a), wherein the VSV-G envelope protein comprises an amino acid substitution at position 331, (c) A viral vector comprising a nucleic acid molecule containing a promoter expressible in the same cell type as in (a).

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

3. The viral vector according to claim 1 or 2, wherein the VSV-G mutation at position 331 is selected from I331E, I331W, I331M, I331L, I331Q, and I331R.

4. The viral vector according to any one of claims 1 to 3, wherein the VSV-G mutation at position 331 is I331E.

5. A further VSV-G mutation is provided at position I52, according to any one of claims 1 to 4, in the viral vector.

6. A further VSV-G mutation is provided at position E355 in the viral vector according to any one of claims 1 to 5.

7. A further VSV-G mutation is provided at the N9 position in the viral vector according to any one of claims 1 to 6.

8. A further VSV-G mutation is provided at the K47 position in the viral vector according to any one of claims 1 to 7.

9. A further VSV-G mutation is provided at position T352, according to any one of claims 1 to 8, in the viral vector.

10. A further VSV-G mutation is provided at the H8 position in the viral vector according to any one of claims 1 to 9.

11. A further VSV-G mutation is provided at the M45 position in the viral vector according to any one of claims 1 to 10.

12. Further VSV-G mutations are provided at the K11 position in the viral vector according to any one of claims 1 to 11.

13. Further VSV-G mutations are provided at position S408 in the viral vector according to any one of claims 1 to 12.

14. Further VSV-G mutations are provided at position R354, according to any one of claims 1 to 13, in the viral vector.

15. Further VSV-G mutations are provided at position Q53 in the viral vector according to any one of claims 1 to 14.

16. Further VSV-G mutations are provided at position E353 in the viral vector according to any one of claims 1 to 15.

17. Further VSV-G mutations are provided at position Q10, according to any one of claims 1 to 16, in the viral vector.

18. Further VSV-G mutations are provided at position K50, according to any one of claims 1 to 17, in the viral vector.

19. The viral vector according to any one of claims 1 to 18, wherein the mutant VSV-G envelope protein comprises mutants I52R, I331E, and E355L.

20. The viral vector according to any one of claims 1 to 19, wherein the mutant VSV-G envelope protein comprises mutants N9G, K47G, I331E, and T352S.

21. The viral vector according to any one of claims 1 to 20, wherein the mutant VSV-G envelope protein comprises mutants H8L, M45L, I331E, and E355R.

22. The viral vector according to any one of claims 1 to 21, wherein the mutant VSV-G envelope protein comprises mutants Q53A, I331E, and T352I.

23. The viral vector according to any one of claims 1 to 22, wherein the mutant VSV-G envelope protein comprises mutants K11A, S408L, I331L, and R354K.

24. The viral vector according to any one of claims 1 to 23, wherein the mutant VSV-G envelope protein comprises mutants N9R, Q53I, I331Q, and R354M.

25. The viral vector according to any one of claims 1 to 24, wherein the mutant VSV-G envelope protein comprises mutants I331E and E353K.

26. The viral vector according to any one of claims 1 to 25, wherein the mutant VSV-G envelope protein comprises mutants Q10V, K50H, I331M, and T352D.

27. The viral vector according to any one of claims 1 to 26, wherein the mutant VSV-G envelope protein comprises mutants N9A, I331E, and E353S.

28. The viral vector according to any one of claims 1 to 27, wherein the VHH-binding domain and the promoter are specific for use in immune cells.

29. The viral vector according to claim 28, wherein the immune cells are selected from T cells, natural killer (NK) cells, and / or macrophages.

30. The viral vector according to any one of claims 1 to 29, wherein the viral vector is a retroviral vector, for example, a lentiviral vector.

31. The viral vector according to any one of claims 1 to 30, wherein the viral envelope substantially does not contain or lacks MHC class I.

32. The viral vector according to any one of claims 1 to 31, wherein the viral vector presents high levels of CD47, agonist-active CD3 scFv, 41BBL, and / or CD80 on the surface of the envelope.

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

34. The viral vector according to any one of claims 1 to 33, wherein the VHH-binding domain is specific to CD3ε, CD8, or T cell receptor (TCR).

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

36. The nucleic acid molecule encodes a transgene, as described in any one of claims 1 to 35.

37. The viral vector according to any one of claims 1 to 36, wherein the nucleic acid molecule encodes a chimeric antigen receptor (CAR).

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

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

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

41. The viral vector according to any one of claims 1 to 40, wherein the promoter is a synthetic promoter comprising a promoter element and an enhancer element.

42. A viral vector according to any one of claims 1 to 41, for use in the diagnosis, prevention, or treatment of a disease.

43. The disease is cancer, the viral vector for use according to claim 42.

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

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

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

47. A method for producing a viral vector according to any one of claims 1 to 41, (a) A step of manipulating cells to express a cell-type specific membrane-anchored VHH-binding domain, (b) A step of manipulating the cells to express a VSV-G envelope protein presented on the outside of the envelope that can promote infection of the same cell type as in (a), wherein the VSV-G envelope protein includes an amino acid substitution at position 331, (c) A step of replicating a nucleic acid vector containing a promoter capable of expressing the same cell type as in (a) within the cell, (d) A method comprising the step of recovering the viral vector from the cells in the form of vesicles.

48. The method according to claim 47, wherein the cells are a cell line derived from 293T.

49. The method according to claim 47 or claim 48, wherein the cells are modified to express or overexpress CD47.

50. The method according to any one of claims 47 to 49, wherein the cells are modified so that the expression of MHC class I is knocked out.

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

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

53. The method according to claim 52, wherein the modification is carried out in vivo or ex vivo, preferably in vivo.

54. The method according to claim 52 or claim 53, wherein the cells to be modified are T cells.

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

56. The method according to claim 54, wherein the VHH binds to the T cell via CD3ε.

57. The method according to claim 52 or claim 53, wherein the cells to be modified are monocytes or macrophages.

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

59. The method according to claim 52 or claim 53, wherein the cells to be modified are NK cells.

60. The method according to claim 59, wherein the VHH binds to the NK cells via CD16, CXCR4, or CCR1.

61. Virus-like particles (VLPs) having a lipid bilayer envelope, (a) A cell-type specific VHH-binding domain presented on the outside of the envelope, (b) A virus-like particle (VLP) comprising a VSV-G envelope protein presented on the outside of the envelope, which can promote infection of the same cell type as in (a), wherein the VSV-G envelope protein includes one or more mutant amino acid substitutions at position 331.

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