Cd7 and cd3 dual targeting fusion proteins and uses thereof
By constructing a pseudo-lentiviral vector using a CD7 and CD3 dual-targeting fusion protein, the problems of difficulty in infecting and over-activation of resting T cells were solved, achieving efficient and safe T cell targeted killing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANYIKANG PHARMACEUTICAL (SHANGHAI) CO LTD
- Filing Date
- 2026-06-01
- Publication Date
- 2026-07-07
Smart Images
Figure CN122344265A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell immunotherapy, specifically relating to a CD7 and CD3 dual-targeting fusion protein and its applications. Background Technology
[0002] In recent years, with the rapid development of tumor immunology and genetic engineering technology, adoptive cell immunotherapy has become an important breakthrough in the field of tumor treatment. Among them, chimeric antigen receptor T-cell (CAR-T) therapy is one of the most representative treatment methods.
[0003] CAR-T cell therapy has shown significant anti-tumor effects in clinical practice, but it still faces many technical bottlenecks that directly affect the quality of cell products, treatment efficacy, and clinical accessibility.
[0004] Compared with the traditional in vitro CAR-T preparation and intravenous infusion method, the in vivo CAR-T cell therapy fully demonstrates its advantages: (1) It greatly shortens the treatment cycle and achieves "immediate treatment". (2) It avoids excessive activation and functional exhaustion of in vitro T cells. (3) It significantly reduces production costs and simplifies the process. (4) In vivo homing and distribution are more physiological, enhancing the infiltration ability of solid tumors, etc.
[0005] However, in vivo CAR-T cell therapy still faces some challenges, such as the difficulty of efficiently infecting resting T cells with lentiviruses. To address this challenge, antibodies with strong affinity and activation ability, such as CD3e and TCR activating antibodies, are often used on the viral envelope. However, the drawback is that strong activating antibodies can lead to over-activation of T cells in vivo, causing rapid cell depletion. At the same time, rapid proliferation of T cells can also cause a series of toxic side effects.
[0006] Therefore, in vivo CAR-T cell therapy urgently needs to develop a new strategy or method to balance T cell activation intensity, infection efficiency, and anti-tumor ability, so as to achieve transient, low-intensity activation of resting T cells, which can both improve viral transduction of resting T cells and reduce a series of toxic side effects caused by excessive T cell activation, while ensuring that CAR-T cells can quickly exert anti-tumor activity. Summary of the Invention
[0007] This invention provides a CD7 and CD3 dual-targeting fusion protein and its applications.
[0008] In a first aspect of the invention, a fusion protein is provided, the fusion protein comprising, from the N-terminus to the C-terminus, the following: (a) Anti-CD7 single-domain antibody (CD7 VHH); (b) Anti-CD3 single-domain antibody (CD3 VHH); and (c) A transmembrane region for anchoring the fusion protein to a cell membrane or viral envelope.
[0009] In another preferred embodiment, the transmembrane region is derived from a protein selected from the group consisting of VSV-G protein, CD8a protein, CD28 protein, or CD4 protein.
[0010] In another preferred embodiment, the viral envelope protein is the VSV-G mut protein.
[0011] In another preferred embodiment, the amino acid sequence of the VSV-G mut protein is shown in SEQ ID NO: 9.
[0012] In another preferred embodiment, the nucleotide sequence of the VSV-G mut protein is shown in SEQ ID NO: 10.
[0013] In another preferred embodiment, the amino acid sequence of the anti-CD7 single-domain antibody is shown in SEQ ID NO: 13.
[0014] In another preferred embodiment, the nucleic acid sequence of the anti-CD7 single-domain antibody is shown in SEQ ID NO: 14.
[0015] In another preferred embodiment, the amino acid sequence of the anti-CD3 single-domain antibody is shown in SEQ ID NO: 17.
[0016] In another preferred embodiment, the nucleic acid sequence of the anti-CD3 single-domain antibody is shown in SEQ ID NO: 18.
[0017] In another preferred embodiment, the amino acid sequence of the transmembrane region is as described in SEQ ID NO: 21.
[0018] In another preferred embodiment, the nucleic acid sequence of the transmembrane region is shown in SEQ ID NO: 22.
[0019] In another preferred embodiment, the anti-CD7 single-domain antibody and the anti-CD3 single-domain antibody are directly linked by peptide bonds or by linking peptides (excluding self-cleaving peptides).
[0020] In another preferred embodiment, the fusion protein further includes a hinge region located between the anti-CD3 single-domain antibody and the transmembrane region.
[0021] In another preferred embodiment, the amino acid sequence of the hinge region is shown in SEQ ID NO: 19.
[0022] In another preferred embodiment, the nucleic acid sequence of the hinge region is shown in SEQ ID NO: 20.
[0023] In another preferred embodiment, the anti-CD7 single-domain antibody and the anti-CD3 single-domain antibody are directly linked by peptide bonds or by linking peptides (excluding self-cleaving peptides).
[0024] In another preferred embodiment, the fusion protein further includes a co-stimulatory molecule.
[0025] In another preferred embodiment, the co-stimulatory molecule is located between the anti-CD3 single-domain antibody and the hinge region.
[0026] In another preferred embodiment, the co-stimulatory molecules are selected from the group consisting of: CD80, 4-1BB, CD27, CD28, OX40, CD30, CD40, CD40L, CD70, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, G ITR, BAFFR, HVEM, SLAMF7, CD7, NKp80, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1(CD11a / CD18), ITGAM, CD11b , ITGAX, CD11c, ITGB1, ITGB2, KLRC2, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TNFRSF18, TNFRSF14, TRANCE / RAN KL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, SELPLG, LTBR, LAT, GADS, SLP-76, PAG / Cbp, HAVCR1, LGALS9, Dap10, DAP12, CDS, ICAM-1, NKG2D, GITR, TLR2, TMIGD2, or combinations thereof.
[0027] In another preferred embodiment, the co-stimulatory molecule is CD80.
[0028] In another preferred embodiment, the amino acid sequence of the co-stimulatory molecule is shown in SEQ ID NO: 23.
[0029] In another preferred embodiment, the nucleic acid sequence of the co-stimulatory molecule is shown in SEQ ID NO: 24.
[0030] In another preferred embodiment, the elements of the fusion protein are directly linked by peptide bonds or by linker peptides.
[0031] In another preferred embodiment, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 29 or 31.
[0032] In a second aspect of the invention, a pseudolentiviral vector targeting T cells is provided, wherein the envelope of the pseudolentiviral vector displays the fusion protein described in the first aspect of the invention.
[0033] In another preferred embodiment, the envelope protein of the pseudolentiviral vector is a modified vesicular stomatitis virus glycoprotein VSV-G mut.
[0034] In another preferred embodiment, the amino acid sequence of the VSV-G mut protein is shown in SEQ ID NO: 9.
[0035] In another preferred embodiment, the pseudo-lentiviral vector further comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR).
[0036] In another preferred embodiment, the chimeric antigen receptor is a chimeric antigen receptor targeting CD20, and its amino acid sequence is shown in SEQ ID NO: 1.
[0037] In another preferred embodiment, the nucleic acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 2.
[0038] In a third aspect of the invention, an isolated nucleic acid molecule is provided, said nucleic acid molecule encoding the fusion protein described in the first aspect of the invention.
[0039] In another preferred embodiment, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 26 or 28.
[0040] In a fourth aspect of the invention, an expression vector is provided, the expression vector comprising the nucleic acid molecule described in the third aspect of the invention.
[0041] In another preferred embodiment, the expression vector is a pMD2.G series vector or a derivative thereof.
[0042] In a fifth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the fourth aspect of the invention, or having a nucleic acid molecule described in the third aspect of the invention integrated into its genome, or expressing the fusion protein described in the first aspect of the invention.
[0043] In a sixth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (a) The pseudo-lentiviral vector described in the second aspect of the present invention.
[0044] In another preferred embodiment, the pharmaceutical composition further comprises (b) a pharmaceutically acceptable carrier.
[0045] In a seventh aspect of the invention, the use of the pseudo-lentiviral vector as described in the second aspect of the invention in the preparation of a medicament for treating cancer or tumors is provided.
[0046] In another preferred embodiment, the tumor is selected from the group consisting of solid tumors (such as colon cancer, gastric cancer, pancreatic cancer, liver cancer), hematologic malignancies (such as lymphoma, multiple myeloma), or combinations thereof.
[0047] In an eighth aspect of the invention, a lentivirus packaging system is provided, the lentivirus packaging system comprising: (a) One or more packaging plasmids containing nucleic acid sequences encoding lentiviral Gag, Pol, and Rev proteins; (b) One or more expression plasmids comprising: (i) The nucleic acid sequence encoding the modified vesicular stomatitis virus glycoprotein VSV-G mut; (ii) A nucleic acid sequence encoding the fusion protein described in the first aspect of the invention; and (iii) Optionally, a nucleic acid sequence encoding a chimeric antigen receptor (CAR).
[0048] In a ninth aspect of the present invention, a method for preparing the pseudo-lentiviral vector described in the second aspect of the present invention is provided, comprising the step of transfecting host cells using the lentiviral packaging system described in the sixth aspect of the present invention.
[0049] In a tenth aspect of the present invention, a method for preparing CAR-T cells is provided, comprising the following steps: (a) Provide T cells; (b) Infect the T cells with the pseudolentiviral vector described in the second aspect of the present invention; The pseudo-lentiviral vector further contains a nucleic acid sequence encoding a chimeric antigen receptor (CAR); (c) Culture the infected T cells from (b) to obtain CAR-T cells.
[0050] In another preferred embodiment, the T cells described in step (a) are resting T cells and are not subjected to additional activation treatment prior to infection.
[0051] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
[0052] In another preferred embodiment, the method is an in vitro method.
[0053] In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 1.
[0054] In another preferred embodiment, the nucleic acid sequence encoding the chimeric antigen receptor is shown in SEQ ID NO: 2.
[0055] In an eleventh aspect of the present invention, a CAR-T cell is provided, which is prepared by the method described in the eighth aspect of the present invention.
[0056] In a twelfth aspect of the invention, the use of the CAR-T cells described in the eleventh aspect of the invention in the preparation of a medicament for treating tumors is provided.
[0057] In another preferred embodiment, the tumor is selected from the group consisting of solid tumors (such as colon cancer, gastric cancer, pancreatic cancer, liver cancer), hematologic malignancies (such as lymphoma, multiple myeloma), or combinations thereof.
[0058] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description
[0059] Figure 1 The structures of the anti-CD20 CAR and EGFP overexpression vectors, as well as schematic diagrams of the structures of different viral envelope fusion proteins, are shown.
[0060] Figure 2 The study demonstrated the preparation of EGFP-overexpressing viruses using different viral envelope fusion proteins, infection of resting T cells, and flow cytometry detection of T cell activation markers CD69 and CD25 after 6 hours.
[0061] Figure 3 The study demonstrated the preparation of EGFP-overexpressing viruses using different viral envelope fusion proteins, infection of resting T cells, flow cytometry detection of T cell activation markers CD69 and CD25 on days 1 and 8, and statistical analysis of T cell activation marker CD25 expression.
[0062] Figure 4 The study demonstrated the preparation of EGFP-overexpressing viruses using different viral envelope fusion proteins, their infection of resting T cells, and the statistical analysis of T cell proliferation.
[0063] Figure 5The study showed the preparation of EGFP-overexpressing viruses using different viral envelope fusion proteins, their infection of resting T cells, and the percentage of EGFP-positive T cells.
[0064] Figure 6 The study demonstrated the preparation of anti-CD20 CAR virus overexpressing different viral envelope fusion proteins, infection of resting T cells, and flow cytometry detection of T cell activation markers CD69 and CD25 after 6 hours.
[0065] Figure 7 The study demonstrated the preparation of anti-CD20 CAR viruses using different viral envelope fusion proteins, their infection of resting T cells, and the detection of CAR expression in T cells by flow cytometry.
[0066] Figure 8 The study demonstrated the preparation of anti-CD20 CAR viruses using different viral envelope fusion proteins, their infection of resting T cells, and the flow cytometry detection of T cell activation markers CD69 and CD25 on days 1 and 8, as well as the statistical analysis of T cell activation marker CD25 expression.
[0067] Figure 9 The study demonstrated the preparation of anti-CD20 CAR virus overexpressing different viral envelope fusion proteins, infection of resting T cells, and statistical analysis of T cell proliferation.
[0068] Figure 10 The study demonstrated the preparation of anti-CD20 CAR viruses using different viral envelope fusion proteins, their infection of resting T cells, and the statistical analysis of the T cell activation marker CD25.
[0069] Figure 11 The study demonstrated the preparation of anti-CD20 CAR virus overexpression using different viral envelope fusion proteins, infection of resting T cells, flow cytometry detection of Raji killing of target cells by CAR-T cells, and statistical analysis of Raji residues in target cells. Detailed Implementation
[0070] Through extensive and in-depth research and screening, the inventors have developed, for the first time, a fusion protein comprising: a high-affinity anti-CD7 single-domain antibody, a weak-affinity and weakly activating anti-CD3 single-domain antibody, a hinge region, and a transmembrane region (which may also include a co-stimulatory molecule). A pseudolentiviral vector displaying this fusion protein on its viral envelope can effectively transfect T cells. When the pseudolentiviral vector contains a chimeric antigen receptor coding sequence targeting a specific antigen, it can significantly enhance the specific targeting and killing ability of the transfected T cells. Based on this, the present invention was completed.
[0071] Specifically, experiments of this invention show that the pVSV-Gm-005, pVSV-Gm-007, and pVSV-Gm-008 structures significantly improve the infection efficiency of the virus in naïve T cells. Furthermore, CAR-T cells prepared using the pVSV-Gm-005 and pVSV-Gm-007 structures exhibit superior cytotoxicity.
[0072] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.
[0073] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values between 99 and 101.
[0074] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.
[0075] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).
[0076] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.
[0077] Chimeric antigen receptor (CAR) As used herein, a chimeric antigen receptor (CAR) comprises an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain. The extracellular domain includes an optional signal peptide and a target-specific binding domain (also known as an antigen-binding domain). When expressed in T cells, the extracellular domain recognizes a specific antigen, which is then transduced through the intracellular domain, leading to cell activation and proliferation, cytotoxicity, and the secretion of cytokines such as IL-2 and IFN-γ. This affects tumor cells, causing them to stop growing, die, or otherwise be affected, resulting in a reduction or elimination of the tumor burden in patients.
[0078] pseudo-lentiviral vector The pseudotyped lentiviral vector (or lentiviral vector or lentiviral-based vector particle) defined in this invention is a pseudotyped lentiviral vector consisting of a vector having an envelope protein derived from a virus different from the vector genome providing the lentiviral vector. Therefore, the envelope protein is a "heterologous" viral envelope protein relative to the vector genome. Hereinafter, when "enveloping protein" is mentioned, it includes any type of envelope protein suitable for carrying out this invention. In particular, the envelope protein displays the fusion protein of this invention, which comprises, from the N-terminus to the C-terminus, the following: (a) Anti-CD7 single-domain antibody (CD7 VHH); (b) Anti-CD3 single-domain antibody (CD3 VHH); and (c) A transmembrane region for anchoring the fusion protein to a cell membrane or viral envelope.
[0079] In another preferred embodiment, the fusion protein further includes a hinge region located between the anti-CD3 single-domain antibody and the transmembrane region.
[0080] In another preferred embodiment, the fusion protein further includes a co-stimulatory molecule located between the anti-CD3 single-domain antibody and the hinge region.
[0081] In another preferred embodiment, the amino acid sequence of the anti-CD7 single-domain antibody is shown in SEQ ID NO: 13, and the amino acid sequence of the anti-CD3 single-domain antibody is shown in SEQ ID NO: 17.
[0082] In another preferred embodiment, the fusion protein comprises: (1) CD7 VHH-CD3 VHH-hinge-TM Its amino acid sequence is shown in SEQ ID NO: 29, and its nucleotide sequence is shown in SEQ ID NO: 30.
[0083] (2) CD7 VHH-CD3 VHH-CD80-hinge-TM Its amino acid sequence is shown in SEQ ID NO: 31, and its nucleotide sequence is shown in SEQ ID NO: 32.
[0084] When “lentivirus” vectors (lentivirus-based vectors) are mentioned in this application, they include, in specific implementations, HIV-based vectors and, in particular, HIV-1-based vectors.
[0085] The lentiviral vector of the present invention is a substitution vector, meaning that the original lentiviral sequence encoding lentiviral proteins is substantially deleted in the genome of the vector or, when present, modified, particularly mutated, particularly truncated, to prevent the expression of biologically active lentiviral proteins. In particular, in the case of HIV, the transfer vector is prevented from expressing functional ENV, GAG, and POL proteins, as well as optionally other structural and / or accessory and / or regulatory proteins of lentiviruses, particularly HIV.
[0086] The "vector genome" of the vector particle is a recombinant vector that also contains a target polynucleotide or transgene encoding a chimeric antigen receptor (CAR). The lentivirus-based sequence of the vector genome and the polynucleotide / transgene are carried by a plasmid vector, thus producing a "transfer vector" also known as a "sequence vector". Therefore, these expressions are used interchangeably in this application.
[0087] According to the present invention, a lentiviral vector is pseudotyped using a heterologous viral envelope protein or a viral polyprotein derived from an RNA virus envelope, wherein the RNA virus is not a lentiviral sequence that provides the genome of the lentiviral vector.
[0088] As an example of a typing envelope protein for preparing a lentiviral vector, the present invention relates to a transmembrane glycosylated (so-called G protein) envelope protein of vesicular stomatitis virus (VSV), said protein being selected, for example, from the VSV-G protein of Indiana strain, the VSV-G protein of New Jersey strain, the VSV-G protein of Cocal strain, the VSV-G protein of Isfahan strain, the VSV-G protein of Kindipura strain, the VSV-G protein of Piri strain, or the VSV-G protein of SVCV strain.
[0089] The VSV-G protein mentioned in this article is the VSV-G mut protein, which is the VSV-G glycoprotein with a mutation at amino acid sites K47 and R354 to Q amino acid; this mutation can significantly reduce the binding of VSV-G glycoprotein to the low-density lipoprotein receptor LDL-R.
[0090] The amino acid sequence and coding sequence of the VSV-G mut protein are shown in SEQ ID NO: 9 and 10, respectively.
[0091] Nucleic Acids and Vectors This invention provides one or more nucleic acids encoding the fusion protein described herein. In some embodiments, the nucleic acid comprises or is composed of DNA and / or RNA.
[0092] In some embodiments, the nucleic acid is or is contained in one or more vectors. That is, the nucleotide sequence of the nucleic acid may be contained in a vector. The fusion protein of the present invention can be transcribed and translated intracellularly via a vector, which encodes the antigen-binding molecule, polypeptide, or fusion protein.
[0093] Therefore, the present invention also provides one or more vectors comprising the nucleic acids or multiple nucleic acids described in the present invention. The vectors facilitate the delivery of nucleic acids encoding the fusion proteins described in the present invention. The vectors may be expression vectors containing elements required for expressing the fusion proteins described in the present invention.
[0094] The nucleic acids and vectors described in this invention can be provided in purified or isolated form, i.e., separated from other nucleic acids or natural biological materials.
[0095] The nucleotide sequence may be contained in a vector (such as an expression vector). As used herein, "vector" refers to a nucleic acid molecule used to transfer exogenous nucleic acids into cells. The vector may be a vector for expressing nucleic acids in cells. The vector may include a promoter sequence operatively linked to the nucleotide sequence to be expressed. The vector may also contain a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express peptides or polypeptides in the vectors described herein.
[0096] The term "operably linked" refers to the covalent linkage of a selected nucleic acid sequence to a regulatory nucleic acid sequence (such as a promoter and / or enhancer) in such a way that the expression of the nucleic acid sequence is influenced or controlled by the regulatory sequence (thus forming an expression cassette). Therefore, if a regulatory sequence can influence the transcription of a nucleic acid sequence, then the regulatory sequence is operably linked to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide.
[0097] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (such as retroviral vectors, for example, gamma-retroviral vectors (such as murine leukemia virus (MLV)-derived vectors, such as SFG vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (such as yeast artificial chromosomes).
[0098] In some embodiments, the vector may be a eukaryotic vector, such as a vector containing elements required for expressing the vector protein in eukaryotic cells. In some embodiments, the vector may be a mammalian vector, such as a vector containing a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.
[0099] The main advantages of this invention include: (a) The chronic disease envelope protein of the present invention enables lentiviruses to effectively target and activate T cells, and can transfect CAR into T cells to achieve effective targeted killing of T cells.
[0100] (b) The present invention uses CD3e VHH nanobody with weak activation intensity to effectively avoid overactivation of T cells, thereby avoiding rapid depletion of T cells.
[0101] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.
[0102] Example 1: Preparation of lentiviruses with different activation intensities and their T-cell infection (1) Vector construction: The sequence includes an overexpression EGFP sequence and an overexpression Anti-CD20 CAR structure sequence (target plasmid, automatically wrapped into the constructed lentivirus), as well as different lentivirus envelope structure sequences. The sequence diagram is shown below. Figure 1 As shown in the figure; specific sequence information is shown in Table 1.
[0103] The Anti-CD20 CAR structure comprises, in sequence: a signal peptide, an anti-CD20 single-chain antibody, a hinge region, a transmembrane region (TM), a co-stimulatory signaling molecule (4-1BB), and a signal transduction domain (CD3z); its specific amino acid and nucleotide sequences are shown in SEQ ID NO: 1 and 2, respectively; specifically, it includes: (1) Signal peptide, the amino acid sequence and nucleotide sequence of which are shown in SEQ ID NO 33 and 34, respectively; (2) The tag, whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO 35 and 36, respectively; (3) Anti-CD20 scfv, whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO 37 and 38, respectively; (4) Hinge region, whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO 39 and 40, respectively; (5) Transmembrane region (TM), whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO 41 and 42, respectively; (6) Co-stimulatory molecule (4-1BB), whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO 43 and 44, respectively; (7) Activation domain (CD3z), whose amino acid and nucleotide sequences are shown in SEQ ID NO 45 and 46, respectively.
[0104] The structures of the lentiviral envelope vectors are shown below: (1) pVSV-G-001: VSV-G WT Including signal peptide (SEQ ID NO: 5) and VSV-G WT (SEQ ID NO: 7); (2) pVSV-Gm-002: VSV-G mut P2A-CD7 VHH Including signal peptide (SEQ ID NO: 5), VSV-G mut (SEQ ID NO: 9), P2A (SEQ ID NO: 11) and CD7 VHH (SEQ ID NO: 13); (3) pVSV-Gm-003: VSV-G mut P2A-CD3 VHH Including signal peptide (SEQ ID NO: 5), VSV-G mut (SEQ ID NO: 9), P2A (SEQ ID NO: 11) and CD3 VHH (SEQ ID NO: 17); (4) pVSV-Gm-004: VSV-G mut -CD7 VHH- P2A-CD3 VHH It includes a signal peptide (SEQ ID NO: 5), VSV-G mut (SEQ ID NO: 9), CD7 VHH (SEQ ID NO: 13), P2A (SEQ ID NO: 11), CD3 VHH (SEQ ID NO: 17), a hinge region (SEQ ID NO: 19), and a transmembrane region (SEQ ID NO: 21); (5) pVSV-Gm-005: VSV-G mut - P2A-CD7 VHH- CD3 VHH It includes a signal peptide (SEQ ID NO: 5), VSV-G mut (SEQ ID NO: 9), P2A (SEQ ID NO: 11), CD7 VHH (SEQ ID NO: 13), CD3 VHH (SEQ ID NO: 17), a hinge region (SEQ ID NO: 19), and a transmembrane region (SEQ ID NO: 21); (6) pVSV-Gm-006: VSV-G mut - CD7 VHH- P2A-CD3 VHH-CD80 It includes a signal peptide (SEQ ID NO: 5), VSV-G mut (SEQ ID NO: 9), CD7 VHH (SEQ ID NO: 13), P2A (SEQ ID NO: 11), CD3 VHH (SEQ ID NO: 17), CD80 (SEQ ID NO: 23), a hinge region (SEQ ID NO: 19), and a transmembrane region (SEQ ID NO: 21); (7) pVSV-Gm-007: CD7 VHH- CD3 VHH It includes a signal peptide (SEQ ID NO: 5), CD7 VHH (SEQ ID NO: 13), CD3 VHH (SEQ ID NO: 17), a hinge region (SEQ ID NO: 19), and a transmembrane region (SEQ ID NO: 21).
[0105] (8) pVSV-Gm-008: CD7 VHH- CD3 VHH-CD80 It includes a signal peptide (SEQ ID NO: 5), CD7 VHH (SEQ ID NO: 13), CD3 VHH (SEQ ID NO: 17), CD80 (SEQ ID NO: 23), a hinge region (SEQ ID NO: 19), and a transmembrane region (SEQ ID NO: 21).
[0106] Table 1 The fusion gene was artificially synthesized by Shanghai Qingke Biotechnology Co., Ltd. using conventional genetic engineering methods. The overexpression vector was cloned into the plasmid pLenti-EF1a, and the lentiviral envelope structure was cloned into the pMD2.G vector. The plasmid extraction was also completed by Shanghai Qingke Biotechnology Co., Ltd., and the plasmid was stored at -80℃ for later use.
[0107] (2) Preparation, packaging and concentration of lentiviruses 293T cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Once the cells reached the logarithmic growth phase, they were digested with trypsin, centrifuged, counted, and resuspended. A total of 20 million cells were then seeded into a new 15 cm dish, mixed, and transferred to a 37°C, 5% CO2 incubator for overnight culture until the virus was packaged.
[0108] The viral packaging systems included four-plasmid and five-plasmid packaging systems. For each packaging system, the plasmids were mixed in 1 ml of serum-free and antibiotic-free Opti-MEM medium, with the plasmid ratios as follows. 120 μL of PEI transfection reagent was added to 880 μL of serum-free and antibiotic-free Opti-MEM medium and mixed thoroughly. The 1 ml of each reagent was then mixed thoroughly and incubated at room temperature for 12 minutes. The lentivirus packaging systems are shown in Tables 2 and 3 below.
[0109] Table 2 Four-plasmid system Table 3 Five-plasm system Take 10 ml of serum-free and antibiotic-free opti-MEM culture medium and add it to the 2 ml mixture described above. Gently pipette and mix thoroughly. Add the final mixture to a 15 cm dish of 293T cells, mix well, and incubate at 37°C with 5% CO2 for 5.5 hours. Discard the supernatant, add 30 ml of high-glucose DMEM medium containing 2% fetal bovine serum, and incubate at 37°C with 5% CO2. Collect the supernatant of 293T cells after 48 hours.
[0110] Approximately 30 ml of 293T cell supernatant was collected in a 50 ml centrifuge tube. After centrifugation at 3000 rpm for 10 minutes, the supernatant was collected and filtered through a 0.22 μm filter flask. The filtrate was transferred to a 35 ml ultracentrifuge tube. This tube was then transferred to a high-speed metal centrifuge tube, vacuum-sealed, and centrifuged at 25000 rpm for 2 hours. The vacuum was removed, the ultracentrifuge tube was removed with forceps, the supernatant was aspirated, and 160 μL of serum-free, antibiotic-free, and cytokine-free Lonza medium was added. The tube was then incubated overnight at 4°C to thaw the virus particles. The next day, the virus suspension was gently pipetted, aliquoted, and the concentrated virus solution was transferred to a -80°C freezer for cryopreservation.
[0111] (3) Sorting, activation and infection of human peripheral blood pan-T cells PBMC cell isolation (provided by Shanghai Saili Biotechnology Co., Ltd.) Leukopark disinfects the outer surface of the incision and transfers it to a 50ml centrifuge tube, with each tube containing no more than 20ml. Take out 20ul of cell suspension, dilute it 50 times, and count the cells. Take a new 50ml centrifuge tube, add 20ml of lymphocyte separation medium to the lower layer, and add 15-17ml of diluted apheresis blood to the upper layer to form density stratification. Carefully transfer to a centrifuge, centrifuge at 600g at room temperature for 25 minutes, increment 4 and decrement 5. Transfer the white membrane layer cells (~10ml) to a new 50ml centrifuge tube, add 40ml washing buffer, invert 5 times to mix the cells, centrifuge at 600g at room temperature for 10 minutes, increment 9 and decrement 9, and collect the cells. Discard the supernatant suspension, resuspend the precipitate in 10 ml washing buffer, and transfer it to a new 50 ml centrifuge tube. Combine 4 tubes into 1 tube; Invert the tube 5 times to mix the cells, take 20 μL, dilute it 50 times, count the cells, and calculate the PBMC cell isolation yield. (4) Pan-T cell sorting Collect cells by centrifuging at 400g at room temperature for 10 min, resuspend the cells with washing buffer, and adjust the cell density to 50 million / ml. Add pan-T isolation antibody cocktail at a ratio of 50ul / ml, mix the cells, and incubate at room temperature for 5 min. Add RapidSphere at a ratio of 40 μL / ml, mix the cells, and place at room temperature for 3 min. Insert a magnet, let stand for 10 min, and aspirate the supernatant into a new 50 ml centrifuge tube. Take a sample, dilute and count, and calculate the Pan-T separation yield. Cells were collected by centrifugation at 400g at room temperature for 10 min. The cells were resuspended in cryopreservation buffer (90% fetal bovine serum and 10% DMSO) and aliquoted into cryovials at a density of 50 million / vial and 20 million / vial. The cells were then transferred to a cryopreservation box and stored at -80°C overnight, and then transferred to a liquid nitrogen tank for long-term storage.
[0112] (5) Pan-T recovery Pan-T cells were taken from liquid nitrogen, thawed in a 37°C water bath, and transferred to a 15ml centrifuge tube. 9ml of preheated Lonza medium at 37°C was added, and the mixture was mixed well. 20ul of AOPI reagent was mixed with the cells and counted. The mixture was centrifuged at 300g for 6 minutes. Resuspend the cells in Lonza medium containing 2% human serum albumin and 300 IU / ml IL2, adjust the cell concentration to 2 million / ml, and culture overnight; (6) pan-T lentivirus infection Take the above T cells, transfer them into a 15ml centrifuge tube, count the cells, adjust the cell density to 1 million / ml, and seed 1ml / well into a 12-well plate; Calculate the required viral load based on an MOI of 2. The formula is as follows: Required viral load = (MOI × number of cells) / viral titer; Remove the virus at -80℃, thaw it rapidly in a 37℃ incubator, add the required amount of virus according to the calculation, mix well, and incubate for 24 hours in a 37℃, 5% carbon dioxide incubator. Daily cell counting, cell expansion, detection of T activation & exhaustion markers, and cell phenotype analysis are performed.
[0113] (7) Flow cytometry detection of T cells Preparation of cell suspension: Collect expanded cultured cells, count them, take 0.1 million cells and add them to a U-shaped 96-well plate, centrifuge at 300g for 6 minutes; Prepare antibody mixture: 0.2ul / test Percp Anti-CD3, 0.25ul / test APC Anti-FMC63, 0.2ul / test PE / CY7 Anti-CD4, 0.2ul / test APC / CY7 Anti-CD8, gently mix with a 1ml pipette, add 50ul to the centrifuged cells (after removing the supernatant), and incubate at room temperature in the dark for 1 hour. Washing: Add 200 μL of PBS buffer to each well, centrifuge at 300g for 6 minutes, wash twice, resuspend cells in 120 μL of PBS buffer, and analyze. FlowJo V-10.10 software is used to analyze data.
[0114] Example 2: Verification of infection efficiency of different viral envelopes in naïve T cells Resting T cells were infected with viruses of different envelope types prepared in Example 1. After 6 hours of viral infection, the early activation marker CD69 and the intermediate-early activation marker CD25 of T cells were detected. Figure 2 Flow cytometry results showed that the viral envelope CD3 VHH antibody could effectively activate naïve T cells. The combination of CD7 VHH and CD3 VHH showed a high proportion of early activation marker CD69 and a strong degree of activation. The combined co-stimulatory ligand CD80 further enhanced T cell activation.
[0115] Meanwhile, the test results also showed that different combinations of CD7 VHH and CD3 VHH also affected the activation of naïve T cells by the virus (e.g., the lower T cell activation of the pVSV-Gm-004 CD7 VHH and CD3 VHH combination).
[0116] Flow cytometry was used to detect the expression of the activation marker CD25 and to statistically analyze changes in the CD25-positive T cell population. Figure 3 The results showed that the pVSV-Gm-005 and pVSV-Gm-007 structures better maintained the expression of the activated marker CD25, which may help improve T cell function.
[0117] Statistical cell proliferation ( Figure 4 The results showed that the pVSV-Gm-005 and pVSV-Gm-007 structures can effectively improve T cell proliferation.
[0118] EGFP expression was detected on day 8 of viral infection. Figure 5 The results showed that the structures pVSV-Gm-005, pVSV-Gm-007, and pVSV-Gm-008 significantly improved the infection efficiency of the virus in naïve T cells.
[0119] Example 3: Validation of in vitro antitumor activity of CAR-T cells prepared with different viral envelopes Resting T cells were infected with different types of viruses prepared in Example 1. The early activation marker CD69 was detected 6 hours after viral infection. Figure 6 The activation efficiencies of pVSV-Gm-005, pVSV-Gm-007, and pVSV-Gm-008 structures are stronger than those of single-target CD3 VHH, but weaker than those of dynabeads.
[0120] Detection of T-cell CAR expression 7 days after viral infection ( Figure 7 The pVSV-Gm-005, pVSV-Gm-007, and pVSV-Gm-008 structures significantly improved viral infection efficiency in naïve T cells. Expression of CAR-T cell activation markers CD69 and CD25 was detected on days 1 and 11. Compared to dynabead activation, the pVSV-Gm-005 and pVSV-Gm-007 structures showed better persistence of CAR-T cell activation. Figure 8 and Figure 10 ).
[0121] Cell proliferation statistics also showed that the pVSV-Gm-005 and pVSV-Gm-007 structures significantly improved CAR-T cell proliferation compared to the dynabeads activation infection method. Figure 9 ).
[0122] Based on the positive CAR test on day 7, in vitro cell-killing functional plating was performed. CAR-T cells and Raji cells were plated at a ratio of 1:2. Flow cytometry was used to detect Raji cell residuals 3 days after cell-killing. Figure 11 (As shown in Table 4), due to the low CAR-T positivity rate after pVSV-Gm-003 infection, no killing plate was performed). CAR-T cells prepared with pVSV-Gm-005 and pVSV-Gm-007 structures have good killing function.
[0123] Table 4 In summary, the experimental results show that the binding and activation of the weak CD3 VHH antibody by the CD7 VHH antibody with strong affinity effectively solves the problem of low infection efficiency of naïve T cells, and the killing function of the prepared CAR-T cells is also improved.
[0124] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. A fusion protein, characterized in that, The fusion protein comprises, from N-terminus to C-terminus, the following: (a) Anti-CD7 single-domain antibody (CD7 VHH); (b) Anti-CD3 single-domain antibody (CD3 VHH); and (c) A transmembrane region for anchoring the fusion protein to a cell membrane or viral envelope; The amino acid sequence of the anti-CD7 single-domain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the anti-CD3 single-domain antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the transmembrane region is shown in SEQ ID NO:
21.
2. The fusion protein as described in claim 1, characterized in that, The fusion protein further includes a hinge region located between the anti-CD3 single-domain antibody and the transmembrane region, and the amino acid sequence of the hinge region is shown in SEQ ID NO:
19.
3. The fusion protein as described in claim 2, characterized in that, The fusion protein further includes a co-stimulatory molecule located between the anti-CD3 single-domain antibody and the hinge region, and the co-stimulatory molecule is CD80. The amino acid sequence of the co-stimulatory molecule is shown in SEQ ID NO:
23.
4. A pseudo-lentiviral vector targeting T cells, characterized in that, The envelope protein of the pseudolentiviral vector is a modified vesicular stomatitis virus glycoprotein VSV-G mut; and the envelope of the pseudolentiviral vector displays the fusion protein as described in claim 1.
5. The pseudo-lentiviral vector as described in claim 4, characterized in that, The pseudo-lentiviral vector also contains a nucleic acid sequence encoding a chimeric antigen receptor (CAR); The chimeric antigen receptor is a chimeric antigen receptor that targets CD20, and the nucleic acid sequence is shown in SEQ ID NO:
2.
6. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein of claim 1.
7. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 6.
8. A host cell, characterized in that, The host cell contains the expression vector of claim 7, or has the nucleic acid molecule of claim 6 integrated into its genome, or expresses the fusion protein of claim 1.
9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) The pseudo-lentiviral vector of claim 4; and (b) Pharmaceutically acceptable carriers.
10. Use of the pseudolentiviral vector as described in claim 4 in the preparation of a medicament for treating cancer or tumors.