WO2024119819A1

WO2024119819A1 - Polypeptide tag and use thereof

Info

Publication number: WO2024119819A1
Application number: PCT/CN2023/106240
Authority: WO
Inventors: 马承宁; 李加国; 刘祥箴; 孙艳; 钱其军
Original assignee: 上海细胞治疗集团股份有限公司; 上海细胞治疗集团药物技术有限公司
Priority date: 2022-12-09
Filing date: 2023-07-07
Publication date: 2024-06-13
Also published as: CN118206620A

Abstract

The present invention relates to a polypeptide tag and the use thereof, and specifically provides a polypeptide tag which has: (1) a sequence as shown in any one of SEQ ID NOs: 1-11, or, (2) a sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of (1). The tag can contribute to the in-vitro specific activation and expansion, isolation and enrichment, in-vivo activation and expansion, inactivation, clearance, etc. of T cells.

Description

A polypeptide tag and its application

Technical Field

The present invention belongs to the field of medical biology, and specifically relates to a polypeptide tag, and CAR, CAR-T cells and applications carrying the tag.

Background technique

Chimeric antigen receptor T cell (CAR-T) therapy technology is a field of tumor immune cell therapy. CAR-T cells express fusion proteins on the cell surface, enabling T lymphocytes to recognize specific antigens in a non-MHC restricted manner, enhancing their ability to recognize and kill tumors. In general, CAR includes an extracellular domain that binds to an activating ligand, a transmembrane domain that participates in the formation of an immune synapse with a "target" cell, and an intracellular domain that responds to the binding of the extracellular domain by activating T cell-related transcriptional responses.

The structure of the chimeric antigen receptor (CAR) was proposed by the Eshhar research team in Israel in 1989. Since then, T cells showing CAR structural cell surface proteins have been shown to have good effects in tumor immunotherapy. The first-generation CAR receptor contains a single-chain variable fragment (scFv), and the intracellular activation signal is transmitted through the CD3ζ (CD3z) signal chain. However, the first-generation CAR receptor lacks a domain that provides T cell co-stimulatory signals, which results in CAR-T cells playing only a transient role, a short time in the body, and less cytokine secretion. The second-generation CAR receptor introduces the intracellular domain of co-stimulatory signal molecules, including, for example, CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell co-stimulator (ICOS), DNAX activation protein 10 (DAP10) and other domains that enhance T cell proliferation and cytokine secretion. The production of IL-2, IFN-γ and GM-CSF is increased, thereby breaking the immunosuppression of the tumor microenvironment, such as activation-induced cell death (AICD). The third-generation CAR receptor adds secondary co-stimulatory molecules, such as 4-1BB, between the co-stimulatory structure CD28 and the ITAM signal chain, thereby producing a three-signal CAR receptor. At present, the commonly used CAR structure in treatment is the second-generation CAR, whose structure can be divided into the following four parts: antibody single-chain variable region (scFv), hinge region, transmembrane region, and intracellular stimulatory signal peptide. The hinge region helps to form the correct conformation and dimer, affecting the ability of CAR to bind to tumor cell surface antigens.

Selecting the right tumor antigen as a target is the key to designing safe and effective CAR-T. Currently, various types of CAR-T cells are being developed for the treatment of hematological malignancies, including the use of anti-CD19, anti-CD20, anti-kappa light chain, anti-CD22, anti-CD23, anti-CD30, anti-CD70 and other antibodies to construct CAR-modified T cell therapies. Among these anti-tumor studies that have been conducted, anti-CD19 and anti-CD20 monoclonal antibodies are the most commonly used antibodies.

However, although CAR-T cells have a strong ability to kill blood tumor cells, their activity is easily inhibited in the tumor microenvironment (TME) of solid tumors. This result stems from the problems of CAR-T cells' proliferation, survival, tumor infiltration, and resistance to the tumor microenvironment in the body after they are infused back into the patient. For example, after entering the body, CAR-T cells may lack direct and sufficient contact with solid tumor cells, resulting in the inability to activate and proliferate in large quantities after infusion, thereby affecting the therapeutic effect. In addition, there is currently a lack of suitable means to detect the progress and whereabouts of CAR-T cells after they enter the body. In addition, in the absence of methods to control overactivated CAR-T cells or eliminate these CAR-T cells, CAR-T cells continue to proliferate and activate uncontrollably, which may cause fatal off-target toxicity, cytokine release syndrome, or neurotoxicity.

In addition, current CAR-T cell-based therapies rely on the in vitro proliferation of CAR-T cells before reinfusion into patients. Although the current CAR-T technology platform can achieve in vitro expansion using anti-CD3/28 stimulatory factors or corresponding antigen stimulation, the specificity and stability of its expansion are limited, and it cannot stably and effectively enrich highly specific positive CAR-T cells, leading to the following problems: 1) The in vitro activation/amplification process is inefficient or unstable: nucleic acid electroporation and anti-CD3/28 activation will result in a low CAR positivity rate of the obtained cells; the use of antigen stimulation methods will result in unstable effects of different antigens; 2) The method of using antigen coating stimulation is usually a non-closed operation, which is difficult to connect to automated equipment; 3) The in vitro expansion culture time is too long, the reinfusion dose is large, and the cost is high.

Summary of the invention

The present invention first provides a polypeptide tag, which comprises an extracellular domain sequence of a protein expressed by normal human cells or a truncated portion of the extracellular domain sequence, or a mutant thereof;

Preferably, the protein expressed by normal human cells is selected from BCMA, BAFFR, CD20, CD40, more preferably BCMA;

Preferably, the polypeptide tag has:

(1) a sequence as shown in any one of SEQ ID Nos: 1-11, or

(2) A sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the sequence of (1).

In one or more embodiments, the polypeptide tag further comprises a connecting fragment (linker) located at the N-terminus or C-terminus for covalently linking to other polypeptides.

In one or more embodiments, the linker is (GGGGS) _N , where N is an integer of 1-10, and a preferred linker is GGGGS (positions 154-171 of SEQ ID No. 13).

In one or more embodiments, the polypeptide tag further comprises a signal peptide located at the N-terminus.

The present invention provides a fusion protein, characterized in that it comprises the above-mentioned polypeptide tag and a functional polypeptide.

In one or more embodiments, the polypeptide tag is located inside the functional polypeptide; preferably, the polypeptide tag is connected to the antigen receptor domain, and the polypeptide tag is located at the N-terminus or C-terminus of the antigen receptor domain, preferably the C-terminus.

In one or more embodiments, the polypeptide tag can also be connected to the outside of the functional polypeptide, that is, separated from the functional polypeptide to form an independent polypeptide tag. Preferably, the polypeptide tag is connected to the C-terminus of the functional polypeptide through a cleavage sequence and a signal peptide sequence, and the C-terminus of the polypeptide tag is connected to an expression auxiliary sequence; preferably, the cleavage sequence comprises a sequence encoding a furin recognition site and a 2A element, and the auxiliary sequence comprises a transmembrane anchor portion and a gap portion between the extracellular tag and the transmembrane structure; preferably, the cleavage sequence is T2A (SEQ ID No.15) or FT2A (SEQ ID No.16), the signal peptide is S3 (SEQ ID No.17) or S5 (SEQ ID No.18) or Sg (SEQ ID No.19) or Sk (SEQ ID No.20), the transmembrane anchor portion is B _TM (SEQ ID No.21), and the gap portion between the extracellular tag and the transmembrane structure is B _GAP (SEQ ID No.22).

In one or more embodiments, the functional polypeptide is a chimeric antigen receptor, the sequence of which includes the following regions:

(1) The extracellular region, which contains the antigen receptor domain and the hinge region;

(2) transmembrane region;

(3) The intracellular region includes the signal transduction domain and/or the co-stimulatory domain.

In one or more embodiments, the antigen receptor domain of the extracellular region comprises a binding molecule for a tumor antigen, such as a ligand, an antibody or an antigen binding fragment thereof for a tumor antigen. Preferably, the tumor antigen is selected from the group consisting of BCMA, BAFFR, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, VEGFR-2, EGFRvIII, ErbB2, ErbB3HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt 4. CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin (MSLN), NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79b and Notch-1-4.

In one or more embodiments, the hinge region is derived from a portion of the extracellular or transmembrane domain of the following proteins: CD8, CD28, CD3, CD15, CD16, CD40, CD27. Preferably, the hinge region is a CD8 hinge. The sequence of the CD8 hinge is shown in SEQ ID No. 12, amino acids 1-55.

In one or more embodiments, the transmembrane region in (2) is selected from the group consisting of: CD28, CD8, CD134, 4-1BB, The transmembrane region of any one of LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-10R, IL-12R, IL-15R, IL-21R, CD226, CD27 and CD40. Preferably, the transmembrane region is a CD28 transmembrane region or a CD8 transmembrane region. The sequence of the CD28 transmembrane region is shown in amino acids 56-83 of SEQ ID No.12, and the sequence of the CD8 transmembrane region is shown in amino acids 266-310 of SEQ ID No.25.

In one or more embodiments, the intracellular signaling domain includes, but is not limited to, the signaling domains of CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10, and DAP12. Preferably, the signaling domain is the signaling domain of CD3ζ; its sequence is shown in amino acids 125-236 of SEQ ID No.12.

In one or more embodiments, the intracellular co-stimulatory domain is selected from any one or more combinations of 4-1BB, ICOS, CD27, OX40, CD28, MYD88, IL1R1, CD70, TNFRSF19L, TNFRSF27, TNFRSF1OD, TNFRSF13B, TNFRSF18, CD134 tumor necrosis factor superfamily. Preferably, the co-stimulatory domain is CD28 or 4-1BB; the sequences thereof are shown in amino acids 84-124 of SEQ ID No.12 and 335-376 of SEQ ID No.25, respectively.

In one or more embodiments, the fusion protein comprises or is, from N-terminus to C-terminus, the following sequence: a binding molecule for a tumor antigen, a polypeptide tag, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and a signal transduction domain; or: a binding molecule for a tumor antigen, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, a signal transduction domain, a cleavage sequence, a tag signal peptide, a polypeptide tag, and an auxiliary expression sequence.

In one or more embodiments, the amino acid sequence of the fusion protein is as shown in SEQ ID No.13.

The present invention also provides a nucleic acid molecule comprising a sequence selected from the following:

(1) a nucleic acid sequence encoding a polypeptide tag or fusion protein as described in any embodiment of the present invention, or a fragment thereof used as an amplification primer or detection probe,

(2) a variant having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with (1),

(3) A complementary sequence of (1) or (2).

The present invention also provides a nucleic acid construct, wherein the nucleic acid construct:

(1) expressing a polypeptide tag or fusion protein as described in any embodiment of the present invention, and/or

(2) comprising a nucleic acid molecule as described herein.

In one or more embodiments, the nucleic acid construct is a vector, preferably a non-viral vector.

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

The present invention also provides a host cell, which comprises, expresses and/or secretes the polypeptide tag or fusion protein described herein.

In one or more embodiments, the host cell comprises the nucleic acid molecule or nucleic acid construct described in any embodiment herein, or the nucleic acid molecule is integrated into the chromosome.

In one or more embodiments, the host cell is an immune effector cell, such as a T cell or a NK cell.

Another aspect of the present invention provides a solid phase carrier coupled with an anti-tag antibody, wherein the anti-tag antibody specifically recognizes the polypeptide tag described herein.

In one or more embodiments, the antibody is selected from one or more of NB-36, NB-100, NB-102, NB-257, and NB-367 in CN202110301079.1. The amino acid sequences of NB-36, NB-100, NB-102, NB-257, and NB-367 are shown in SEQ ID No. 27-31.

In one or more embodiments, the solid support is magnetic particles/microspheres.

In one or more embodiments, the coupling is by direct chemical connection, by specific antigen-antibody binding, by specific biotin group-avidin group binding or other indirect connection.

The present invention also provides a cell binding/labeling/detection/stimulation/sorting kit for stimulating or sorting cells expressing the polypeptide tag or fusion protein described herein, wherein the kit comprises an anti-tag antibody or a solid phase carrier coupled with an anti-tag antibody, and the anti-tag antibody specifically recognizes the polypeptide tag described herein.

In one or more embodiments, the antibody is selected from one or more of NB-36, NB-100, NB-102, NB-257, and NB-367 in CN202110301079.1.

In one or more embodiments, the detection kit further comprises a polypeptide tag, fusion protein, nucleic acid molecule or nucleic acid construct as described in any embodiment herein.

Another aspect of the present invention provides a method for preparing T cells, comprising:

(1) expressing the polypeptide tag and fusion protein described in any embodiment of the present invention in T cells, such as introducing the nucleic acid construct described in any embodiment of the present invention into T cells, and

(2) Activating the T cells obtained in step (1) using an anti-tag antibody or a solid phase carrier coupled with an anti-tag antibody cells for stimulation or sorting.

In one or more embodiments, the T cells are CD3+ and/or CD28+ T cells.

In one or more embodiments, the method further comprises the step of expressing the chimeric antigen receptor in T cells, such as the step of introducing a nucleic acid construct containing a chimeric antigen receptor encoding sequence into T cells.

Another aspect of the present invention provides a pharmaceutical composition, comprising any one or more of the polypeptide tag, fusion protein, nucleic acid molecule, nucleic acid construct, solid phase carrier and host cell described in any embodiment of the present invention and pharmaceutically acceptable excipients. As a preferred example, the solid phase carrier is used to prepare T cells expressing the polypeptide tag or fusion protein described herein.

Another aspect of the present invention provides the use of any one or more of the polypeptide tags, fusion proteins, nucleic acid molecules, nucleic acid constructs, host cells, and solid phase carriers described in any embodiment of the present invention in the preparation of a drug for treating tumors. As a preferred example, the drug comprises T cells expressing the polypeptide tags or fusion proteins described herein. In one or more embodiments, the T cells are activated and/or sorted by anti-tag antibodies or solid phase carriers coupled to anti-tag antibodies described herein.

The present invention also provides a method for treating tumors, comprising administering T cells expressing the polypeptide tag or fusion protein described herein to a patient.

In one or more embodiments, in one or more embodiments, the T cells are activated and/or sorted by anti-tag antibodies or solid phase carriers coupled with anti-tag antibodies as described herein. The method also includes the step of activating and/or sorting the T cells by anti-tag antibodies or solid phase carriers coupled with anti-tag antibodies.

The present invention also provides a method for activating T cells expressing the polypeptide tag or fusion protein described herein, comprising the steps of activating and/or sorting the T cells using an anti-tag antibody or a solid phase carrier coupled to the anti-tag antibody described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: BL label (B-label) design and its high-affinity VHH screening assay.

A, schematic diagram of BL tag design based on truncated BCMA-ECD;

B, ELISA detection of the binding of antibody (-Fc) to BL (BLΔ0/1/2/3/4 and BLΔ0mut)-M044 fusion peptide;

C, ELISA detection of the binding of antibody (-Fc) to BLΔ0 peptide;

D, ELISA detection of the binding of BL (BLΔ0/2/5 and BLΔ0mut) (-Fc) to antibody NB-36.

Figure 2: ELISA detection of the binding of antibody NB-36 to BLΔ2D2G, BLΔ2L2G, BLΔ2I2G and BLΔ0mut.

Figure 3: Microscopic image of the microspheres coupled with NB-36 antibody binding to BL-CAR-T cells. Dynabeads coupled with NB-36 by Biotin: M2339-BLΔ0-CAR-T (Day 13) = 1:1

Figure 4: Construction of (BL-)CAR vector and (BL-)CAR-T cells targeting mesothelin-positive cancer cells.

A, schematic diagram of the BL-CAR structure targeting mesothelin-positive cancer cells;

B-C, in vitro preparation and expansion of (BL-)CAR-T cells prepared from PBMCs of one of the human sources (B) and detection of CAR positive rate (C);

D-E, in vitro preparation and expansion of (BL-)CAR-T cells prepared from human PBMC (D) and CAR positive rate detection (E);

F, in vitro preparation and expansion of CAR-T cells containing point mutations BL (BLΔ2D2G, BLΔ2L2G, BLΔ2I2G);

Figure 5: Microspheres coupled to NB-36 antibody activated and expanded BL-CAR-T cells in vitro.

A, Schematic diagram of the principle of in vitro activation and expansion of BL-CAR-T cells by microspheres coupled to NB-36;

B, BL-CAR-T cells activated and expanded in vitro by microspheres conjugated with different antibodies were prepared and expanded in vitro;

Figure 6: Validation, characterization, and quality control of BL-CAR-T cells prepared by coated ligand stimulation.

A-E, BLΔ0/2-CAR-T activated by coated mesothelin or NB-36 (plus CD28 antibody) typing (CD4/8 ratio, A), stemness (Tem/Tcm ratio, B), activation (CD25 positivity, C) and exhaustion phenotype (PD-1/TIM3 positivity, D, E), derived from PBMC of one of the human donors;

F-J, BLΔ0/2-CAR-T activated by coated mesothelin or NB-36 (plus CD28 antibody) typing (CD4/8 ratio, F), stemness (Tem/Tcm ratio, G), activation (CD25 positivity, G) and exhaustion phenotype (PD-1/TIM3 positivity, I, J) detection, derived from PBMC of the second human donor;

Figure 7: Validation, characterization, and quality control of BL-CAR-T cells prepared by stimulation with antibody-conjugated microspheres.

AH, CAR positivity (A), activation (CD25/69 positivity, B, C), typing (CD4/8 ratio, D), exhaustion (PD-1/TIM3/LAG-3 positivity, EG) and stemness (Tem/Tcm ratio, H) phenotype detection of BLΔ2-CAR-T activated by antibody-coupled microspheres;

Figure 8: Validation, characterization and quality control of point mutation BL-CAR-T cells prepared by stimulation of antibody-conjugated microspheres.

A-H, CAR positivity (A), activation (CD25/69 positivity, B, C), typing (CD4/8 ratio, D), exhaustion (PD-1/TIM3/LAG-3 positivity, E-G) and stemness (Tem/Tcm ratio, H) phenotype detection of BLΔ2D2G/L2G/I2G-CAR-T activated by antibody-coupled microspheres;

Figure 9: Detection of the killing effect of BL-labeled mesothelin CAR-T cells on mesothelin-positive cancer cells (XCelligence RTCA).

A-B, Detection of the killing effect of conventionally activated MSLN-CAR-T cells with BLΔ0 tag on ovarian cancer cells SK-OV3 (A)/lung cancer cells NCI-H226 (B);

C-D, Detection of the killing effect of BLΔ2 (or BLΔ2L2G)-tagged MSLN-CAR-T cells activated by microspheres coupled to NB-36 antibody on ovarian cancer cells SK-OV3 (C)/lung cancer cells NCI-H226 (D);

Figure 10: Construction of independent BL tag-CAR vector and CAR-T cells.

A, Schematic diagram of two different forms of independent BL tag-CAR structures targeting CD19/22-positive cancer cells;

B, schematic diagram of the sequence composition of the above CAR structure;

C, Comparison of the expansion process of independent BL-tagged-CD19/22CAR-T cells with different structures (signal peptide and cleavage sequence);

D, Comparison of CAR positivity and activation phenotype (CD25/69) detection of independent BL tags-CD19/22CAR-T cells with different structures (signal peptide and cleavage sequence);

Figure 11: Validation and characterization of independent BL tag-CAR vectors and CAR-T cells.

A-C, Comparison of the expansion (A), typing (CD4/CD8 ratio, B) and stemness (Tcm/Tem ratio, C) phenotype detection during the preparation process of independent BL-labeled CD19/22CAR-T cells with different structures;

D, Detection of CAR positivity, BL positivity, and CD25 and CD69 positivity of independent BL-labeled-CD19/22CAR-T cells prepared from PBMCs of three different human donors;

Detailed ways

The present invention discloses a specific polypeptide tag (BL) and a chimeric antigen receptor T cell (BL-CAR-T) carrying the tag, which can be used for specific activation and amplification, labeling-separation and enrichment, activity shutoff and clearance of CAR-T cells. The fusion protein expressed by the BL-CAR-T comprises a polypeptide tag and a functional polypeptide. The functional polypeptide is a chimeric antigen receptor, and its sequence includes the following regions: (1) an extracellular region comprising an antigen receptor domain and a hinge region; (2) a transmembrane region; (3) an intracellular region comprising a signal transduction domain and/or a co-stimulatory domain. The polypeptide tag is located inside the functional polypeptide, or the polypeptide tag is connected to the outside of the functional polypeptide and can be separated from the functional polypeptide to form an independent polypeptide tag. The polypeptide tag comprises the extracellular domain of a human cell protein target or a truncated portion of the extracellular domain. Preferably, the polypeptide tag Contains the BCMA extracellular domain or a truncated portion of the extracellular domain.

The specific polypeptide tag of the present invention can be used for non-viral vectors and viral vectors, and is particularly suitable for non-viral vectors. Compared with viral vectors, the transduction efficiency of non-viral vectors is usually lower, and the use of electrotransfection will also have a certain impact on cells, so special activation and amplification methods are required. The use of the specific polypeptide tag of the present invention can solve the problems of activation and amplification of CAR-T prepared by non-viral vectors.

In addition, the present invention also discloses a CAR-T expression vector including the above-mentioned label and a construction method thereof, a solid phase carrier coupled with an anti-label antibody (microspheres that specifically recognize and bind to the label), and the use of the label in the preparation of CAR-T cells and in tumor treatment. The labeled CAR-T cells of the present invention cooperate with the solid phase carrier coupled with the anti-label antibody to enable the BL-CAR-T cells to be efficiently and specifically activated and amplified during the in vitro culture preparation process, and have a good tumor killing effect without affecting the cell phenotype, thereby improving the specificity, effectiveness and safety of BL-CAR-T cell therapy.

As described herein, "chimeric antigen receptor (CAR)" is an artificially engineered protein that binds to specific molecules, such as tumor cell surface antigens, and stimulates the proliferation program in immune cell-type effector cells. CARs generally include antigen binding domains (or antigen receptor domains) in the order from amino to carboxyl, such as the antigen binding region of a single-chain antibody; an optional (but usually present) hinge region; a transmembrane region; and an intracellular signaling region.

As used herein, "domain" refers to a region of a polypeptide that folds into a specific structure independently of other regions.

"VHH" may refer to the variable domains of a single heavy chain antibody ("VHH antibody"), such as a camel antibody. "Single-chain antibody" (SCA) is a single-chain polypeptide, generally comprising a number of relatively conserved domains, which are combined together to form a framework region (FR region) when the polypeptide is folded, and a variable region, which is combined together to form a variable antigen domain. Therefore, VHH antibody is a SCA. According to this term, the variable domain present in a natural single heavy chain antibody will also be referred to herein as a "VHH domain" to distinguish it from the heavy chain variable domain present in a traditional four-chain antibody (referred to herein as a "VH domain") and the light chain variable domain present in a traditional four-chain antibody (referred to herein as a "VL domain"). The isolated single variable domain polypeptide preferably has the full antigen binding ability of its cognate SCA and is a stable polypeptide in an aqueous solution. Stable antigen-binding single-chain polypeptides include one or more domains (FR or variable region origin) derived from mammalian antibody domains or similar to mammalian antibody domains (e.g., VH domains), and are also included in the "single-chain antibody" herein.

The term "coding sequence" is defined herein as a portion of a nucleic acid sequence that encodes the amino acid sequence of a polypeptide product (e.g., a CAR, a single-chain antibody, or a domain thereof). The boundaries of the coding sequence are usually determined by the ribosome binding site (for prokaryotes) upstream of the 5' open reading frame of the coding mRNA and the transcription termination sequence downstream of the 3' open reading frame of the coding mRNA. The coding sequence may include, but is not limited to, DNA, cDNA, recombinant nucleic acid sequences, RNA.

The term "Fc" (crystallizable fragment) is a part of a mammalian antibody and refers to the peptide located at the end of the "Y" structure handle of the antibody molecule, including the CH2 and CH3 domains of the antibody heavy chain constant region. It is the site of interaction for many molecules and cells and provides some biological effects of mammalian antibodies.

The term "co-stimulatory molecule" refers to a molecule that is present on the surface of antigen-presenting cells and binds to the co-stimulatory molecule receptor on Th cells to produce a co-stimulatory signal. The proliferation of lymphocytes requires not only the binding of antigens, but also the signal of co-stimulatory molecules. Co-stimulatory signals are mainly transmitted to T cells by binding to the co-stimulatory molecule CD80 on the surface of antigen-presenting cells, and CD86 binds to the CD28 molecule on the surface of T cells. B cells receive co-stimulatory signals, which can be transmitted through common pathogen components such as LPS, complement components, or activated antigen-specific Th cell surface protein CD40L.

The term "linker" is a polypeptide fragment that connects different proteins or polypeptides, and its purpose is to maintain the spatial relationship of the connected proteins or polypeptides to maintain the function or activity of the proteins or polypeptides, such as by relieving the steric inhibition of ligand binding. Exemplary linkers include linkers containing G and/or S, and, for example, the Furin 2A peptide.

The term "specific binding" refers to the reaction of a binding protein with a ligand, such as an antibody or antigen-binding fragment and the antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) means that the antibody-antigen affinity is characterized by a binding constant Kd of less than about ^10-5 M, such as less than about ^10-6 M, ^10-7 M, ^10-8 M, ^10-9 M or ^10-10 M or less. "Specific recognition" has a similar meaning.

The term "effective amount" refers to a dosage that can achieve treatment, prevention, alleviation and/or relief of a disease or condition in a subject as described herein.

The term "disease and/or condition" refers to a physical state of a subject associated with the diseases and/or conditions described herein.

The term "subject" or "patient" may refer to patients or other animals, especially mammals, such as humans, dogs, monkeys, cows, horses, etc., who receive the pharmaceutical composition of the present invention to treat, prevent, improve and/or alleviate the diseases or conditions of the present invention.

Peptide tags and fusion proteins

The present invention provides a polypeptide tag, which can be fused with other sequences to be used for T cell-specific activation and expansion, labeling-separation and enrichment, activity shut-off and clearance.

In some embodiments, the polypeptide tag comprises an extracellular domain sequence of a protein expressed by normal human cells or a truncated portion of the extracellular domain sequence, or a mutant thereof. In a preferred embodiment, the protein expressed by normal human cells is a protein specifically expressed by a small group of cells in normal human tissue or blood, such as BCMA, BAFFR, CD20, CD40, etc.

In some embodiments, the tag has a sequence as shown in any one of SEQ ID Nos: 1-11, or The sequence has at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. The polypeptide tag is connected to other parts (such as functional polypeptides) in the fusion protein through a connecting fragment (linker) at its N-terminus or C-terminus. Among them, SEQ ID NO: 2-7 are truncated sequences that can retain the function of the polypeptide tag while avoiding the impact on the chimeric antigen receptor; SEQ ID NO: 8-11 are mutant sequences that are intended to avoid the binding of existing antibodies in cells or the human body to the polypeptide tag, making the activation, labeling-separation enrichment, shut-down activity and clearance of the polypeptide tag more controllable.

In some embodiments, the fusion protein is a chimeric antigen receptor (CAR) comprising a polypeptide tag, comprising an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region comprises an antigen receptor domain and a hinge region, and the intracellular region comprises a signaling domain and/or a co-stimulatory domain. The polypeptide tag is located at the N-terminus or C-terminus of the antigen receptor domain. The polypeptide tag located at the N-terminus is conducive to the activation of the polypeptide tag, but it will affect the binding of the antigen receptor domain and reduce the therapeutic effect of CAR-T; the polypeptide tag located at the C-terminus does not affect the antigen receptor domain, but is located inside the fusion protein, and activation may be difficult, and the truncation or mutation of the polypeptide tag is required to be high. In the present invention, the therapeutic effect of CAR-T is mainly considered, preferably the C-terminus. In some embodiments, by selecting a specific polypeptide tag (SEQ ID No: 1-11), the therapeutic effect and activation of CAR-T can be guaranteed at the same time.

The antigen receptor domain comprises a binding molecule for a tumor antigen, such as a ligand, an antibody or an antigen binding fragment thereof for a tumor antigen. The sequences of tumor antigens and their binding molecules are readily available to those skilled in the art. Preferably, the tumor antigen is selected from the group consisting of BCMA, BAFFR, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, VEGFR-2, EGFRvIII, ErbB2, ErbB3HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt 4. CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin (MSLN), NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79b and Notch-1-4.

The hinge region of the chimeric antigen receptor can be selected from any sequence commonly used as a hinge in the art. For example, the hinge region can be derived from a portion of the extracellular or transmembrane domain of the following proteins: CD8, CD28, CD3, CD15, CD16, CD40, CD27. The sequence of the CD8 hinge is shown in SEQ ID No. 12, amino acids 1-55.

The transmembrane region of the chimeric antigen receptor can be selected from: CD28, CD8, CD134, 4-1BB, LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-10R, IL-12R, IL-15R, IL-21R, CD226, CD27 The sequences of the exemplary transmembrane regions of CD28 and CD8 are shown in amino acids 56-83 of SEQ ID No. 12 and 266-310 of SEQ ID No. 25.

In the present invention, the intracellular signaling domain includes, but is not limited to, any one or more combinations of the signaling domains of CD3ζ, CD3γ, CD38, CD3ε, FcRγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10, and DAP12. The intracellular co-stimulatory domain includes, but is not limited to, any one or more combinations of the tumor necrosis factor superfamily of 4-1BB, ICOS, CD27, OX40, CD28, MYD88, IL1R1, CD70, TNFRSF19L, TNFRSF27, TNFRSF1OD, TNFRSF13B, TNFRSF18, and CD134. The chimeric immune cell co-receptor of the present invention can be constructed using one or more of these signaling domains or co-stimulatory domains or variants thereof that retain the biological function of transmitting signals. The amino acid sequence of an exemplary CD3ζ signaling domain is shown in amino acids 125-236 of SEQ ID No. 12. Exemplary amino acid sequences of CD28 and 4-1BB are shown in SEQ ID No. 12, amino acids 84-124, and SEQ ID No. 25, amino acids 335-376.

In one or more embodiments, the fusion protein comprises or is, from the N-terminus to the C-terminus, a tumor antigen binding molecule, a polypeptide tag, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and a signal transduction domain. Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID No. 13.

In other embodiments, the polypeptide tag in the fusion protein can also be connected to the outside of the functional polypeptide, that is, separated from the functional polypeptide to form an independent polypeptide tag. Preferably, the polypeptide tag is connected to the C-terminus of the functional polypeptide through a cleavage sequence and a tag signal peptide sequence, and the C-terminus of the polypeptide tag is connected to an expression auxiliary sequence. In one or more embodiments, the fusion protein comprises or is sequentially from the N-terminus to the C-terminus: a binding molecule for a tumor antigen, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, a signal transduction domain, a cleavage sequence, a tag signal peptide, a polypeptide tag, and an expression auxiliary sequence.

"Tag signal peptide" refers to a signal peptide that guides a polypeptide tag, which is located at the N-terminus of the polypeptide tag. Exemplary signal peptides or tag signal peptides are S3 (SEQ ID No. 17) or S5 (SEQ ID No. 18) or Sg (SEQ ID No. 19) or Sk (SEQ ID No. 20).

"Closing element/sequence" refers to an element that can induce an mRNA molecule to produce two different polypeptides separated by the cleavage element during the process of mRNA translation and protein expression. The cleavage element can be a self-cleaving or co-cleaving peptide that enables a transcription product to produce multiple proteins, such as a 2A element, including but not limited to T2A elements, P2A elements, E2A elements and F2A elements. The cleavage element can also be a sequence that can independently initiate translation, such as an IRES element. The amino acid sequence encoded by a cleavage element (such as a 2A element) is called a "cleaving peptide" or "cleaving sequence (cleaving peptide)". Preferably, the cleavage sequence is T2A, the sequence is shown in SEQ ID No. 15, and its N-terminus or C-terminus may also be connected to a sequence that can be recognized by furin protease to obtain a cleaner cleavage end, preferably FT2A (i.e., furin+T2A), the sequence is shown in SEQ ID NO: 16.

The "expression auxiliary sequence" includes the transmembrane anchor portion and the gap between the extracellular tag and the transmembrane structure. In some embodiments, the transmembrane anchor portion is B _TM (SEQ ID No.21), and the gap portion between the extracellular tag and the transmembrane structure is B _GAP (SEQ ID No.22). It should be understood that the various domains, polypeptides, and proteins described herein include their functional fragments. "Functional fragment" refers to a fragment that retains the desired biological function. For example, the functional fragment of the intracellular domain described herein refers to a fragment that retains the biological function of the costimulatory signal molecule to transmit costimulatory signals and activate immune cells. The functional fragments of each extracellular domain and each intracellular domain suitable for the present invention can be easily determined by a person skilled in the art in combination with the existing technical means in the art.

The "mutants" described herein include mutants of various domains, polypeptides, and proteins, as long as the mutants retain the corresponding biological functions of the domains, polypeptides, and proteins. For example, mutants suitable for the polypeptide tags of the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the polypeptide tags used as a comparison; mutants suitable for the extracellular domain of the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the extracellular domain used as a comparison; mutants suitable for the transmembrane region of the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the transmembrane region used as a comparison; mutants suitable for the intracellular domain of the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the intracellular domain used as a comparison. Alternatively, compared to the sequence used as a comparison, the mutant of the present invention has one or more (e.g., within 20, within 15, within 10, within 8, within 5 or within 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted or deleted. For example, in the art, conservative substitution with amino acids with similar or similar properties usually does not change the function of the protein or polypeptide. "Amino acids with similar or similar properties" include, for example, families of amino acid residues having similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The present invention includes mutants having one or more (such as within 20, within 15, within 10, within 8, within 5 or within 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted or deleted compared to the polypeptide tag or fusion protein described above. Such mutants retain the biological functions of the polypeptide tag or fusion protein described in the present invention, including but not limited to the functions of CAR-T cell-specific activation and amplification, labeling-separation and enrichment, shutting down activity and clearance.

The polypeptides described herein may be modified polypeptides. Modifications (usually without changing the primary structure) include: Chemical derivatization forms of polypeptides such as acetylation or carboxylation in vitro or in vivo. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of polypeptides or in further processing steps. This modification can be completed by exposing the polypeptide to an enzyme that performs glycosylation (such as mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their anti-proteolytic properties or optimize solubility.

Polynucleotide molecules

The present invention provides a polynucleotide molecule encoding the polypeptide tag or fusion protein of the present invention or a fragment thereof as an amplification primer or a detection probe. The present invention also provides a complementary sequence of the coding sequence. The polynucleotide molecule can be a recombinant nucleic acid molecule or a synthetic one; it can contain DNA, RNA and PNA (peptide nucleic acid) and can be a hybrid thereof.

Also provided is an expression frame of the polypeptide tag or fusion protein of the present invention, which is a nucleic acid construct containing a promoter, a polypeptide tag or fusion protein coding sequence and a Poly A tailing signal sequence. The nucleic acid construct may also contain other elements required for expression, including but not limited to enhancers, etc.

The nucleic acid molecule encoding the fusion protein can be: a coding sequence of a chimeric antigen receptor (CAR) protein comprising a polypeptide tag, comprising a coding sequence of a chimeric antigen receptor extracellular region, a transmembrane region, and an intracellular region. The extracellular region coding sequence comprises an antigen receptor domain and a hinge region coding sequence, and the intracellular region includes a signaling domain and/or a co-stimulatory domain coding sequence. The polypeptide tag coding sequence is located at the 5' end or 3' end of the antigen receptor domain coding sequence, preferably the 3' end.

The nucleic acid molecule encoding the fusion protein can also be: the coding sequence of the functional polypeptide (such as a chimeric antigen receptor) is connected to the coding sequence of the polypeptide tag through the coding sequence of the cleavage element, and then a nucleic acid molecule is formed. Exemplarily, the coding sequence of the independently expressed polypeptide tag containing the tag signal peptide, the polypeptide tag, and the expression auxiliary sequence portion can be connected to the coding sequence of the CAR through a cleavage element (e.g., the coding sequence of F2A, FT2A, P2A, T2A or E2A or IRES), and the two are in an expression frame. After being expressed by the same promoter, they are cut by the cleavage sequence to form a separate CAR protein and an independently expressed polypeptide tag protein.

A vector is also provided, which contains the polynucleotide molecules, expression cassettes or nucleic acid constructs described herein. The vector can be a plasmid, a cosmid, a virus and a phage. The vector can be a viral vector or a non-viral vector, preferably a non-viral vector. The vector can be a cloning vector, an integration vector, or an expression vector. The expression vector can be a transposon vector. In certain embodiments, the expression vector is one or more selected from the following transposon vectors: piggybac, sleeping beauty, frog prince, Tn5 and Ty. In addition to the polynucleotide molecules described in the present invention, the expression vector usually contains other elements that are usually contained in the vector, such as multiple cloning sites, resistance genes, replication initiation sites, etc. In certain embodiments, the recombinant expression vector uses a vector well known in the art as a backbone vector. In certain embodiments, the present invention uses the pNB338B vector constructed by CN109988759A.

In some embodiments, the same vector encodes both the fusion protein of the present invention and the CAR. The vector may be a bicistronic vector. The coding sequence of the CAR may be disposed at the 5' or 3' end of the coding sequence of the fusion protein body. The expression of the CAR and the fusion protein may be under the guidance of the same or different regulatory sequences.

In the case where the polynucleotide sequence is known, each polynucleotide molecule can be prepared by conventional methods in the art and the corresponding vector can be constructed. Recombinant vectors can be constructed using methods familiar to those skilled in the art, see, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory). Vectors containing nucleic acid molecules of the present invention can be transferred to host cells by well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al. (see above).

Host cells

Herein, when expressing a heterologous nucleic acid sequence, a "host cell" is one that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell may be "transfected" or "transformed," which refers to the process of transfection or transduction of an exogenous nucleic acid into a host cell. Transformed cells include primary subject cells and their progeny. The terms "engineered" and "recombinant" cells or host cells used herein often refer to cells into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Specifically, the present invention provides cells carrying a polypeptide tag or fusion protein of the present invention and/or its coding sequence. The cells of the present invention are preferably immune cells, including T cells (e.g., CD3+ and/or CD28+ T cells), CTL cells, NK cells, NKT cells, CAR-T, CAR-NK, TCR-T, CIK, TIL, DN T cells; and other immune cells that can induce effector functions.

In one or more embodiments, the host cell expresses a fusion protein as described herein, which comprises a functional polypeptide (chimeric antigen receptor) and a polypeptide tag located internally or connected to the outside thereof by a cleavage element.

The nucleic acid construct/recombinant expression vector of the present invention can be transferred into the cell of interest. The method of transfer is a conventional method in the art, including but not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation and electrotransformation. In certain embodiments, electrotransformation is used to transfer the nucleic acid construct or recombinant expression vector.

Cells carrying the polypeptide tag or fusion protein and/or its coding sequence described in the present invention can be activated and/or sorted using anti-tag antibodies. Therefore, the present invention provides a method for preparing T cells, comprising: (1) the step of expressing the polypeptide tag and fusion protein described in any embodiment of the present invention in T cells, such as the step of introducing the nucleic acid construct described in any embodiment of the present invention into T cells, and (2) using anti-tag antibodies or solid phase carriers coupled with anti-tag antibodies to activate the T cells obtained in step (1) to achieve activation or sorting. The anti-tag antibody specifically recognizes the polypeptide tag described herein. In one or more embodiments, the antibody is selected from one or more of NB-36, NB-100, NB-102, NB-257, and NB-367 in CN202110301079.1, which is incorporated herein by reference in its entirety. In one or more embodiments, the method further comprises expressing a chimeric antigen in a T cell. The step of introducing a nucleic acid construct containing a chimeric antigen receptor coding sequence into T cells is described in detail below.

The anti-tag antibody can be coupled to a solid phase carrier for easy sorting. Exemplarily, the solid phase carrier is a magnetic particle/microsphere; the coupling is directly connected by chemical means, by specific binding of antigen-antibody, by specific binding of biotin group-avidin group or other indirect connection.

Pharmaceutical composition

On the other hand, the present invention provides a pharmaceutical composition, including any one or more of the polypeptide tags, fusion proteins, nucleic acid molecules, nucleic acid constructs, solid phase carriers and host cells described in any embodiment of the present invention and a pharmaceutically acceptable carrier. Herein, "pharmaceutical composition" refers to a composition for administration to an individual and covers a composition of cells for immunotherapy. The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers. For example, pH regulators include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride. Compositions containing such excipients can be formulated by well-known conventional methods.

These pharmaceutical compositions can be administered to a subject at a suitable dose. The dosage regimen can be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on a variety of factors, including the patient's size, body surface area, age, specific compound to be administered, sex, administration time and route of administration, overall health status, and other drugs administered simultaneously.

The compositions of the present invention can be administered topically or systemically. In certain embodiments, the compositions provided herein (e.g., cells expressing chimeric immune cell co-receptors of the present invention) can be administered parenterally, such as intravenously, intra-arterially, intrathecally, subdermally, or intramuscularly.

Methods and uses

The fusion protein, host cell and pharmaceutical composition comprising these substances described in the present invention can be used to prevent, treat or alleviate cancer, especially cancers in which corresponding tumor antigens are expressed on the surface of cancer cells, or for preparing drugs for preventing, treating or alleviating cancer. On the other hand, the present invention provides the use of any one or more of the polypeptide tags, fusion proteins, nucleic acid molecules, nucleic acid constructs and host cells described in any embodiment of the present invention in the preparation of drugs for preventing, treating or alleviating tumors. As a preferred example, the drug comprises T cells expressing the polypeptide tags or fusion proteins described herein, so that the T cells can be activated and/or sorted by anti-tag antibodies or solid phase carriers coupled with anti-tag antibodies as described herein.

In addition, the present invention provides a method for preventing, treating or alleviating cancer, comprising the following steps: The subject is given an effective amount of cells, the cells carry the polypeptide tag, fusion protein or T cell described in the present invention and/or generated by the method of the present invention. The T cells are activated and/or sorted by anti-tag antibodies or solid phase carriers coupled with anti-tag antibodies as described herein. Therefore, the method also includes the step of activating and/or sorting the T cells by anti-tag antibodies or the solid phase carriers coupled with anti-tag antibodies.

Herein, cancer includes various solid tumors and blood tumors, including but not limited to lung cancer (such as non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma and brain glioma, etc. More specifically, cancer herein includes but is not limited to breast, prostate, lung and colon cancer or epithelial cancer, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, melanoma; genital-urinary tract cancer, such as ovarian cancer, endometrial cancer, cervical cancer; kidney cancer, lung cancer, gastric cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, etc. Administration of the composition of the present invention can be used for all stages and types of cancer, including for example, minimal residual disease, early cancer, advanced cancer and/or metastatic cancer and/or cancer that is difficult to treat.

The present invention also provides a cell binding/labeling/detection/stimulation/sorting kit for stimulating or sorting cells expressing the polypeptide tag or fusion protein described herein, the kit comprising the anti-tag antibody described herein or a solid phase carrier coupled with the anti-tag antibody, the anti-tag antibody specifically recognizes the polypeptide tag described herein. The detection kit may also comprise the polypeptide tag, fusion protein, nucleic acid molecule or nucleic acid construct described in any embodiment of the present invention.

The embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area (for example, reference to "Molecular Cloning Experiment Guide" by J. Sambrook, Huang Peitang, etc., the third edition, Science Press), corresponding references, or according to product specifications are carried out. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

Example

Example 1, BL tag design and high affinity VHH screening

This example describes the identification and characterization of the label of the present invention, and the label can identify and bind to VHH antibodies such as NB-36. In order to make the tagged CAR-T cells safer for treatment, we chose the extracellular sequence of the natural human BCMA protein to design the label, as shown in Figure 1, A. BCMA-FL (184aa, sp|Q02223), BCMA-EC (54aa, sp|Q02223|1-54), BLΔ0-5 represent the full-length sequence, extracellular domain sequence, and truncated sequence of the human BCMA protein, respectively, and BLΔ0mut is the site mutation sequence of BLΔ0. The specific sequences are shown in SEQ ID No. 1-8, respectively. For high affinity VHH for human BCMA protein, refer to patent CN202110301079.1. As shown in the patent, NB-36, 100, 257, 102, 367, etc. have better expression and affinity; NB-36 has high affinity for BCMA antigen, with a KD value of 2.92E-10; NB-36 also shows good specific binding affinity for HEK293T-BCMA cell line.

ELISA (enzyme-linked immunosorbent assay) was used to detect and screen high-affinity VHHs with different BL tags (BLΔ0/1/2/3/4/5 and BLΔ0mut). The BL tag fusion peptide (M044 connected to the C-terminus via a 3×GGGGS linker, disclosed in WO2022143550A1, and with a Myc-His tag at the C-tail) was synthesized by Biointron (Suzhou, China). The fusion peptide was fixed to the bottom of the plate by anti-Myc antibody and incubated with different VHH-Fc (human IgG4-Fc was used as an isotype control). After washing, an anti-Fc antibody conjugated to HRP was used as a secondary antibody for incubation and washing. Finally, HRP substrate was added, and the samples were detected and quantitatively analyzed by an ELISA reader. As shown in Figure 1, B, all VHHs tested can bind to the BLΔ0/1/2 fusion peptide (ELISA affinity: NB-367>36>102>100), and NB-36, 90, 100, 102, 257, and 367 specifically bind to the BLΔ3/4 tag (NB-100/36/102/367 showed loss of binding to BLΔ3 compared to BLΔ0/1). In comparison, as we predicted, all VHHs showed no binding to the BLΔ0mut fusion peptide. In the results of Figure 1, C-D, NB-36, 100, 102, and 367 can specifically bind to BLΔ0.

The binding affinity of BLΔ0 fusion peptide to these VHHs was detected by surface plasmon resonance (SPR). The binding affinity of human BCMA natural ligands BAFF, APRIL to BLΔ0 fusion peptide was detected to determine the safety of BL tag. First, VHH-Fc (Biointron, Suzhou, China) was passed through a sensor chip pre-immobilized with Protein-A and captured by Protein-A. Then, five different concentrations of BLΔ0 fusion peptide were used as mobile phase, and the binding time and dissociation time were 30min and 60min, respectively. The opening rate (Kon or Ka), closing rate (Koff or Kb) and equilibrium constant (KD or Kd) were analyzed using Biacore Evaluation Software 2.0 (GE, USA). As shown in Table 1, NB-36/101/367 has a specific affinity; NB-36 has a high affinity for BLΔ0 fusion peptide, with a KD of 3.63E-9. As a control, there was no specific affinity between BAFF, APRIL and BLΔ0 fusion peptide ("-": not detected).

Table 1: Kinetics of binding of ligands or VHHs to BLΔ0 fusion peptides

Based on the above results, NB-36, NB-102, NB367, etc. can be combined with BCMA-FL, BCMA-EC, BLΔ0, BLΔ1, BLΔ2, BLΔ3, BLΔ4 and BLΔ5 bind (Figure 1B, D). In addition, as shown in Figure 1C, NB-36 has strong binding to the cell line, EC50 = 0.3829nM. These results show that NB-36 (as well as NB-102, NB367, etc.) binds to BLΔ0/2/5 with high affinity and can be used as a BL tag binding antibody (BL-binding-VHH), and BLΔ0/2/5 can be used as a tag on CAR-T cells.

Example 2, optimized site mutation BL tag and related verification

The surface plasmon resonance (SPR) results in Example 1 show that BLΔ2 does not interact with BAFF/APRIL. In order to use the BL tag in vivo, it is necessary to limit its ability to bind to non-target, non-specific proteins. Referring to Bossen C, Schneider P. BAFF, APRIL and their receptors: structure, function and signaling [C] // Seminars in immunology. Academic Press, 2006, 18 (5): 263-275. and other literature, D15, L17, I22, etc. in the BΔ2 region are important amino acid sites for the peptide/protein to maintain a three-dimensional structure and exert binding activity on factors such as BAFF/APRIL. Mutating them separately may prevent them from binding to related factors, which improves the safety of BL in vivo application. Therefore, we designed three site mutations of the BLΔ2 tag: BLΔ2D2G, BLΔ2L2G and BLΔ2I2G (D15, L17, I22 mutated to G, SEQ ID No. 9-11). As shown in Figure 2, NB-36, which specifically binds to BLΔ2, cannot bind to BLΔ2D2G, BLΔ2L2G, and BLΔ2I2G, suggesting that the mutation of key active amino acid sites may cause its binding to related factors and even previously screened antibodies to be greatly reduced. The BL-CAR-T formed may need to be further stimulated and activated using traditional general activation methods, and the mutant BL can be used as a safer candidate label in vivo and become a target for the next generation of antibody screening.

Example 3, Construction of a solid phase carrier (BL tag-bound microspheres) coupled with an anti-tag antibody and BL- CAR-T cell marker sorting

1 mL (4 × 10 ⁸ ) Dynabeads M-450 Tosylactivated (Invitrogen, USA)/Dynabead Biotin Binder (Invitrogen, USA) was used to couple about 200 μg of anti-tag antibody (biotin-conjugated anti-tag antibody is required for Biotin Binder) to produce NB-36-beads (direct coupling of Dynabeads M-450 Tosylactivated) or NB-36-SAbeads (biotin coupling of Biotin Binder). The washed magnetic beads were placed in the magnet for 1 minute and the supernatant was discarded. The tube was removed from the magnet, the beads were resuspended in 0.1 M sodium phosphate buffer (pH 7.4-8.0), and 200 μg of antibody (NB-36 or biotin-conjugated NB-36 with anti-CD28) was added during mixing to reach a total coupling volume of 1 mL. Incubate at room temperature for 16-24 hours with gentle tilting and rotation. Place the tube in the magnet for 1 minute and discard the supernatant. Add 1 mL of Buffer W (Ca ²⁺ and Mg ²⁺ free phosphate buffered saline (PBS) supplemented with 0.1% bovine serum albumin (BSA) and 2 mM EDTA, pH 7.4) and mix, incubate at 2°C to 8°C for 5 minutes with gentle tilting and rotation. Place the tube in the magnet for 1 minute and discard the supernatant, remove from the magnet and repeat the wash step once. Add 1 mL of Buffer W, mix and incubate at 2°C to 8°C. Incubate for 5 minutes with gentle tilting and rotation. Remove the beads from the magnet and resuspend the coated beads in Buffer W.

Add 25 μL of pre-coupled, washed and resuspended magnetic beads to 1 mL of prepared sample (1×10 ⁷ transfected T cells). Incubate at 4°C for 20 minutes with gentle tilting and rotation. Place the tube in the magnet for 2 minutes and discard the supernatant. Wash the magnetic bead-bound cells 2-4 times. The proportion of BL-CAR-T cells was detected by flow cytometry (Beckman, USA) using biotin-conjugated mesothelin (or biotin-conjugated NB-36 for specific detection of BL-tagged CAR-T) and PE-conjugated streptavidin secondary antibody (BD Bio., USA). As shown in Figure 3, Biotin-NB-36-conjugated Dynabead Biotin Binder magnetic beads can effectively bind to BL-CAR-T cells: (M2339-BLΔ0-CAR-T: coupled magnetic beads = 1:1).

Example 4. Construction of BL-CAR vector and BL-CAR-T cells targeting mesothelin-positive cancer cells

The tag CAR gene is composed of an antigen receptor domain sequence (antibody to tumor antigen), a BL tag sequence, a hinge and a transmembrane sequence (CD28TM), a CD28 or 4-1BB intracellular co-stimulatory signal domain sequence (CD28/4-1BB-IC), and a CD3ζ activation domain sequence from 5' to 3' (Figure 4, A). Here, the antigen receptor domain M2339 (VHH, an antibody to MSLN) for mesothelin (MSLN) is described in patent disclosure WO2021130535A (incorporated herein in its entirety by reference and for all purposes). According to the patent, M2339 (VHH) binds to mesothelin-full, mesothelin I, and mesothelin II+III domains with different affinities. The mesothelin II+III domain is well recognized by M2339, with a KD value of 4.32E-11M, which has similar affinity to the complete mesothelin polypeptide. The tag CAR gene was replaced by the antiCD19 (CD19 antibody) gene to generate a non-specific control construct plasmid (NS). The tag-CAR gene was amplified by PCR and cloned into the piggyBac transposon vector pNB338B (see patent CN109988759A) to obtain a plasmid. In order to produce different tag CAR-Ts, different vectors were constructed, as shown in Table 2. As a preferred example, SEQ ID No. 14 shows the M2339-BLΔ2-CAR fusion protein gene (SP-M2339-BLΔ2-CD8h-CD28TM-CD28IC-CD3ζ) of the M2339-BLΔ2 vector. All vectors encode proteins with the same hinge-transmembrane-intracellular structure, including CD28 and CD3ζ intracellular parts (SEQ ID No: 12).

Table 2. Tag CAR vectors

Human peripheral blood mononuclear cells (PBMCs) collected from healthy donors were purchased from AllCells (Shanghai, China). PBMCs were cultured in AIM-V CTS medium (Gibco, USA) supplemented with 2% fetal bovine serum (FBS; Gibco, USA) at 37°C in a 5% CO2 humidified incubator for 0.5-1 h, then harvested and washed twice with PBS. PBMCs were counted and electroporated using the Amaxa Human T Cell Nucleo(ector Kit (Lonza, Switzerland) in an electroporator (Lonza, Switzerland) according to its instructions. Thereafter, transfected T cells ( Example 5) or stimulated with anti-mesothelin or anti-CD3 plus anti-CD28 antibody (5+5 μg/mL) coated on the bottom of the plate in advance for 4-5 days, and then cultured for 7-9 days to produce a sufficient number of effector T cells. The transfection efficiency of CAR to T cells was determined by flow cytometry (Beckman, USA) using biotin-conjugated mesothelin (or biotin-conjugated NB-36 for BL-CAR-T specific expression detection expressing BL tag) and PE-conjugated streptavidin secondary antibody (BD Bio., USA).

As shown in Figure 4, B, C, D, and E, the BLΔ0/BLΔ2-CAR-T cells cultured in vitro can be effectively expanded by coating NB-36-Fc (containing antiCD28) for 5, 8, and 13 days of activation, and the PBMCs of different donors reached a peak 10-12 after transfection. This is consistent with the classic M2339-CAR-T activated by coating mesothelin (with anti-CD28). In addition, the CAR positivity rate of total product T cells was also not significantly different from that of specific mesothelin (with anti-CD28) activation. These prove that BL-CAR-T cells were successfully constructed and that the amplification using the coated tag-binding antibody was effective. In addition, among the three site mutations of BLΔ2-CAR-T, the preparation (activated by coating mesothelin + anti-CD28) results showed that the amplification and construction of the BLΔ2-L2G form were successful, while the amplification of the D2G and I2G forms of BL-CAR-T was poor, indicating that BLΔ2-L2G can be used as a preferred solution for improving the safety of mutated BL in vivo and applied to BL-CAR-T, as shown in Figure 4, F.

Example 5: In vitro activation and amplification of solid phase carriers (BL tag-bound microspheres) coupled with anti-tag antibodies BL-CAR-T cells

During the in vitro culture of CAR-T cells, BL-CAR-T cells can be stimulated by specific activation through recognition and binding to a solid phase carrier (BL tag-bound microspheres) coupled to an anti-tag antibody, as shown in Figure 5A. In AIM-V medium containing 2% FBS and 100U/mL recombinant human interleukin-2, transfected T cells were stimulated in 6-well plates with specific BL tag-bound micromagnetic beads (magnetic beads: cells were 1:1 (1x), 1:4 (0.25x) or 4:1 (4x)) for 4-5 days, and then cultured for 7-9 days to produce a sufficient number of effector T cells. In order to verify the rapid activation and expansion of BL-CAR-T cells by micromagnetic beads, biotin-coupled mesothelin (or biotin-coupled NB-36 for specific expression detection of BL-CAR-T expressing BL tags) and PE-coupled streptavidin secondary antibodies (BD Bio., USA) were used to label and flow cytometry (Beckman, USA) was used to measure the expression of T cell CAR genes. The transduction efficiency.

As shown in Figure 5, B, in the presence of the activation beads (NB-36 directly coupled to the beads, or Biotin-NB-36 coupled to Biotin Binder/SA-micromagnetic beads, both coupled to anti-CD28), BLΔ0/BLΔ2-CAR-T cells prepared from PBMCs of different donors can be effectively activated and expanded to the peak number 10-12 days after transfection. This is consistent with the activation of the commonly used antiCD3/28 dynabeads (Invitrogen, USA), and is significantly improved compared to the specific activation effect of coated mesothelin (plus anti-CD28), proving that the expansion effect of the anti-LB tag activation beads is very good. The bead: cell ratio of 1:1 (1x) showed better activation effect than 1:4 (0.25x) or 4:1 (4x). In addition, the CAR-positive rate of total product T cells was also consistent with the activation of specific coated mesothelin (plus anti-CD28), and was significantly better than the commonly used nonspecific stimulator antiCD3/28 dynabeads (Figure 6, C). These results prove that the activation and enrichment of CAR-T cells based on BL tag-corresponding activation microbeads is effective.

Example 6, Validation, Characterization and Quality Control of BL-CAR-T Cells

After the expansion of the tagged CAR-T cells, the modified T cells were validated and characterized by a series of tests, including activation phenotype (positive ratio of CD25 and CD69), exhaustion phenotype (positive ratio of PD-1 and TIM3), CD4/8 positive ratio in CD3 positive cells, effector memory T (Tem, CCR7- and CD62L-)/central memory T cells (Tcm, CCR7+ and CD62L+) ratio in memory T cells (Tm, CD45RO+). The positive ratio of CD3, CD4, CD8, CD25, CD69, PD-1, TIM3, CD45RA, CCR7, and CD62L on the surface of T cells was determined by flow cytometry, and each fluorescein-conjugated antibody (BD Bio., USA) was used for staining according to the instructions of the staining reagent.

As shown in Figure 6, the CD4/8 ratio of BLΔ0/BLΔ2-CAR-T cells prepared from PBMCs of different donor sources and activated by coating NB-36 or mesothelin (with anti-CD28) varied between 0.23 and 0.78, with central memory T cells accounting for the majority. In addition, the CD25, PD-1, and TIM3 positive ratios of BLΔ0/BLΔ2-CAR-T cells activated by coating NB-36 or mesothelin (with anti-CD28) were consistent with those of classical M2339-CAR-T cells activated by coating mesothelin (with anti-CD28). This indicates that the phenotype of BLΔ0/BLΔ2-CAR-T cells activated by coated ligands is consistent with that of unlabeled CAR-T, that is, the BL label does not affect the typing, stemness, activation, and exhaustion of CAR-T cells.

Figure 7 detected the CAR positivity, activation, stemness, and exhaustion phenotypes of BLΔ2-CAR-T cells activated by different antibody-coupled microbeads, proving that the microbeads coupled to NB-36 were effective in activating and enriching BLΔ2-CAR-T cells and produced a good phenotype.

For mutant BLΔ2-CAR-T cells, as shown in Figure 8, the CD25, PD-1, TIM3 positive ratios and CD4/8 ratios and effector/central memory T cell ratios of BLΔ2L2G-CAR-T cells activated by coating mesothelin (with anti-CD28) (7 days and 13 days) were consistent with those of wild-type BLΔ2-CAR-T cells. The phenotype of CAR-T cells is consistent with the results of in vitro CAR-T cell culture/construction (Example 5). These indicate that the site mutation tag of BLΔ2L2G does not affect the typing, stemness, activation and exhaustion of CAR-T.

Example 7: Toxicity of labeled CAR-T cells to cancer cells

Target tumor cells, human ovarian cancer cells SK-OV3 and human lung cancer cells NCI-H226 (ATCC, USA), were cultured in DMEM or RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C in an atmosphere of 5% CO2. Cytotoxicity/killing effects were determined by an impedance-based xCELLigence RTCA TP instrument (ACEA Bio., USA). Target tumor cells were seeded at 10,000 cells per well in an impedance-measurable 96-well plate bottom (ACEA Bio., USA) and cultured overnight (more than 16 hours) on an RTCA TP instrument in a cell culture incubator. The prepared transformed T cells (effector cells) were then added and incubated with target tumor cells at different ratios (E:T) for about 72-120 hours (the end point depends on the killing efficiency). The cytolytic effects of effector cells on target tumor cells were compared at three different E:T ratios of 4:1, 1:1, and 1:4. During the experiment, the tumor cell viability index value was closely related to its adhesion. The lower the degree of cell adhesion, the higher the effector cell toxicity. The RTCA system collects this data every 5 minutes. The real-time toxicity killing curve is automatically generated by the system software. The experiment also used the endpoint data (co-cultured for 72 hours) to calculate the specific cell lysis (%) of each transformed T cell [specific cell lysis = (cell index of tumor cells only - cell index of transformed T cells co-cultured with tumor cells) / cell index of tumor cells only].

As shown in Figure 9, A and B, M2339-CAR-T (activated by coated mesothelin plus anti-CD28 antibody), M2339-BLΔ0-CAR-T (activated by coated mesothelin plus anti-CD28 antibody), and M2339-BLΔ0-CAR-T (activated by coated NB-36 plus anti-CD28 antibody) had strong specific killing effects on target tumor cells SK-OV3 and NCI-H226, while CD19-CAR-T cells (activated by CD19 plus anti-CD28 antibody) as nonspecific T cells (N.S.) had no obvious cell killing effect on SK-OV3 and NCI-H226. Under different E∶T, the EC50 values of M2339-BLΔ0-CAR-T were the same as those of classic M2339-CAR-T, and the EC50 for SK-OV3 was 0.028 (E∶T＝4∶1), 0.024 (E∶T＝1∶1) and 0.022 (E∶T＝1∶4); the EC50 for NCI-H226 was 0.028 (E∶T＝4∶1), 0.024 (E∶T＝1∶1) and 0.022 (E∶T＝1∶4).

As shown in Figure 9, C and D, the killing effects of M2339-BLΔ2-CAR-T activated by different methods on target tumor cells were compared. The results showed that the cell killing effect of M2339-BLΔ2-CAR-T activated by NB-36-coupled microspheres (directly coupled or coupled by Botin-SA) on target tumor cells SK-OV3 and NCI-H226 was not significantly different from that of the classic activation method of coated mesothelin plus anti-CD28 antibody, and had a certain statistically significant advantage over the commonly used anti-CD3/28 dynabeads activation method on SK-OV3 cells.

Example 8 CAR vector and CAR-T with independent BL tag for CD19/22 positive cancer cells Cell construction

In order to achieve easy labeling and sorting of CAR-positive cells, we also developed a CAR structure with an independent BL tag (sBL) based on the above-mentioned BL tag and tag-bound microbeads. Figures 10A, B and Table 3 describe the construction of the sBL-CAR-T vector. C1922 represents a CAR domain sequence that can simultaneously target CD19 and CD22, comprising an anti-CD19 VHH antibody domain sequence 1902 and an anti-CD22 VHH antibody domain sequence 2205 connected thereto by a linker (SEQ ID No. 25: 22-265). For the expression of the independent BL tag, the BL tag sequence is separated from the CAR structure part sequence C1922-H-TM-IC (SEQ ID No. 25: 22-488) by a segmentation sequence T2A (abbreviated as "T", SEQ ID No. 15) or Furin-T2A (abbreviated as "FT", SEQ ID No. 16), and is guided by another signal peptide (SP) sequence. S3 is the natural signal peptide of human IgM-Vh4 protein (O95973), S5 is the natural signal peptide of human azurocidin protein (P20160), Sg is the natural signal peptide of human GMCSFRa protein (P15509), and Sk is the natural signal peptide of human IgK protein (P01624). These sequences are listed in SEQ ID No.17-20. In order to anchor the independent BLΔ2 tag peptide to the cell membrane after expression, we added the transmembrane part of human BCMA protein (B _TM , SEQ ID No: 22) after the BLΔ2 sequence to form the BLΔ2-M sequence ("M" represents B _TM ), see SEQ ID No.23. In addition, in order to make the expression, folding and localization of BLΔ2 and B _TM more ideal, we designed to add the extracellular space part of the natural BCMA protein (B _GAP , SEQ ID No: 21) to the BLΔ2 sequence and B _TM to form a BLΔ2-GM sequence ("GM" stands for B _GAP -B _TM ), see SEQ ID No. 24. As a preferred example, SEQ ID No. 26 shows the independent BLΔ2-C1922-CAR gene of the C1922-FTS3-BLΔ2-GM vector. The design of other parts of the vector is the same as in Example 4.

Table 3. CAR vectors with independent BLΔ2 tags

The construction process of (BL-)CAR expression vector and CAR-T cells is the same as in Example 4. It is worth noting that the activation method of label-bound microbeads (Example 5) cannot be used to activate CAR-T cells with independent BL. Use biotin-coupled CD19 (or biotin-coupled NB-36 for CAR-T detection expressing BL label) and PE-coupled The cells were labeled with streptavidin secondary antibody (BD Bio., USA) and flow cytometry (Beckman, USA) was used to measure the transduction efficiency of T cell CAR gene.

As shown in Figure 10C, the preparation and basic phenotypes of CAR-T cells with independent BL in different forms were compared. The expansion and activation phenotypes (CD25/69+ ratio) of C1922-FTS3-BLΔ2-M/GM, C1922-FTS5-BLΔ2-M, C1922-FTSk-BLΔ2-M, C1922-S3-BLΔ2-M (without Furin-T2A), and C1922-F-BLΔ2-M (without T2A-signal peptide) CAR-T (activated by coated CD19 plus anti-CD28) were basically the same as those of the control group without label C1922-CAR-T (activated by coated antiCD3/CD28), and the CAR positive ratio was significantly higher than that of the control group.

As shown in Figure 11, independent BL-CAR-T cells with different structures (except for the group labeled αCD3/28, which was activated by coated antiCD3/CD28, all were activated by coated CD19 plus anti-CD28), showing the comprehensive results of CAR-T prepared from PBMCs of three donors. The expansion and activation of CAR-T cells (activated by coated CD19 plus anti-CD28), C1922-FTS3-BLΔ2-M/GM, C1922-FTSg-BLΔ2-M/GM, and C1922-F-BLΔ2-M (without T2A-SP) showed good CD4/8 ratio memory phenotype, while only C1922-FTS3-BLΔ2-GM and C1922-FTSg-BLΔ2-GM showed significantly higher label expression (basically the same as the CAR positive ratio). These results indicate that the "-M" form lacking B _GAP may have a key problem for label expression, and B _GAP is necessary for independent BL labeling. Finally, the preferred decision for the independent BL-tagged CAR structure was C1922-FT3-BLΔ2-ET CAR.

*Statistical Analysis

T-test was used to evaluate the differences between two independent groups. One-way ANOVA was used to compare whether there were any statistically significant differences between three or more independent groups. Two-way ANOVA was used to determine the effects of two nominal predictor variables on continuous outcome variables. All statistical analyses were performed using Graphpad Prism version 7 software (LaJolla, CA). All data with error bars are presented as mean ± SD. Statistically significant differences were considered: P ≥ 0.05 (no significant difference, ns), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 00001 (****).

Claims

A polypeptide tag, characterized in that it comprises:

(1) a sequence as shown in any one of SEQ ID NOs: 1-11, or:

(2) A sequence having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the sequence of (1).
The polypeptide tag according to claim 1, characterized in that the polypeptide tag further comprises a linker fragment located at the N-terminus or the C-terminus for covalent connection with other polypeptides,

Preferably, the linker is (GGGGS) N , wherein N is an integer of 1-10.
A fusion protein, characterized in that it comprises a polypeptide tag and a functional polypeptide,

The polypeptide tag comprises the extracellular domain sequence of a protein expressed by normal human cells or a truncated portion of the extracellular domain sequence, or a mutant thereof;

The functional polypeptide is a chimeric antigen receptor, and its sequence includes the following regions:

(1) The extracellular region, which contains the antigen receptor domain and an optional hinge region;

(2) transmembrane region;

(3) The intracellular region includes the signal transduction domain and/or the co-stimulatory domain.
The fusion protein according to claim 3, characterized in that the normal human cell expressed protein is selected from BCMA, BAFFR, CD20, CD40, preferably BCMA; more preferably, the polypeptide tag is the polypeptide tag of claim 1 or 2.
The fusion protein according to claim 3, characterized in that the polypeptide tag is located inside the functional polypeptide,

Preferably, the polypeptide tag is connected to the antigen receptor domain, and the polypeptide tag is located at the N-terminus or C-terminus of the antigen receptor domain, preferably the C-terminus.
The fusion protein according to claim 3, characterized in that the polypeptide tag and the functional The peptide is externally connected and can be separated from the functional peptide to form an independent peptide tag.

Preferably, the polypeptide tag is connected to the N-terminus or C-terminus of the functional polypeptide via a cleavage sequence, and a signal peptide sequence is further included between the polypeptide tag and the cleavage sequence;

Preferably, the polypeptide tag is connected to the C-terminus of the functional polypeptide via a cleavage sequence, and the C-terminus of the polypeptide tag is connected to an expression auxiliary sequence;

Preferably, the cleavage sequence comprises a sequence encoding a furin recognition site and a 2A element, and the auxiliary sequence comprises a transmembrane anchor portion and a gap portion between the extracellular tag and the transmembrane structure;

Preferably, the cleavage sequence is T2A (SEQ ID No.15) or FT2A (SEQ ID No.16), the signal peptide is S3 (SEQ ID No.17) or S5 (SEQ ID No.18) or Sg (SEQ ID No.19) or Sk (SEQ ID No.20), the transmembrane anchor portion is B TM (SEQ ID No.21), and the gap portion between the extracellular tag and the transmembrane structure is B GAP (SEQ ID No.22).
The fusion protein according to any one of claims 3 to 6, characterized in that

The antigen receptor domain comprises a binding molecule for a tumor antigen; preferably, the tumor antigen is selected from the group consisting of BCMA, BAFFR, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, VEGFR-2, EGFRvIII, ErbB2, ErbB3HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, F lt1, KDR, Flt4, CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin (MSLN), NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79b, and Notch-1-4;

The hinge region is selected from the following proteins: partial extracellular or transmembrane domains: CD8, CD28, CD3, CD15, CD16, CD40, CD27;

The transmembrane region is selected from the group consisting of CD28, CD8, CD134, 4-1BB, LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-10R, IL-12R, IL-15R, IL-21R, CD226, CD27 and the transmembrane region of any of CD40;

The intracellular signaling domain includes but is not limited to: signaling domains of CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10, and DAP12.

The intracellular co-stimulatory domain is selected from any one or more combinations of 4-1BB, ICOS, CD27, OX40, CD28, MYD88, IL1R1, CD70, TNFRSF19L, TNFRSF27, TNFRSF1OD, TNFRSF13B, TNFRSF18, and CD134 tumor necrosis factor superfamily.
The fusion protein according to any one of claims 3 to 6, characterized in that it comprises or is, from the N-terminus to the C-terminus:

Antigen receptor domain, peptide tag, hinge region, transmembrane region, intracellular co-stimulatory domain, signal transduction domain, or:

Antigen receptor domain, hinge region, transmembrane region, intracellular co-stimulatory domain, signal transduction domain, cleavage sequence, tag signal peptide, polypeptide tag and expression auxiliary sequence.
The fusion protein according to claim 3 is characterized in that the amino acid sequence of the fusion protein is shown in SEQ ID NO: 13.
A nucleic acid molecule, characterized in that it comprises a sequence selected from the following:

(1) a nucleic acid sequence encoding the polypeptide tag according to claim 1 or 2 or the fusion protein according to any one of claims 3 to 9, or a fragment thereof used as an amplification primer or detection probe,

(2) a variant having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with (1),

(3) A complementary sequence of (1) or (2).
A nucleic acid construct, characterized in that:

(1) expressing the polypeptide tag of claim 1 or 2 or the fusion protein of any one of claims 3 to 9, and/or

(2) comprising the nucleic acid molecule according to claim 10.
The nucleic acid construct according to claim 11, wherein the nucleic acid construct is a vector, such as a non-viral vector.
The nucleic acid construct according to claim 11, wherein the nucleic acid construct is a cloning vector or an expression vector.
A host cell, characterized in that it contains, expresses and/or secretes the polypeptide tag according to claim 1 or 2 or the fusion protein according to any one of claims 3 to 9;

Preferably, the host cell comprises the nucleic acid molecule according to claim 10 or the nucleic acid construct according to claim 11, or the nucleic acid molecule according to claim 10 or the nucleic acid construct according to claim 11 is integrated into the chromosome.

More preferably, the host cell is an immune effector cell, such as a T cell or a NK cell.
A solid phase carrier, characterized in that the solid phase carrier is a particle or microsphere with or without magnetism and has the function of binding to the polypeptide tag according to claim 1.
The solid phase carrier according to claim 15, characterized in that it is coupled with an anti-tag antibody, which specifically recognizes the polypeptide tag according to claim 1,

Preferably, the antibody is selected from one or more of NB-36, NB-100, NB-102, NB-257, and NB-367, whose amino acid sequences are shown in SEQ ID NOs: 27-31, respectively.
The solid phase carrier as claimed in claim 15 is characterized in that the coupling is directly connected by chemical means, by specific binding of antigen-antibody, by specific binding of biotin group-avidin group or other indirect connection.
A cell labeling/detection/stimulation/sorting kit, characterized in that it is used for binding, labeling, stimulating, sorting or detecting the host cell described in claim 14.
The kit according to claim 18, characterized in that it comprises the solid phase carrier according to any one of claims 15 to 17.
The kit according to claim 18, characterized in that it further comprises the polypeptide tag according to claim 1 or 2, or the fusion protein according to any one of claims 3-9, or the nucleic acid molecule according to claim 10, or the nucleic acid construct according to any one of claims 11-13.
A pharmaceutical composition, characterized in that it comprises a pharmaceutically acceptable excipient and one or more selected from the following: the polypeptide tag according to claim 1 or 2, the fusion protein according to any one of claims 3 to 9, the nucleic acid molecule according to claim 10, the nucleic acid construct according to any one of claims 11 to 13, the host cell according to claim 14, and the solid phase carrier according to any one of claims 15 to 17.
Use of any one or more of the polypeptide tag according to claim 1 or 2, the fusion protein according to any one of claims 3 to 9, the nucleic acid molecule according to claim 10, the nucleic acid construct according to any one of claims 11 to 13, the host cell according to claim 14, and the solid phase carrier according to any one of claims 15 to 17 in the preparation of a drug for treating tumors.