Recombinant viral particles

Abstract

Fusion proteins comprising a T cell targeting element and a co-stimulatory molecule and combinations thereof are provided. VSVG virus glycoprotein variants are provided. Also provided is a recombinant virus particle comprising the fusion protein and a VSVG variant. Further provided are uses of the recombinant viral particles for in vivo immune cell therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to recombinant viral particles for in vivo immune cell therapy. Background Technology

[0002] In vivo CAR-T therapy is an engineered technology that uses T-cell targeting vectors to precisely transduce nucleic acid elements encoding chimeric antigen receptors (CARs) into T cells within the recipient's body. This technology directly transforms the recipient's own T cells into CAR-T cells within the body, avoiding the cumbersome procedures of in vitro preparation and addressing the potential immunogenicity issues of existing universal CAR-Ts, possessing the excellent characteristics of being "affordable, ready to use, and universal." However, the in vivo transduction environment is complex, and the efficacy of in vivo CAR-T therapy is critically influenced by the targeting specificity of the vector to T cells, as well as the efficiency of T-cell activation and transduction.

[0003] T cells possess various receptors and adhesion molecules on their surface, capable of generating co-stimulatory signals and playing important auxiliary roles in immune cell activation and killing functions. Among these, CD80 / CD86 is considered one of the most important co-stimulatory molecules. A phase I clinical trial of in vivo in situ CAR-T using the gene delivery platform VivoVec has been reported. This platform is based on a third-generation lentiviral vector with an envelope modified by a co-viral fusion glycoprotein and a membrane-binding fusion protein. The membrane-binding fusion protein, formed by the fusion of the CD58 extracellular domain, a single-stranded variable fragment of CD3, and the full-length CD80 via a GS repeat sequence, is considered to provide key T cell tropism and activation signals (see, for example, PCT international application publication number WO2023 / 215848). The effects of using CD86 and other T cell co-stimulatory molecules as T cell targeting elements are still unclear.

[0004] The viral fusion protein VSVG plays a crucial role in the fusion of recombinant viral vectors into target immune cells in vivo. The COCAL glycoprotein, derived from another member of the vesicular virus genus within the rhabdomyovirus family, also exhibits similar effects to VSVG. Both COCAL and VSVG bind to the low-density lipoprotein receptor (LDLR), which is widely distributed on the surface of various cells. K47Q and R354A mutations have been reported to eliminate the extensive binding of VSVG to the LDLR but retain its ability to fuse with the cell membrane (Nikolic J, Belot L et al., Nat Commun. 2018 Mar 12; 9(1):1029, PMID:29531262). Summary of the Invention

[0005] In vivo, the vast majority of T lymphocytes in peripheral blood are in a resting state and have not yet been activated by antigens. Their LDLR levels on the cell surface are extremely low. Therefore, lentiviral vectors based on pseudoglycoprotein-mediated envelope fusion such as VSVG are extremely difficult to bind effectively to resting T cells and carry out subsequent infection. Furthermore, unlike in vitro pure cultures, blood cells contain only a very small number of T lymphocytes. If the viral vector cannot efficiently transduce T cells, or cannot rapidly induce the transduced T cells to enter a proliferative state to avoid T cell depletion, then a therapeutically effective quantity of CAR-T cells cannot be generated in vivo to achieve tumor-killing effects.

[0006] The inventors of this application constructed a series of fusion protein molecules assembled from different T cell adhesion molecules and co-stimulatory molecules, and tested their effects on viral packaging titer, T cell infection efficiency and activation degree of recombinant virus, as well as the killing activity of transduced T cells on target cells. The results showed that these fusion protein molecules can simultaneously exert the effects of targeting T cells and activating T cells.

[0007] The inventors of this application have also discovered that using different fusion proteins, especially fusion proteins containing different co-stimulatory signaling elements, is more conducive to synergistic targeting and activation of T cells, thereby making the recombinant virus significantly superior to the expression of a single fusion protein in terms of transduction efficiency, activation efficiency, and / or killing activity.

[0008] The inventors of this application have also discovered a novel variant of the viral glycoprotein VSVG, which exhibits low LDLR binding activity. When used in combination with molecules targeting specific cells (such as T cells), it can effectively improve the targeting and transduction activity of enveloped viral vectors.

[0009] Therefore, a first aspect of the present invention provides a T-cell-targeting fusion protein comprising two or more elements directly or indirectly linked, said elements being selected from any one of CD58 or its active fragment, OX40L or its active fragment, an antibody or ligand of CD3, an antibody or ligand of CD7, an antibody or ligand of CD4, an antibody or ligand of CD8, IL-2, CD80, CD86 or CD275.

[0010] In some embodiments, the fusion protein includes CD58 or an active fragment thereof as the extracellular domain of CD58. In a preferred embodiment, the CD58 extracellular domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:1. In some embodiments, the CD58 extracellular domain has the amino acid sequence described in SEQ ID NO:1.

[0011] In some embodiments, the fusion protein includes IL-2 which comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:96. In some embodiments, IL-12 has the amino acid sequence described in SEQ ID NO:96.

[0012] In some embodiments, the fusion protein comprises an OX40L active fragment that is the OX40L extracellular domain. In a preferred embodiment, the OX40L active fragment comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:97. In some embodiments, the OX40L active fragment has the amino acid sequence described in SEQ ID NO:97.

[0013] In some embodiments, the CD3 antibody included in the fusion protein is an activating antibody or an endocytosis-promoting antibody. In some embodiments, the CD3 antibody included in the fusion protein has the following amino acid sequences: HCDR1 as described in SEQ ID NO:3, HCDR2 as described in SEQ ID NO:4, HCDR3 as described in SEQ ID NO:5, LCDR1 as described in SEQ ID NO:6, LCDR2 as described in SEQ ID NO:7, and LCDR3 as described in SEQ ID NO:8. In a preferred embodiment, the CD3 antibody includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:2. In some embodiments, the CD3 antibody has the amino acid sequence described in SEQ ID NO:2. In some embodiments, the fusion protein comprises a CD3 antibody having HCDR1 as described in SEQ ID NO:121, HCDR2 as described in SEQ ID NO:122, HCDR3 as described in SEQ ID NO:123, LCDR1 as described in SEQ ID NO:124, LCDR2 as described in SEQ ID NO:125, and LCDR3 as described in SEQ ID NO:126. In a preferred embodiment, the CD3 antibody comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:101. In some embodiments, the CD3 antibody has the amino acid sequence described in SEQ ID NO:101.

[0014] In some embodiments, the CD7 antibody included in the fusion protein is an activating antibody or a endocytosis-promoting antibody. In some embodiments, the CD7 antibody included in the fusion protein has the following amino acid sequences: HCDR1 as described in SEQ ID NO:103, HCDR2 as described in SEQ ID NO:104, HCDR3 as described in SEQ ID NO:105, LCDR1 as described in SEQ ID NO:106, LCDR2 as described in SEQ ID NO:107, and LCDR3 as described in SEQ ID NO:108. In a preferred embodiment, the CD7 antibody comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:102. In some embodiments, the CD7 antibody has the amino acid sequence described in SEQ ID NO:102.

[0015] In some embodiments, the CD7 ligand is K12 or an active fragment thereof. In a preferred embodiment, the K12 active fragment is the extracellular domain of K12. In a more preferred embodiment, the K12 active fragment comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:98. In some embodiments, the K12 active fragment has the amino acid sequence described in SEQ ID NO:98.

[0016] In some embodiments, the CD8 antibody included in the fusion protein is an activating antibody or an endocytosis-promoting antibody. In some embodiments, the CD8 antibody included in the fusion protein has the following amino acid sequences: HCDR1 as described in SEQ ID NO:160, HCDR2 as described in SEQ ID NO:161, HCDR3 as described in SEQ ID NO:162, LCDR1 as described in SEQ ID NO:163, LCDR2 as described in SEQ ID NO:164, and LCDR3 as described in SEQ ID NO:165. In a preferred embodiment, the CD8 antibody comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:158. In some embodiments, the CD8 antibody has the amino acid sequence described in SEQ ID NO:158.

[0017] In some embodiments, the fusion protein includes CD80 which comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:94. In some embodiments, CD80 has the amino acid sequence described in SEQ ID NO:94.

[0018] In some embodiments, the fusion protein includes CD86 which comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:9. In some embodiments, CD86 has the amino acid sequence described in SEQ ID NO:9.

[0019] In some embodiments, the fusion protein includes CD275, which comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:95. In some embodiments, CD275 has the amino acid sequence described in SEQ ID NO:95.

[0020] In some embodiments, the elements comprising the T-cell-targeting fusion protein are covalently linked by a linker. In some embodiments, the linker is a flexible linker. In some embodiments, the flexible linker is a GS-type linker. In some embodiments, the flexible linker has an amino acid sequence as described in any one of SEQ ID NO:7, 85 to 89, or 109.

[0021] In some embodiments, the T-cell targeting fusion protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of any one of SEQ ID NO: 11, 19 to 50, 139 to 148. In some embodiments, the T-cell targeting fusion protein comprises an amino acid sequence of any one of SEQ ID NO: 11, 19 to 50, 139 to 148 with one or more deleted, substituted, and / or added amino acid sequences. In some embodiments, the number of deleted, substituted, and / or added amino acids is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a preferred embodiment, the T-cell targeting fusion protein has an amino acid sequence as described in any one of SEQ ID NO: 11, 19 to 50, 139 to 148.

[0022] A second aspect of this disclosure provides recombinant enveloped viral particles that express one or more T-cell-targeting fusion proteins of this disclosure.

[0023] In some embodiments, the recombinant viral particles comprise at least two different fusion proteins described above. In some embodiments, the recombinant viral particles comprise two or more amino acid sequences selected from SEQ ID NO: 11, 19 to 50, 139 to 148. In some embodiments, the two or more amino acid sequences are expressed separately by independent nucleic acid constructs (e.g., plasmids) or by different expression cassettes within the same nucleic acid construct. In some embodiments, the recombinant viral particles comprise fusion protein A having the amino acid sequence shown in SEQ ID NO: 29 and fusion protein B having the amino acid sequence shown in SEQ ID NO: 37. In some embodiments, the recombinant viral particles comprise fusion protein A having the amino acid sequence shown in SEQ ID NO: 29 and fusion protein B having the amino acid sequence shown in SEQ ID NO: 44. In some embodiments, the recombinant viral particles comprise fusion protein A having the amino acid sequence shown in SEQ ID NO: 29 and fusion protein B having the amino acid sequence shown in SEQ ID NO: 45. In some embodiments, the recombinant viral particles comprise fusion protein A having the amino acid sequence shown in SEQ ID NO: 29 and fusion protein B having the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the recombinant viral particles comprise fusion protein A having the amino acid sequence shown in SEQ ID NO:29 and fusion protein B having the amino acid sequence shown in SEQ ID NO:43.

[0024] In some embodiments, the recombinant viral particles of this disclosure further comprise VSVG viral glycoprotein variants, including mutants K47Q and R354A, and also include any one selected from K66T, S162T, or T230N.

[0025] In some embodiments, the mutation position of the VSVG viral glycoprotein variant is determined relative to SEQ ID NO:138. In some embodiments, the VSVG viral glycoprotein variant comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from any of SEQ ID NO:17, 128 to 132. In a preferred embodiment, the VSVG viral glycoprotein variant has an amino acid sequence selected from any of SEQ ID NO:17, 128 to 132.

[0026] In some embodiments, the recombinant enveloped viral particle further comprises a payload. In some embodiments, the payload comprises a nucleotide sequence encoding a target polypeptide and / or a nucleotide sequence encoding a non-coding RNA. In some embodiments, the target polypeptide is selected from chimeric antigen receptors (CARs), fluorescent proteins, antibodies, or gene-editing enzymes. In some embodiments, the gene-editing enzyme is selected from zinc finger nucleases or Cas proteins. In some embodiments, the non-coding RNA is selected from siRNA, miRNA, shRNA, or sgRNA. In some embodiments, the chimeric antigen receptor comprises an antigen-binding domain specifically targeting any cancer-associated antigen selected from CD19, BCMA, GPRC5D, ROR1, FcRL5, alpha-fetoprotein, Her2, MUC1, or GPC3. In a preferred embodiment, the chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO:83.

[0027] In some implementations, the recombinant enveloped viral particles are lentiviral particles or retroviral particles.

[0028] A second aspect of this disclosure provides a pharmaceutical composition comprising the recombinant viral particles of this disclosure and a pharmaceutically acceptable carrier.

[0029] A third aspect of this disclosure provides a novel T-cell-targeting fusion protein and its uses. The T-cell-targeting fusion protein comprises two or more selected from CD58 or its active fragment, OX40L or its active fragment, an antibody or ligand for CD3, an antibody or ligand for CD7, an antibody or ligand for CD4, an antibody or ligand for CD8, IL-2, CD80, CD86, or CD275. Its uses are selected from one or more of the following: A. prolonging the half-life of recombinant viral particles or transgenic immune cells generated therefrom; B. increasing the safety of recombinant viral particles or transgenic immune cells generated therefrom; and / or C. enhancing the tumor-killing activity of recombinant viral particles or transgenic immune cells generated therefrom.

[0030] In some implementations, the immune cells are selected from T cells or NK cells.

[0031] A fourth aspect of this disclosure provides novel VSVG viral glycoprotein variants and their uses. The VSVG viral glycoprotein variants disclosed herein comprise mutants K47Q and R354A, and also comprise any one selected from K66T, S162T, or T230N. The VSVG variants disclosed herein can be used to increase the infection efficiency of recombinant viruses on immune cells, increase the infection specificity of recombinant viruses on immune cells, and / or increase serum tolerance to recombinant viruses.

[0032] A fifth aspect of this disclosure provides a method for transducing a population of T cells in a subject, comprising administering a viral particle or a pharmaceutical composition of this disclosure to a subject in need. In some embodiments, the subject's T cell population expresses the payload contained in the viral particle after administration.

[0033] A sixth aspect of this disclosure provides a method for treating a disease or condition in a subject in need, comprising administering to the subject the viral particles of this disclosure or the pharmaceutical composition of this disclosure.

[0034] The seventh aspect of this disclosure provides the use of viral particles in the preparation of a medicament for treating a disease or condition, wherein in some embodiments, the disease or condition is selected from any one of acute lymphoblastic leukemia, large B-cell lymphoma, multiple myeloma, relapsed / refractory B-cell non-Hodgkin lymphoma, gastric cancer / gastroesophageal junction cancer, liver cancer, pancreatic cancer, biliary tract cancer, malignant pleural mesothelioma / lung cancer, refractory neurological autoimmune diseases (such as multiple sclerosis, myasthenia gravis), or systemic lupus erythematosus. Attached Figure Description

[0035] Figure 1 This demonstrates the infection of hPBMC cells with 250 ng of different recombinant viruses (initial seeding volume 1 × 10⁻⁶). 5 The percentage of GFP+CD3 double-positive cells, GFP+CD7 double-positive cells, or GFP+CD3+CD7 triple-positive cells in the total number of cells on day 2 after (cells / well).

[0036] Figure 2 This demonstrates the infection of hPBMC cells with 250 ng of different recombinant viruses (initial seeding volume 1 × 10⁻⁶). 5 On day 2 after loading (cells / well), the differences in the number of GFP+CD3 double-positive cells, GFP+CD7 double-positive cells, or GFP+CD3+CD7 triple-positive cells were observed. In the figure, the Y-axis represents the number of cells contained in each μl of flow cytometry loading solution.

[0037] Figure 3 This demonstrates the infection of hPBMC cells with 50 ng of different recombinant viruses (initial seeding volume 5 × 10⁶). 4 The percentage of GFP and CD3 double-positive cells in the total number of cells on day 6 after (cells / well);

[0038] Figure 4 and Figure 5 This demonstrates the infection of hPBMC cells with 50 ng of different recombinant viruses (initial seeding volume 5 × 10⁶). 4 The percentage of GFP and CD3 double-positive cells in the total number of cells on day 4 after (cells / well);

[0039] Figure 6 This demonstrates the infection of hPBMC cells with 50 ng of different recombinant viruses (initial seeding volume 5 × 10⁶). 4 The percentage of GFP-positive cells in the total number of cells on day 4 after (cells / well);

[0040] Figure 7 The results show the infection of hPBMC cells with 1690 ng of recombinant virus (initial seeding volume 1×10⁻⁶). 6 The percentage of GFP-positive cells in the total number of cells on day 4 after (cells / well);

[0041] Figure 8 The copy number of the CAR gene in the genome of 100 ng CAR-T cells after infection with a recombinant lentivirus carrying CD19CAR as the payload is shown.

[0042] Figure 9 The exemplary CD19-CAR-T cells of this disclosure exhibit multiple rounds of cytotoxic activity against the B-lymphoblastic leukemia cell line Nalm-6-Luciferase. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments.

[0044] definition

[0045] As used in the specification of this invention, the following words and phrases are generally considered to have the meanings set forth below, unless otherwise specified in the context in which they are used.

[0046] As used herein, the terms “comprising” or “including” are used interchangeably and are open-ended, meaning that one or more items mentioned after the term are not an exhaustive list of this or those items, nor are they limited to the listed items. “Mainly composed of”, when used to define compositions and methods, should exclude any other components that are obviously important to the composition. Therefore, compositions defined herein as mainly composed of these components will not exclude trace contamination from separation and purification methods and pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, etc. “Composed of” should exclude trace components of other ingredients used in the application of the compositions of the invention and substantial methods of application. Examples defined by these provisional terms are all within the scope of this invention.

[0047] Unless otherwise stated herein, the listing of numerical ranges herein is intended only as a shorthand method for individually referring to each individual value falling within that range, and each individual value is incorporated into the specification as if it were listed separately herein. For example, in this specification, if the concentration range is expressed as 1 to 10 μM, it is intended to explicitly list values ​​such as 2 to 9 μM, 5 to 6 μM, or 1 to 5 μM. These are merely examples of specific intentions, and all possible combinations of values ​​between and including the listed minimum and maximum values ​​will be considered explicitly stated in this disclosure. The use of the word “about” to describe a particular listed quantity or range of quantities means indicating that a value very close to the listed quantity is included in that quantity, such as values ​​that can or naturally be considered due to manufacturing tolerances, instrumental and human errors in the formation of the measurement. For example, a value described herein as “about” means covering ±10% of the indicated value. In some cases, “about” may mean ±20%, or ±5%, or ±1%. A value or parameter described herein as “about” includes the value or parameter itself.

[0048] As used in this article, the singular form “one” or “this” includes a plural referent unless the context clearly indicates otherwise.

[0049] As used herein, the term "viral particle" refers to a viral particle consisting of at least one viral capsid protein and a capsid-encapsulated polynucleotide vector. If the particle contains a payload to be delivered to a target cell, it may also be referred to as a "viral vector particle." In some embodiments, the viral particle used herein may be selected from, for example, lentiviral particles or retroviral particles, with lentiviral particles being preferred.

[0050] As used herein, the term "fusion protein" refers to the use of genetic expression or protein synthesis methods that encode protein polynucleotides to linearly link two or more proteins or fragments thereof (such as domains) through their respective peptide backbones.

[0051] CD86, a member of the B7 family, interacts with CD28 to activate T cells, promoting their proliferation, differentiation, and cytokine secretion, thus initiating the initial immune response. However, its interaction with the CTLA-4 receptor inhibits T cell activation, helping to maintain immune tolerance (Greenwald RJ et al., The B7 family revisited. Annu Rev Immunol. 2005; 23:515-48). Compared to CD80, another member of the family, CD86 binds to CD28 more quickly (rapid response), but with lower affinity (KD–4 μM).

[0052] As used herein, the term "extracellular domain" refers to a region of a membrane protein (e.g., a transmembrane protein) located outside the cell membrane. Extracellular domains often contain binding domains that specifically bind ligands or cell surface receptors. The term "extracellular binding domain" refers to an extracellular domain or a portion of an extracellular domain capable of specifically binding to a target protein.

[0053] As used herein, the term "flexible linker" refers to an amino acid sequence that connects domains to provide a degree of movement or interaction. Such linkers are typically composed of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, but may also contain polar amino acids such as Lys and Glu, for example, to improve solubility. The small size of these amino acids provides flexibility and allows the connected functional domains to be mobile. As a non-limiting example, the flexible linker described herein may be, for example, an amino acid sequence having 1 to 50, preferably 1 to 30, such as 1 to 15 amino acid residues. Those skilled in the art can determine suitable linkers for use in antibodies of the present invention based on the disclosure herein, optionally after limited conventional experiments. In some instances, the linker comprises a (GlyxSeryXAAz)n type linker, where x is any integer from 1 to 4, y is any integer from 1 to 3, z is 0 or 1, and n is any integer from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, or 8, as in (Gly4Ser)3 or (Gly3Ser2)3.

[0054] As used herein, the term "payload" can refer to siRNA, miRNA, shRNA, non-coding RNA (e.g., guide RNA of the CRISPR system), peptide, polypeptide, protein, viral genome, or any combination thereof. In some embodiments, the polypeptide is a chimeric antigen receptor.

[0055] As used herein, the terms "chimeric antigen receptor" and "CAR" are used interchangeably, referring to an antigen-binding domain fused directly or indirectly (e.g., via a hinge or transmembrane domain) to an intracellular signaling domain capable of activating or stimulating immune cells. Most commonly, the extracellular binding domain of a CAR consists of a single-chain variable fragment (scFv) fused to a variable heavy chain and light chain region derived from a murine or humanized monoclonal antibody. Alternatively, a scFv derived from Fab (rather than an antibody derived from, for example, a Fab library) may be used. In various embodiments, this scFv is fused to a transmembrane domain and then to an intracellular signaling domain. However, the antigen-binding domain can be any molecule capable of binding to a target on the cell. For example, the antigen-binding domain of a CAR can be an antibody, an scFv antibody, an antigen-binding domain, an ankyrin repeat sequence (e.g., DARPIN), a VHH domain antibody, a nanobody, a single-domain antibody, an FN3 domain, or any combination thereof. In some embodiments, CARs include those that provide only CD3ζ signaling upon antigen binding. In some embodiments, the CAR includes those that provide both co-stimulation (e.g., CD28 or CD137) and activation (CD3ζ). In some embodiments, the CAR includes those that provide multiple co-stimulations (e.g., CD28 and CD137) and activation (CD3ζ). In various embodiments, the CAR is selected to have high affinity or co-existence with the antigen. In some embodiments, the antigen-binding domain binds to CD20. In some embodiments, the antigen-binding domain comprises a CD20 antibody or a fragment thereof. In some embodiments, the antibody fragment is as provided herein, such as, but not limited to, scFv antibodies, antigen-binding domains, ankyrin repeat sequences (e.g., DARPIN), VHH domain antibodies, nanobodies, single-domain antibodies, FN3 domains, or any combination thereof.

[0056] Example

[0057] The preferred embodiments for carrying out the present invention will now be described. It should be noted that the embodiments given below are merely illustrative of the invention and can serve as a guide for further improvements by those skilled in the art. However, the present invention is not limited to these embodiments.

[0058] Unless otherwise specified, the methods used in the examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the examples are commercially available. Unless otherwise specified, the quantitative experiments in the examples are the average values ​​of three repeated experiments.

[0059] Experimental materials, reagents, instruments and experimental methods

[0060] cell:

[0061] hPBMC cells: purchased from Shanghai Ruian Biotechnology Co., Ltd.

[0062] Culture medium:

[0063] HEK293T medium: DMEM (Gibco, C11995500BT) medium containing 10% fetal bovine serum (Gibco, A5256701);

[0064] hPBMC medium: 1640 (Gibco, C11875500BT) medium containing 10% fetal bovine serum (Excell, FSP500);

[0065] Methodology:

[0066] Virus Packaging and Purification

[0067] HEK293T cells (ATCC) were cultured at 37°C and 5% CO2 until 80% to 90% confluence. Then, according to the manufacturer's instructions, a mixture of lentiviral packaging plasmids (pMDlg / PRRE, pRSV / REV, purchased from Addgene) and PEI (purchased from Polyplus) was added to the culture medium. After culturing the cells at 37°C and 5% CO2 for another 4 hours, the medium was changed with DMEM complete medium and cultured for another 2 days. The culture supernatant containing the virus was then harvested by centrifugation.

[0068] Viral precipitate was collected from the supernatant by ultracentrifugation (25000 rpm, 2 h). The precipitate was resuspended in PBS buffer (Sijing Biotechnology, SJ00522.1) to obtain recombinant lentivirus solution for transduction.

[0069] Virus titer determination

[0070] Viral titers were determined using the p24 kit (Genscript, catalog number: L00938) as follows. Specifically, 5 μL of lentivirus concentrate was diluted 10 μL with 1× wash buffer. 6 To obtain the viral dilution for titer determination, add 10 μL of lysis buffer and 100 μL of dilution buffer or standard to each well of a 96-well plate. Mix thoroughly at 25°C and incubate for 60 minutes. Then, label the virus p24 with biotinylated anti-p24 antibody (100 μL / well) at 25°C. After thorough washing, add 100 μL of streptavidin-HRP to each well and incubate at 25°C for 10 minutes. After thorough washing, add 100 μL of TMB chromogenic solution to each well and incubate at 25°C in the dark for 15 minutes. Finally, stop the reaction by adding 50 μL of stop solution and immediately measure the absorbance at 450 nm using a microplate reader.

[0071] T cell transduction

[0072] Fully resuscitated hPBMCs were resuspended in 1640 medium containing 10% fetal bovine serum, followed by the addition of 250 ng or 50 ng of lentiviral concentrate to infect T cells. The cells were cultured at 37°C and 5% CO2 for 24 h, then the medium was replaced with IL-2 containing 100 U / mL. The cells were then cultured at 37°C and 5% CO2 for 2–6 days to obtain virus-transduced recombinant cells (CAR-T).

[0073] Flow cytometry

[0074] After transduction, cells were collected and seeded into 96-well plates at a density of 1E5 cells per well. After washing twice with 1% BSA / PBS, 100 μL of diluted primary antibody (CD3-PE, Invitrogen, 12-0037-42; CD69-PE-cy7, Dubio, S0339; CD25-APC, Dubio, S0114; all antibodies were diluted 1:100 with 1% BSA / PBS) was added to each well, and the cells were incubated at 4°C for 1 hour. Cells were collected, washed twice, and resuspended in 80–100 μL of 1% BSA / PBS. Then, 1 μL of 7-AAD nucleic acid dye (Invitrogen, 00-6993-50) was added to each well to label cell viability, and the cells were incubated at 4°C for 10 minutes. Finally, the cells were analyzed using a Cytek Aurora full-spectrum flow cytometer.

[0075] Cell killing activity assay

[0076] According to the manufacturer's instructions, prepare the One-Lite assay reagent (Novazia, DD1203) using One-Lite Luciferase Assay Buffer and One-Lite Luciferase Assay Substrate. Add an equal volume of the assay reagent equilibrated to room temperature to a pre-equilibrated cell culture plate (Jing'an Biotechnology, J09602). Incubate at room temperature for at least 3 minutes to allow for complete cell lysis before measuring the absorbance at 380-780 nm.

[0077] Example 1

[0078] This example illustrates the construction of recombinant viral plasmids.

[0079] 1. Constructing a plasmid encoding a T-cell targeting fusion protein

[0080] By using segmented commissioned synthesis, the polynucleotide sequence encoding the following fusion protein was obtained:

[0081]

[0082]

[0083] Wherein, 7 represents CD7scFv (SEQ ID NO: 90), 8Stalk represents the transmembrane and intracellular regions of CD8 (SEQ ID NO: 91), VSVGStalk represents the transmembrane region of CD8 and the transmembrane region of wild-type VSVG (SEQ ID NO: 92), VSVG430Stalk represents a fragment of the transmembrane region of CD8 and the transmembrane region of wild-type VSVG (F430-K521) (SEQ ID NO: 93), 58 represents the extracellular domain of CD58 (SEQ ID NO: 1), 86 represents a fragment of CD86 with the N-terminal signal peptide removed (SEQ ID NO: 9), 80 represents a fragment of CD80 with the N-terminal signal peptide removed (SEQ ID NO: 94), 3 represents a CD3-promoted endocytosis antibody (SEQ ID NO: 2), 275 represents a fragment of CD275 with the N-terminal signal peptide removed (SEQ ID NO: 95), IL-2 represents the full-length IL-2 (SEQ ID NO: 96), and OX40L represents the natural ligand of OX40 (SEQ ID NO: 95). SEQ ID NO: 97), K12 represents the K12 fragment with the signal peptide removed (SEQ ID NO: 98), 86-truncation represents the fragment with the upstream (N-terminus) of the CD86 Ig-like V-type region removed (SEQ ID NO: 99), V80C86T80 represents a fusion protein composed of the Ig-like V-type region of CD80, the Ig-like C2-type region of CD86, and the transmembrane and intracellular regions of CD80 (SEQ ID NO: 100), 3-tep represents the CD3scFv with the generic name Teplizumab (SEQ ID NO: 101), and LGS represents a GS linker (SEQ ID NO: 10). PMD2.G plasmid was used as the donor plasmid. The PMD2.G plasmid, digested with restriction endonucleases XbaⅠ and XhoⅠ, was purified by gel electrophoresis to obtain a linearized donor plasmid fragment. The above-encoded polynucleotide was ligated with the linearized donor plasmid fragment to obtain the corresponding plasmid, named PMD2.G- (abbreviation for fusion protein).

[0084] 2. Constructing a plasmid encoding a variant of the VSVG viral glycoprotein

[0085] Using the same method as described in Section 1 of this embodiment, a polynucleotide (SEQ ID NO:17) encoding VSVGmut-SR-2 (with K47Q, R354A, S162T mutations) was ligated to the linearized plasmid PMD2.G to obtain the plasmid pMD2.G-mut-SR2.

[0086] 3. Packaging of recombinant enveloped viruses

[0087] HEK293T cells were seeded at a density of 1.5E7 in 15cm culture dishes (Jetbio), with 18mL of 293T medium added per well. Cells were cultured at 37°C and 5% CO2 until 80–90% confluence. Using the PEIpro kit (Polyplus), each PMD2.G plasmid encoding the fusion protein was co-transfected into HEK293T cells with pMD2.G-mut-SR2 (9μg), packaging plasmid pRSV.REV (7.26μg), pMDlg / pRRE (30μg), and pCCL-EF1α-GFP (30μg) (Addgene). Unless otherwise specified, the transfection dose of PMD2.G- (fusion protein abbreviation) is 9μg. After incubation for 12–16 hours, the medium was replaced with fresh 293T medium. Cells were then cultured at 37°C. At 24 and 48 hours, the culture supernatant was collected, centrifuged, filtered, and the supernatant was discarded. The virus precipitate was collected and resuspended in PBS buffer to obtain a concentrated recombinant virus solution expressing each fusion protein and the VSVGmut-SR-2 variant protein on the envelope surface.

[0088] 4. Detecting virus titer

[0089] The results of the determination of P24 protein content in the concentrated virus solution are shown in Tables 1 and 2.

[0090] Table 1

[0091]

[0092] *: The numbers in () indicate the amount of plasmid used for transfection. For example, the transfection amounts of PMD2.G(58-3-275) and PMD2.G(58-3-86) are 3 μg and 6 μg, respectively; this expression method will be used in the following text.

[0093] Table 2:

[0094]

[0095] 5. Detect the infection efficiency of recombinant viruses

[0096] hPBMC cells were resuspended in RPMI 1640 complete medium (purchased from Gibco) and then 1×10⁻⁶ cells were added. 5 Or 5×10 4Cells / well were seeded into 96-well plates (purchased from Jetech Biotechnology). The concentrated virus solution was diluted with RPMI 1640 complete medium, and 250 ng or 50 ng of virus was added per well. The plates were incubated at 37°C and 5% CO2 for 24 hours to allow viral infection. The medium was then replaced with RPMI 1640 complete medium containing 100 U / ml IL-2. On days 2, 4, or 6 post-infection, the proportion of GFP-positive cells in the total cell count was determined by flow cytometry based on the anti-human CD3-PE antibody (Biolegend). Results are shown below. Figure 1 , Figure 3 , Figure 4 , Figure 5 and Figure 6 Simultaneously, the number of GFP-positive cells in each group was measured, and the results are shown in... Figure 2 .

[0097] Figures 1 to 6 The results showed that the various fusion proteins tested and their combinations were able to target and deliver recombinant viruses to T cells. Transduction efficiency was affected by the specific type, number, and linkage order of the co-stimulatory molecules used to construct the fusion proteins.

[0098] Figures 3 to 6 The results showed that, compared to using a single fusion molecule, co-expressing fusion proteins targeting different T cell surface molecules in combination typically significantly improves the efficiency of recombinant viral transfection of T cells. The highest transduction efficiency was observed in recombinant viruses expressing either the combination of 58-7-86 and 58-3-tep-275, or the combination of K12-7-86 and 58-3-tep-275.

[0099] Figure 4 , Figure 5 The influence of the ratio of fusion protein expression plasmids on the transduction efficiency of recombinant viruses was also investigated. It is evident that for recombinant viruses expressing fusion protein combinations, the ratio of fusion protein expression plasmids and / or the ratio of fusion protein expression levels also affect transduction efficiency. In the case of recombinant viruses containing dual fusion proteins, the highest transduction efficiency can be achieved by controlling the ratio of the recombinant plasmid encoding the CD275 fusion protein to the plasmid encoding the other fusion protein at 1:2.

[0100] Example 2

[0101] This embodiment uses a chimeric antigen receptor or its encoding nucleotide sequence as an example of a payload to describe the construction of a recombinant virus carrying a payload and its transfection effect.

[0102] A DNA fragment (SEQ ID NO:83) encoding the anti-CD19 chimeric antigen receptor (CD19CAR) was synthesized by a commissioned team, and the expression vector pCCL-EF1α-CD19CAR was constructed.

[0103] Following the same method as described in Example 1, each PMD2.G plasmid encoding the fusion protein was co-transfected with pMD2.G-mut-SR2, packaging plasmid pRSV.REV, pMDlg / pRRE, and expression vector pCCL-EF1α-CD19CAR into HEK293T cells to obtain concentrated solutions of the corresponding recombinant viruses. The viral titers are shown in Tables 3 and 4 below.

[0104] Table 3

[0105]

[0106]

[0107] **: The blank control does not contain the expression plasmid of CD19-CAR.

[0108] Table 4:

[0109]

[0110] Using the same method as in Example 1, the virus concentrate shown in Table 4 was used to infect hPBMC cells at the indicated p24 concentration. The percentage of GFP-positive cells produced after infection is shown in Table 5. Figure 7 .

[0111] Table 5

[0112]

[0113]

[0114] Depend on Figure 7 It is evident that when the recombinant virus expresses the combination of fusion proteins 58-3-tep-275 and 58-3-tep-86, or the combination of 58-3-tep-275 and IL-2-3-tep-86, it exhibits the highest T cell transduction efficiency at various dosages. This may be because the combination of different co-stimulatory molecules can more effectively deliver co-stimulatory signals to T cells, thereby more effectively activating resting T cells.

[0115] hPBMCs were collected on day 7 post-infection, and genomic DNA was extracted. The copy number of the CAR gene in 100 ng of genomic DNA was detected by qPCR. The results are shown in... Figure 8 .Depend on Figure 8It is evident that recombinant viruses expressing various fusion protein combinations can effectively express the payload molecules in infected cells. Among them, the expression levels of 58-3-tep-275 and 58-3-tep-86, 58-3-tep-275 and 58-3-86, K12-3-tep-275 and 58-3-tep-86, or K12-7-275 and 58-3-86 are superior to other single fusion proteins or combinations of fusion proteins.

[0116] Example 3

[0117] This embodiment measured the multi-round killing activity of the exemplary CD19-CAR-T cells prepared in Example 2 against tumor cells in vitro. hPBMCs cells infected on day 7 were divided into 5 aliquots and dispensed into the wells of a 24-well plate after medium change. Nalm-6-Luciferase (Shanghai Jinyuan Biotechnology Co., Ltd.) B-lymphocytic leukemia cell line was used as the target cells, seeded at a density of 1E5 / well and mixed with the dispensed cell culture. After 12 to 18 hours, the killing activity of CAR-T cells against the target cells was detected using a One-Lite assay kit. After 24 hours of killing, 1E5 / well of Nalm-6-Luciferase target cells was added again for the next round of killing. Five rounds of killing were repeated, and the results of the five consecutive rounds are shown in Table 6. Figure 9 .

[0118] Table 6

[0119]

[0120] It is evident that all the recombinant viruses tested effectively produced CAR-T cells and exhibited significant cytotoxic activity against target cells. Among them, the CAR-T cells produced by the recombinant viruses combining 58-3-tep-275 and 58-3-tep-86, and 58-3-tep-275 and 58-7-86, showed the strongest and most stable cytotoxic activity.

Claims

1. A T-cell-targeting fusion protein, characterized in that, The fusion protein comprises two or more elements that are directly or indirectly linked to each other, the elements being selected from any one of CD58 or its active fragment, OX40L or its active fragment, an antibody or ligand of CD3, an antibody or ligand of CD7, an antibody or ligand of CD4, an antibody or ligand of CD8, IL-2, CD80, CD86 or CD275.

2. The fusion protein according to claim 1, wherein, The CD58 or its active fragment is the CD58 extracellular domain, preferably containing an amino acid sequence that has at least 90% identity with the amino acid sequence described in SEQ ID NO:1; Optionally, the IL-2 comprises an amino acid sequence having at least 90% identity with the amino acid sequence described in SEQ ID NO:96; Optionally, the OX40L active fragment is the OX40L extracellular domain, preferably containing an amino acid sequence that has at least 90% identity with the amino acid sequence described in SEQ ID NO:97; Optionally, the CD3 antibody has HCDR1 as described in SEQ ID NO:3, HCDR2 as described in SEQ ID NO:4, HCDR3 as described in SEQ ID NO:5, LCDR1 as described in SEQ ID NO:6, LCDR2 as described in SEQ ID NO:7, and LCDR3 as described in SEQ ID NO:8, and preferably includes an amino acid sequence that has at least 90% identity with the amino acid sequence described in SEQ ID NO:2; Optionally, the CD3 antibody has HCDR1 as described in SEQ ID NO:121, HCDR2 as described in SEQ ID NO:122, HCDR3 as described in SEQ ID NO:123, LCDR1 as described in SEQ ID NO:124, LCDR2 as described in SEQ ID NO:125, and LCDR3 as described in SEQ ID NO:126, and preferably includes an amino acid sequence that has at least 90% identity with the amino acid sequence described in SEQ ID NO:101; Optionally, the CD7 antibody has HCDR1 as described in SEQ ID NO:103, HCDR2 as described in SEQ ID NO:104, HCDR3 as described in SEQ ID NO:105, LCDR1 as described in SEQ ID NO:106, LCDR2 as described in SEQ ID NO:107, and LCDR3 as described in SEQ ID NO:108, and preferably includes an amino acid sequence that has at least 90% identity with the amino acid sequence described in SEQ ID NO:102; Optionally, the CD7 ligand is K12 or an active fragment thereof; preferably, it is the extracellular domain of K12, more preferably, it comprises an amino acid sequence having at least 90% identity with the amino acid sequence described in SEQ ID NO: 98; Optionally, CD80 comprises an amino acid sequence having at least 90% identity with the amino acid sequence described in SEQ ID NO:94; Optionally, CD86 comprises an amino acid sequence having at least 90% identity with the amino acid sequence described in SEQ ID NO:9; Optionally, CD275 comprises an amino acid sequence having at least 90% identity with the amino acid sequence described in SEQ ID NO:95; Optionally, the CD8 antibody has HCDR1 as described in SEQ ID NO:160, HCDR2 as described in SEQ ID NO:161, HCDR3 as described in SEQ ID NO:162, LCDR1 as described in SEQ ID NO:163, LCDR2 as described in SEQ ID NO:164, and LCDR3 as described in SEQ ID NO:165, and preferably includes an amino acid sequence having at least 90% identity with the amino acid sequence described in SEQ ID NO:

158.

3. The fusion protein according to claim 1, wherein, The components are covalently connected by flexible joints; Optionally, the flexible joint is a GS type joint; Optionally, the flexible connector has an amino acid sequence as described in any one of SEQ ID NO:7, 85 to 89, or 109.

4. The fusion protein according to claim 1, comprising an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NO: 11, 19 to 50, 139 to 148; Preferably, the T-cell targeting fusion protein has an amino acid sequence as described in any one of SEQ ID NO:11, 19 to 50, 139 to 148.

5. Recombinant enveloped viral particles, characterized in that, The viral particles express one or more of the T-cell-targeting fusion proteins according to any one of claims 1 to 4.

6. The recombinant enveloped virus particle according to claim 5, further expressing a VSVG virus glycoprotein variant; in, The VSVG viral glycoprotein variants include mutants K47Q and R354A, and also include any one of K66T, S162T or T230N.

7. The recombinant enveloped virus particle according to claim 6, wherein, The VSVG virus glycoprotein variant comprises an amino acid sequence that has at least 90% identity with the amino acid sequence selected from any of SEQ ID NO: 17, 128 to 132; Preferably, the VSVG viral glycoprotein variant has an amino acid sequence selected from any of SEQ ID NO:17, 128 to 132.

8. The recombinant enveloped virus particle according to claim 5, further comprising a payload; Optionally, the payload comprises a nucleotide sequence encoding a target polypeptide and / or a nucleotide sequence encoding a non-coding RNA; Optionally, the target polypeptide is selected from chimeric antigen receptors (CARs), fluorescent proteins, antibodies, or gene-editing enzymes; Optionally, the gene-editing tool enzyme is selected from zinc finger nucleases or Cas proteins; Optionally, the non-coding RNA is selected from siRNA, miRNA, shRNA, or sgRNA.

9. The recombinant enveloped virus particle according to claim 8, wherein, The chimeric antigen receptor includes an antigen-binding domain that specifically targets any cancer-associated antigen selected from CD19, BCMA, GPRC5D, ROR1, FcRL5, alpha-fetoprotein, Her2, MUC1, or GPC3. Preferably, the chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO:

83.

10. The recombinant enveloped virus particle according to any one of claims 5 to 9, wherein it is a lentiviral particle or a retroviral particle.

11. A pharmaceutical composition comprising any one of claims 5 to 10 recombinant enveloped viral particles and a pharmaceutically acceptable carrier.

12. Use of the T-cell-targeting fusion protein according to any one of claims 1 to 4, wherein it is used for one or more of the following purposes: A. Prolonging the half-life of recombinant viral particles or transgenic immune cells produced from them; B. To increase the safety of recombinant viral particles or transgenic immune cells derived therefrom; and / or C. Enhances the tumor-killing activity of recombinant viral particles or transgenic immune cells produced by them.

13. A method for transducing a population of T cells in a subject, comprising administering to a subject in need a viral particle according to any one of claims 5 to 10 or a pharmaceutical composition according to claim 11; Optionally, the subject's T cell population expresses the payload contained in the viral particles after administration.

14. A method of treating a disease or condition in a subject in need, comprising administering to the subject a viral particle according to any one of claims 5 to 10 or a pharmaceutical composition according to claim 11.

15. Use of the virus particles according to any one of claims 5 to 10 in the preparation of a medicament for treating a disease or ailment. Optionally, the disease or condition is selected from any one of acute lymphoblastic leukemia, large B-cell lymphoma, multiple myeloma, relapsed / refractory B-cell non-Hodgkin lymphoma, gastric cancer / gastroesophageal junction cancer, liver cancer, pancreatic cancer, biliary tract cancer, malignant pleural mesothelioma / lung cancer, refractory neurological autoimmune diseases (such as multiple sclerosis, myasthenia gravis), or systemic lupus erythematosus.

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