EGFR polypeptide and application thereof

Abstract

The invention relates to the technical field of biology, in particular to EGFR polypeptide and application thereof. The novel truncated EGFR polypeptide is composed of extracellular structural domains III and IV, a transmembrane region and an intracellular proximal membrane terminal amino acid sequence, does not contain an extracellular ligand binding structural domain and an intracellular tyrosine kinase structural domain, and is named as JxM-EGFRt. Compared with the traditional EGFRt, the JxM-EGFRt improves the expression efficiency and the expression abundance of the EGFR polypeptide while improving the safety, on one hand, the ADCC efficiency is enhanced, the safety is better, and on the other hand, the truncated EGFR polypeptide can be used as a selection marker, and the marking efficiency is improved. Besides, CAR-T cells expressing the JxM-EGFRt polypeptide have a higher initial / immature T cell proportion, in-vivo and in-vitro storage of target cells is enhanced, and the in-vivo and in-vitro anti-tumor effect is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to EGFR polypeptides and applications thereof. Background Art

[0002] Chimeric Antigen Receptor-T cell (CAR-T) T cells are genetically modified T cells that can recognize specific target antigens in an MHC-unrestricted manner and continuously activate and proliferate. Chimeric antigen receptor (CAR) is the core component of CAR-T, which gives T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-modified T cells to recognize a wider range of targets compared to natural T cell surface receptors TCR.

[0003] One of the great advantages of CAR-T cells is that they are active drugs. Once injected, physiological mechanisms regulate the balance, memory formation, and antigen-driven expansion of T cells. However, this treatment has not yet been perfected, and CAR-T cell therapy is often accompanied by a variety of toxic side effects, such as cytokine release syndrome (CRS), neurotoxicity, and B cell aplasia. While ensuring the targeted killing function of CAR-T cells, it is necessary to add a "safety switch" to CAR-T cells, which can induce CAR-T cell apoptosis by activating suicide genes in the early stage of acute toxic effects, thereby increasing the safety of CAR-T cell therapy.

[0004] Cetuximab, with the English trade name ERBITUX and the Chinese trade name Erbitux, is an IgG1 monoclonal antibody targeting the EGF receptor. After the two specifically bind, they inhibit the tyrosine kinase (TK) that binds to the EGF receptor, blocking the intracellular signal transduction pathway, thereby inhibiting the proliferation of cancer cells and inducing apoptosis of cancer cells. Using its mechanism of action, researchers in the field of cell therapy have designed a combination of the CAR structure and the truncated EGFRt to form a "safety switch" for CAR-T. When CAR-T cells are "out of control" in the body and side effects need to be controlled, cetuximab is given to recognize the truncated EGFR (EGFRt) expressed on the surface of CAR-T cells, thereby inducing apoptosis of CAR-T cells, stopping the CAR-T cell response, and controlling side effects. This effect is mainly produced by the antibody-mediated ADCC effect. ADCC refers to the binding of the Fab segment of the antibody to the antigenic epitope of virus-infected cells or tumor cells, and the binding of its Fc segment to the FcR on the surface of killer cells (NK cells, macrophages, neutrophils, etc.), mediating the direct killing of target cells by killer cells, which is an important mechanism for the action of anti-tumor therapeutic antibody drugs. The ADCC effect requires three basic components, namely effector cells, antibodies and target cells. The ADCC effect can only be exerted when effector cells, antibodies and target cells are present at the same time. The traditional EGFRt safety switch has not been popularized in clinical applications. The reason for this is that on the one hand, the effectiveness of CAR-T cells is not enough overall, so the exposed toxic side effects are not strong enough, so there is no need to clear CAR-T cells; on the other hand, the traditional EGFRt clearance efficiency is low, and the actual value is not great in clinical applications. Summary of the invention

[0005] In view of this, the present invention provides EGFR polypeptides and applications thereof. This study invented a truncated EGFR novel polypeptide, which consists of extracellular domains III and IV, a transmembrane domain (TM) and an intracellular juxtamembrane domain (JxM), does not contain an extracellular ligand binding domain and an intracellular tyrosine kinase domain, and is named JxM-EGFRt. Compared with traditional EGFRt, JxM-EGFRt improves the expression efficiency and abundance of EGFR polypeptides while improving safety. On the one hand, it enhances ADCC efficiency and has better safety. On the other hand, the truncated EGFR polypeptide can be used as a screening marker to improve the labeling efficiency. In addition, CAR-T cells expressing JxM-EGFRt polypeptides have a higher ratio of initial / naive T cells, enhance the in vivo and in vitro survival of target cells, and help improve the anti-tumor effects in vivo and in vitro.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a JxM-EGFRt polypeptide, which includes the extracellular domain III of EGFR, the extracellular domain IV of EGFR, the transmembrane region of EGFR, and the intracellular proximal membrane end, excluding the extracellular ligand binding domain and the intracellular tyrosine kinase domain;

[0008] One or more amino acids are added to the intracellular membrane-proximal end of the JxM-EGFRt polypeptide;

[0009] The plurality includes 2, 3, 4, 5, 10 or 19.

[0010] In some specific embodiments of the present invention, the one amino acid added near the intracellular membrane includes R;

[0011] The two amino acids added near the intracellular membrane include RR;

[0012] The three amino acids added near the intracellular membrane include RRR;

[0013] The four amino acids added near the intracellular membrane include RRRH;

[0014] The five amino acids added near the intracellular membrane include RRRHI;

[0015] The 10 amino acids added near the intracellular membrane include RRRHIVRKRT;

[0016] The 19 amino acids added near the intracellular membrane include RRRHIVRKRTLRRLLQERE.

[0017] In some specific embodiments of the present invention, the JxM-EGFRt polypeptide has:

[0018] (I), an amino acid sequence as shown in SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37, SEQ ID No.38, SEQ ID No.39 or SEQ ID No.40; or

[0019] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0020] (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (I).

[0021] The present invention also provides a mutant of the JxM-EGFRt polypeptide, comprising the JxM-EGFRt polypeptide;

[0022] Any one or more amino acid mutations among the three amino acids added to the intracellular membrane-proximal end of the JxM-EGFRt polypeptide;

[0023] The three amino acids include a first amino acid, a second amino acid and a third amino acid.

[0024] In some specific embodiments of the present invention, the mutation of the first amino acid includes but is not limited to mutation to a basic and polar amino acid, an acidic and polar amino acid or a hydrophilic and polar amino acid; and / or

[0025] The mutation of the second amino acid includes but is not limited to mutation to a hydrophobic and non-polar amino acid, a neutral and polar amino acid, an acidic and polar amino acid or a basic and polar amino acid; and / or

[0026] The mutation of the third amino acid includes, but is not limited to, mutation to a basic and polar amino acid, an acidic and polar amino acid, a hydrophilic and polar amino acid, or a hydrophobic and non-polar amino acid.

[0027] In some specific embodiments of the present invention, the mutation of the first amino acid includes but is not limited to mutation to K, R or H; and / or

[0028] The mutation of the second amino acid includes, but is not limited to, mutation to S, N, C, Q, G, T, Y, A, I, L, M, F, P, W, V, D, E, R, K or H; and / or

[0029] The mutation of the third amino acid includes but is not limited to mutation to K, R or H.

[0030] In some specific embodiments of the present invention, the first amino acid is mutated to K or R;

[0031] The second amino acid is mutated to S, F, D, K or R;

[0032] The third amino acid is mutated to K or R.

[0033] In some specific embodiments of the present invention, the mutant has:

[0034] (IV), an amino acid sequence as shown in SEQ ID No.41, SEQ ID No.42, SEQ ID No.43, SEQ ID No.44, SEQ ID No.45, SEQ ID No.46, SEQ ID No.47, SEQ ID No.48, SEQ ID No.49, SEQ ID No.50, SEQ ID No.51 or SEQ ID No.52; or

[0035] (V) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (IV); or

[0036] (VI) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (IV).

[0037] Based on the above research, the present invention also provides a nucleic acid molecule encoding any of the following:

[0038] (VII), the JxM-EGFRt polypeptide; and / or

[0039] (VIII), the mutant.

[0040] In some specific embodiments of the present invention,

[0041] (1) The nucleic acid molecule of the JxM-EGFRt polypeptide has a nucleotide sequence as shown in SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No.14; or

[0042] (2) The nucleic acid molecule of the mutant has a nucleotide sequence as shown in SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.24, SEQ ID No.25 or SEQ ID No.26; or

[0043] (3) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (1) or (2) but is different from the nucleotide sequence shown in (1) or (2) due to the degeneracy of the genetic code; or

[0044] (4) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in any one of (1) to (3), and having the same or similar functions as the nucleotide sequence shown in any one of (1) to (3); or

[0045] (5) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (1) to (4).

[0046] The present invention also provides a protein combination comprising any of the following and a chimeric antigen receptor:

[0047] (VII), the JxM-EGFRt polypeptide; and / or

[0048] (VIII), the mutant.

[0049] In some specific embodiments of the present invention, the chimeric antigen receptor comprises an antigen recognition domain, a transmembrane domain, an intracellular signaling domain and / or a hinge region;

[0050] The antigen recognition domain includes, but is not limited to, ScFv targeting one or more of CD19, CD20, CD22, CD33, CLL-1, CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY, L1CAM, MAGE, MAGE-A1 or TAG-72;

[0051] The intracellular signaling domain includes any of the following:

[0052] (iii), immunoreceptor tyrosine-based activation motifs; and / or

[0053] (iv), the primary intracellular signaling domain of an ITAM; and / or

[0054] (v), a primary intracellular signaling domain and one or two or more co-stimulatory signaling domains;

[0055] The primary intracellular signaling domain of the ITAM includes but is not limited to CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS, FcεRI, CD66d, DAP10 or DAP12;

[0056] The co-stimulatory signaling domain is selected from one or more of D28, 41BB, OX40, CD27, DAP10, 2B4, CD3γ, CD3δ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80, signaling lymphocyte activation molecule, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE, LFA-1, GITR, BAFFR, LIGHT or HVEM;

[0057] The transmembrane domain is selected from the group consisting of a type I transmembrane protein of a T cell receptor, an alpha, beta or zeta chain of a T cell receptor, CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD357, Toll-like receptor 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, NKp44 or NKp46;

[0058] The hinge region includes, but is not limited to, 8hinge, 8h(dc) hinge or CD8(1) hinge derived from human CD8.

[0059] In some specific embodiments of the present invention, the antigen recognition domain comprises a ScFv targeting CD123, PSCA or CLL-1;

[0060] The transmembrane domain includes CD8TM derived from human CD8 transmembrane or CD28TM derived from CD28 transmembrane;

[0061] The co-stimulatory signal transduction domain is 41BB;

[0062] The primary intracellular signaling domain of the ITAM is CD3ζ or a CD3ζ mutant.

[0063] In some specific embodiments of the present invention,

[0064] (①), the ScFv targeting CD123 and PSCA has the amino acid sequences shown in SEQ ID No. 28 and 61 respectively; or

[0065] (②), the 8hinge, 8h(dc) hinge and CD8(1) hinge have the amino acid sequences shown in SEQ ID No.29, 53 and 58 respectively; or

[0066] (③), the CD8TM and CD28TM have the amino acid sequences shown in SEQ ID No. 30 and 54 respectively; or

[0067] (④), the 4-1BB has an amino acid sequence as shown in SEQ ID No.31 or 56; or

[0068] (⑤), the CD3ζ has the amino acid sequence shown in SEQ ID No.32; or

[0069] (⑥), the CD3ζ mutant has an amino acid sequence as shown in SEQ ID No. 57 or 60; or

[0070] (⑦) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in any one of (①) to (⑥); or

[0071] (⑧) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of (①) to (⑦).

[0072] In some specific embodiments of the present invention, the CD3ζ mutant includes CD3z(3) and / or CD3z(1).

[0073] In some specific embodiments of the present invention, the chimeric antigen receptor is a fusion protein combination of a tumor suppressor molecule, a cytokine, or a T cell activation molecule;

[0074] The molecules that reverse tumor suppressor signals include but are not limited to PD1, CTLA-4, PD-L1, LAG-3, TIM-3, BTLA or CD47;

[0075] The cytokines include but are not limited to membrane-bound IL-15, membrane-bound IL12 or membrane-bound IL21;

[0076] The T cell activation molecules include but are not limited to CD80 or CD86.

[0077] The present invention also provides genetic elements, including the nucleic acid molecules and nucleic acid molecules encoding chimeric antigen receptors.

[0078] In some specific embodiments of the present invention, the chimeric antigen receptor comprises an antigen recognition domain, a transmembrane domain, an intracellular signaling domain and / or a hinge region;

[0079] The antigen recognition domain includes, but is not limited to, ScFv targeting one or more of CD19, CD20, CD22, CD33, CLL-1, CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY, L1CAM, MAGE, MAGE-A1 or TAG-72;

[0080] The intracellular signaling domain includes any of the following:

[0081] (iii), immunoreceptor tyrosine-based activation motifs; and / or

[0082] (iv), the primary intracellular signaling domain of an ITAM; and / or

[0083] (v), a primary intracellular signaling domain and one or two or more co-stimulatory signaling domains;

[0084] The primary intracellular signaling domain of the ITAM includes but is not limited to CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS, FcεRI, CD66d, DAP10 or DAP12;

[0085] The co-stimulatory signaling domain is selected from one or more of D28, 41BB, OX40, CD27, DAP10, 2B4, CD3γ, CD3δ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80, signaling lymphocyte activation molecule, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE, LFA-1, GITR, BAFFR, LIGHT or HVEM;

[0086] The transmembrane domain is selected from the group consisting of a type I transmembrane protein of a T cell receptor, an alpha, beta or zeta chain of a T cell receptor, CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD357, Toll-like receptor 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, NKp44 or NKp46;

[0087] The hinge region includes, but is not limited to, 8hinge, 8h(dc) hinge or CD8(1) hinge derived from human CD8.

[0088] In some specific embodiments of the present invention, the antigen recognition domain comprises a ScFv targeting CD123, PSCA or CLL-1;

[0089] The transmembrane domain includes CD8TM derived from human CD8 transmembrane or CD28TM derived from CD28 transmembrane;

[0090] The co-stimulatory signal transduction domain is 41BB;

[0091] The primary intracellular signaling domain of the ITAM is CD3ζ or a CD3ζ mutant.

[0092] In some specific embodiments of the present invention,

[0093] (①), the ScFv encoding the CD123 and PSCA targets has the nucleotide sequences shown in SEQ ID No. 2 and 70 respectively; or

[0094] (②), the nucleic acid molecules encoding the 8hinge, 8h(dc) hinge and CD8(1) hinge have the nucleotide sequences shown in SEQ ID No. 3, 62 and 67 respectively; or

[0095] (③), the nucleic acid molecules encoding the CD8TM and CD28TM have the nucleotide sequences shown in SEQ ID No. 4 and 63 respectively; or

[0096] (④), the nucleic acid molecule encoding the 41BB has a nucleotide sequence as shown in SEQ ID No. 5 or 65; or

[0097] (⑤), the nucleic acid molecule encoding the CD3ζ has the nucleotide sequence shown in SEQ ID No.6; or

[0098] (⑥), the nucleic acid molecule encoding the CD3ζ mutant has a nucleotide sequence as shown in SEQ ID No.66 or 69; or

[0099] (⑦) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence as shown in any one of (①) to (⑥), and having the same or similar functions as the nucleotide sequence as shown in any one of (①) to (⑥); or

[0100] (⑧) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (①) to (⑦).

[0101] The present invention also provides an expression vector, comprising the gene element.

[0102] The present invention also provides a virus particle, comprising the expression vector.

[0103] The present invention also provides engineered cells expressing any of the following:

[0104] (A), the JxM-EGFRt polypeptide; and / or

[0105] (B), the mutant; and / or

[0106] (C), the nucleic acid molecule; and / or

[0107] (D), the protein combination; and / or

[0108] (E), the genetic element; and / or

[0109] (F), the expression vector; and / or

[0110] (G) The viral particles.

[0111] In some specific embodiments of the present invention, the engineered cells include but are not limited to T cells, NK cells, macrophages, DC cells and related precursor cells, mesenchymal stem cells, human embryonic stem cells and / or iPS-derived stem cells.

[0112] The present invention also provides the use of any of the following as a screening marker:

[0113] (J), the JxM-EGFRt polypeptide; and / or

[0114] (K), the mutant; and / or

[0115] (L), the nucleic acid molecule; and / or

[0116] (M), the protein combination; and / or

[0117] (N), the genetic element; and / or

[0118] (O), the expression vector.

[0119] The present invention also provides screening markers, including any of the following:

[0120] (R), the JxM-EGFRt polypeptide; and / or

[0121] (S), the mutant; and / or

[0122] (T), the nucleic acid molecule; and / or

[0123] (U), the protein combination; and / or

[0124] (V), the genetic element; and / or

[0125] (W), the expression vector.

[0126] The present invention also provides the use of any of the following items in the preparation of a drug for preventing and / or treating tumors:

[0127] 1) the JxM-EGFRt polypeptide; and / or

[0128] 2) the mutant; and / or

[0129] 3) the nucleic acid molecule; and / or

[0130] 4) the protein combination; and / or

[0131] 5) the genetic element; and / or

[0132] 6) the expression vector; and / or

[0133] 7) the virus particles; and / or

[0134] 8) The engineered cells.

[0135] The present invention also provides a drug comprising any of the following items and a pharmaceutically acceptable adjuvant or auxiliary agent:

[0136] A), the JxM-EGFRt polypeptide; and / or

[0137] B), the mutant; and / or

[0138] C), the nucleic acid molecule; and / or

[0139] D), the protein combination; and / or

[0140] E), the genetic element; and / or

[0141] F), the expression vector; and / or

[0142] G), the viral particle; and / or

[0143] H), the engineered cells.

[0144] The present invention also provides a drug combination, comprising the drug and any other effective ingredients.

[0145] The present invention also provides a method for treating tumors, comprising administering the drug or the drug combination to a subject.

[0146] The present invention provides EGFR polypeptides and applications thereof. The beneficial effects of the present invention are: 1) The present invention provides a truncated EGFR novel polypeptide JxM-EGFRt, which has higher expression efficiency and stronger expression abundance compared with traditional EGFRt, and improves its use value as a screening marker; 2) The present invention provides a truncated EGFR novel polypeptide JxM-EGFRt, which has stronger ADCC efficiency and higher safety than the traditional safety switch EGFRt; 3) The CAR structure containing the truncated EGFR novel polypeptide JxM-EGFRt provided by the present invention has a more primitive phenotype, a higher proportion of Tnaive cell subpopulations, and enhances its ability to survive in vivo and in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0148] Figure 1 Shown are wild-type EGFR, traditional EGFRt and novel JxM-EGFRt;

[0149] Figure 2 Shows the sequence comparison of wild-type EGFR, traditional EGFRt and novel JxM-EGFRt;

[0150] Figure 3 The EGFR expression abundance detection in Example 1 is shown;

[0151] Figure 4 The results of CAR-1 and CAR-4 EGFRt positive rate detection in Example 2 are shown;

[0152] Figure 5 The results of the detection of CAR-1 and CAR-4 EGFRt expression abundance in Example 2 are shown;

[0153] Figure 6 The comparison of ADCC efficiency of CAR-1 and CAR-4 at different cetuximab concentrations in Example 3 is shown;

[0154] Figure 7 The comparison of ADCC efficiency of CAR-1 and CAR-4 at different NK / CAR-T cell ratios in Example 3 is shown;

[0155] Figure 8 The comparison of ADCC efficiency between CAR-1 and CAR-4 at different NK / CAR-T cell co-culture times in Example 3 is shown;

[0156] Fig. 9 The CAR-1 and CAR-4 copy number detection results in Example 4 are shown;

[0157] Fig.10 The phenotypes of CAR-1 and CAR-4 CAR-T cells in Example 5 on the 7th day of culture are shown;

[0158] Fig.11 The phenotypes of CAR-1 and CAR-4 CAR-T cells in Example 5 on the 12th day of culture are shown;

[0159] Fig.12 The comparison of the in vitro killing ability of CAR-1 and CAR-4 CAR-T cells in Example 6 is shown;

[0160] Fig.13 The comparison of IFN-γ secretion levels between CAR-1 and CAR-4 CAR-T cells in Example 6 is shown;

[0161] Fig.14 The effect of the replacement / optimization of the intracellular proximal membrane amino acid sequence in Example 8 on the EGFRt expression efficiency of CAR-T cells containing JxM-EGFRt is shown;

[0162] Fig.15 The effect of the replacement / optimization of the intracellular proximal membrane amino acid sequence in Example 8 on the abundance of EGFRt expression in CAR-T cells containing JxM-EGFRt is shown;

[0163] Fig.16 The effect of the replacement / optimization of the intracellular proximal membrane amino acid sequence in Example 8 on the abundance of EGFRt expression in CAR-T cells containing JxM-EGFRt is shown;

[0164] Fig.17 The effect of different numbers of proximal amino acids on the expression abundance of EGFR polypeptide in Example 9 is shown;

[0165] Fig.18 The effect of the replacement / optimization of the intracellular proximal membrane amino acid sequence in Example 8 on the abundance of EGFRt expression in CAR-T cells containing JxM-EGFRt is shown;

[0166] Fig.19The ADCC effect detection of CAR-T cells containing JxM-EGFRt in Example 10 is shown;

[0167] Fig. 20 The ADCC effects of different numbers of intracellular proximal membrane amino acids of JxM-EGFRt in Example 10 are shown;

[0168] Fig.21 The ADCC results are shown in Example 10 when the number of amino acids near the intracellular membrane of JxM-EGFRt is 3, 5 and 10 respectively;

[0169] Fig. 22 The effectiveness of PSCA CAR-T cells constructed with different EGFRt in Example 10 is shown;

[0170] Fig.23A The ADCC effect statistics of CAR-21 and CAR-22 in Example 10 are shown in FIG. 10 and FIG. 11 and FIG. 12 are shown in FIG. 13 and FIG. 14 are shown in FIG. 15;

[0171] Fig. 23B The ADCC effect statistics of CAR-T constructed by CAR-23 and CAR-24 in T cells from donor #526 in Example 10 are shown;

[0172] Fig.23C The ADCC effect statistics of CAR-T constructed by CAR-23 and CAR-24 in T cells derived from #602 donor in Example 10 are shown;

[0173] Fig.24 In Example 11, CAR-23 is used as a screening marker in CAR-T constructed from T cells derived from donor #526. DETAILED DESCRIPTION

[0174] The present invention discloses EGFR polypeptides and their applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0175] The complete EGFR includes four extracellular domains (named I, II, III and IV) and an intracellular tyrosine kinase domain. The N-terminal extracellular domains I and II of EGFR are the binding sites of ligands (EGF and TGF-α). When the ligand binds to this site, the inactive receptor tyrosine kinase monomer can be converted into an activated dimer form. The extracellular domain III contains the binding site of cetuximab and / or other EGFR monoclonal antibodies. The traditional EGFRt (The truncatedEGF receptor, EGFRt) polypeptide contains extracellular domains III and IV, a transmembrane domain (TM), does not contain an extracellular ligand binding domain, and does not contain an intracellular tyrosine kinase domain, and does not transmit other signals to T cells.

[0176] At present, traditional EGFRt contains the extracellular structure of wild-type EGFR and III and IV, as well as the transmembrane region. When clearing the target cells, it is often necessary to give cetuximab treatment multiple times. On the one hand, it reflects the limited clearance ability of traditional EGFRt, including the speed and clearance ratio when clearing cells. It is speculated that the main reason is the low expression efficiency and / or expression abundance of EGFR, especially the expression abundance of EGFRt, which is more critical for antibody-dependent cell-mediated cytotoxicity. The higher the expression efficiency and expression abundance of the truncated EGFR polypeptide, the more significant the ADCC effect. Therefore, we designed a new truncated EGFR polypeptide, which contains the extracellular domains III and IV, the transmembrane domain (TM) and the intracellular juxtamembrane domain (JxM), but does not contain the intracellular tyrosine kinase domain, which improves the expression efficiency and expression abundance of EGFR polypeptide while improving safety.

[0177] The new truncated EGFR peptide we designed contains the extracellular domains III and IV, the transmembrane domain (TM) and the intracellular juxtamembrane domain (JxM), but does not contain the intracellular tyrosine kinase domain and will not transmit other signals to T cells. The truncated EGFR peptide is named JxM-EGFRt because it contains the intracellular juxtamembrane amino acid sequence JxM. Compared with traditional EGFRt, JxM-EGFRt significantly improves the expression ratio and abundance of EGFR peptides. On the one hand, it can significantly improve the ADCC efficiency, quickly shut down the side effects of target cells, and enhance safety; on the other hand, JxM-EGFRt can be used as a cell screening marker to improve the screening efficiency of target cells and better realize the role of cell identification and tracing. In addition, our study also found that CAR-T cells expressing JxM-EGFRt peptides have a higher proportion of initial / naive T cells ( T cells), which can enhance the survival of CAR-T cells.

[0178] This study invented a new truncated EGFR polypeptide, which consists of extracellular domains III and IV, a transmembrane domain (TM) and an intracellular juxtamembrane domain (JxM), but does not contain an extracellular ligand binding domain and an intracellular tyrosine kinase domain, and is named JxM-EGFRt. Compared with traditional EGFRt, JxM-EGFRt improves the expression efficiency and abundance of EGFR polypeptides while improving safety. On the one hand, it enhances ADCC efficiency and has better safety. On the other hand, the truncated EGFR polypeptide can be used as a screening marker to improve the labeling efficiency. In addition, CAR-T cells expressing JxM-EGFRt polypeptides have a higher ratio of initial / naive T cells, which enhances the survival of target cells in vivo and in vitro, and helps to improve the anti-tumor effect in vivo and in vitro.

[0179] Sequences used in the present invention:

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187] Amino Acid Sequence:

[0188]

[0189]

[0190]

[0191]

[0192] BBZ is the abbreviation of 4-1BB-CD3ζ, and BB here refers to 4-1BB cytoplasm.

[0193] CD3ζ mutants include CD3z(3) and CD3z(1).

[0194] The EGFR polypeptide provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0195] The present invention will be further described below in conjunction with embodiments:

[0196] Example 1 Comparison of expression between JxM-EGFRt containing intracellular proximal membrane amino acid sequence and traditional EGFRt

[0197] 1. The traditional EGFRt contains the extracellular domains III and IV of the wild-type EGFR, as well as the transmembrane region. On this basis, we constructed a truncated EGFR peptide containing the intracellular proximal membrane amino acid sequence. The structure is shown in the following table:

[0198] Table 1 Truncated EGFR polypeptide vector information

[0199] Plasmid number Plasmid construction Remark ER-1 pL-GMR-EGFR(335) Traditional EGFRt ER-4 pL-GMR-EGFR(338) JxM-EGFRt

[0200] EGFR (335) is a traditional truncated EGFR commonly used in the field to design a CAR-T "safety switch", that is, a traditional EGFRt; FR (338) is a truncated EGFR polypeptide designed by the present invention comprising an intracellular proximal membrane amino acid sequence, also known as JxM-EGFRt.

[0201] 2. The above-mentioned truncated EGFR polypeptide sequence was obtained from the NCBI website database. The human EGFR extracellular region gene sequence information, NCBI sequence number: NP_005219.2, was obtained by PCR. The following two fragments, EGFR (335) and EGFR (338), were obtained by double digestion with restriction endonucleases NheI (Thermo) and EcoRI (Thermo), and then connected to the lentiviral vector with T4 ligase. After enzyme digestion and sequencing verification, the plasmid was extracted and purified using a large extraction purification kit (large extraction purification kit, manufacturer: Omega, product number: D6926-04).

[0202] 3. The above-mentioned target plasmid completes virus preparation:

[0203] The present embodiment uses the calcium phosphate method to package the lentivirus, referring to the Molecular Cloning Experiment Guide (3rd edition, J. Sambrook et al.), specifically: 293T cells are cultured to a better state with DMEM medium containing 10% FBS (w / v), and the packaging plasmid (RRE:REV:2G) and the expression plasmid are added to a 1.5mL centrifuge tube in a certain ratio, CaCl2 and 2×HBS are added, mixed and allowed to stand at room temperature, and then added to the treated 293T cell culture fluid, and the fluid is changed again to 10mL DMEM medium containing 10% FBS after 3-5h, and the cell supernatant is collected after 48h or 72h. The virus supernatant is collected, centrifuged and filtered, and then transferred to a new centrifuge tube; according to the amount of virus supernatant, PEG6000 and NaCl are used to purify the virus, and the purified virus is resuspended with 200μL DMEM medium containing 10% FBS, and 1.5mL EP tubes are packaged and stored at -80℃ for later use. The virus infected 293T cells, and 72 hours after infection, the genome was extracted using the QIAampDNA Blood Mini Kit (purchased from Qiagen, Cat. No. 511004). The kit instructions were followed. The qRT-PCR was used to determine the virus titer. The data were analyzed using analysis software, and the virus titer was calculated based on the standard curve. The results were expressed in TU / mL. The titer detection is shown in Table 4.

[0204] 4. Lymphocytes were separated by gradient centrifugation to obtain human PBMC cells or cryopreserved PBMCs. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, and then infected with activated PBMCs at 5 MOI (multiplicity of infection, in some embodiments, the multiplicity of infection can be adjusted according to specific experiments, such as 1-10 MOI or even higher). The expression efficiency and abundance of EGFR were detected on the 7th day of virus infection. The detection method was flow cytometry: the virus-infected T cells cultured for 7-10 days were centrifuged at 300 g / min for 5 minutes, and the supernatant was discarded to collect the cells; the cells were resuspended with PBS solution containing 1% fetal bovine serum by volume, and the cell density was adjusted to 1×10 6 / ml; the collected cells were divided into final labeled antibodies, incubated at 4℃ for 30min, washed twice with PBS solution, and detected by flow cytometry. The antibodies were: CD3 PE-Cy7, EGFR-APC, where CD3 represents the proportion of T cells. The expression abundance of the truncated JxM-EGFRt polypeptide ( Figure 3 and Table 2), where #103 and #158 are the donor numbers.

[0205] Table 2 EGFR expression abundance detection

[0206] EGFRtMFI #103 #158 ER-1 54338 51081 ER-4 97524 108645

[0207] Above Figure 3 As can be seen from Table 2, the truncated JxM-EGFRt polypeptide ER-4 has a higher expression level (intensity) in PBMC or.

[0208] Example 2 Detection of expression intensity of JxM-EGFRt containing intracellular proximal membrane amino acid sequence and traditional EGFRt combined with CAR

[0209] CD123(ScFv)-8h-8TM-BBz is a classic CAR structure. Based on this, this study constructed a series of CARs containing different forms of truncated EGFR peptides and optimized intracellular amino acid sequences. The CAR structures are shown in Table 3:

[0210] EGFR (335) is a traditional truncated EGFR commonly used in the field to design a CAR-T "safety switch", that is, a traditional EGFRt; EGFRt (336) is a truncated EGFR polypeptide designed by the present invention comprising one amino acid sequence near the intracellular membrane end; EGFRt (337) is a truncated EGFR polypeptide designed by the present invention comprising two amino acid sequences near the intracellular membrane end; EGFR (338) is a truncated EGFR polypeptide designed by the present invention comprising three amino acid sequences near the intracellular membrane end; EGFRt (339) is a truncated EGFR polypeptide designed by the present invention A truncated EGFR polypeptide comprising 4 amino acid sequences near the intracellular membrane end, EGFRt(340) is a truncated EGFR polypeptide designed by the present invention comprising 5 amino acid sequences near the intracellular membrane end, EGFRt(345) is a truncated EGFR polypeptide designed by the present invention comprising 10 amino acid sequences near the intracellular membrane end, EGFRt(354) is a truncated EGFR polypeptide designed by the present invention comprising 19 amino acid sequences near the intracellular membrane end, and the above EGFRt(336) to EGFRt(354) are all JxM-EGFRt designed by the present invention.

[0211] In addition to selecting the number of intracellular proximal membrane amino acids, the present invention also mutated the three proximal membrane amino acids of EGFR (338) (a truncated EGFR polypeptide comprising three amino acid sequences proximal to the intracellular membrane), forming a series of mutated JxM-EGFRts such as EGFRt (338RSK), (338RFK), EGFRt (338RDR), EGFRt (338RSR), EGFRt (338RKR), EGFRt (338RDK), EGFRt (338RRK), EGFRt (338RKK), EGFRt (338KDK), EGFRt (338KSK), EGFRt (338KRK), and EGFRt (338LYC). The above-mentioned multiple possible JxM-EGFRt and traditional EGFRt were combined with a CAR structure to construct an artificial vector for verifying the function and effectiveness of the EGFRt designed by the present invention. The CAR structure takes ScFv-8h-8TM-BBz as an example, and the specific vector structure is as follows:

[0212] Table 3 Structural information of CD123 CAR containing EGFR intracellular amino acid sequence

[0213]

[0214] Wherein 2A is a connecting peptide, connecting JxM-EGFRt or traditional EGFRt and CAR gene to construct an artificial vector, GMR is a signal peptide gene, and such a signal peptide gene also exists before JxM-EGFRt or traditional EGFRt gene in the above vector to guide the expression of JxM-EGFRt or traditional EGFRt and CAR gene. In some embodiments, the signal peptide can also be derived from CD8α.

[0215] The method of Example 1 was used to prepare a virus containing the above-mentioned JxM-EGFRt or traditional EGFRt and the vector gene of the CAR gene. The titer of the above-mentioned virus is shown in Table 4:

[0216] Table 4 List of CD123 CAR lentivirus titers containing EGFR intracellular amino acid sequences

[0217]

[0218]

[0219] Both the traditional EGFRt and JxM-EGFRt designed viral vectors can complete infection.

[0220] The above-mentioned vector transfection T cells were used to prepare CAR-T cells using the method of Example 1. The CAR-T cells expressed traditional EGFRt or JxM-EGFRt polypeptides and also expressed CAR proteins; the naming of CAR-T cells and the vectors transduced and expressed by them were the same, and the subsequent effectiveness and functional verification of CAR-1, CAR-4, etc. were CAR-T cells expressing CAR-1 and CAR-4 vectors. In CAR-T cells, the CAR structure is the same, and the only difference is the traditional EGFRt or JxM-EGFRt polypeptide, so the function and effectiveness of different CAR-Ts reflect the function and effectiveness of traditional EGFRt or different JxM-EGFRt polypeptides.

[0221] The expression efficiency and abundance of EGFR were detected on the 7th day of virus infection by flow cytometry and the antibodies were: CD3 PE-Cy7, EGFR-APC, where CD3 represented the proportion of T cells.

[0222] The experimental results showed that the expression efficiency of CAR-4 from 9 batches of different donors was better than that of CAR-1, with smaller dispersion, and there was a significant difference, P < 0.01 (P = 0.0012, **). Figure 4 and Table 5) as shown:

[0223] Table 5 CAR-1 and CAR-4EGFRt positive rate detection results

[0224]

[0225] The experimental results showed that the test results of 9 batches of different donors showed that the expression abundance of CAR-4EGFRt was significantly higher than that of CAR-1. The average expression intensity of CAR-4EGFRt was 2.14 times that of CAR-1, ranging from 1.84 to 2.46 times. (Note: #N2 was tested with different instruments, so the fluorescence values ​​were quite different, but the trends were the same). The expression abundance of CAR-4 and CAR-1 is shown in ( Figure 5 and Table 6) as shown:

[0226] Table 6 CAR-1 and CAR-4 EGFRt expression abundance detection results

[0227]

[0228]

[0229] Figure 5 As shown in Table 6, the expression intensity (strength) of the truncated JxM-EGFRt polypeptide designed by the present invention is significantly better than that of the traditional EGFRt, which also suggests that the combined design of the JxM-EGFRt polypeptide and the CAR structure will have a more "sensitive" responsiveness and the combined CAR-T cells will be safer.

[0230] Example 3 Verification of the safety switch function of JxM-EGFRt containing intracellular proximal membrane amino acid sequence and traditional EGFRt

[0231] 1. NK cells (Miltenyi NK isolation kit) were isolated from PBMC as effector cells, co-cultured with CAR-1 or CAR-4 CART at a ratio of 1:1, and cetuximab (Cetuximab, Erbitux) was added or not, with the final concentration of cetuximab being 10 μg / mL and 50 μg / mL, respectively. After 24 hours, CD3 and CAR expression were detected by flow cytometry. ADCC percentage = (1-CAR positive rate in monoclonal antibody group / CAR positive rate without cetuximab) × 100%.

[0232] The results showed that when the concentration of cetuximab was 10μg / mL and 50μg / mL, the effector-target ratio was 1:1, that is, NK / CAR-T cells were 1:1, and the action time was 24h, the ADCC efficiency of CAR-4 cells containing JxM-EGFRt was significantly higher than that of CAR-1 cells containing traditional EGFRt. Figure 6 , Table 7:

[0233] Table 7

[0234] Antibody concentration CAR1 CAR4 Cetuximab 10 μg / mL 39.46％ 61.03％ Cetuximab 50 μg / mL 38.57％ 59.68％

[0235] 2. In addition to the above-mentioned verification of different cetuximab concentrations, the present invention also verifies the ability to "turn off" the function of CAR-T cells at different effector-target ratios: NK cells (Miltenyi NK isolation kit) were isolated from PBMC as effector cells, co-cultured with CAR-1 or CAR-4CART at a ratio of 1:1 or 4:1, with or without the addition of cetuximab (Cetuximab, Erbitux), and the final concentration of cetuximab was 10μg / mL, respectively. After 24 hours, CD3 and CAR expression were detected by flow cytometry. ADCC percentage = (1-CAR positive rate in the monoclonal antibody group / CAR positive rate in the non-antibody group) × 100%. The results showed that when the cetuximab concentration was 10μg / mL, the NK / CAR ratio was 1:1 and 4:1, respectively, and the action time was 24 hours, the ADCC efficiency of CAR-4 cells containing JxM-EGFRt was significantly higher than that of CAR-1 cells containing traditional EGFRt at two effector-target ratios. See the results for details. Figure 7 .

[0236] 3. We further verified the reactivity of JxM-EGFRt peptide and traditional EGFRt peptide in a short period of time after the addition of cetuximab to prove the sensitivity and safety strength of the "safety switch". NK cells (Miltenyi NK isolation kit) were isolated from PBMC as effector cells and co-cultured with CAR-1 or CAR-4CART cells at a ratio of 1:1, with or without the addition of cetuximab (Cetuximab, Erbitux), with a final concentration of cetuximab of 10 μg / mL, and cultured for 6h and 24h, respectively, and CD3 and CAR expression were detected by flow cytometry. ADCC percentage = (1-CAR positivity rate in monoclonal antibody group / CAR positivity rate in non-antibody group) × 100%

[0237] The results showed that the ratio of NK / CAR-T cells was 1:1. In an environment with a cetuximab concentration of 10 μg / mL, the ADCC efficiency of CAR-4 cells containing JxM-EGFRt was significantly higher than that of CAR-1 cells containing traditional EGFRt after 6 hours of action, P = 0.04 (*). Moreover, this difference increased further with the extension of the action time. When the cetuximab concentration was 10 μg / mL and continued to act for 24 hours, the ADCC efficiency of CAR-4 cells was significantly higher than that of CAR-1 cells, P = 0.0052 (**).

[0238] The results are shown in Figure 8 And Table 8, Table 9:

[0239] Table 8 Comparison of ADCC efficiency of CAR-1 and CAR-4 after 6 h of NK / CAR-T cell co-culture

[0240]

[0241] Table 9 Comparison of ADCC efficiency of CAR-1 and CAR-4 after 24h co-culture of NK / CAR-T cells

[0242]

[0243]

[0244] The above results show that the CAR structure containing the JxM-EGFRt design is more sensitive to the ADCC effect caused by cetuximab, and can "turn off" the CAR-T cell response more quickly and effectively when the CAR-T cells react "out of control" in the body, and is a more advantageous "safety switch".

[0245] Example 4 CAR constructed by JxM-EGFRt has higher expression ability

[0246] Gradient centrifugation was used to separate lymphocytes and obtain human PBMC cells. After the obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, the activated PBMCs were infected at 5 MOI (multiplicity of infection, in some embodiments the multiplicity of infection can be adjusted according to the specific experiment, such as 1-10 MOI or even higher), and the expression efficiency of CAR was detected on the 7th day of viral infection. The detection method was RT to detect the copy number, and the detection index was BBz. Specific detection method: Probe working solution: 10 μL probe (100 μM) + 90 μL ultrapure water (can be prepared in proportion according to experimental requirements);

[0247] Primer working solution (mix): 10μL FP+10μL RP+80μL ultrapure water (can be prepared in proportion according to experimental requirements).

[0248] Editing board settings

[0249] The qPCR reaction conditions used in this verification were: 50°C, 2 min, 95°C, 10 min; 40 cycles: 95°C, 15 s, 60°C, 1 min, and the reaction system was 20 μL.

[0250] The results of multiple batches of experiments showed that the CAR copy number of CAR-4 cells containing JxM-EGFRt was significantly higher than that of CAR-1 cells containing traditional EGFRt, P = 0.018 (*). This result shows that JxM-EGFRt CAR-4CAR-4 cells will not weaken the expression of CAR while ensuring higher ADCC efficiency, laying a good foundation for the function of CAR-T cells. Fig. 9 And Table 10:

[0251] Table 10 CAR-1 and CAR-4 copy number detection results

[0252]

[0253] Example 5 Phenotypic distribution of CAR-T cells constructed with different EGFRt

[0254] Gradient centrifugation was used to separate lymphocytes and obtain human PBMC cells. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, and then infected with activated PBMC at 5 MOI (multiplicity of infection). The phenotype of CAR-T cells was detected on the 7th and 12th days of virus infection, respectively. The detection method was flow cytometry, and the antibodies were: CD3 PE-Cy7, PL-PE, CD4-FITC, CD8-BV510, CD45RA-BV421, CD45RO PerCP-Cy5.5, CD197-APC.

[0255] The experimental results showed that when CAR-1 and CAR-4CAR-T cells were cultured to day 7, the proportion of Tnaive cells in CAR-4CAR-T cells was significantly higher than that in CAR-1 cells in the four batches of donors, P = 0.0023 (**) ( Fig.10 In addition, the experimental results also showed that when CAR-1 and CAR-4 CAR-T cells were cultured to day 12, the phenotype of CAR-4 CAR-T cells was still better than that of CAR-1 CAR-T cells, which was specifically reflected in the higher proportion of Tnaive cells ( Fig.11 ).

[0256] Table 11 Phenotypes of CAR-1 and CAR-4 CAR-T cells on day 7 of culture

[0257]

[0258]

[0259] The above results indicate that the CAR constructed by the EGFRt JxM-EGFRt designed in the present invention is beneficial to the maintenance of the CAR-T Tnaive phenotype and can have better efficacy at low-dose CAR-T.

[0260] Example 6 Validation of the effectiveness of CAR-T cells constructed with different EGFRt

[0261] CD123+MV-4-11-Luc-GFP, THP-1-Luc-GFP, MOLM-13-Luc-GFP cells (target cells are derived from the corresponding cells purchased from the ADCC official website) are used as positive target cells, and Raji-Luc-GFP is used as negative target cells. CAR-T cells are spread on target cells at a ratio of 1:1, and killing is detected by luciferase after 24 hours. Luciferase principle: During the detection, after the target cells are lysed with lysis buffer, the luciferase in them will decompose the substrate to emit fluorescence. The formula for analyzing the results using fluorescence value is: CAR-T cell killing rate = 1-(fluorescence value of experimental group ÷ fluorescence value of blank control group) × 100%.

[0262] The results of multiple batches of experiments showed that the killing ability of CAR-4CAR-T cells against three positive target cells MV-4-11-Luc-GFP, THP-1-Luc-GFP, MOLM-13-Luc-GFP and negative target cells was comparable to that of CAR-1CAR-T cells, with no significant difference between the two. This result shows that CAR-4 cells improve ADCC efficiency without affecting the killing function of CAR-T cells. Fig.12 :

[0263] THP-1-Luc-GFP cells were used as positive target cells. CAR-T cells were plated on target cells at a ratio of 1:1. Cell supernatants were collected 24 hours after killing to detect the IFN-γ secretion ability of CAR-T cells after stimulation by target cells. The collected supernatant was used to detect the secretion of IFN-γ by ELISA (enzyme-linked immunosorbent assay), where IFN-γ was purchased from BD Company, catalog number: 555142. The specific detection method was operated according to the instructions.

[0264] The results of multiple batches of experiments showed that the level of IFN-γ secreted by CAR-4CAR-T cells after killing positive target cells THP1 E0K0F1 was comparable to that of CAR-1CAR-T cells, with no significant difference between the two ( Fig.13 ).

[0265] Example 7 Comparison of expression efficiency, expression abundance and ADCC efficiency of JxM-EGFRt-CAR-T cells with other different targets and traditional EGFRt-CAR-T cells

[0266] The present invention also proves in Example 1 that the JxM-EGFRt peptide designed by the present invention can be expressed independently and has a higher expression ratio and expression abundance (intensity) than traditional EGFRt. As a "safety switch" of the CAR structure, although the above embodiments all use the CAR structure of the CD123 target to verify the effectiveness and sensitivity of JxM-EGFRt as a "safety switch", those skilled in the art should also know that the JxM-EGFRt peptide designed by the present invention is suitable for CAR structures of any target, including but not limited to: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM 5. One or more of CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma associated antigen 1) or TAG-72.

[0267] In addition to the hinge structure and transmembrane structure derived from human CD8, the intracellular co-stimulatory signal derived from CD137 (41BB), and the primary cytoplasmic signaling sequence CD3ζ, 8h-8TM-BBz were used in the above examples.

[0268] The above-mentioned CAR structure, "chimeric antigen receptor" or "CAR" comprises: an antigen recognition domain (such as ScFv), a hinge structure (such as 8hinge derived from human CD8), a transmembrane structure (such as CD8TM derived from human CD8 transmembrane) and an intracellular signaling domain, wherein the intracellular signaling domain can be a primary intracellular signaling domain containing only an immunoreceptor tyrosine-based activation motif or ITAM: Examples of primary cytoplasmic signaling sequences containing ITAM include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS (CD278 ), FcεRI, CD66d, DAP10 and DAP12, etc., and may also contain one or two or more co-stimulatory signaling domains in addition to the primary intracellular signaling domain, and the co-stimulatory signaling domain may be selected from any one or more of the following molecules and their derived functional variants: CD28, 41BB, OX40, CD27, DAP10, 2B4 (SLAMF4, CD244), CD3γ, CD3δ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, S LAMF7, NKp80 (KLRF1), signaling lymphocyte activation molecule (SLAM protein), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, LFA-1 (CD11a / CD18), GITR, BAFFR, LIGHT, HVEM (LIGHTR), etc. The transmembrane region (TM) connects the extracellular domain of CAR and the intracellular activation signal domain. The transmembrane region in the present invention can be selected from the type I transmembrane protein of T cell receptor. one of the proteins, α, β or ζ chain of the T cell receptor, CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9, NKp44 or NKp46.The chimeric antigen receptor can also be combined with fusion proteins that reverse tumor suppressor molecules, cytokines, T cell activation molecules, etc. The molecules that reverse tumor suppressor signals can be fusion proteins / polypeptides of any structure such as PD1, CTLA-4, PD-L1, LAG-3, TIM-3, BTLA, CD47, etc. Cytokines can be membrane-bound IL-15, membrane-bound IL12, membrane-bound IL21, etc.; T cell activation molecules can be CD80, CD86, etc. The chimeric antigen receptor can be applicable to a variety of structures.

[0269] The JxM-EGFRt and CAR structure designed by the present invention can be designed as

[0270] In addition to expressing an expression vector such as EGFRt(338)-CD123(ScFv)-8h-8TM-BBz on the surface of T cells, the JxM-EGFRt peptide and CAR structure can also be designed into vectors to prepare viruses for T cell transfection.

[0271] Example 8 Effect of intracellular proximal membrane amino acid sequence substitution / optimization on EGFRt expression efficiency and abundance in CAR-T cells containing JxM-EGFRt

[0272] The experimental results of Example 2 and Example 3 show that CAR-4 cells containing JxM-EGFRt have significant advantages in the expression efficiency and abundance of EGFR polypeptides, and the ADCC efficiency is also significantly higher than that of CAR-1 cells containing traditional EGFRt. Compared with CAR-1, the difference between CAR-4 and CAR-1 is only the difference in the three amino acids near the intracellular membrane. This study prompts us to think about the effect of replacement or optimization of the amino acid sequence near the intracellular membrane on the expression efficiency and abundance of EGFR polypeptides. In order to further explore the effect of replacement or optimization of the amino acid sequence near the intracellular membrane on the expression efficiency and abundance of EGFR polypeptides, we designed a series of sequence optimization or replacement of the three amino acids near the intracellular membrane. The CAR plasmids were named CAR-9-CAR-20, among which CAR-4 added the near-membrane amino acid sequence RRR on the basis of traditional EGFRt (EGFRt used by CAR1). See Tables 3 and 4 for relevant structure and virus information.

[0273] Gradient centrifugation was used to separate lymphocytes and obtain human PBMC cells. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, and then infected with activated PBMC at 5 MOI (multiplicity of infection). The expression efficiency and abundance of EGFR were detected on the 7th day of virus infection. The detection method was flow cytometry, and the antibodies were: CD3 PE-Cy7, EGFR-APC, where CD3 represented the proportion of T cells.

[0274] The results in Table 4 show that CAR9-CAR-20 can successfully prepare viruses, and the prepared viruses have the ability to infect target cells, indicating that the CAR structure after the replacement or optimization of the three amino acid sequences near the intracellular membrane can be normally expressed on the surface of immune cells. When the multiplicity of infection reaches a certain value, the expression efficiency of the truncated EGFR polypeptide is high enough, and there is little difference between the groups ( Fig.14 , Table 13). At this time, the expression abundance of EGFR polypeptide has a greater impact on ADCC efficiency. Therefore, this study focuses on the effect of the three amino acid sequences near the intracellular membrane on the expression abundance of EGFR ( Figure 15-16 , Tables 14-15).

[0275] According to the properties of amino acids, optimization or replacement tests were conducted on the three amino acid sequences near the membrane, and a total of 12 CAR plasmids were designed. The results showed that the amino acid sequence near the intracellular membrane was optimized or replaced by RSK, RFK, RDR, RSR, RKR, RDK, RRK, RKK, KDK, KSK, and KRK, which could significantly increase the expression abundance of EGFR. When RRR was optimized or replaced by LYC, it had an inhibitory effect on the expression abundance of EGFR polypeptide. The table corresponding to the properties of amino acids is shown in Table 12:

[0276] Table 12 Optimization of intracellular membrane-proximal amino acid sequence and effect test

[0277]

[0278] According to the acidity and alkalinity and hydrophilicity of amino acids, the first amino acid is preferably a basic / polar amino acid, such as K, R, and H, and the rest can also be acidic / polar amino acids. Hydrophilic / polar amino acids can achieve the purpose of increasing the abundance of EGFRt expression and their combinations, and the use of hydrophobic / non-polar amino acids has a poor effect; the second amino acid is a hydrophilic / polar amino acid, such as S, N, C, Q, G, T, and Y; the second amino acid can also be a hydrophobic / non-polar amino acid, such as A, I, L, M, F, P, W, and V; the second amino acid can also be an acidic / polar amino acid, such as D and E; the second amino acid can also be a basic / polar amino acid, such as R, K, and H; the third amino acid is preferably a basic / polar amino acid, such as K, R, and H, and the rest can also be selected from acidic / polar amino acids, hydrophilic / polar amino acids, and other hydrophobic / non-polar amino acids that can achieve the purpose of increasing the abundance of EGFRt expression and their combinations.

[0279] Table 13

[0280] Carrier structure EGFRt expression efficiency % CAR-1 81.21 CAR-4 90.74 CAR-9 83.91 CAR-10 89.66 CAR-11 90.43 CAR-12 86.18 CAR-13 91.28 CAR-14 88.97 CAR-15 90.13 CAR-16 90.15 CAR-17 84.68 CAR-18 88.98 CAR-19 86.48 CAR-20 84.13

[0281] Table 14

[0282]

[0283]

[0284] Table 15

[0285] Carrier structure EGFRt expression abundance Enhancement percentage compared with CAR1 CAR-1 65477 —— CAR-4 95966 46.56 CAR-9 126395 93.04 CAR-10 143454 119.09 CAR-11 121404 85.41 CAR-12 133158 103.37 CAR-13 144887 121.28 CAR-14 135621 107.13 CAR-15 170158 159.87 CAR-16 149801 128.78 CAR-17 139390 112.88 CAR-18 127038 94.02 CAR-19 115006 46.56 CAR-20 41581 Lower than CAR-1

[0286] The intracellular membrane-proximal amino acid sequence was replaced / optimized. According to the properties of the amino acids, CAR-1, CAR-4, CAR-9, CAR-10, CAR-12, CAR-14, CAR-15, CAR-16, and CAR-17 were randomly selected for multiple batches of validation and statistical analysis. The results are shown in the figure. Fig.18 As shown in Table 16, CAR-1 and CAR-4 have extremely significant differences, P < 0.0001 (****); in addition, CAR-1 has significant differences with CAR-9, CAR-10, CAR-12, CAR-14 and CAR-16, P < 0.05 (*). CAR-15 and CAR-17 have a significant effect on the abundance of EGFRt expression in the same batch of donors. Due to individual differences in donors, there is no significant difference in the results of these 5 batches, but there is a clear trend of enhancement.

[0287] Table 16 Effect of substitution / optimization of intracellular proximal membrane amino acid sequence on EGFRt expression abundance in CAR-T cells containing JxM-EGFRt

[0288] CAR structure EGFRtMFI (mean±SD) N (number of donors) Compared with CAR-1, P value Significant difference CAR-1 46955.538±24788.689 13 / / CAR-4 85646.286±36224.828 14 ＜0.0001 P＜0.0001，**** CAR-9 119071±52558.835 6 0.0165 P＜0.05，* CAR-10 122070±54184.832 6 0.038 P＜0.05，* CAR-12 99222.6±39081.212 5 0.0461 P＜0.05，* CAR-14 111198.2±48022.842 5 0.0348 P＜0.05，* CAR-15 123160.4±58191.153 5 0.0586 / CAR-16 119951.6±49849.285 5 0.0364 P＜0.05，* CAR-17 101157.8±32329.684 5 0.0785 /

[0289] The above results showed that the EGFRt expression abundance of CAR-4, CAR-9, CAR-10, CAR-12, CAR-14, CAR-15, CAR-16, and CAR-17 cells containing JxM-EGFRt peptide was significantly higher than that of CAR-1 cells containing traditional control peptide EGFRt, further verifying the sensitivity and safety strength of the intracellular proximal membrane amino acid sequence as a "safety switch" after replacement / optimization. The above 9 CARs belong to 5 categories respectively, among which CAR-1 does not contain the intracellular membrane-proximal amino acid sequence, CAR-4, CAR-15 and CAR-16 represent the three intracellular amino acids with properties of basic / polar-basic / polar-basic / polar, CAR-9 represents the three intracellular amino acids with properties of basic / polar-neutral / polar-basic / polar, CAR-10 represents the three intracellular amino acids with properties of basic / polar-hydrophobic / non-polar-basic / polar, and CAR-14 and CAR-17 represent the three intracellular amino acids with properties of basic / polar-acidic / polar-basic / polar.

[0290] Example 9 Effects of different numbers of proximal amino acids on the expression abundance of EGFR polypeptides

[0291] Gradient centrifugation was used to separate lymphocytes and obtain human PBMC cells. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, and then infected with activated PBMC at 5 MOI (multiplicity of infection). The expression efficiency and abundance of EGFR were detected on the 7th day of virus infection. The detection method was flow cytometry, and the antibodies were: CD3 PE-Cy7, EGFR-APC, where CD3 represented the proportion of T cells.

[0292] This study first verified the effects of 1, 2, and 3 intracellular amino acids near the membrane on the abundance of EGFRt expression in donor #406. The results showed that containing 3 amino acids near the membrane had the most significant effect on increasing the abundance of EGFRt expression. Subsequently, the range of intracellular amino acids was further expanded, and the number of intracellular amino acids near the membrane was set to 1, 2, 3, 4, 5, 10, and 19, respectively, among which the intracellular amino acids did not contain receptor tyrosine kinase activity (the maximum number of intracellular amino acids was 19). The experimental results of different donor batches #406, #N2, #820, #755, and #364 showed that the number of amino acids contained in the cell can be 1, 2, 3, 4, 5, and 10, which all have a significant effect on increasing the abundance of EGFRt expression. Only when the number of intracellular amino acids is 19, the expression abundance of EGFRt is inhibited ( Fig.17 , Table 17).

[0293] Table 17 Effect of different numbers of amino acids near the membrane end on the expression abundance of EGFR polypeptide

[0294]

[0295] Example 10: Detection of ADCC effect of CAR-T cells containing JxM-EGFRt

[0296] NK cells (Miltenyi NK isolation kit) were isolated from PBMC as effector cells and co-cultured with CAR-1, CAR-4, CAR-9, CAR-10, CAR-12, CAR-14, CAR-15, CAR-16 or CAR-17 cells at a ratio of 1:1, with or without cetuximab (Cetuximab, Erbitux), with a final concentration of 10ug / mL, and co-cultured for 24h, and CD3 and CAR expression were detected by flow cytometry. ADCC percentage = (1-CAR positive rate in monoclonal antibody group / CAR positive rate in non-antibody group) × 100%. The results are shown in Fig.19 As shown in Table 18:

[0297] Table 18 Effect of substitution / optimization of intracellular proximal membrane amino acid sequence on ADCC efficiency of CAR-T cells containing JxM-EGFRt

[0298] CAR structure ADCC efficiency (%) Enhancement percentage compared with CAR1 CAR-1 13.37 —— CAR-4 32.06 139.80 CAR-9 32.48 142.92 CAR-10 19.61 46.66 CAR-12 29.44 120.19 CAR-14 33.27 148.83 CAR-15 58.26 335.78 CAR-16 37.47 180.25 CAR-17 26.81 100.51

[0299] The results showed that the ratio of NK / CAR-T cells was 1:1. In an environment with a cetuximab concentration of 10ug / ml, after 24 hours of action, the ADCC efficiency of CAR-4, CAR-9, CAR-10, CAR-12, CAR-14, CAR-15, CAR-16, and CAR-17 cells containing JxM-EGFRt was higher than that of CAR-1 cells containing traditional EGFRt peptides. The four types of CAR structures with different amino acid properties had consistent ADCC efficiency.

[0300] The sensitivity and safety strength of the "safety switch" were further verified when the number of intracellular proximal membrane amino acids was 3 and 10, respectively. NK cells (Miltenyi NK isolation kit) were isolated from PBMC as effector cells and co-cultured with CAR-1, CAR-4 or CAR-7 cells at an effector-target ratio of 1:1, with or without cetuximab (Cetuximab, Erbitux), with a final concentration of cetuximab of 10ug / mL. The cells were co-cultured for 24 hours, and CD3 and CAR expression were detected by flow cytometry. ADCC percentage = (1-CAR positivity rate in the monoclonal antibody group / CAR positivity rate in the non-antibody group) × 100%. The results are shown in Fig. 20 As shown in Table 19:

[0301] Table 19 ADCC effect when the number of amino acids near the intracellular membrane of JxM-EGFRt is 3 and 10 respectively

[0302]

[0303] The results showed that the ratio of NK / CAR-T cells was 1:1. In an environment with a cetuximab concentration of 10ug / ml, after 24 hours of action, the average fluorescence intensity of EGFRt in CAR-4 cells containing 3 amino acids near the intracellular membrane and CAR-7 cells containing 10 amino acids near the intracellular membrane were stronger than that in CAR-1, with increases of 48.24% and 90.96%, respectively. In addition, the ADCC efficiency was also significantly higher than that in CAR-1 cells, with increases of 55.02% and 111.98%, respectively.

[0304] In another independent donor, the sensitivity and safety strength of the "safety switch" were further verified when the number of intracellular proximal membrane amino acids was 3, 5, and 10, respectively. NK cells (Miltenyi NK isolation kit) were isolated from PBMC as effector cells and co-cultured with CAR-1, CAR-4, CAR-6, or CAR-7 cells at a ratio of 1:1, with or without cetuximab (Cetuximab, Erbitux), with a final concentration of 10ug / mL. The cells were co-cultured for 24 hours, and CD3 and CAR expression were detected by flow cytometry. ADCC percentage = (1-CAR positive rate in the monoclonal antibody group / CAR positive rate in the non-antibody group) × 100%. The results are shown in Fig.21 As shown in Table 20:

[0305] Table 20 ADCC effect when the number of intracellular proximal amino acids of JxM-EGFRt is 3, 5 and 10 respectively

[0306]

[0307] The results showed that the ratio of NK / CAR-T cells was 1:1. In an environment with a cetuximab concentration of 10ug / ml, after 24 hours of action, the average fluorescence intensity of EGFRt in CAR-4 cells containing 3 amino acids near the intracellular membrane, CAR-6 cells containing 5 amino acids near the intracellular membrane, and CAR-7 cells containing 10 amino acids near the intracellular membrane were all stronger than that in CAR-1, with increases of 73.36%, 136.41%, and 136.09%, respectively. In addition, the ADCC efficiency was also significantly higher than that in CAR-1 cells, with increases of 139.80%, 231.59%, and 309.26%, respectively.

[0308] The JxM-EGFRt of the present invention can be combined with any target and any structure of CAR and expressed in immune cells. In addition to the CAR structure of the CD123 target in the above embodiment, in this section, the inventors also verified the effectiveness of the combined application of JxM-EGFRt with different structures (ScFv-8h(dc)-28TM-28(1)BB(2)Z(3), ScFv-8h(1)-28TM-28z(1)) of CAR targeting PSCA and CLL-1 for different indications of solid tumors and hematological malignancies, including pancreatic cancer, prostate cancer, bladder cancer, acute myeloid leukemia. The carrier structure of JxM-EGFRt combined with different CAR structures is shown in Table 21 below:

[0309] Table 21

[0310] serial number structure EGFRt sequence CAR-21 EGFRt(335)-2A-ScFv(PSCA)-8h(dc)-28TM-28(1)BB(2)Z(3) ER-1 CAR-22 EGFRt(338)-2A-ScFv(PSCA)-8h(dc)-28TM-28(1)BB(2)Z(3) ER-4 CAR-23 EGFRt(335)-2A-ScFv(CLL-1)-8h(1)-28TM-28z(1) ER-1 CAR-24 EGFRt(338)-2A-ScFv(CLL-1)-8h(1)-28TM-28z(1) ER-4

[0311] Viral particles containing the above-mentioned vector were prepared according to the scheme shown in Example 1, and the virus titer was detected, as shown in Table 22 below:

[0312] Table 22

[0313] serial number structure Virus titer (Tu / ml) CAR-21 EGFRt(335)-2A-ScFv(PSCA)-8h(dc)-28TM-28(1)BB(2)Z(3) 7.02E+08 CAR-22 EGFRt(338)-2A-ScFv(PSCA)-8h(dc)-28TM-28(1)BB(2)Z(3) 5.52E+08 CAR-23 EGFRt(335)-2A-ScFv(CLL-1)-8h(1)-28TM-28z(1) 6.03E+08 CAR-24 EGFRt(338)-2A-ScFv(CLL-1)-8h(1)-28TM-28z(1) 7.08E+08

[0314] The results show that any vector constructed by combining JxM-EGFRt with CARs of different targets and structures described in the present invention can be normally prepared for virus and can achieve normal expression. JxM-EGFRt described in the present invention can be applied to any CAR structure of any target.

[0315] Verify the effectiveness of PSCA CAR-T cells constructed with different EGFRt: Gradient centrifugation was used to separate lymphocytes and obtain human PBMC cells. After the obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, the activated PBMCs were infected with the above-prepared virus particles at 5MOI (multiplicity of infection) to prepare CAR-T cells. PC-3-PSCA-Luc-GFP (numbered PC-3E7K0F1) (target cells were derived from the corresponding cells purchased from the ADCC official website) was used as the positive target cell. CAR-T cells were plated on the target cells at a ratio of 1:1, and killing was detected by luciferase after 24 hours. Luciferase principle: During the detection, the target cells were lysed with lysis buffer, and the luciferase therein would decompose the substrate to emit fluorescence. The fluorescence value analysis result formula is: CAR-T cell killing rate = 1-(fluorescence value of the experimental group ÷ fluorescence value of the blank control group) × 100%. The results are as follows Fig. 22 and 23:

[0316] Table 23 Effectiveness of PSCA CAR-T cells constructed with different EGFRt

[0317]

[0318] The results of three batches of experiments showed that CAR-22CAR-T cells had stronger killing ability against positive target cells PC-3-PSCA-Luc-GFP (numbered PC-3E7K0F1) than CAR-21CAR-T cells, and there was a significant difference between the two, P=0.0245(*).

[0319] Contains validation of ADCC effects of CAR-T cells with different EGFRt, different CAR structures, and different targets:

[0320] The sensitivity and safety strength of PSCA CAR-T cells containing intracellular proximal amino acid sequences as a "safety switch" were further verified. NK cells (Miltenyi NK isolation kit) were isolated from PBMC as effector cells, and co-cultured with CAR-21, CAR-22, CAR-23, and CAR-24 cells at a ratio of 1:1, with or without cetuximab (Cetuximab, Erbitux), with a final concentration of 10ug / mL, and co-cultured for 24h, respectively, and CD3 and CAR expression were detected by flow cytometry. ADCC percentage = (1-CAR positive rate in monoclonal antibody group / CAR positive rate in non-antibody group) × 100%. The results are shown in Tables 24, 25 and Figure 23:

[0321] Table 24 CAR-21, CAR-22 ADCC effects

[0322]

[0323] Table 25 CAR-23, CAR-24 ADCC Effect

[0324]

[0325] Fig.23A Statistical graph of ADCC effect of CAR-T constructed by CAR-21 and CAR-22 in T cells from three different donors: #526, #602, and #755; Fig. 23B Statistical graph of ADCC effect of CAR-T constructed by CAR-23 and CAR-24 in T cells derived from donor #526; Fig.23C The ADCC effect statistics of CAR-T constructed by CAR-23 and CAR-24 in T cells from donor #602. Fig.23A As shown in Table 24: After CAR-T cells containing different lengths of EGFRt (335), EGFRt (338) and targeted PSCACAR were exposed to cetuximab, the EGFRt containing different lengths were able to play the role of a "safety switch", initiate the ADCC effect, and prompt NK cells to kill CAR-T cells, among which the ADCC efficiency induced by EGFRt (338) was significantly higher than that induced by EGFRt (335), and the ADCC effect induced by EGFRt (338) was enhanced by 97.25% compared with EGFRt (335), among which EGFRt (335) was the traditional EGFRt.

[0326] like Fig. 23B , Fig.23CAs shown in Table 25, after CAR-T cells containing different lengths of EGFRt (335), EGFRt (338) and CLL-1 targeting CAR were exposed to cetuximab, the EGFRt of different lengths were able to play the role of a "safety switch", initiate the ADCC effect, and prompt NK cells to kill CAR-T cells. Among them, the ADCC efficiency induced by EGFRt (338) was significantly higher than that induced by EGFRt (335). The ADCC effect induced by EGFRt (338) was enhanced by 73.88% compared with that of EGFRt (335), among which EGFRt (335) was the traditional EGFRt.

[0327] From the above Figure 23 and Tables 24 and 25, it can be seen that the ability of the JxM-EGFRt EGFRt (338) designed by us to induce ADCC is significantly better than that of the traditional EGFRt EGFRt (335), and the combination of CAR molecules with different targets and different CAR structural elements shows a better ADCC induction effect than the traditional EGFRt. In summary, the JxM-EGFRt designed by the present invention has the ability to construct safe and controllable CAR-T cells in combination with CAR molecules of any target and any CAR structural element.

[0328] Example 11: CAR-T cells containing JxM-EGFRt as a screening marker

[0329] In order to further prove that JxM-EGFRt can be used as a cell screening marker, improve the screening efficiency of target cells, and better realize the role of cell identification and tracing. In this embodiment, a randomly selected CAR-23 is used as an example, and lymphocytes are separated by gradient centrifugation to obtain human PBMC cells. Three batches of PBMC cells obtained, #526, #602 and #755, are selected, and after 24 hours of activation with anti-CD3 and CD28 monoclonal antibodies, CAR-23 is infected with activated PBMC at 5MOI (multiplicity of infection), and the EGFRt expression and CAR positive expression of CAR-23CAR-T cells are detected on the 7th day of viral infection. The detection method is flow cytometry, and the antibodies are: CD3 PE-Cy7, PL-PE, EGFR-APC.

[0330] The experimental results showed that when CAR-23CAR-T cells were cultured to the 7th day, in the three batches of donors, the CAR positivity rate of CAR-23CAR-T cells and the EGFRt expression positivity rate were highly consistent, and co-positivity was completely achieved, indicating that the JxM-EGFRt described in the present invention can be applied to the screening marker of any CAR structure of any target.

[0331] The results are shown in Table 26 and Fig.24 As shown:

[0332] Table 26 CAR-23JxM-EGFRt as a screening marker

[0333] White film batch CD3+(%) PL+(%) EGFR+(%) PL+EGFR+(%) #526 99.42 85.57 87.38 84.96 #602 99.37 79.44 81.02 78.69 #755 98.20 83.87 85.55 82.75

[0334] Although the targets of the CAR-T used here are CD123, PSCA, and CLL-1, and the CAR structures used are scFv-8h-8TM-BBz, ScFv-8h(dc)-28TM-28(1)BB(2)Z(3), and ScFv-8h(1)-28TM-28z(1) structures for verification, based on the above results, the contents disclosed in the present invention, and the understanding of CAR-T and safety switches by those skilled in the art, it can be known that CAR structures designed based on any other targets such as PSMA, IL13Rα2, EGFRvIII, EGFR, EPCAM, GD2, MUC1, HER2, GPC3, Meso (MSLN), CD133, NKG2D, CD138, LeY, kappa light chain, ROR1, CD70, BCMA, CD20, CD22, PSCA, CLL-1, CD70, CD47, Claudin, ERBB, etc. The CAR structure can be a dual-target CAR structure design, such as CAR1-2A-CAR2 tandem or other C involved in patents 202010254194.3, 202010254435.4, 202010254451.3, 202010257762.5, 202010259458.4, 202010260796.X, 202010260805.5, 202010260849.8, etc. AR structure; it can also be a CAR structure that can be induced to activate by antibodies or small molecule reagents; it can also be a CAR structure containing a hypoxia promoter; it can also be a scFv-8h-28TM-28z structure or a third-generation CAR structure such as: scFv-8h-28TM-28BBz and all possible CAR structure designs can be combined with the JxM-EGFRt described in the present invention as a safety switch for the design / application of engineered immune cells or compositions for the treatment of malignant tumors.

[0335] In addition to designing the JxM-EGFRt and CAR disclosed in the present invention in one expression vector and expressing them in T cells through connecting peptides such as 2A, the JxM-EGFRt and CAR genes disclosed in the present invention can also be expressed in two expression vectors respectively. This expression method can obtain the same effect as the examples of the present application.

[0336] It should be noted that although the present invention takes T cells and CAR-T cells as examples, based on the content disclosed in the present invention, a person skilled in the art can obviously infer that the JxM-EGFRt of the present invention can also be applied to other immune cells such as NK cells, macrophages, DC cells and related precursor cells. The JxM-EGFRt of the present application can be expressed as a safety switch peptide in T cells, NK cells, macrophages, DC cells and related precursor cells, mesenchymal stem cells, human embryonic stem cells, and stem cells derived from iPS; it can be co-expressed with CAR molecules of any structure in T cells, NK cells, macrophages, DC cells and related precursor cells, mesenchymal stem cells, human embryonic stem cells, and stem cells derived from iPS, or the JxM-EGFRt of the present application can be expressed as a safety switch peptide in immune cells such as CAR-T, TCR-T, CAR-NK, CAR-M, CAR-DC, CAR-B, and mesenchymal stem cells, human embryonic stem cells, and stem cells derived from iPS, to enhance the efficacy and safety of the above cells.

[0337] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A JxM-EGFRt polypeptide, characterized in that It includes the extracellular domain III of EGFR, the extracellular domain IV of EGFR, the transmembrane region of EGFR, and the intracellular proximal membrane end, excluding the extracellular ligand binding domain and the intracellular tyrosine kinase domain; One or more amino acids are added to the intracellular membrane-proximal end of the JxM-EGFRt polypeptide; The plurality includes 2, 3, 4, 5, 10 or 19.

2. The JxM-EGFRt polypeptide according to claim 1, characterized in that The one amino acid added near the intracellular membrane includes R; The two amino acids added near the intracellular membrane include RR; The three amino acids added near the intracellular membrane include RRR; The four amino acids added near the intracellular membrane include RRRH; The five amino acids added near the intracellular membrane include RRRHI; The 10 amino acids added near the intracellular membrane include RRRHIVRKRT; The 19 amino acids added near the intracellular membrane include RRRHIVRKRTLRRLLQERE.

3. The JxM-EGFRt polypeptide according to claim 1 or 2, characterized in that It has: (I), an amino acid sequence as shown in SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37, SEQ ID No.38, SEQ ID No.39 or SEQ ID No.40; or (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (I).

4. A mutant of the JxM-EGFRt polypeptide, characterized in that Comprising the JxM-EGFRt polypeptide according to any one of claims 1 to 3; Any one or more amino acid mutations among the three amino acids added to the intracellular membrane-proximal end of the JxM-EGFRt polypeptide; The three amino acids include a first amino acid, a second amino acid and a third amino acid.

5. The mutant according to claim 4, characterized in that The mutation of the first amino acid includes but is not limited to mutation to a basic and polar amino acid, an acidic and polar amino acid or a hydrophilic and polar amino acid; and / or The mutation of the second amino acid includes but is not limited to mutation to a hydrophobic and non-polar amino acid, a neutral and polar amino acid, an acidic and polar amino acid, or a basic and polar amino acid; and / or The mutation of the third amino acid includes, but is not limited to, mutation to a basic and polar amino acid, an acidic and polar amino acid, a hydrophilic and polar amino acid, or a hydrophobic and non-polar amino acid.

6. The mutant according to claim 4 or 5, characterized in that The mutation of the first amino acid includes but is not limited to mutation to K, R or H; and / or The mutation of the second amino acid includes, but is not limited to, mutation to S, N, C, Q, G, T, Y, A, I, L, M, F, P, W, V, D, E, R, K or H; and / or The mutation of the third amino acid includes but is not limited to mutation to K, R or H.

7. The mutant according to any one of claims 4 to 6, characterized in that It has: (IV), an amino acid sequence as shown in SEQ ID No.41, SEQ ID No.42, SEQ ID No.43, SEQ ID No.44, SEQ ID No.45, SEQ ID No.46, SEQ ID No.47, SEQ ID No.48, SEQ ID No.49, SEQ ID No.50, SEQ ID No.51 or SEQ ID No.52; or (V) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (IV); or (VI) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (IV).

8. A nucleic acid molecule encoding any of the following: (VII), the JxM-EGFRt polypeptide according to any one of claims 1 to 3; and / or (VIII) The mutant according to any one of claims 4 to 7.

9. The nucleic acid molecule according to claim 8, characterized in that (1) The nucleic acid molecule of the JxM-EGFRt polypeptide has a nucleotide sequence as shown in SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No.14; or (2) The nucleic acid molecule of the mutant has a nucleotide sequence as shown in SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.24, SEQ ID No.25 or SEQ ID No.26; or (3) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (1) or (2) but is different from the nucleotide sequence shown in (1) or (2) due to the degeneracy of the genetic code; or (4) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in any one of (1) to (3), and having the same or similar functions as the nucleotide sequence shown in any one of (1) to (3); or (5) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (1) to (4).

10. A protein combination, characterized in that Includes any of the following and chimeric antigen receptors: (VII), the JxM-EGFRt polypeptide according to any one of claims 1 to 3; and / or (VIII) The mutant according to any one of claims 4 to 7.

11. The protein combination according to claim 10, characterized in that The chimeric antigen receptor comprises an antigen recognition domain, a transmembrane domain, an intracellular signaling domain and / or a hinge region; The antigen recognition domain includes, but is not limited to, ScFv targeting one or more of CD19, CD20, CD22, CD33, CLL-1, CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY, L1CAM, MAGE, MAGE-A1 or TAG-72; The intracellular signaling domain includes any of the following: (iii), immunoreceptor tyrosine-based activation motifs; and / or (iv), the primary intracellular signaling domain of an ITAM; and / or (v), a primary intracellular signaling domain and one or two or more co-stimulatory signaling domains; The primary intracellular signaling domain of the ITAM includes but is not limited to CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS, FcεRI, CD66d, DAP10 or DAP12; The co-stimulatory signaling domain is selected from one or more of D28, 41BB, OX40, CD27, DAP10, 2B4, CD3γ, CD3δ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80, signaling lymphocyte activation molecule, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE, LFA-1, GITR, BAFFR, LIGHT or HVEM; The transmembrane domain is selected from the group consisting of a type I transmembrane protein of a T cell receptor, an alpha, beta or zeta chain of a T cell receptor, CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD357, Toll-like receptor 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, NKp44 or NKp46; The hinge region includes, but is not limited to, 8hinge, 8h(dc) hinge or CD8(1) hinge derived from human CD8.

12. The protein combination according to claim 11, characterized in that The antigen recognition domain includes a ScFv targeting CD123, PSCA or CLL-1; The transmembrane domain includes CD8TM derived from human CD8 transmembrane or CD28TM derived from CD28 transmembrane; The co-stimulatory signal transduction domain is 41BB; The primary intracellular signaling domain of the ITAM is CD3ζ or a CD3ζ mutant.

13. The protein combination according to claim 12, characterized in that (①), the ScFv targeting CD123 and PSCA has the amino acid sequences shown in SEQ ID No. 28 and 61 respectively; or (②), the 8hinge, 8h(dc) hinge and CD8(1) hinge have the amino acid sequences shown in SEQ ID No.29, 53 and 58 respectively; or (③), the CD8TM and CD28TM have the amino acid sequences shown in SEQ ID No. 30 and 54 respectively; or (④), the 4-1BB has an amino acid sequence as shown in SEQ ID No.31 or 56; or (⑤), the CD3ζ has the amino acid sequence shown in SEQ ID No.32; or (⑥), the CD3ζ mutant has an amino acid sequence as shown in SEQ ID No. 57 or 60; or (⑦) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in any one of (①) to (⑥); or (⑧) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of (①) to (⑦).

14. A genetic element, characterized in that The method comprises the nucleic acid molecule according to claim 8 or 9 and a nucleic acid molecule encoding a chimeric antigen receptor.

15. The genetic element according to claim 14, wherein The chimeric antigen receptor comprises an antigen recognition domain, a transmembrane domain, an intracellular signaling domain and / or a hinge region; The antigen recognition domain includes, but is not limited to, ScFv targeting one or more of CD19, CD20, CD22, CD33, CLL-1, CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY, L1CAM, MAGE, MAGE-A1 or TAG-72; The intracellular signaling domain includes any of the following: (iii), immunoreceptor tyrosine-based activation motifs; and / or (iv), the primary intracellular signaling domain of an ITAM; and / or (v), a primary intracellular signaling domain and one or two or more co-stimulatory signaling domains; The primary intracellular signaling domain of the ITAM includes but is not limited to CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS, FcεRI, CD66d, DAP10 or DAP12; The co-stimulatory signaling domain is selected from one or more of D28, 41BB, OX40, CD27, DAP10, 2B4, CD3γ, CD3δ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80, signaling lymphocyte activation molecule, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE, LFA-1, GITR, BAFFR, LIGHT or HVEM; The transmembrane domain is selected from the group consisting of a type I transmembrane protein of a T cell receptor, an alpha, beta or zeta chain of a T cell receptor, CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD357, Toll-like receptor 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, NKp44 or NKp46; The hinge region includes, but is not limited to, 8hinge, 8h(dc) hinge or CD8(1) hinge derived from human CD8.

16. The genetic element according to claim 15, wherein The antigen recognition domain includes a ScFv targeting CD123, PSCA or CLL-1; The transmembrane domain includes CD8TM derived from human CD8 transmembrane or CD28TM derived from CD28 transmembrane; The co-stimulatory signal transduction domain is 41BB; The primary intracellular signaling domain of the ITAM is CD3ζ or a CD3ζ mutant.

17. The genetic element according to claim 16, wherein (①), the ScFv encoding the CD123 and PSCA targets has the nucleotide sequences shown in SEQ ID No. 2 and 70 respectively; or (②), the nucleic acid molecules encoding the 8hinge, 8h(dc) hinge and CD8(1) hinge have the nucleotide sequences shown in SEQ ID No. 3, 62 and 67 respectively; or (③), the nucleic acid molecules encoding the CD8TM and CD28TM have the nucleotide sequences shown in SEQ ID No. 4 and 63 respectively; or (④), the nucleic acid molecule encoding the 4-1BB has a nucleotide sequence as shown in SEQ ID No. 5 or 65; or (⑤), the nucleic acid molecule encoding the CD3ζ has the nucleotide sequence shown in SEQ ID No.6; or (⑥), the nucleic acid molecule encoding the CD3ζ mutant has a nucleotide sequence as shown in SEQ ID No.66 or 69; or (⑦) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence as shown in any one of (①) to (⑥), and having the same or similar functions as the nucleotide sequence as shown in any one of (①) to (⑥); or (⑧) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (①) to (⑦).

18. An expression vector, characterized in that Comprising the genetic element according to any one of claims 14 to 17.

19. A virus particle, characterized in that Comprising the expression vector as claimed in claim 18.

20. An engineered cell, characterized in that Express any of the following: (A), the JxM-EGFRt polypeptide according to any one of claims 1 to 3; and / or (B) a mutant according to any one of claims 4 to 7; and / or (C) a nucleic acid molecule as described in claim 8 or 9; and / or (D) a protein combination as described in any one of claims 10 to 13; and / or (E) a genetic element as claimed in any one of claims 14 to 17; and / or (F), the expression vector according to claim 18; and / or (G) The virus particle according to claim 19.

21. The engineered cell according to claim 20, characterized in that The engineered cells include, but are not limited to, T cells, NK cells, macrophages, DC cells and related precursor cells, mesenchymal stem cells, human embryonic stem cells and / or iPS-derived stem cells.

22. Use of any of the following as screening markers: (J), the JxM-EGFRt polypeptide according to any one of claims 1 to 3; and / or (K), a mutant according to any one of claims 4 to 7; and / or (L), a nucleic acid molecule as described in claim 8 or 9; and / or (M), a protein combination as described in any one of claims 10 to 13; and / or (N), a genetic element as claimed in any one of claims 14 to 17; and / or (O) The expression vector according to claim 18.

23. A screening marker, characterized in that Include any of the following: (R), the JxM-EGFRt polypeptide according to any one of claims 1 to 3; and / or (S), a mutant according to any one of claims 4 to 7; and / or (T), a nucleic acid molecule as described in claim 8 or 9; and / or (U), a protein combination as described in any one of claims 10 to 13; and / or (V) a genetic element as claimed in any one of claims 14 to 17; and / or (W), the expression vector according to claim 18.

24. Use of any of the following in the preparation of a drug for preventing and / or treating tumors: 1) The JxM-EGFRt polypeptide according to any one of claims 1 to 3; and / or 2) A mutant according to any one of claims 4 to 7; and / or 3) A nucleic acid molecule according to claim 8 or 9; and / or 4) A protein combination according to any one of claims 10 to 13; and / or 5) A genetic element according to any one of claims 14 to 17; and / or 6) The expression vector according to claim 18; and / or 7) The viral particle according to claim 19; and / or 8) The engineered cell according to claim 20 or 21.

25. A drug, characterized in that Includes any of the following items and pharmaceutically acceptable excipients or adjuvants: A), the JxM-EGFRt polypeptide according to any one of claims 1 to 3; and / or B), a mutant according to any one of claims 4 to 7; and / or C), a nucleic acid molecule as claimed in claim 8 or 9; and / or D), a protein combination as claimed in any one of claims 10 to 13; and / or E), a genetic element as claimed in any one of claims 14 to 17; and / or F), the expression vector according to claim 18; and / or G), the viral particle as claimed in claim 19; and / or H) The engineered cell according to claim 20 or 21.

26. A pharmaceutical combination, characterized in that Comprising the drug as claimed in claim 25 and any other active ingredients.

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