Membrane surface protein containing a gpi anchor

Abstract

The present application relates to membrane surface proteins containing GPI anchor region, and specifically provides fusion proteins comprising functional domains and GPI anchor region and uses thereof. The fusion proteins can effectively activate and proliferate immune cells, and enhance the effector function of immune cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a membrane surface protein containing a GPI anchor region. BACKGROUND

[0002] Transmembrane proteins are usually anchored on the membrane surface by a transmembrane domain, i.e. a transmembrane polypeptide. However, in addition to the common polypeptide transmembrane domain, proteins can also be localized on the membrane surface by lipid modification that occurs post-translationally. Most lipid modifications occur in the cytoplasm, helping to localize the protein to the membrane, and in some cases to specific membrane microdomains. Another type of lipid modification, glycosylphosphatidylinositol (GPI) modification, occurs on the luminal side of the endoplasmic reticulum membrane. After the core structure of GPI is synthesized on the endoplasmic reticulum membrane, it is transferred to the precursor protein that has just completed ER translocation, forming a nascent GPI-anchored protein. The nascent GPI-anchored protein undergoes several steps of modification in the endoplasmic reticulum and Golgi apparatus, forming a mature GPI-anchored protein, which is finally transported to the cell membrane surface to become a membrane surface protein integrated on the cell membrane lipid bilayer by the GPI structure.

[0003] The GPI portion of the GPI-anchored protein is composed of a conserved core polysaccharide, phosphatidylinositol, and a polysaccharide branch. Its core polysaccharide structure is EtNP-6Manα2-Manα6-(EtNP)2Manα4-GlNα6-Myo-Inositol-P-lipid. The protein part is basically a hydrophobic polypeptide with a missing transmembrane region, and the GPI covalently connected at the C-terminus is anchored on the cell membrane. For molecules that need to function by forming dimers or multimers, such as cytokines, antibodies, etc., if they are anchored on the membrane surface by the structure of GPI-anchored proteins, these molecules may be able to better exert their respective functions. SUMMARY

[0004] The present application provides a fusion protein comprising a functional domain and a GPI anchor region.

[0005] In one or more embodiments, the functional domain exercises its function by dimerization or multimerization.

[0006] In one or more embodiments, the functional domain comprises an antibody or an antigen-binding fragment thereof, and / or an amino acid-based cytokine or a functional fragment thereof.

[0007] In one or more embodiments, the cytokine comprises interleukin or its receptor, chemokine or its receptor, interferon, and TNF-α.

[0008] In one or more embodiments, the interleukin or its receptor comprises one or more selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, CD25, IL-7R, IL-12Rβ1, IL-12Rβ2, and IL-15R.

[0009] In one or more embodiments, the chemokine or its receptor comprises one or more selected from the following: CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL 15, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CCR2, CCR5, CCR1, CCR3, CCR4, CCR6, CCR7, CCR8, CCR9, CCR10 and XCR1.

[0010] In one or more embodiments, the interferon is IFN-γ.

[0011] In one or more embodiments, the antibody includes blocking antibodies and activating antibodies.

[0012] In one or more embodiments, the antibody is an antibody that targets tumor-associated antigens, such as antibodies that target HER2, HER3, CD137, or PD-1.

[0013] In one or more embodiments, the GPI anchoring region includes one or more of the following or their GPI anchoring domains: CD44, CD56, CD73, CD55, Thy1, AchE, IAP, ALPP, CD59, CD14, CD16, CD24, CD28, CD48, CD52, CD58, CD66a, CD66c, CD66d, CD66e, CD67, CD87, CD108, CD157, uPAR, JMH protein, GDNFR, CNTFR, TAG-1, PrP, phosphatidylinositol protein, serotonin 7, CEA, GFR, Ly6G, transferrin receptor, contactin (F3), and T-cadherin.

[0014] In one or more embodiments, the GPI anchor region comprises one or more of, or a GPI anchor domain of, a protein selected from the group consisting of: CD52, CD48, CD55, ALPP, CD90.

[0015] In one or more embodiments, the GPI anchor region has the sequence set forth in SEQ ID NO: 2 at positions 232-268, or a variant thereof having at least 90% sequence identity and having a function of forming a GPI anchor structure.

[0016] In one or more embodiments, the functional domain is located in the N-terminal direction of the GPI anchor region.

[0017] In one or more embodiments, the fusion protein further optionally comprises a spacer region located between the functional domain and the GPI anchor region.

[0018] In one or more embodiments, the spacer region has a length of 12-66 aa, for example 12 aa, 54 aa, 55 aa, or 66 aa.

[0019] In one or more embodiments, the spacer region comprises: a linker and / or a protein extracellular region.

[0020] In one or more embodiments, the linker has the sequence set forth in SEQ ID NO: 4 at positions 177-188, or a sequence having at least 90% sequence identity thereto.

[0021] In one or more embodiments, the protein extracellular region is a CD8 extracellular region and / or a BCMA extracellular region.

[0022] In one or more embodiments, the protein extracellular region has the sequence set forth in SEQ ID NO: 2 at positions 177-231 or SEQ ID NO: 4 at positions 189-242, or a sequence having at least 90% sequence identity thereto.

[0023] In one or more embodiments, the fusion protein further comprises a signal peptide.

[0024] In one or more embodiments, the signal peptide is a signal peptide of a protein corresponding to the GPI anchor region.

[0025] In one or more embodiments, the signal peptide is any one or more of a CD52, CD48, CD55, ALPP, CD90 protein signal peptide.

[0026] The present application also provides a nucleic acid molecule comprising a sequence selected from the group consisting of:

[0027] (1) a coding sequence of the fusion protein according to the first aspect of the present application or a fragment thereof as an amplification primer or a detection probe,

[0028] (2) a variant having at least 80% sequence identity to (1),

[0029] (3) a complement of (1) or (2).

[0030] Another aspect of the present application provides a nucleic acid construct comprising the nucleic acid molecule according to any of the embodiments herein.

[0031] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector or an integration vector.

[0032] Another aspect of the present application provides a host cell, which:

[0033] (1) comprises, expresses and / or secretes the fusion protein according to any of the embodiments of the first aspect of the present application, and / or

[0034] (2) comprises any one or more of the nucleic acid molecules and nucleic acid constructs according to any of the embodiments herein.

[0035] In one or more embodiments, the host cell is a lymphocyte, such as a T cell, an NK cell, a TIL cell, etc.

[0036] In one or more embodiments, the lymphocyte is a T cell, an NK cell or a tumor infiltrating lymphocyte (TIL).

[0037] In one or more embodiments, the T cell or TIL cell further expresses an exogenous TCR and / or CAR.

[0038] In one or more embodiments, the T cell comprises: a CAR-T cell, a TCR-T cell.

[0039] The present application also provides a method for preparing the fusion protein according to any of the embodiments of the first aspect of the present application, comprising incubating the host cell according to any of the embodiments herein.

[0040] Another aspect of the present application provides a pharmaceutical composition comprising any one or more of the fusion protein, the nucleic acid molecule, the nucleic acid construct and the cell according to any of the embodiments herein, and a pharmaceutically acceptable excipient.

[0041] The present application also provides the use of any one or more of the fusion protein, the nucleic acid molecule, the nucleic acid construct, the cell and the pharmaceutical composition according to any of the embodiments herein in the preparation of an antitumor medicament.

[0042] The present application also provides a method for treating and / or preventing tumors, comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of any one or more of the fusion proteins, nucleic acid molecules, nucleic acid constructs and cells described in any of the embodiments herein.

[0043] Advantages of the present application:

[0044] 1) Anchoring cytokines or antibodies on the surface of immune cells through GPI-anchored proteins can effectively activate and proliferate immune cells, and enhance the effector function of immune cells;

[0045] 2) Exogenous proteins such as cytokines and antibodies anchored on the surface of the cell membrane through GPI-anchored proteins have significantly improved expression strength compared to conventional amino acid transmembrane domain fixation;

[0046] 3) Compared with the GPI signal sequence disclosed in CN110709416A, the CD52 used in the present application is a complete protein, and the CD52-anchored membrane surface protein of the present application does not introduce potential new antigen sequences, has low immunogenicity, and is more conducive to subsequent in vivo application; BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 Microscopic bright field morphology of T cells expressing cytokines on the membrane surface through GPI-anchoring regions.

[0048] Figure 2 Number of T cells expressing cytokines on the membrane surface through GPI-anchoring regions.

[0049] Figure 3 Viability of T cells expressing cytokines on the membrane surface through GPI-anchoring regions.

[0050] Figure 4 Flow cytometry results of T cells expressing cytokines on the membrane surface through GPI-anchoring regions.

[0051] Figure 5 Cytokine secretion level of T cells expressing cytokines on the membrane surface through GPI-anchoring regions.

[0052] Figure 6 Flow cytometry results of T cells expressing cytokines and TCR on the membrane surface through GPI-anchoring regions.

[0053] Figure 7 Killing effect of T cells expressing cytokines and TCR on the membrane surface through GPI-anchoring regions on target cells under the condition of effector-to-target ratio of 1:1.

[0054] Figure 8Killing effect of T cells expressing HER2 antibody on homologous pairing tumor primary cells.

[0055] Figure 9 Killing effect of T cells expressing HER2 antibody on homologous pairing tumor primary cells.

[0056] Figure 10 Killing effect of T cells expressing HER2 antibody on homologous pairing tumor primary cells.

[0057] Figure 11 Number of T cells expressing cytokines anchored on the membrane surface by different GPI proteins.

[0058] Figure 12 Viability of T cells expressing cytokines anchored on the membrane surface by different GPI proteins.

[0059] Figure 13 Killing effect of T cells expressing cytokines and TCR anchored on the membrane surface by different GPI proteins on target cells at 0.5:1 effector-target ratio.

[0060] Figure 14 Tumor volume change of tumor-bearing mice administered with TCR-T and TCR-T cells expressing cytokines anchored on the membrane surface by GPI proteins in vivo.

[0061] Figure 15 Fluorescence value change of tumor of tumor-bearing mice administered with TCR-T and TCR-T cells expressing cytokines anchored on the membrane surface by GPI proteins in vivo.

[0062] Figure 16 Fluorescence in vivo imaging of tumor of tumor-bearing mice administered with TCR-T and TCR-T cells expressing cytokines anchored on the membrane surface by GPI proteins in vivo.

[0063] Figure 17 Number of human lymphocytes in peripheral blood of tumor-bearing mice administered with TCR-T and TCR-T cells expressing cytokines anchored on the membrane surface by GPI proteins in vivo. DETAILED DESCRIPTION

[0064] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987 and periodic updates thereto); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0065] GPI-anchored proteins are connected to lipid rafts on the cell membrane through lipid-lipid or glycan-glycan interactions. The inventors have found that for molecules that need to function by forming dimers or multimers, such as cytokines, antibodies, etc., anchoring these molecules to the membrane surface through the structure of GPI-anchored proteins can allow these molecules to better perform their respective functions, such as effectively increasing the proliferation and activation of immune cells and the level of killing of target cells.

[0066] The present application first provides a fusion protein comprising a functional domain and a GPI-anchoring region. The GPI-anchoring region is used to anchor the functional domain to the cell surface (such as an immune cell) to facilitate dimerization or multimerization of the functional domain. The functional domain is preferably located in the N-terminal direction of the GPI-anchoring region.

[0067] The GPI anchor region comprises one or more of, or a GPI anchor domain thereof (also referred to herein as a GPI signal sequence) selected from the group consisting of CD44, CD56, CD73, CD55, Thy1, AchE, IAP, ALPP, CD59, CD14, CD16, CD24, CD28, CD48, CD52, CD58, CD66a, CD66c, CD66d, CD66e, CD67, CD87, CD108, CD157, uPAR, JMH protein, GDNFR, CNTFR, TAG-1, PrP, phosphatidylinositol protein, Semaphorin 7, CEA, GFR, Ly6G, transferrin receptor, Contactin (F3), and T-cadherin; preferably, the GPI anchor region is a CD52 protein or a GPI anchor domain thereof. The sequence of a GPI anchor protein or its anchor domain is known in the art. Moreover, one skilled in the art can readily obtain the sequence of the anchor domain from the sequence of a GPI anchor protein.

[0068] In one or more embodiments, the GPI anchor region has the sequence set forth in SEQ ID NO: 2 at positions 232-268, or a variant thereof having at least 90% sequence identity and having the function of binding GPI. Alternatively, the GPI anchor region can be a CD48 GPI domain, a CD55 GPI domain, an ALPP GPI domain, and a CD90 GPI domain, the amino acid sequences of which are set forth in SEQ ID NOs: 15-18, respectively, as recited in the specification of CN110709416A at page 14.

[0069] Herein, a functional domain includes any polypeptide or protein that can exercise its function through dimerization or multimerization. In some embodiments, the functional domain includes a cytokine or a functional fragment thereof.

[0070] The cytokines that can be the functional domains described herein are primarily amino acid-based polypeptide cytokines, including but not limited to interleukins or their receptors, chemokines or their receptors, interferons, and TNF-a. The interleukins or their receptors include one or more selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, CD25, IL-7R, IL-12Rβ1, IL-12Rβ2, and IL-15R. The chemokines or their receptors include one or more selected from the group consisting of CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCR1, CXCR2, CXCR3A, CXCR3B, CXCR4, CXCR5, CXCR6, CCR2, CCR5, CCR1, CCR3, CCR4, CCR6, CCR7, CCR8, CCR9, CCR10, and XCR1. The interferon is, for example, IFN-γ. The above-mentioned cytokines include any form thereof known in the art, such as subtypes, derivatives. The amino acid sequences of these cytokines are within the routine knowledge of a person skilled in the art.

[0071] The functional domain can also be an antibody or an antigen-binding fragment thereof. Herein, a broad "antibody" can be any type of specific binding molecule against an antigen, such as a full-length antibody (which has an immunoglobulin Fc region), a heavy chain antibody, a nanobody, a minibody, an affibody, a target binding region of a receptor, a cell adhesion molecule, a ligand, etc. In some embodiments, the antibody includes a monoclonal antibody, an antibody composition with polyepitopic specificity, a multispecific antibody (e.g., a bispecific antibody), a diabody and a single chain molecule, as well as an antibody fragment, especially an antigen-binding fragment, such as Fab, F(ab')2, and Fv. The antibody includes a blocking antibody and an activating antibody. In embodiments against tumors, the antibody is preferably an antibody targeting a tumor-associated antigen, such as an antibody targeting HER2, HER3, CD137, or PD-1. Illustratively, the antibody targeting HER2 is a single chain antibody, preferably having the sequence shown in SEQ ID NO: 8 at positions 25-268. Alternatively, illustratively, the antibody targeting CD137 is a single chain antibody, preferably having the sequence shown in SEQ ID NO: 10 at positions 25-269.

[0072] The fusion protein also optionally includes a spacer between the functional domain and the GPI anchoring region. Whether or not to use a spacer is a routine choice for one of skill in the art, and thus it is not necessarily present. Typically, the spacer is 10-70 aa in length, preferably 12-66 aa, such as 12 aa, 54 aa, 55 aa, or 66 aa, or a range between any two of the foregoing. The spacer can comprise a linker, such as a polypeptide sequence composed of G and / or S, such as GS, GSG, GSSG, SGS, GGGGS repeated n times (e.g., 3 times). An exemplary linker has the sequence set forth in SEQ ID NO:4 at positions 177-188, or a sequence having at least 90% sequence identity thereto. The spacer can comprise an extracellular region of a protein. For example, a CD8 extracellular region, a BCMA extracellular region. An exemplary linker protein extracellular region has the sequence set forth in SEQ ID NO:2 at positions 177-231 or SEQ ID NO:4 at positions 189-242, or a sequence having at least 90% sequence identity thereto.

[0073] The fusion proteins of the application can be modified to affect function. The application includes fusion proteins having modified glycosylation patterns. Alternatively, modifications can be made to remove undesired glycosylation sites. The fusion proteins can also have modifications that facilitate expression, secretion, purification, such as signal peptides. In one or more embodiments, the signal peptide is the signal peptide of the protein to which the GPI anchoring region corresponds. Exemplarily, the signal peptide of the fusion proteins herein is any one or more of the CD52, CD48, CD55, ALPP, CD90 protein signal peptides.

[0074] In particular embodiments, the fusion proteins of the application have, from N- to C-terminus, a structure selected from any one of: (1) IL-7, CD8 extracellular region, CD52, and the N-terminus optionally has a CD52 signal peptide, (2) IL-7, linker, BCMA extracellular region, CD52, and the N-terminus optionally has a CD52 signal peptide, (3) Her2 antibody, linker, CD52, and the N-terminus optionally has a CD52 signal peptide, (4) CD137 antibody, linker, CD52, and the N-terminus optionally has a CD52 signal peptide, (5) IL-7, CD8 extracellular region, CD48 GPI anchoring domain, and the N-terminus optionally has a CD48 signal peptide, (6) IL-7, CD8 extracellular region, CD55 GPI anchoring domain, and the N-terminus optionally has a CD55 signal peptide, (7) IL-7, CD8 extracellular region, ALPP GPI anchoring domain, and the N-terminus optionally has an ALPP signal peptide, (8) IL-7, CD8 extracellular region, CD90 GPI anchoring domain, and the N-terminus optionally has a CD90 signal peptide.

[0075] The fusion proteins of the present application also include variants of the sequences shown therein having the same function. These variants include, but are not limited to, deletions from, and insertions into, and / or substitutions of, residues within the sequence shown herein, as well as in the addition of one or more (typically 1-20, preferably 1-10, more preferably 1-5) amino acids to or on the C- and / or N- terminus of the sequences shown herein. For example, in the art, it is recognized that certain conservative amino acid substitutions can be made in a protein without appreciable loss of function. In the art, such conservative changes typically involve the substitution of one amino acid for another within the same family of amino acids, as defined in the art. Such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Also, in the art, it is recognized that one or more amino acids can be added to or removed from the N- and / or C- terminus of a polypeptide or protein without appreciable loss of function. Conservative substitutions of non-coded amino acids are known in the art. Other conservative substitutions of non-coded amino acids can be determined based on a comparison of their physical properties with those of the genetically coded amino acids.

[0076] Any polypeptide that is highly homologous to the fusion proteins shown herein (e.g., 70% or more homologous to the sequence shown in SEQ ID NO: 2 or 4; preferably, 80% or more homologous; more preferably, 90% or more homologous, such as 95%, 98% or 99% homologous) and has similar or the same function as the fusion proteins is also included in the present application. The "same or similar function" refers primarily to the function of the functional domain of the fusion protein (e.g., anti-tumor) and the function of the GPI anchoring region (i.e., binding to GPI and thereby anchoring to the membrane).

[0077] The present application also encompasses analogs of the claimed fusion proteins. These analogs can differ from the original fusion protein by differences in the amino acid sequence, by differences in the form of the modification that does not affect the sequence, or by both. These analogs of the proteins include naturally or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, by site-directed mutagenesis, or other known molecular biology techniques. Analogues also include those with residues other than naturally occurring L-amino acids (e.g., D-amino acids), as well as analogues with non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is understood that the proteins of the present application are not limited to the representative proteins exemplified above.

[0078] The polypeptide fragments, derivatives or analogs of the present application can also be: (i) a polypeptide formed by fusion of the mature polypeptide to another compound, such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol; or (ii) a polypeptide formed by fusion of additional amino acid sequences to the polypeptide sequence, such as leader sequences or secretory sequences or sequences or proteins that serve to purify the polypeptide or prosequences. These fragments, derivatives and analogs are within the scope of those skilled in the art according to the definitions herein.

[0079] The present application also relates to polynucleotide sequences encoding the fusion proteins of the present application or variants, analogs, derivatives thereof, and nucleic acid molecules comprising the sequences. The polynucleotides can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0080] The present application also relates to variants of the above nucleic acid molecules that encode fusion proteins having the same amino acid sequence as the fusion proteins of the present application or fragments, analogs and derivatives thereof. The variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a variant of a polynucleotide, which can differ from a reference sequence by one or more nucleotides, but which does not change the functional properties of the encoded polypeptide in a substantial way. As used herein, a degenerate variant is a nucleic acid sequence that encodes the fusion proteins of the present application, but differs in sequence due to the degeneracy of the code. A "polynucleotide that encodes a polypeptide" can be a polynucleotide that includes only the coding sequence for the polypeptide, or a polynucleotide that includes additional coding and / or non-coding sequences.

[0081] The present application also relates to polynucleotides that hybridize (complement) to the sequences described above and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under lower ionic strength and higher temperature, such as 0.2x SSC, 0.1% SDS, 60°C; or (2) addition of denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95% or more. In some embodiments, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 1 or 2.

[0082] The sequences of the variants described herein can have at least 95%, 96%, 97%, 98%, or 99% identity to the sequence from which they are derived. The sequence identity described herein can be measured using sequence analysis software. For example, using the computer program BLAST, in particular BLASTP or BLASTN, with default parameters.

[0083] The full-length sequence of the fusion protein coding sequence of the present application or fragments thereof (e.g., primers or probes) can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the disclosed amino acid or nucleotide sequences, in particular the open reading frame sequences, of the present application, and a commercially available DNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequences. In addition, artificial synthesis can be used to synthesize the relevant sequences, in particular when the length of the fragments is relatively short, such as primers or probes. Methods known in the art for designing primer and probe sequences can be used herein, for example, through the software Primer Express.

[0084] The present application also relates to nucleic acid constructs comprising the polynucleotide sequences described herein, and one or more control sequences operably linked to the sequences. The polynucleotides described herein can be manipulated in a variety of ways to produce the fusion proteins of the present application. Manipulation of the nucleic acid constructs prior to their insertion into a vector can be used to control the expression of the fusion proteins. Techniques for modifying polynucleotide sequences using recombinant DNA methods are known in the art.

[0085] The regulatory sequences can be appropriate promoter sequences. A promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutated, truncated, and hybrid promoters, and can be derived from genes either homologous or heterologous to the host cell. The regulatory sequences can also be a suitable transcription terminator sequence, a sequence recognized by a host cell for termination of transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice can be used in the present application. The regulatory sequences can also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence which is functional in the host cell of choice can be used in the present application.

[0086] In certain embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or an integration vector. The polynucleotides of the present application can be cloned into a number of types of vectors, e.g., plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide the coding sequence of the fusion proteins of the present application. Expression vectors can be provided to cells in the form of viral vectors. Expression of the polynucleotides of the present application is typically achieved by operably linking the polynucleotide of the present application to a promoter and incorporating the construct into an expression vector. Integration vectors are used to integrate the expression cassettes described herein into the host genome, e.g., integration vectors such as pNB, pNC, which integrate genes via the PB transposition system. Typically, a suitable vector will comprise an origin of replication functional in at least one organism, a promoter sequence, transcriptional and translational stop signals, regions of

[0087] To assess expression of the protein, the expression vector introduced into the cell can also comprise a selectable marker gene and / or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. The selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

[0088] The polynucleotides described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each amplification in the correct order. Alternatively, the nucleic acid molecules described herein can be synthesized directly.

[0089] Methods of introducing genes into cells and expressing genes into cells are known in the art. Vectors can be readily introduced into host cells, e.g., mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0090] Physical methods of introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods of introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, and beads; and lipid- based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0091] Herein, host cells contain, express, and / or secrete the fusion proteins described herein. Herein, when referring to a cell containing or comprising, expressing, secreting a molecule such as a polypeptide, "containing" means that the molecule is contained within or on the surface of the cell; "expressing" means that the cell produces the molecule; and "secreting" means that the cell secretes the expressed molecule outside the cell. Host cells include both immune cells (e.g., T cells) that are ultimately used for therapeutic purposes, and various cells used in the process of producing immune cells, such as E. coli cells, for example, to provide a coding sequence for a protein of the application or to provide a vector described herein. The proteins expressed and / or secreted by the cells can be purified by methods known in the art.

[0092] T cells suitable for use in the application can be various types of T cells of various origins. For example, the T cells can be derived from PBMCs of healthy individuals. In certain embodiments, provided herein is a TCR-T cell, a CAR-T cell, a TIL cell that stably expresses a fusion protein described herein. Methods of obtaining these cells are known to those skilled in the art. In certain embodiments, after obtaining the T cells, they can be activated by stimulation with an appropriate amount (e.g., 30-80 ng / ml, such as 50 ng / ml) of CD3 antibody, and then cultured in a medium containing an appropriate amount (e.g., 30-80 IU / ml, such as 50 IU / ml) of IL2 for later use.

[0093] Thus, in certain embodiments, the present application provides a genetically modified cell (e.g., an immune cell) that contains a nucleic acid molecule described herein, or that contains a vector described herein, or that is made using a method described herein, or that stably expresses a fusion protein described herein.

[0094] Also provided herein is a pharmaceutical composition containing any one or more of the fusion proteins, nucleic acid molecules, nucleic acid constructs, and cells described herein, and a pharmaceutically acceptable excipient. As used herein, a pharmaceutically acceptable excipient refers to a carrier and / or vehicle with which the active ingredient is combined in order to facilitate administration of a subject. In certain embodiments, the excipient is preferably nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the pharmaceutical composition can contain such materials as agents for improving, maintaining or preserving the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, release, absorption, or penetration of the composition. These materials are present in minor amounts and do not affect the efficacy of the immune cells, particularly T cells, of the composition. Exemplary of such materials are pH adjusting agents, surfactants, ion exchangers, bulking agents, binding agents, emulsifiers, solubilizers, preservatives, and / or adjuvants. More particularly, suitable pharmaceutically acceptable excipients can be those used in the art for the administration of immune cells, particularly T cells. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed., A. R. Genrmo, ed., 1990, Mack Publishing Company. The optimal pharmaceutical composition will be determined by the desired route of administration and concentration of the active ingredient.

[0095] The pharmaceutical compositions of the present application can be selected for parenteral delivery. Alternatively, the compositions can be selected for inhalation or delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations that include the immune cells, particularly T cells, in sustained or controlled release delivery formulations. Techniques for formulation of a variety of other sustained or controlled delivery means, such as liposomal carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. The cell-containing pharmaceutical compositions can be injected directly into a tumor, lymph node, or site of infection.

[0096] Pharmaceutical compositions for in vivo administration are typically provided in sterile form. Sterilization is achieved by filtration through a sterile filtration membrane. Compositions for parenteral administration can be stored in lyophilized form or in solution (e.g., a cryopreservation formulation). Parenteral compositions are typically placed into a container having a sterile access port, for example, a vial or ampule having a stopper pierceable by a hypodermic injection needle.

[0097] The pharmaceutical compositions, once formulated, can be stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, cryoprecipitates, or as dehydrated or lyophilized powders. The pharmaceutical formulations (e.g., cryopreserved formulations) can be stored in a ready-to-use form or in a form that is further formulated prior to administration. For example, a suitable delivery of the pharmaceutical compositions described herein can be a cryopreserved formulation, which can be resistant to damage from long distance transport. In addition to the cells themselves, the cryopreserved formulation typically includes components such as a cell cryopreservation solution, human serum albumin (HSA), and the like. The cryopreserved pharmaceutical compositions are stored at a low temperature (e.g., in liquid nitrogen) prior to administration (e.g., intravenous infusion). The cryopreserved formulation can be thawed and administered directly or formulated into an infusion composition for infusion into a patient. Those of skill in the art are aware of the components and concentrations of conventional cryopreservation solutions. For example, the cryopreservation solution or infusion composition can also include sodium chloride, dextrose, sodium acetate, potassium chloride, or magnesium chloride, among others, at concentrations that can be determined by those of skill in the art (e.g., an experienced physician) based on the cells, the disease, the patient, and the like. The present application also provides kits for generating a single dose administration unit. The kits of the present application can each contain a first container having a cell cryopreservation solution or a cryopreserved formulation and, optionally, a second container having an infusion formulation. In certain embodiments of the present application, kits containing single and multiple chamber pre-filled infusion devices (e.g., liquid infusion devices) are provided.

[0098] Also provided herein is a kit containing the nucleic acid construct described herein. The kit can also contain various reagents suitable for transfecting the nucleic acid construct into a cell, and optionally, instructions for a person of skill in the art to transfect the recombinant expression vector into a cell.

[0099] The present application also provides methods of treating a disease in a patient by administering the fusion protein, cell, or pharmaceutical composition described in any of the embodiments of the present application. The diseases described herein include various types of cancer (tumors), including solid tumors and blood tumors, such as adenocarcinoma, lung cancer, colon cancer, large bowel cancer, breast cancer, ovarian cancer, cervical cancer, stomach cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer, and prostate cancer, among solid tumors, and leukemia and lymphoma, such as B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myelogenous leukemia, among others. Preferably, the disease is a tumor that benefits from dimerization or multimerization of the functional domain (antibody or cytokine described herein).

[0100] As used herein, the terms "patient," "individual," "subject" are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., a rat, a mouse, a dog, a cat, a rabbit, etc.), and most preferably a human. "Treatment" means the use of a therapeutic regimen described herein on a subject to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of cancer cell infiltration into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth). Typically, a therapeutically effective amount of cells is contained in a pharmaceutical composition. A therapeutically effective amount means an amount that can effect treatment, prevention, attenuation, and / or alleviation of a disease or condition in a subject. A therapeutically effective amount can be determined based on age, sex, condition being treated, the severity of the condition, the general health of the patient, and like factors. As used herein, a subject or patient generally refers to a mammal, and more particularly to a human.

[0101] The therapeutically effective amount of a pharmaceutical composition containing a fusion protein or immune cell of the present application to be employed will depend, for example, on the therapeutic effect desired and the target. Those of ordinary skill in the art will ascertain an appropriate therapeutic regimen for treating a patient by considering factors such as the molecule being delivered, the indication, the route of administration, and the size (weight, body surface, or organ size) and / or condition (age, general health, the ability of the therapy to elicit an anti-cancer response in the subject) of the patient. In certain embodiments, a clinician can titrate the dose and vary the route of administration to achieve optimal therapeutic effects.

[0102] The frequency of dosing will depend on the pharmacokinetic parameters of the immune cells in the formulation being used. The clinician typically will administer the composition until a dosage is reached that achieves the desired effect. The composition thus can be administered as a single dose, or as two or more doses (which can or can not contain the same amounts of the desired molecules) over time, or by an implantation device or a continuous infusion via a catheter.

[0103] The route of administration of the pharmaceutical composition is generally known methods, e.g., oral, by injection at intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal, or intralesional routes; by sustained release systems, or by implantation devices.

[0104] In some embodiments of the present application, the fusion proteins, cells, or pharmaceutical compositions of the present application can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressive agents. For example, the treatment can be combined with a radiation or chemotherapy agent known in the art for treating a tumor antigen-mediated disease.

[0105] The present application also provides the use of any one or more of the fusion proteins, nucleic acid molecules, nucleic acid constructs, cells, and pharmaceutical compositions described herein in the manufacture of an anti-tumor medicament. In the present context, "anti-tumor" refers to a biological effect that can be indicated by a reduction in tumor volume, a reduction in tumor cell number, a reduction in metastasis number, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

[0106] The present application is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the present application should in no way be interpreted as being limited to the following examples, but rather should be interpreted to include any and all examples that become evident as a result of the teachings provided herein. The methods and reagents used in the examples are routine in the art, unless otherwise specified.

[0107] Example

[0108] Antibodies and instruments

[0109] OKT3 purchased from: ThermoFisher, Cat. No. 14-0037-82

[0110] CD28 antibody purchased from: ThermoFisher Cat. No. 14-0281-82

[0111] Electroporator: Lonza Nucleofector 2b purchased from Lonza Cat. No. AAB-1001

[0112] Example 1, Construction of expression vector of CD52 linker-anchored membrane surface protein

[0113] The following coding sequences for membrane surface proteins comprising a CD52 linker anchor were synthesized:

[0114] 1. Amino acid sequence and coding sequence of a fusion protein of IL-7 and CD52 reading frames:

[0115] IL-7-CD52-1: CD52 signal peptide - IL-7 - CD8 ectodomain - CD52, coding nucleic acid sequence: SEQ ID NO: 1; protein sequence: SEQ ID NO: 2;

[0116] IL-7-CD52-2: CD52 signal peptide - IL-7 - linker - BCMA ectodomain - CD52, coding nucleic acid sequence: SEQ ID NO: 3; protein sequence: SEQ ID NO: 4;

[0117] IL-7-CD8TM: CD8 signal peptide-IL-7-CD8 extracellular region-CD8 transmembrane region, encoding nucleic acid sequence: SEQ ID NO: 5; protein sequence: SEQ ID NO: 6

[0118] 2. Amino acid sequence and encoding sequence of fusion protein of HER2 antibody and CD52 reading frame:

[0119] aHer2-CD52: CD52 signal peptide-aHer2-linker-CD52, encoding nucleic acid sequence: SEQ ID NO: 7, protein sequence SEQ ID NO: 8;

[0120] 3. Amino acid sequence and encoding sequence of fusion protein of CD137 activating antibody and CD52 reading frame:

[0121] aCD137-CD52: CD52 signal peptide-aCD137-linker-CD52 sequence: encoding nucleic acid sequence: SEQ ID NO: 9; protein sequence SEQ ID NO: 10

[0122] The pKB20 vector was constructed according to the method described in Example 1 of page 21 of the specification of PCT application WO2022078310A1. The pKB20 vector containing the expression frame of IL-7-CD52-1, IL-7-CD52-2, IL-7-CD8TM, aHer2-GPI and aCD137-GPI was constructed according to the method described in the example, and was named pKB20-IL-7-CD52-1, pKB20-IL-7-CD52-2, pKB20-IL-7-CD8TM, pKB20-aHer2-CD52 and pKB20-aCD137-CD52, respectively. The vector pKB20-EGFP expressing EGFP was constructed according to the method described in the same example. The obtained recombinant plasmid was transformed into E. coli (DH5c), and after sequencing, the high-quality plasmid of each recombinant expression vector was obtained by using the Qiagen plasmid purification kit to extract and purify the plasmid.

[0123] Example 2, Preparation of tumor antigen NY-ESO-1 TCR expression vector

[0124] The DNA sequence encoding the alpha chain and beta chain of the TCR recognizing the NY-ESO-1 antigen peptide SLLMWITQC (HLA-*A02:01) was synthesized, both linked by a DNA sequence encoding a P2A peptide segment, the spliced sequence being shown as SEQ ID NO: 11. The DNA sequence encoding EGFP was then linked to the 3' end of SEQ ID NO: 11 by a DNA sequence encoding a P2A peptide segment, the resulting sequence being shown as SEQ ID NO: 12. SEQ ID NO: 12 was synthesized by a commissioned company and cloned into the prepared pKB20 vector according to the method described in Example 1, page 21 of the specification of PCT application WO2022078310A1, named pKB20-TCR. The vector pKC20 without the PB transposase expression frame was prepared according to the method described in Example 1, page 21 of the specification of PCT application WO2022078310A1. SEQ ID NO: 12 was cloned into the prepared pKC20 vector according to the method described in the same example, named pKC20-TCR. The obtained recombinant plasmids were transformed into E. coli (DH5c), and after sequencing, the plasmids were extracted and purified using the Qiagen plasmid purification kit, obtaining high-quality plasmids of each recombinant expression vector.

[0125] Example 3, Preparation and culture of T cells expressing CD52 linker-anchored membrane surface IL-7

[0126] The PBMCs were electroporated with the expression vector in Example 1 to prepare CD52 linker-anchored T cells expressing membrane surface IL-7, and the PBMCs used were purchased from AllCells company, from the peripheral blood of healthy adults.

[0127] 1) Collect the suspended cells into a 50 ml centrifuge tube, centrifuge at 1200 rpm for 3 min;

[0128] 2) Discard the supernatant, resuspend with physiological saline, centrifuge at 1200 rpm for 3 min, discard the physiological saline, and repeat this step, and count the cells;

[0129] 3) Take 5 1.5 ml centrifuge tubes, add 5 x 10 6 cells to each tube, centrifuge at 1200 rpm for 3 min;

[0130] 4) Discard the supernatant, take the electroporation kit (purchased from Lonza Company), add 18 μL of solution I reagent and 82 μL of solution II reagent, add 5 μg of pKB20-IL-7-CD52-1 plasmid to the first tube, add 5 μg of pKB20-IL-7-CD52-2 plasmid to the second tube, add 5 μg of pKB20-IL-7-CD8TM plasmid to the third tube, and add 5 μg of pKB20-EGFP plasmid to the fourth and fifth tubes;

[0131] 5) Transfer the cell suspension mixed with the plasmid in the centrifuge tube to the electroporation cup, place it in the electroporator, select the T020 program, and perform the electric shock;

[0132] 6) Use the micropipette in the kit to transfer the electroporated cell suspension to the wells of the 6-well plate containing AIM-V culture solution (AIM-V culture solution containing 2% FBS), mix well, and place it in a 37°C, 5% CO2 incubator for 4-6 hours of static culture; at the same time, coat 5 wells of the 6-well plate with a mixture containing 5 μg / mL OKT-3 and 5 μg / mL CD28 antibodies, add 1 mL of the mixture to each well, and place the 6-well plate in a 37°C incubator;

[0133] 7) After 6 hours, remove the antibody coating mixture and wash twice with PBS. Transfer the cells that have been statically cultured in the 37°C, 5% CO2 incubator after electroporation to the 6-well plate coated with OKT-3 and CD28 antibodies, add culture solution to each well to 3 mL, do not add IL-2 to the culture solution of cells electroporated with pKB20-IL-7-CD52-1, pKB20-IL-7-CD52-2, and pKB20-IL-7-CD8TM, do not add IL-2 to the culture solution of cells in one of the two wells of cells electroporated with pKB20-EGFP (pKB20-EGFP-IL-2-), and add IL-2 to the culture solution of cells in the other well to a final concentration of 500 U / mL (pKB20-EGFP-IL-2+), and observe the growth of cells in each group, respectively.

[0134] The results are shown in Figure 1 , Figure 2 and Figure 3 . Figure 1 The morphology of T cells electroporated with pKB20-IL-7-CD52-2 (IL-7-CD52-2) and T cells electroporated with pKB20-EGFP and then normally cultured with IL-2 (EGFP-IL-2+) under a microscope. Compared with the T cells in the EGFP-IL-2+ group, the aggregation of T cells in the IL-7-CD52-2 group is more obvious, and the aggregation of T cells in growth is usually related to the increase of the level of self-activation. Figure 2It is shown that the cell number of pKB20-IL-7-CD52-1 group and pKB20-IL-7-CD52-2 group is significantly higher than that of pKB20-IL-7-CD8TM group after culture for 13 days after electroporation, and the cell number of the above three groups is significantly higher than that of pKB20-EGFP-IL-2+ group. No significant cell proliferation is observed in pKB20-EGFP-IL-2- group. Figure 3 It is shown that the cell viability of pKB20-IL-7-CD52-1 group, pKB20-IL-7-CD52-2 group and pKB20-IL-7-CD8TM group is similar, all above 90%, slightly higher than that of pKB20-EGFP-IL-2+ group. The cell viability of pKB20-EGFP-IL-2- group starts to decrease after culture for 7 days after electroporation, and has decreased to about 20% after culture for 13 days after electroporation. Figure 1-3 The results show that the membrane surface-anchored IL-7 can replace IL-2 in the cell culture medium to produce significant activation and proliferation effects on PBMC cells, and the stimulation and proliferation effect of IL-7 anchored on the cell membrane surface through the GPI anchor of CD52 is better than that of the conventional polypeptide transmembrane domain.

[0135] Example 4, Detection of proportion of positive T cells expressing CD52 linker-anchored membrane surface IL-7

[0136] A, indirect method for detecting the proportion of cells positive for expressing membrane surface-anchored IL-7, the method is as follows:

[0137] 1) Collect pKB20-IL-7-CD52-1, pKB20-IL-7-CD52-2 and pKB20-IL-7-CD8TM group cells respectively, collect 1×10 6 cells for each, centrifuge at 1000 rpm for 3 min;

[0138] 2) Discard the supernatant, resuspend the cells with physiological saline, centrifuge at 1000 rpm for 3 min;

[0139] 3) Discard the supernatant, resuspend the cells with 100 μL of physiological saline, add 1 μL of biotin-labeled anti-IL-7 antibody (purchased from Biolegend, item number: 506602) to each tube, and incubate at 4°C for 30 min;

[0140] 4) Add appropriate amount of physiological saline, centrifuge at 1000 rpm for 3 min, wash twice, and discard the supernatant;

[0141] 5) Resuspend the cells with 100 μL of physiological saline, add 1 μL of PE-labeled streptavidin (purchased from Thermo Fisher, item number: S20982) to each tube, mix well, and incubate at 4°C for 30 min;

[0142] 6) Add physiological saline to each, centrifuge at 1000 rpm for 3 min, wash twice, and discard the supernatant;

[0143] 7) Resuspend with 400 μL of physiological saline, and detect with a flow cytometer.

[0144] The flow cytometry results show that the proportion of positive cells for extracellular gene expression in the pKB20-IL-7-CD52-1, pKB20-IL-7-CD52-2, and pKB20-IL-7-CD8TM groups is more than 30%.

[0145] B, the BCMA extracellular domain contained in the extracellular region of the pKB20-IL-7-CD52-2 group cells is used as a tag, and the proportion of cells positive for exogenous gene expression in the pKB20-IL-7-CD52-2 group cells obtained in Example 3 is detected by a flow cytometer by a direct method, as follows:

[0146] 1) Collect pKB20-IL-7-CD52-2 group cells, and collect 1 x 10 6 cells at 1000 rpm for 3 min;

[0147] 2) Discard the supernatant, resuspend the cells with physiological saline, centrifuge at 1000 rpm for 3 min;

[0148] 3) Discard the supernatant, resuspend the cells with 100 μL of physiological saline, add 2 μL of BCMA flow cytometry antibody (purchased from Biolegend, item number: 357504) to each tube, and incubate at room temperature for 30 min;

[0149] 4) Add physiological saline to each, centrifuge at 1000 rpm for 3 min, wash twice, and discard the supernatant;

[0150] 5) Resuspend with 400 μL of physiological saline, and detect with a flow cytometer.

[0151] The results are shown in Figure 4 . The proportion of BCMA+ cells in the pKB20-IL-7-CD52-2 group cells is 33.62%, indicating that the proportion of positive cells in the pKB20-IL-7-CD52-2 group cells is more than 30%. This is basically consistent with the results of indirect detection.

[0152] Example 5, Detection of IFN-γ secretion of T cells expressing CD52 linker-anchored membrane surface IL-7

[0153] Harvest the cells of each group prepared in Example 3, and detect the IFN-γ secretion level using the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, item number: 62HIFNGPET) according to the method recorded in the instructions. The results are shown in Figure 5 .Figure 5 The results show that the concentration of IFN-γ secreted by each group of cells into the supernatant is more than 25000 pg / mL. Among them, the IFN-γ secretion level of the pKB20-IL-7-CD52-2 group of cells is higher than that of the pKB20-IL-7-CD52-1 group and the pKB20-IL-7-CD8TM group, and is equivalent to the secretion level of the pKB20-EGFP-IL-2+ group. It is indicated that the expression of CD52 anchoring on the surface of T cells achieves a similar cytokine secretion effect as incubation with IL-2.

[0154] Example 6, Preparation of TCR-T expressing CD52 linker-anchored membrane surface IL-7

[0155] Preparation of human activated T cells:

[0156] Coat the six-well plate chamber with coating solution containing 5 μg / ml of anti-CD3 antibody and 5 μg / ml of anti-CD28 antibody at room temperature for 2-4 hours, wash the well plate with normal saline for 1-3 times after removing the coating solution, and add 2% FBS AIM-V medium for standby; human peripheral blood PBMC (HLA-*02:01, purchased from ALLCELLS) is recovered in a 37°C water bath, and the adherent cells are cultured for 2-4 hours. The non-adherent cells in suspension are the initial T cells. The suspension cells are collected into a 15 ml centrifuge tube, centrifuged at 1200 rpm for 3 min, the supernatant is discarded, normal saline is added, centrifuged at 1200 rpm for 3 min, the normal saline is discarded, and the step is repeated. Then the washed initial T cells are transferred to the antibody coated wells containing the standby medium, and the subsequent experiments are carried out after 3-4 days of culture at 37°C and 5% CO2.

[0157] Preparation of TCR-T cells expressing NY-ESO-1 TCR-EGFP and NY-ESO-1 TCR-EGFP+CD52 linker anchored TCR-T cells expressing membrane surface IL-7 by electroporation:

[0158] 1) Preheat the AIM-V medium to 37°C in a 5% CO2 cell culture incubator for 1 hour before adding it to 2 wells of a 12-well plate, 2 mL per well, and then transferring it to the cell culture incubator;

[0159] 2) Prepare the electroporation solution according to the following table, a total of 2 wells:

[0160] Table 1

[0161] 100 μL Nucleocuvette TM Strip (μL) Nucleofector TM volume of the solution 82 Electrotransfection supplement solution 18

[0162] 3) Take the obtained activated T cells to 2 EP tubes, add 5×10 6 cells to each EP tube, centrifuge at 1200 rpm for 5 min, discard the supernatant, then resuspend the cells with 500 μL of normal saline, repeat the centrifugation step to wash the cell pellet;

[0163] 4) Add 6 μg of plasmid pKB20-TCR to one of the two wells of the prepared electrotransfection solution in 2), and add 3 μg of pKB20-TCR and 3 μg of pKB20-IL-7-CD52-2 to the other well, then stand at room temperature for no more than 30 min;

[0164] 5) Resuspend the activated T cells in two tubes with 100 μL of the prepared electrotransfection solution containing plasmids in 4), carefully transfer the cell suspension into the LONZA 100 μL electrotransfection cup, and place the electrotransfection cup into the LONZA Nucleofector TM 2b electrotransfection tank, start the electrotransfection program, and select T-020 for the electrotransfection program;

[0165] 6) After the completion of electrotransfection, carefully remove the electrotransfection cup, transfer the cell suspension into an EP tube, add 200 μL of preheated AIM-V medium to each tube, then transfer the cell suspension into the wells containing preheated AIM-V medium in the 12-well plate in 1), and culture at 37°C and 5% CO2. After 1 h of culture, add compound G150 (purchased from MedChemExpress) to a final concentration of 5 μM, then continue to culture for 13 days, and subculture according to the cell proliferation during the culture period. After 13 days, detect the number of cells and the cell viability of each electrotransfection sample.

[0166] The cells expressing exogenous genes prepared by the above method are respectively named TCR-T and IL-7-CD52-TCR-T.

[0167] Obtain the above-mentioned cells and detect the proportion of EGFP-positive cells by flow cytometry (Beckman Cytoflex). For TCR-T, EGFP-positive cells can be regarded as cells with positive expression of NY-ESO-1 TCR gene. For cells co-transfected with IL-7-CD52-2 and TCR, because the PB transposase expression frame is not included in the pKC20 vector, EGFP-positive indicates that the PB transposase required for the successful integration and expression of TCR-EGFP comes from the pKB20 vector expressing the IL-7-CD52-2 gene, and therefore in the cells co-transfected with IL-7-CD52-2 and TCR, EGFP-positive cells can be considered as double integration and expression of IL-7-CD52-2 and NY-ESO-1 TCR.

[0168] The flow cytometry detection results of the obtained cells are shown in Figure 6 . Figure 6 The first row in the table is the flow cytometry detection results of TCR-T, and the second row is the flow cytometry detection results of IL-7-CD52-TCR-T. Figure 5The results show that the proportion of cells positive for TCR integration expression in TCR-T is 27.92% by the proportion of GFP positive cells, and the proportion of cells positive for IL-7-CD52-2 and TCR double integration expression in IL-7-CD52-TCR-T is 13.48% by the proportion of GFP positive cells.

[0169] Example 7, Killing of target cells by TCR-T expressing CD52 linker-anchored membrane surface IL-7

[0170] The NY-ESO-1 expression positive HLA-A*02:01 melanoma cell line A375 (purchased from the American Type Culture Collection ATCC, Cat. CRL-1619) was selected as the target cell, and the real-time label-free cell function analyzer (RTCA) of Eisen Company was used to detect the in vitro killing activity of the TCR-T and IL-7-CD52-TCR-T cells prepared in Example 6. The specific steps are as follows:

[0171] (1) Zero setting: add 50 μL of DMEM or 1640 culture solution to each well, place it in the instrument, select step 1, and zero set;

[0172] (2) Target cell plating: melanoma cells A375 were plated at 10 4 cells / 50 μL per well in a plate containing a detection electrode, and placed for a few minutes to allow the cells to stabilize, then placed in the instrument, started step 2, and cultured the cells;

[0173] (3) Add effector cells: after the target cells were cultured for 24 h and the cell index reached 1.0, step 2 was paused, 50 μL of effector cells was added to each well, and the number of effector cells was added according to the positive cell ratio in Example 6 at an effector to target ratio of 1:1 and 0.5:1, respectively, step 3 was started, and the co-culture was continued for more than 80 h, and the cell proliferation curve was observed.

[0174] The results are shown in Figure 7 and Figure 8 . Figure 7 and Figure 8 The results show that under the conditions of effector to target ratio of 1:1 and 0.5:1, the killing rate of IL-7-CD52-TCR-T on target cells A375 is significantly higher than that of TCR-T, indicating that the expression of CD52 linker anchoring IL-7 can significantly improve the recognition of TCR-T cells to antigen and the killing power of target cells.

[0175] Example 8, Preparation and in vitro killing detection of tumor infiltrating lymphocyte cells expressing CD52 linker-anchored membrane surface anti-HER2 antibody Figure 9

[0176] Preparation of tumor infiltrating lymphocytes (TIL) of breast tumor tissue:

[0177] Collect newly removed HER2+ breast cancer specimens and process them immediately under aseptic conditions. The specific method is as follows: remove the normal tissue and necrotic areas surrounding the breast tumor specimen, and remove 1-2 mm specimens from different areas of the specimen. 3 Small tissue fragments were placed in each well of a 24-well plate. 2 mL of complete culture medium (AIM-V medium containing 10% FBS) and 3000 IU / mL IL-2 were added to each well. The 24-well plate was incubated at 37°C in a 5% CO2 incubator. On days 5-6 after initiation, half the medium was replaced in all wells. Thereafter, half the medium was replaced every 1-2 days depending on TIL growth. Once the wells were confluent with TILs and all adherent cells had been removed, the TILs from each confluent well were collected.

[0178] Preparation of TILs expressing CD52 linker-anchored membrane-surface anti-HER2 antibodies and their in vitro killing effect on primary tumor cells:

[0179] Following the method described in Example 3, the breast tumor TIL cells prepared above were electroporated with the pKB20-αHer2-CD52 vector prepared in Example 1 to obtain αHer2-CD52-TIL. A portion of identical breast tumor tissue was taken, enzymatically digested into a single-cell suspension, and then seeded into RTCA analyzer wells according to the method described in Example 7. Based on the tumor cell growth rate, the time for adding effector cells was adjusted to 16 hours after target cell seeding. The cells were divided into a TIL group and an αHer2-CD52-TIL group. The effector-to-target ratio for the TIL group was set to 0.5:1, and the αHer2-CD52-TIL group was divided into three effector-to-target ratio groups: 0.5:1, 1:1, and 2:1. The differences in killing effects among the different groups were observed.

[0180] The results are as follows Example 9, Preparation of TCR-T expressing CD52 linker-anchored membrane surface CD137 antibody and in vitro killing of target cells As shown, at the same effector-to-target ratio (0.5:1), αHer2-CD52-TIL exhibited significantly higher cytotoxicity against primary breast cancer cells than unmodified TIL. Furthermore, with increasing effector-to-target ratio, the cytotoxicity of αHer2-CD52-TIL against primary breast cancer cells also significantly increased, indicating that expression of αHer2-CD52 on the TIL surface can significantly enhance the cytotoxicity of TIL against its corresponding homologous target cells.

[0181] Figure 10 Example 10, Construction of expression vector of other GPI protein-anchored membrane surface IL-7

[0182] The pKB20-aCD137-CD52 vector and the pKC20-TCR vector prepared in Example 1 were co-transfected into T cells in the manner described in Example 6 to obtain aCD137-CD52-TCR-T, and the T cells used were the same batch as those used in Example 6. The target cells A375 were subjected to RTCA killing detection by aCD137-CD52-TCR-T and TCR-T prepared in Example 6 in the manner described in Example 7. According to the growth rate of the plated A375, the time of adding the effector cells was adjusted to 16 hours after the target cells were plated, and the cells were divided into a TCR-T group and an aCD137-CD52-TCR-T group, the effector-to-target ratio of the TCR-T group was set to 0.5:1, and the effector-to-target ratio of the aCD137-CD52-TCR-T group was set to 0.5:1, 1:1 and 2:1, respectively, and the killing effect of different groups was observed.

[0183] The results are shown in Table 1. Example 11, Preparation and culture of T cells expressing other GPI protein-anchored membrane surface IL-7 As shown in Table 1, under the same effector-to-target ratio (0.5:1), the killing effect of aCD137-CD52-TCR-T on target cells A375 was significantly higher than that of unmodified TCR-T. And with the increase of the effector-to-target ratio, the killing effect of aCD137-CD52-TCR-T on A375 cells also increased significantly, indicating that the expression of aCD137-CD52 on the surface of TCR-T can significantly improve the killing effect of TCR-T on target cells.

[0184] Figure 11

[0185] The following IL-7-GPI anchor protein fusion proteins were constructed respectively

[0186] IL-7-CD48: CD48 signal peptide-IL-7-CD8 extracellular region-CD48 GPI signal sequence, the coding nucleic acid sequence thereof is SEQ ID NO: 13, and the protein sequence thereof is SEQ ID NO: 14.

[0187] IL-7-CD55: CD55 signal peptide-IL-7-CD8 extracellular region-CD55 GPI signal sequence, the coding nucleic acid sequence thereof is SEQ ID NO: 15, and the protein sequence thereof is SEQ ID NO: 16.

[0188] IL-7-ALPP: ALPP signal peptide-IL-7-CD8 extracellular region-ALPP GPI signal sequence, the coding nucleic acid sequence thereof is SEQ ID NO: 17, and the protein sequence thereof is SEQ ID NO: 18.

[0189] IL-7-CD90: CD90 signal peptide-IL-7-CD8 ectodomain-CD90 GPI signal sequence, encoding nucleic acid sequence: SEQ ID NO: 19; protein sequence: SEQ ID NO: 20

[0190] wherein the amino acid sequences of the CD48 GPI signal sequence, the CD55 GPI signal sequence, the ALPP GPI signal sequence, and the CD90 GPI signal sequence are SEQ ID NOs: 15-18, respectively, as described in the specification of CN110709416A on page 14.

[0191] According to the method described in Example 1, the pKB20 vector containing the expression frame of IL-7-CD48, IL-7-CD55, IL-7-ALPP, and IL-7-CD90 was constructed, and named as pKB20-IL-7-CD48, pKB20-IL-7-CD55, pKB20-IL-7-ALPP, and pKB20-IL-7-CD90, respectively. After the obtained recombinant plasmid was transformed into E. coli (DH5c) and sequencing was correct, the plasmid was extracted and purified using the plasmid purification kit of Qiagen company, and high-quality plasmid of each recombinant expression vector was obtained.

[0192] Figure 12

[0193] According to the method described in Example 3, the PBMCs from a healthy human donor were electroporated with pKB20-IL-7-CD48, pKB20-IL-7-CD55, pKB20-IL-7-ALPP, and pKB20-IL-7-CD90, and the T cells expressing IL-7-CD48, IL-7-CD55, IL-7-ALPP, and IL-7-CD90 were prepared without adding IL-2 to the cell culture solution after electroporation. The growth conditions of the cells in each group were observed, respectively, and compared with the T cells expressing IL-7-CD52 prepared in Example 3, and the T cells expressing IL-7-CD8TM were used as a control group.

[0194] The results are shown in Figure 11 and Figure 12 . Example 12, Preparation and in vitro killing detection of TCR-T expressing other GPI protein-anchored membrane surface IL-7 It is shown that the proliferation level of the T cells expressing IL-7-CD90 is basically the same as that of the control group expressing IL-7-CD8TM. The proliferation levels of the T cells expressing IL-7-CD48, IL-7-CD55, and IL-7-ALPP are significantly higher than that of the IL-7-CD8TM control group. The proliferation level of the T cells expressing IL-7-CD52-2 is higher than that of all other groups. Figure 13 It is shown that the survival rates of the T cells in each group electroporated with different membrane surface IL-7 vectors are basically the same at different time points.

[0195] The above results show that T cells expressing GPI protein-anchored membrane surface IL-7 can enhance the activation and proliferation of T cells to varying degrees, and the degree of enhancement of T cells expressing CD52-anchored membrane surface IL-7 is more obvious than that of T cells expressing CD48, CD55, ALPP and CD90-anchored membrane surface IL-7.

[0196] Example 13, Killing effect of TCR-T expressing CD52 linker-anchored membrane surface IL-7 on tumor tissue in vivo

[0197] According to the method described in Example 6, pKC20-TCR+pKB20-IL-7-CD48, pKC20-TCR+pKB20-IL-7-CD55, pKC20-TCR+pKB20-IL-7-ALPP and pKC20-TCR+pKB20-IL-7-CD90 were co-transfected respectively to prepare IL-7-CD48-TCR-T, IL-7-CD55-TCR-T, IL-7-ALPP-TCR-T and IL-7-CD90-TCR-T. The positive cell proportions of IL-7-CD48-TCR-T, IL-7-CD55-TCR-T, IL-7-ALPP-TCR-T and IL-7-CD90-TCR-T were 15.09%, 16.74%, 12.53% and 17.66% respectively by flow cytometry.

[0198] According to the method described in Example 7, A375 cell line was selected as target cells, and IL-7-CD48-TCR-T, IL-7-CD55-TCR-T, IL-7-ALPP-TCR-T, IL-7-CD90-TCR-T and TCR-T prepared in Example 6, IL-7-CD52-TCR-T were selected as effector cells. The killing detection was carried out at an effector-target ratio of 0.5:1 based on the number of living cells. After the effector cells and target cells were co-cultured for more than 90 hours, the cell proliferation curve was observed.

[0199] The results are shown in Table 1. Figure 14-17The killing effect of each group of TCR-T expressing GPI-anchored membrane surface IL-7 on target cells A375 was obviously better than that of TCR-T not expressing membrane surface IL-7 when the effector-to-target ratio was 0.5:1. Among the groups of TCR-T expressing GPI-anchored membrane surface IL-7, the killing effect of IL-7-CD52-TCR-T expressing IL-7-CD52-2 on target cells was obviously better than that of IL-7-CD48-TCR-T, IL-7-CD55-TCR-T, IL-7-ALPP-TCR-T and IL-7-CD90-TCR-T. This indicates that expressing GPI-anchored membrane surface IL-7 can more effectively improve the killing effect of TCR-T on target cells, and the expression of CD52-anchored membrane surface IL-7 is more obvious than other GPI proteins in improving the killing effect of TCR-T on target cells.

[0200] Figure 14

[0201] Experimental animals

[0202] Immunodeficient B-NDG mice (purchased from Baoaisituo) were selected as the experimental animals for CDX model construction.

[0203] Experimental design and grouping: as shown in Table 2 below

[0204] Table 2: Dosing regimen and grouping of mice

[0205]

[0206]

[0207] The cells administered to the experimental animals were TCR-T and IL-7-CD52-TCR-T prepared in Example 6. Before administration, the cells were centrifuged and resuspended in PBS to prepare a cell suspension with a cell density of 1 x 10 8 / mL PBS.

[0208] Animal feeding

[0209] After purchasing the required amount of B-NDG mice, they were fed in the SPF level experimental animal room for an adaptation period of 7-10 days.

[0210] Environment: The mice will be placed in transparent resin plastic cages in the animal room. The mouse cage bedding is autoclaved wood shavings and corn cob bedding, which is replaced regularly. The animal room is equipped with a high-efficiency air filter, and the temperature will be maintained at 20-26°C (68-79°F), and the relative humidity is 40-70%. The temperature and humidity are continuously observed and recorded. The lighting conditions are 12 hours of daylight lamp illumination and 12 hours of no illumination per day.

[0211] Food and water: Experimental mice can get unlimited special mouse food (irradiated and sterilized, Shanghai Slek Experimental Animal Responsibility Co., Ltd., China), and can access sterile clean drinking water at any time without obstacles.

[0212] Tumor cell inoculation

[0213] Each mouse was inoculated with A375-Luc2 tumor cells (purchased from ATCC, Cat. CRL-1619-LUC2) on the right side of the subcutaneous, 2x10 6 Cell inoculation, inoculated in 0.1 mL of medium containing 50% matrix to promote tumor growth.

[0214] Animal grouping and administration

[0215] When the tumor volume reached ~50mm 3 left and right, 18 animals were randomly grouped according to tumor volume, n=6, to ensure that all groups were comparable at baseline. The day of grouping was recorded as P0. During the experiment, the body weight and tumor volume of the animals were measured twice a week, and the clinical symptoms of the animals were observed daily. The tumor volume was expressed in mm 3 , and the formula was: V=0.5x a x b 2 , where a and b were the long and short diameters of the tumor, respectively. On the day of the experiment, peripheral blood was drawn from each group of mice, and flow cytometry detection technology was performed after staining with human CD45 flow cytometry antibodies (purchased from Biolegend Cat. 304036). On the same day, each group of mice was injected with luciferase substrate luciferin and then imaged in vivo on a small animal live imaging instrument to observe the size of the tumor burden in each group of mice and record the fluorescence value.

[0216] Results

[0217] The experimental results are shown in Figure 15 . The differences in tumor volume, tumor fluorescence value and lymphocyte blood density between groups were statistically analyzed by unpaired two-tailed t test. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 16 It is shown that, by the 21st day after administration, the tumor volume of mice in the TCR-T cell administration group was significantly inhibited compared with tumor-bearing mice injected with PBS control group, and the tumor volume of IL-7-CD52-2 TCR-T administration group was further significantly smaller than that of TCR-T administration group. Figure 17 It is shown that the tumor fluorescence value and the tumor volume change trend are consistent. The tumor fluorescence value of the TCR-T administration group was significantly lower than that of the PBS group after 21 days of administration; while the fluorescence value of the IL-7-CD52-2 TCR-T administration group was close to 0. ​The fluorescence of A375-Luc2 tumor cells in vivo in the mice of the IL-7-CD52-2 TCR-T administration group was basically not visible. ​ As shown, 21 days after administration, the density of human lymphocytes in the mice of the TCR-T administration group was increased compared with the PBS administration group, and the density of human lymphocytes in the peripheral blood of the mice of the IL-7-CD52-2 TCR-T administration group was very sharply increased compared with the TCR-T group.

[0218] The above results show that the expression of IL-7-CD52-2 can significantly improve the killing effect of TCR-T cells on tumors in vivo.

[0219] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details in accordance with all the teachings disclosed herein, and such changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A fusion protein comprising a functional domain and a GPI-anchored region, the functional domain comprising IL-7, the GPI-anchored region comprising CD52 or its GPI-anchored region, the functional domain being located at the N-terminus of the GPI-anchored region, the fusion protein further comprising a spacer region between the functional domain and the GPI-anchored region, the spacer region comprising an extracellular region of protein, the extracellular region comprising a CD8 extracellular region or a BCMA extracellular region, wherein... The GPI anchoring region has the sequence shown in bits 232-268 of SEQ ID NO:

2. The extracellular region of CD8 is shown in positions 177-231 of SEQ ID NO:

2. The extracellular region of the BCMA is shown in the sequence of positions 189-242 of SEQ ID NO:

4.

2. The fusion protein as described in claim 1, characterized in that, The functional domains perform their functions through dimerization or multimerization.

3. The fusion protein as described in claim 1, characterized in that, The spacer area contains a connector.

4. The fusion protein as described in claim 3, characterized in that, The connector has the sequence shown in bits 177-188 of SEQ ID NO:

4.

5. The fusion protein as described in claim 1, characterized in that, The fusion protein also contains a signal peptide.

6. The fusion protein as described in claim 5, characterized in that, The signal peptide is the signal peptide of the protein corresponding to the GPI anchoring region.

7. The fusion protein as described in claim 5, characterized in that, The signal peptide is any one or more of the following protein signal peptides: CD52, CD48, CD55, ALPP, and CD90.

8. A nucleic acid molecule comprising a sequence selected from: (1) The coding sequence of the fusion protein according to any one of claims 1-7, or a fragment thereof used as an amplification primer or detection probe. (2)(1) complementary sequences.

9. A nucleic acid construct comprising the nucleic acid molecule of claim 8.

10. The nucleic acid construct as described in claim 9, characterized in that, The nucleic acid construct is a cloning vector, expression vector, or integration vector.

11. A cell, wherein the cell: (1) Containing, expressing, and / or secreting the fusion protein of any one of claims 1-7, and / or (2) It comprises any one or more of the nucleic acid molecules of claim 8 and the nucleic acid constructs of claim 9 or 10.

12. The cell as claimed in claim 11, characterized in that, The cells in question are lymphocytes.

13. The cell as described in claim 12, characterized in that, The lymphocytes are T cells, NK cells, or TIL cells.

14. The cell as claimed in claim 13, characterized in that, The T cells or TIL cells also express exogenous TCR and / or CAR.

15. A pharmaceutical composition comprising any one or more of the fusion protein of any one of claims 1-7, the nucleic acid molecule of claim 8, the nucleic acid construct of claim 9 or 10, and cells of any one of claims 11-14, and pharmaceutically acceptable excipients.

16. The use of any one or more of the following in the preparation of an antitumor drug: the fusion protein of any one of claims 1-7, the nucleic acid molecule of claim 8, the nucleic acid construct of claim 9 or 10, the cell of any one of claims 11-14, and the pharmaceutical composition of claim 15, wherein the tumor comprises one or more selected from the group consisting of melanoma, ovarian cancer, gastric cancer, intestinal cancer, lung cancer, cervical cancer, glioma, pancreatic cancer, and prostate cancer.

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