Truncated EGFR polypeptide and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a truncated EGFR polypeptide and application thereof. The invention provides the truncated EGFR polypeptide, and the CAR structure containing the EGFR polypeptide has a higher initial / immature T cell proportion, a lower depletion marker expression level and a stronger multi-round stimulation ability, can effectively enhance the in-vivo and in-vitro surviving and killing ability of target cells, and is beneficial to improving the in-vivo and in-vitro anti-tumor effect. The truncated EGFR polypeptide is used as a hinge region of a CAR structure, or simultaneously used as a hinge and a transmembrane region, can be bridged with a bispecific antibody, can simultaneously recognize an EGFR antigen and other specific antigens on target cells, and can enhance the in-vivo and in-vitro anti-tumor capability of immune cells. The EGFR polypeptide in the CAR structure can also be used as a universal detection and selection marker to achieve the purposes of detecting the positive rate of the CAR, monitoring the in-vivo distribution of the CAR positive cells and screening the CAR positive cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to truncated EGFR polypeptides and their applications. Background Art

[0002] Chimeric Antigen Receptor-T cell (CAR-T) refers to T cells that, after genetic modification, can recognize specific target antigens in an MHC-independent manner and continuously activate and expand. The chimeric antigen receptor (CAR) is the core component of CAR-T, endowing T cells with the ability to recognize tumor antigens in an HLA-independent manner. CAR is a modular synthetic receptor composed of four main components: (1) an extracellular target antigen-binding domain, (2) a hinge region, (3) a transmembrane domain, and (4) one or more intracellular signaling domains.

[0003] The function of the hinge region is to provide flexibility to overcome spatial obstacles and, through its length, allow the antigen-binding domain to access the target epitope. Differences in the length and composition of the hinge region can affect the expression, flexibility, signal transduction, epitope recognition, etc. of the CAR molecule, ultimately affecting the function of the CAR molecule.

[0004] Immune cell therapy represents a promising treatment method. Safety, effectiveness, and controllability are not only the most basic requirements for clinical applications but also common problems faced in clinical treatment. For example, cytokine storms exposed during clinical use and tumor recurrence caused by CAR-T cell exhaustion, etc. Therefore, there is still a need to continue developing safe and efficient CARs to promote the improvement of immune cell therapy. Summary of the Invention

[0005] In view of this, the present invention provides truncated EGFR polypeptides and their applications.

[0006] The present invention provides truncated EGFR polypeptides and their applications. The beneficial effects of the present invention are as follows:

[0007] Truncated EGFR can be applied to the construction of engineered receptors, and the engineered receptors include EGFR mutants as hinge or fusion proteins of hinge and transmembrane structures, chimeric antigen receptors, or costimulatory receptors (CCRs, chimeric costimulatory receptors).

[0008] Truncated EGFR, as a chimeric antigen receptor (CAR) constructed with a hinge or a hinge and transmembrane structure, has a higher proportion of naive T cells, a lower expression level of exhaustion markers, and a stronger multi-round tumor re-killing ability (In vitro tumor cell rechallenge assay) compared with traditional CAR structures. It can effectively enhance the survival and killing ability of target cells in vitro and in vivo, and contribute to improving the anti-tumor effect in vitro and in vivo. Moreover, this truncated EGFR polypeptide, as the hinge region of the CAR structure or simultaneously as the hinge and transmembrane regions, can bridge bispecific antibodies to simultaneously recognize EGFR antigens and other specific antigens on target cells, enhancing the anti-tumor ability of immune cells in vitro and in vivo. In addition, the EGFR polypeptide in the CAR structure can also be used as a universal detection and screening marker to detect the positive rate of CAR, monitor the in vivo distribution of CAR-positive cells, and screen CAR-positive cells. In addition, the CAR structure containing the EGFR polypeptide enhances safety while improving effectiveness. When strong side reactions occur after the target cells are infused into the body, cetuximab can be administered to mediate antibody-dependent cell-mediated cytotoxicity (ADCC).

[0009] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0010] The present invention provides an EGFR mutant, which has:

[0011] (I) an amino acid sequence as shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5; or

[0012] (II) a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids based on the amino acid sequence as shown in (I); or

[0013] (III) an amino acid sequence having at least 80% sequence homology with the amino acid sequence as shown in (I).

[0014] The present invention also provides a nucleic acid molecule encoding the EGFR mutant.

[0015] In some specific embodiments of the present invention, the nucleic acid molecule has:

[0016] (IV) a nucleotide sequence as shown in SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 or SEQ ID No.12; or

[0017] (V), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (IV), but different from the nucleotide sequence shown in (IV) due to the degeneracy of the genetic code; or

[0018] (VI), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (IV) or (V), and having the same or similar function as the nucleotide sequence shown in (IV) or (V); or

[0019] (VII), a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (IV) to (VI).

[0020] Based on the above research, the present invention also provides the following applications of any item as a hinge region or simultaneously as a hinge region and a transmembrane region in constructing an engineered receptor:

[0021] (1), the EGFR mutant; and / or

[0022] (2), the nucleic acid molecule.

[0023] The present invention also provides an engineered receptor, which comprises any of the following:

[0024] (3), an antigen recognition region, the EGFR mutant as described in claim 1, a transmembrane region and an intracellular signaling region; and / or

[0025] (4), an antigen recognition region, the EGFR mutant as described in claim 1 and an intracellular signaling region.

[0026] In some specific embodiments of the present invention, the engineered receptor comprises a fusion protein, a chimeric antigen receptor or a co-stimulatory receptor (CCR, chimeric costimulatory receptor).

[0027] In some specific embodiments of the present invention, the intracellular signaling region comprises a primary signaling domain and / or a co-stimulatory signaling domain.

[0028] In some specific embodiments of the present invention, the intracellular signaling region comprises any of the following:

[0029] 1), a primary signaling domain; or

[0030] 2), a co-stimulatory signaling domain; or

[0031] 3), a co-stimulatory signaling domain and a primary signaling domain; or

[0032] 4), multiple co-stimulatory domains and 1 primary signaling domain.

[0033] The primary signal domain described in the present invention may also be referred to as an activation signal region.

[0034] In some specific embodiments of the present invention, the engineered receptor is a chimeric antigen receptor;

[0035] The antigen recognition region of the chimeric antigen receptor includes an antibody fragment, single-chain antibody (ScFv), ligand, receptor, or artificial structure that can recognize and bind to a target antigen.

[0036] In some specific embodiments of the present invention, the target antigen is one or more;

[0037] The antigen-binding fragment or single-chain antibody may be one or more antigen-binding fragments, single-chain antibodies, or a fusion polypeptide or protein formed thereof that recognize different antigens or different epitopes of the same antigen.

[0038] In some specific embodiments of the present invention, the antigen recognition region includes, but is not limited to, ScFv of one or more targets such as CD123, CD19, CEA, or CD22;

[0039] The transmembrane region includes CD8TM derived from human CD8 transmembrane or CD28TM derived from CD28 transmembrane;

[0040] The intracellular signal region includes the signal transduction structure CD3ζ and / or the co-stimulatory signal transduction domain 4-1BB.

[0041] In some specific embodiments of the present invention,

[0042] (5), the ScFv targeting CD123 has an amino acid sequence as shown in SEQ ID No. 27 or SEQ ID No. 28; or

[0043] (6), the ScFv targeting CD19 has an amino acid sequence as shown in SEQ ID No. 29; or

[0044] (7), the ScFv targeting CEA has an amino acid sequence as shown in SEQ ID No. 30; or

[0045] (8), the ScFv targeting CD22 has an amino acid sequence as shown in SEQ ID No. 38; or

[0046] (9), the CD8TM or CD28TM has an amino acid sequence as shown in SEQ ID No. 21 or SEQ ID No. 25; or

[0047] (10), the CD3ζ has an amino acid sequence as shown in SEQ ID No. 23; or

[0048] (11) The co-stimulatory signaling domain 4-1BB has the amino acid sequence shown in SEQ ID No. 22; or

[0049] (12) A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in any one of (5) to (11); or

[0050] (13) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of (5) to (12).

[0051] In some specific embodiments of the present invention, the engineered receptor further includes SIRPγ;

[0052] The SIRPγ has:

[0053] (14) The amino acid sequence shown in SEQ ID No. 24; or

[0054] (15) A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in (14); or

[0055] (16) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (14).

[0056] In some specific embodiments of the present invention, the engineered receptor includes CAR-3, CAR-4, CAR-8, CAR-10, and CAR-11;

[0057] (A) The CAR-3, CAR-4, CAR-8, CAR-10, and CAR-11 sequentially have the amino acid sequences shown in SEQ ID No. 31, 32, 33, 34, and 35; or

[0058] (B) A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in (A); or

[0059] (C) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (A) or (B).

[0060] The present invention also provides a genetic element, including the nucleic acid molecule.

[0061] In some specific embodiments of the present invention, the genetic element includes CAR-3, CAR-4, CAR-8, CAR-10, and CAR-11;

[0062] (D), the CAR-3, CAR-4, CAR-8, CAR-10, and CAR-11 successively have nucleotide sequences as shown in SEQ ID No. 13, 14, 15, 16, and 17; or

[0063] (E), a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (D), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (D); or

[0064] (F), a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in (D) or (E).

[0065] The present invention also provides an artificial vector, comprising the gene element and an acceptable adjuvant or vector.

[0066] The present invention also provides a viral particle, comprising the artificial vector.

[0067] The present invention also provides an engineered cell, comprising or expressing any of the following:

[0068] (a), the EGFR mutant; and / or

[0069] (b), the nucleic acid molecule; and / or

[0070] (c), the engineered receptor; and / or

[0071] (d), the gene element; and / or

[0072] (e), the artificial vector; and / or

[0073] (f), the viral particle.

[0074] In some specific embodiments of the present invention, the engineered cell includes, but is not limited to, NK cells and T cells.

[0075] The present invention also provides the use of any of the following in enhancing the in vivo and in vitro survival ability of target cells, bridging bispecific antibodies, and / or simultaneously recognizing EGFR antigen and other specific target antigens on target cells:

[0076] (a), the EGFR mutant; and / or

[0077] (b), the nucleic acid molecule; and / or

[0078] (c), the engineered receptor; and / or

[0079] (d), the gene element; and / or

[0080] (e), the artificial vector; and / or

[0081] (f), the virus particles; and / or

[0082] (g), the engineered cells.

[0083] In some specific embodiments of the present invention, the EGFR mutant has the amino acid sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5.

[0084] The present invention also provides the use of any of the following as a detection and / or screening marker:

[0085] (a), the EGFR mutant; and / or

[0086] (b), the nucleic acid molecule; and / or

[0087] (c), the engineered receptor; and / or

[0088] (d), the gene element; and / or

[0089] (e), the artificial vector.

[0090] In some specific embodiments of the present invention, the EGFR mutant has the amino acid sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5.

[0091] The present invention also provides a detection marker and / or a screening marker, including any of the following:

[0092] (a), the EGFR mutant; and / or

[0093] (b), the nucleic acid molecule; and / or

[0094] (c), the engineered receptor; and / or

[0095] (d), the gene element; and / or

[0096] (e), the artificial vector.

[0097] The present invention also provides the use of any of the following in the preparation of a drug for preventing and / or treating a disease:

[0098] (a), the EGFR mutant; and / or

[0099] (b), the nucleic acid molecule; and / or

[0100] (c), the engineered receptor; and / or

[0101] (d), the gene element; and / or

[0102] (e), the artificial vector; and / or

[0103] (f), the virus particle; and / or

[0104] (g), the engineered cell.

[0105] In some specific embodiments of the present invention, the EGFR mutant has the amino acid sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5.

[0106] In some specific embodiments of the present invention, the disease includes but is not limited to tumors.

[0107] In some specific embodiments of the present invention, the tumors include acute myeloid leukemia, acute lymphoblastic leukemia, human Burkitt's lymphoma, colorectal cancer, lung cancer, pancreatic cancer and / or liver cancer.

[0108] The present invention also provides a drug, comprising any one of the following and a pharmaceutically acceptable excipient or adjuvant:

[0109] (a), the EGFR mutant; and / or

[0110] (b), the nucleic acid molecule; and / or

[0111] (c), the engineered receptor; and / or

[0112] (d), the gene element; and / or

[0113] (e), the artificial vector; and / or

[0114] (f), the virus particle; and / or

[0115] (g), the engineered cell.

[0116] In some specific embodiments of the present invention, the EGFR mutant has the amino acid sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5.

[0117] The present invention also provides a drug combination, comprising the drug and any other active ingredient.

[0118] In some specific embodiments of the present invention, the any other active ingredient includes a multi-target antibody drug or an ADC drug containing an EGFR inhibitor.

[0119] In some specific embodiments of the present invention, the multi-target antibody drug includes a bispecific antibody that recognizes PSCA / EGFR, a bispecific antibody that recognizes non-PSCA / EGFR targets, or a multispecific antibody that recognizes non-PSCA / EGFR targets;

[0120] The ADC drug containing an EGFR inhibitor includes an antibody containing EGFR or an inhibitor containing EGFR.

[0121] The present invention provides a truncated EGFR polypeptide and its applications. 1) The present invention provides a new truncated EGFR polypeptide; 2) The present invention provides the application of a new truncated EGFR polypeptide in a hinge structure; 3) The present invention provides an engineered receptor comprising a truncated EGFR polypeptide as a hinge or a hinge and a transmembrane structure, and the engineered receptor may be a chimeric antigen receptor, and the new chimeric antigen receptor structure improves the CAR expression level and stability; 4) Compared with the traditional CAR structure, the CAR structure containing the hinge region and / or transmembrane region of the EGFR polypeptide is expressed in immune cells, and the constructed engineered immune cells or CAR-T cells have a higher proportion of naive / naive T cells, effectively enhancing the in vivo and in vitro survival ability of the target engineered cells; 5) Compared with the traditional CAR structure, the CAR structure containing the hinge region and / or transmembrane region of the EGFR polypeptide is expressed in immune cells, and the constructed engineered immune cells or CAR-T cells have a lower expression level of exhaustion markers, reducing immune escape and enhancing the killing function; 6) Compared with the traditional CAR structure, the CAR structure containing the hinge region and / or transmembrane region of the EGFR polypeptide is expressed in immune cells, and the constructed engineered immune cells or CAR-T cells have a stronger anti-tumor effect in vivo and in vitro; 7) The CAR structure containing the hinge region and / or transmembrane region of the EGFR polypeptide can bridge a bispecific antibody, simultaneously recognize the EGFR antigen and other specific target antigens on the target cells, enhance the ability of immune cells to recognize and kill tumors, and reduce immune escape; 8) The EGFR polypeptide in the CAR structure can be used as a universal detection and screening marker, with an expression efficiency highly consistent with that of ScFv, serving the purpose of detecting the CAR positive rate, monitoring the in vivo distribution of CAR-positive cells, and screening CAR-positive cells; 9) While improving the effectiveness, the CAR structure containing the EGFR polypeptide also enhances the safety. When strong side reactions occur after the target cells are infused into the body, cetuximab can be administered to mediate antibody-dependent cell-mediated cytotoxicity (ADCC).

[0122] Definition:

[0123] Engineered receptor: refers to an artificially modified / simulated protein structure, which can be one or more proteins, polypeptide mutants, mutants of protein functional domains, artificial recognition molecules; it can also be a fusion protein formed by one or more proteins, polypeptide mutants, protein functional domains and another one or more proteins, polypeptides or protein functional domains, or a chimeric antigen receptor, or a co-stimulatory receptor (CCR, chimeric costimulatory receptor). In some embodiments, the engineered receptor comprises an antigen recognition region, a hinge region, a transmembrane region and an intracellular signaling region, and the hinge region or the hinge and transmembrane regions are the EGFR mutant peptides; in some embodiments, the intracellular signaling region only has a primary signaling domain (the primary signaling domain can also be referred to as an activation signaling region); in some embodiments, the intracellular signaling region only has a co-stimulatory signaling domain; in some embodiments, the intracellular signaling region has both co-stimulatory and primary signaling domains; in some embodiments, the intracellular signaling region has multiple co-stimulatory domains and primary signaling domains.

[0124] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a group of engineered polypeptides or proteins that, when present in an immune effector cell, bind to a specific antigen contained on a target cell and generate an intracellular signal upon recognition of the specific antigen, activating downstream pathways in the cell in which the receptor is located to initiate killing of the target cell by the immune effector cell. The immune effector cells include, but are not limited to, NK cells, macrophages, neutrophils, T cells, etc. CARs generally comprise at least one extracellular antigen-binding domain, a transmembrane domain and a cytoplasmic signaling domain. The extracellular antigen-binding domain can specifically recognize an antigen, non-limiting examples of which include single-chain variable fragments (scFv) derived from antibodies, fragment antigen-binding regions (Fab) selected from libraries, single-domain fragments or a combination with the natural ligand that engages its cognate receptor, a target-specific recognition domain that is artificially designed to recognize a specific target, such as a fibronectin type III (FN3) domain, and designed ankyrin repeat proteins (DARPins) that recognize a specific target. In some embodiments, the extracellular antigen-binding region can comprise scFv, Fab or a natural ligand, and any derivatives thereof. The extracellular antigen-binding domain can refer to a molecule other than a complete antibody, which can comprise a part of a complete antibody and can bind to the antigen bound by the complete antibody. Examples of antibody fragments can include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bispecific antibodies, linear antibodies; single-chain antibody molecules (e.g., scFv), and the scFv can be a murine antibody or a fully human antibody or a human-mouse chimeric antibody scFv, or a single-domain antibody such as a shark, alpaca or camel antibody; and multispecific antibodies formed by antibody fragments.

[0125] In some embodiments, the extracellular antigen-binding region of the "chimeric antigen receptor" or "CAR" structure can recognize target molecules expressed on the surface of solid tumors and hematological tumor cells / tissues, and the target molecules include but are not limited to: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PD-L1, 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, etc.

[0126] In some embodiments, the structure referred to as "chimeric antigen receptor" or "CAR" comprises: an antigen recognition domain (such as ScFv), a hinge structure (such as the 8hinge derived from human CD8), a transmembrane structure (such as the CD8TM derived from the transmembrane region of human CD8), and an intracellular signaling domain. The intracellular signaling domain can be a primary intracellular signaling domain that only has immunoreceptor tyrosine-based activation motifs or ITAMs: examples of primary cytoplasmic signaling sequences containing ITAMs 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. It can also contain one or two or more co-stimulatory signaling domains in addition to the primary intracellular signaling domain. The co-stimulatory signaling domains can 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, SLAMF7, NKp80 (KLRF1), signaling lymphocytic 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 derived functional molecules are amino acid or nucleic acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity with the above molecules.

[0127] "Chimeric antigen receptor" or "CAR" can have various structures, such as those containing cytokine, antibody gene sequences that can be secreted or expressed on the membrane; those containing structures that can be regulated to be activated or inactivated, and the structures for such regulation of activation or inactivation include: suicide switches such as inducible caspase-9 (iCasp9), thymidine kinase in herpes simplex virus (HSV-TK), and suicide epitopes, truncated EGFR (EGFRt), Fas-FasL apoptotic structure; inducible CAR (Inducible CAR) structures: Peptide neo-epitope (PNE), fluorescein (FITC), 10 amino acids (5B9tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv, Leucine ZipFv linked to antibody, Streptavidin 2 (mSA2) biotin-binding domain, VIPER CAR induction structure, biotin-binding immune receptor (BBIR) system; "logic gate" regulation system combined with SynNotch receptor, etc.

[0128] In some embodiments, the structure of the "chimeric antigen receptor" or "CAR" may further include a chimeric fusion protein, which includes an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular antigen recognition domain of the chimeric fusion protein can be the complete and continuous extracellular segment of the expressed molecule / polypeptide on the cell membrane, or a fusion form of the expressed molecule and other polypeptides such as polypeptides derived from human CD8 and CD4.

[0129] The construction method of the above CAR structure does not affect the uses protected by the present invention. Those skilled in the art can obtain the engineered cells described in the present invention by using any publicly known or unknown cell preparation protocols. Therefore, regardless of the above-mentioned CAR structure, as long as it contains the truncated EGFR described in the present invention as a hinge structure and / or a hinge and transmembrane structure, it can be applied to construct engineered immune cells such as T cells, NK cells, macrophages, etc. described herein.

[0130] Engineered cells: refer to cells obtained by modifying the original target cells with or without changing their functions. The engineered cells can be engineered immune cells, such as CAR-T cells, TCR-T cells, immune cells expressing cell adapter molecules, cells expressing antigen couplers (TAC), etc. The CAR-T cells are T cells expressing chimeric antigen receptors or CARs.

[0131] In some embodiments, the engineered cells described herein are immune cells expressing a chimeric antigen receptor (CAR) comprising the truncated EGFR described in the present invention as a hinge structure and / or hinge and transmembrane structure, and the immune cells include but are not limited to T cells, NK cells, macrophages, etc. In some embodiments, the engineered cells described herein, in addition to expressing a CAR comprising the truncated EGFR described in the present invention as a hinge structure and / or hinge and transmembrane structure, also express a CAR or fusion protein structure comprising one or more antigen-binding domains, transmembrane domains, and intracellular signal transduction domains. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0133] Figure 1a Showing the CAR structure constructed with the EGFR mutant as the hinge and transmembrane in Example 1;

[0134] Figure 1b Showing the CAR structure constructed with the EGFR mutant as the hinge region in Example 1;

[0135] Figure 2a Showing the detection results of the CAR positive rates of CAR-1, CAR-3, and CAR-4 in Example 3;

[0136] Figure 2b Showing the detection results of the CAR positive rates of CAR-1, CAR-3, and CAR-4 of T cells from different donors in Example 3;

[0137] Figure 3 Showing the expression of the CAR structures constructed with ERt-1 and ERt-3 in NK cells in Example 4;

[0138] Figure 4 Showing the detection results of the CAR positive rates of CAR-12 and CAR-13 in Example 5;

[0139] Figure 5 Showing the detection results of the CAR positive rates of CAR-15 and CAR-16 in Example 5;

[0140] Figure 6 Showing the proportion of CAR-3 and CAR-6 T naive cells in Example 7;

[0141] Figure 7 Showing the proportion of CAR-7 and CAR-8 CAR-T cells in naive effector cells in Example 7;

[0142] Figure 8Show the proportion of CAR-9 and CAR-10 CAR-T cell naive phenotype cells in Example 7;

[0143] Figure 9 Show the proportion of CAR-4 and CAR-6 CAR-T cell naive phenotype cells in Example 7;

[0144] Figure 10 Show the expression levels of exhaustion markers in CAR-3 and CAR-6 CAR-T cells in Example 8;

[0145] Figure 11 Show the expression levels of exhaustion markers in CAR-4 and CAR-6 CAR-T cells in Example 8;

[0146] Figure 12 Show the multi-round tumor re-killing ability of CAR-3 and CAR-6 targeting CD123 with THP1-Luc-GFP as target cells in Example 9;

[0147] Figure 13 Show the multi-round tumor re-killing ability of CAR-3 and CAR-6 targeting CD123 with THP1-Luc-GFP as target cells in Example 9;

[0148] Figure 14 Show the ADCC efficiency of CAR-3 in Example 10;

[0149] Figure 15 Show the ADCC efficiency of CAR-4 and CAR-6 in Example 10;

[0150] Figure 16 Show the in vitro killing effect of CAR-11 on target cells DLD1-CEA-Luc-GFP in Example 11;

[0151] Figure 17 Show the in vivo anti-tumor effect of CAR-11 on CEA+ cells A549-CEA-Luc-GFP in Example 11;

[0152] Figure 18 Show the anti-tumor effect of CAR-11 on CEA+ pancreatic cancer PDX tumors in Example 11;

[0153] Figure 19 Show the anti-tumor effect of CAR-11 on CEA+ liver cancer PDX tumors in Example 11;

[0154] Figure 20 Show the extended application of EGFR polypeptide as the hinge and / or transmembrane domain of CAR in Example 12;

[0155] Figure 21It is shown that the bispecific antibody in Example 12 can recognize the PSCA antigen;

[0156] Figure 22 It is shown that the bispecific antibody in Example 12 has a high consistency with Protein L in detecting the CAR positive rate;

[0157] Figure 23 It is shown that in Example 12, PSCA+HPAC-Luc-GFP cells are used as positive target cells to detect the CEA positive rate. Detailed implementation manners

[0158] The present invention discloses a truncated EGFR polypeptide and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate modifications and combinations to 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.

[0159] The present invention designs a novel EGFR polypeptide as the CAR hinge region and / or transmembrane domain, which can simultaneously achieve the functions of the hinge and / or transmembrane, as well as the safety switch. On the one hand, it can increase the proportion of naive T cells in CAR, enhance the ability of multi-round stimulation, and effectively enhance the survival and killing ability of target cells in vivo and in vitro. On the other hand, it can reduce the expression level of exhaustion marker molecules and reduce immune escape caused by the exhaustion of target cells, thereby leading to tumor recurrence. In addition, when CAR-T is infused into the patient's body, if strong side effects occur, cetuximab can be administered to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) to improve safety.

[0160] The present study invented a new truncated EGFR polypeptide, which can be used as the hinge region of the CAR structure, or simultaneously as the hinge region and transmembrane region. The structure of the CAR can be A-B-C-I, or A-B-I, where A is an antibody fragment, single-chain antibody (ScFv), ligand, receptor or artificial structure that can specifically bind to the target antigen. The target antigen can be one or more. The antigen-binding fragment and single-chain antibody can be a fusion polypeptide / protein formed by multiple antigen-binding fragments and single-chain antibodies that recognize different antigens or different epitopes of the same antigen; B is the new truncated EGFR polypeptide of the present invention, which can be used as the hinge region of the CAR structure; C is the transmembrane structure; I is the intracellular signal region, which can include the signal transduction structure CD3ζ, and co-stimulatory signals can be added on this basis.

[0161] Specifically, in the embodiments of the present invention, the structures used are: ScFv-EGFR(t)-TM-ICD (intracellular signaling region) or ScFv-EGFR(t)-ICD (intracellular signaling region).

[0162] More specifically, the new truncated EGFR polypeptides developed in the present invention are: ERt-2, ERt-3, ERt-4, ERt-5, wherein ERt-3 and ERt-4 can be adapted to either of the two structures of ScFv-EGFR(t)-TM-ICD (intracellular signaling region) or ScFv-EGFR(t)-ICD (intracellular signaling region) (CAR-3, CAR-4, CAR-8, CAR-10, CAR-11, CAR-12, CAR-13, CAR-15, CAR-16).

[0163] 1), The present invention provides a new hinge region and / or transmembrane region of the CAR structure, and a truncated EGFR polypeptide, which can be used as the hinge region of the CAR structure, or simultaneously as the hinge region and the transmembrane region, improving the CAR expression level and stability;

[0164] 2), Compared with the traditional CAR structure, the CAR structure containing the hinge region and / or transmembrane region of the EGFR polypeptide has a higher proportion of naive / naive T cells, effectively enhancing the in vivo and in vitro survival ability of target cells;

[0165] 3), Compared with the traditional CAR structure, the CAR structure containing the hinge region and / or transmembrane region of the EGFR polypeptide has a lower expression level of exhaustion markers, reducing immune escape and enhancing the killing function;

[0166] 4), Compared with the traditional CAR structure, the CAR structure containing the hinge region and / or transmembrane region of the EGFR polypeptide has a stronger anti-tumor effect in vivo and in vitro;

[0167] 5), The CAR structure containing the hinge region and / or transmembrane region of the EGFR polypeptide can bridge bispecific antibodies, simultaneously recognize EGFR antigen and other specific target antigens on target cells, enhance the ability of immune cells to recognize and kill tumors, and reduce immune escape;

[0168] 6), The EGFR polypeptide in the CAR structure can be used as a universal detection and screening marker, having an expression efficiency highly consistent with that of ScFv, serving the purposes of detecting the CAR positive rate, monitoring the in vivo distribution of CAR-positive cells, and screening CAR-positive cells;

[0169] 7) The CAR structure containing the EGFR polypeptide enhances safety while improving efficacy. When strong side effects occur after the target cells are infused into the body, cetuximab can be administered to mediate antibody-dependent cell-mediated cytotoxicity (ADCC).

[0170] The truncated EGFR polypeptide provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0171] The present invention will be further described below in conjunction with embodiments:

[0172] Example 1 Construction of CAR plasmids containing CD123 with different hinges and transmembrane regions

[0173] For EGFR, we designed a novel EGFR mutant peptide as the CAR hinge region and / or transmembrane domain, which can simultaneously achieve the functions of hinge and transmembrane, as well as a safety switch, and solve the problem of inconsistent CAR function and safety switch effect faced by traditional safety switches.

[0174] The gene sequence information of the extracellular region of human EGFR was obtained from the NCBI website database, NCBI accession number: NP_005219.2. Based on this, four mutant sequences, ERt-2 to ERt-5, were obtained. The publicly disclosed EGFR mutant peptide ERt-1 was selected as a control. ERt-1: EGFRvIII, epidermal growth factor receptor isoform iprecursor, NCBI accession number: NP_001333870.1, belongs to the EGFR control mutant.

[0175] ERt-1 (EGFRvIII, EGFR control mutant) (SEQ ID No.1)

[0176] LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0177] ERt-2 (SEQ ID No.2)

[0178] NCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRR

[0179] ERt-3 (SEQ ID No.3)

[0180] YEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRR

[0181] ERt-4 (SEQ ID No.4)

[0182] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRR

[0183] ERt-5 (SEQ ID No.5)

[0184] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKAT

[0185] Design the CAR structure using the obtained EGFR mutants ERt1 - ERt5. The CAR structure includes an antigen recognition region, a hinge region, a transmembrane region, and an intracellular signaling region. The EGFR mutant peptide can be designed as the hinge region of the CAR structure or as the hinge and transmembrane regions of the CAR structure. The specific structure is shown in Figure 1a 、 Figure 1b 。

[0186] Construction of the CAR vector:

[0187] After double digestion with the restriction enzymes NheI (Thermo) and EcoRI (Thermo), the sequence was ligated into a lentiviral vector containing CD123ScFv, CD19 ScFv, CD22 ScFv, CEA ScFv, CD5 ScFv, and intracellular co - stimulatory signals, thus obtaining CAR plasmids targeting CD123, CD19, CD22, CEA, and CD5 with different hinges and transmembrane regions. After verification by enzyme digestion and sequencing, the plasmids were extracted and purified using a large - scale purification kit (Large - scale purification kit, manufacturer: Omega, product number: D6926 - 04).

[0188] Table 1 Sequence Listing

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197] Example 2 Preparation of CAR Lentivirus

[0198] In this example, the lentivirus was packaged using the calcium phosphate method. Referring to the Molecular Cloning: A Laboratory Manual (Third Edition, J. Sambrook et al.), specifically: 293T cells were cultured in DMEM medium containing 10% FBS (w / v) until in good condition. The above-mentioned target plasmid was mixed with three lentiviral packaging plasmids, pMDLg / pRRE, pRSV-Rev, and pMD2.G, in appropriate proportions, and CaCl 2 and 2×HBS were added. After mixing, the mixture was allowed to stand at room temperature and then added to the treated 293T cell culture medium. After 4 - 6 hours, the medium was changed again to 10 mL of DMEM medium containing 10% FBS. The cell supernatant was collected after 48 hours or 72 hours. The virus supernatant was collected and the virus was purified. The purified virus was aliquoted into 1.5 mL EP tubes and stored at -80 °C for later use.

[0199] For the concentrated lentivirus, the titer was detected using 293T and / or CHO cells. 1×10 5 293T and / or CHO cells were seeded into a 24-well plate. The concentrated virus was used to infect 293T and / or CHO cells at volumes of 1, 2.5, and 10 μL per well (DEAE transfection enhancer needed to be added). Two days after infection, the infected 293T and / or CHO cells were collected for flow cytometry detection. Protein-L was used to detect the total CAR expression and calculate the virus titer. The titer calculation formula is: Titer (TU / mL) = 1×10 5 ×positive rate × dilution factor ÷ virus volume × 1000. The virus titers of the above CAR constructs are shown in Table 2.

[0200] Table 2 CAR Lentivirus Titer List

[0201] Plasmid number CAR structure Virus titer (Tu / mL) CAR-1 CD123(ScFv1)-ERt-1(HTM)-BBz 4.35E+08 CAR-2 CD123(ScFv1)-ERt-2(HTM)-BBz 6.70E+06 CAR-3 CD123(ScFv1)-ERt-3(HTM)-BBz 4.91E+08 CAR-4 CD123(ScFv1)-ERt-4(HTM)-BBz 2.00E+08 CAR-5 CD123(ScFv1)-ERt-5-CD8(h)-8TM-BBz 7.80E+06 CAR-6 CD123(ScFv1)-8h-8TM-BBz 2.40E+08 CAR-7 CD123(ScFv2)-8h-8TM-BBz 2.76E+08 CAR-8 CD123(ScFv2)-ERt-3(HTM)-BBz 1.95E+08 CAR-9 CD19(ScFv)-8h-8TM-BBz 3.40E+08 CAR-10 CD19(ScFv)-ERt-3(HTM)-BBz 1.51E+08 CAR-11 CEA(ScFv)-ERt-4(h)-8TM-BBz-P2A-SIRPγ-28TM-28 8.56E+08 CAR-12 CD22(ScFv)-ERt-3(HTM)-BBz 2.08E+08 CAR-13 CD22(ScFv)-ERt-4(HTM)-BBz 5.52E+08 CAR-14 CD22(ScFv)-ERt-1(HTM)-BBz 7.89E+08 CAR-15 CD22(ScFv)-ERt-3(h)-8TM-BBz 6.04E+08 CAR-16 CD22(ScFv)-ERt-4(h)-8TM-BBz 2.66E+08 CAR-17 CD22(ScFv)-ERt-1(h)-8TM-BBz 4.53E+08

[0202] In some embodiments, the gene sequence of the above CAR construct further contains a signal peptide derived from the GM-CSF receptor or a signal peptide derived from CD8a. In the above CAR construct, BB represents the intracellular signaling domain derived from CD137, Z represents the CD3ζ chain, and HTM represents the EGFR mutant (ERt) as the hinge and transmembrane part of the CAR construct, i.e., the A - B - I structure; ERt-4(h) and ERt-3(h) are different EGFR mutants as the hinge part of the CAR construct, i.e., the A - B - C - I structure.

[0203] The results showed that the CAR-2 constructed using ERt-2 and the CAR-5 constructed using ERt-5 could not be normally expressed, with low virus titers, while the other structures could be normally expressed. This indicates that not all mutant peptides can be used to design CAR structures. For example, the CAR structures designed with ERt-2 and ERt-5 could not be normally expressed. ERt-1 (CAR-1, CAR-14, CAR-17) derived from the extracellular region of the EGFR natural mutant EGFRvIII (epidermal growth factor receptor isoform i precursor, NCBI accession number: NP_001333870.1) and the mutant peptides ERt-3 and ERt-4 (CAR-3, CAR-4, CAR-8, CAR-10, CAR-11, CAR-12, CAR-13, CAR-15, CAR-16) of the present invention can be used for CAR structure construction. CAR-6 and CAR-7 are conventional control CAR structures with conventional hinge and transmembrane structures targeting CD123 from different extracellular recognition regions.

[0204] Example 3 Preparation of T cells modified with CAR-1, CAR-3 and CAR-4 and detection of CAR positive rate

[0205] Lymphocytes were separated by gradient centrifugation. After centrifugation, the second white lymphocyte layer was taken and washed with physiological saline to obtain human PBMC cells. The obtained PBMC cells were activated with magnetic beads coated with anti-CD3 and CD28 antibodies for 24 h, and then infected with the activated PBMC at a certain multiplicity of infection (3 - 5 MOI). The CAR positive rate was detected on the 7th day after virus infection. The detection method was flow cytometry, and the antibodies used were: CD3 PE-Cy7 (Biolegend, catalog number: 300420), EGFR-APC (Biolegend, catalog number: 352906), Protein-L-PE (SinoBiological, catalog number: 11044-H07E-P). Among them, the CD3 positive rate represents the proportion of T cells, the EGFR positive rate represents the hinge expression efficiency, and the PL positive rate represents the ScFv expression efficiency. Protein-L-PE, where Protein-L can recognize the light chain of the antibody, and the light chain of the ScFv sequence in the CAR antigen recognition region can be recognized by Protein-L. Therefore, Protein-L can be used to detect the CAR positive rate and CAR expression intensity. ERt-1 is the extracellular domain of the EGFR control mutant EGFRvIII. The results are shown in Table 3, Figure 2a and Figure 2b as shown Figure 2aTo express CAR-1, CAR-3, and CAR-4 in T cells from donor #901, and detect their CAR expression, the detection rate of EGFR mutant peptides, and the positive rate of co-expression of CAR and EGFR mutant peptides; Figure 2b : To express CAR-1, CAR-3, and CAR-4 in T cells from donor #428, and detect their CAR expression, the detection rate of EGFR mutant peptides, and the positive rate of co-expression of CAR and EGFR mutant peptides.

[0206] Table 3 Detection Results of CAR Positivity Rates of CAR-1, CAR-3, and CAR-4 (Batch #901)

[0207]

[0208] The results showed that the same trend results were obtained in the two donors. The expression efficiency of the CAR structure designed by ERt-1 was extremely low, inconsistent with the EGFR expression efficiency. Compared with CAR-1 constructed by ERt-1, the expressions of CAR-3 and CAR-4 both showed obvious advantages, and the CAR positive rate was highly consistent with the hinge positive rate.

[0209] It indicated that not all EGFR mutant peptides that could be normally expressed could be used as CAR structures. This result also showed that the EGFR mutant peptides in CAR-3 constructed with ERt-3 as the hinge and transmembrane structure and CAR-4 constructed with ERt-4 as the hinge and transmembrane structure had the function of a tag, which could be used to detect the CAR positive rate and trace the distribution of CAR-T cells, and was significantly superior to the existing ERt-1 design.

[0210] Example 4 Preparation of CAR-1- and CAR-3-Modified NK Cells and Detection of CAR Positivity Rate

[0211] In Example 3 above, the expression levels of CAR-1, CAR-3, and CAR-4 in T cells were tested. Among them, CAR-3 and CAR-4 could be well expressed in CAR-T cells prepared from randomly selected 2 batches of PBMC, and the CAR positive rate was highly consistent with the hinge positive rate. However, the results of both batches showed that the CAR of CAR-1 could not be effectively expressed, excluding donor individual differences. To further prove whether the expression efficiency of CAR-1 is related to specific immune cells, we continued to test in other immune cells besides T cells, such as NK cells, with the aim of excluding whether the expression of this CAR structure is limited only in T cells.

[0212] Lymphocytes were isolated by gradient centrifugation. After centrifugation, the second layer of white lymphocyte layer was taken and washed with physiological saline to obtain human PBMC cells. Magnetic sorting was performed using Microbeads labeled with CD56 antibody to obtain CD56-positive NK cells. After 24 hours of NK cell activation, lentiviral transduction was carried out at an MOI of 3. On the 8th day after transduction, the positive rate of CAR-NK was detected using CD123-6×His (Human IL-3R alpha / CD123 Protein, His Tag (MALS&SPR verified), ACRO-Biosystems, catalog number: ILA-H52H6-100μg). NK cells were labeled with CD3-PE / Cy7 (BioLegend, catalog number: 300420) and CD56-BV510 (BioLegend, catalog number: 318340) to detect the positive rate.

[0213] The detection results are shown in Table 4 and Figure 3 as follows. The expression efficiency of CAR-1 was significantly poor in 3 batches of CAR-NK, and the positive rates were 4.67%, 8.04% and 10.36% respectively. CAR-3 could be well expressed in 3 randomly selected batches of CAR-NK cells, and the positive rate was about 29%. The CAR positive rate was stable among different donors and had a high consistency.

[0214] Table 4 Detection results of CAR positive rates of CAR-1 and CAR-3 in 3 batches of CAR-NK (#N45, #N46 and #N1018 batches)

[0215]

[0216] Example 3 and Example 4 respectively demonstrated that there were certain limitations in the expression of CAR-1 in both CAR-T and CAR-NK cells, further indicating that not all mutant peptides that can normally express EGFR are suitable as the structure of CAR. CARs constructed with ERt-3 and ERt-4 as the hinge and transmembrane structures can be highly expressed in both T cells and NK cells. CARs constructed with ERt-3 and ERt-4 as the hinge and transmembrane structures can be used for the construction of engineered immune cells. Engineered immune cells can be CAR-T, CAR-NK, CAR-macrophage, various immune cells induced by iPSC, etc. The prepared engineered immune cells such as CAR-T and CAR-NK have a high CAR expression rate.

[0217] Example 5 Preparation of CAR-modified T cells constructed with EGFR mutant peptides and detection of CAR positive rate

[0218] Previously, it was separately demonstrated that the CAR-3 constructed with ERt-3 and the CAR-4 constructed with ERt-4 targeting CD123 could be effectively expressed in T cells. Moreover, it was verified that the CAR-3 constructed with ERt-3 could also be effectively expressed in NK cells. Based on this, we constructed CAR-12 with CD22 as the target and ERt-3 as the hinge and transmembrane structure, and CAR-13 with ERt-4 as the hinge and transmembrane structure, to further test whether ERt-3 and ERt-4 could be effectively expressed as CAR structures on the CD22 target. The CAR-T preparation protocol refers to Example 3.

[0219] The results are as Figure 4 shown in Table 5: Figure 4 For the detection of the expression of ERt-3 and ERt-4 as the hinge and transmembrane (A-B-I structure) of the CAR structure in T cells by flow cytometry, the abscissa represents the expression status.

[0220] Table 5 Detection results of the positive rates of CAR-12 and CAR-13

[0221]

[0222] In summary, in Examples 3-5, on the CD123 and CD22 targets, the ScFv-EGFR(t)-ICD (intracellular signaling region) CAR structure (A-B-I) constructed with the EGFR mutant peptides ERt-3 and ERt-4 as the hinge and transmembrane of the CAR structure is feasible, can be normally expressed on T cells, and can be used for the general detection method of CAR expression in CAR-T.

[0223] CAR is a modular synthetic receptor composed of four main components: an extracellular target antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain. The ScFv-EGFR(t)-ICD (intracellular signaling region) CAR structure constructed with the EGFR mutant peptides ERt-3 and ERt-4 as the hinge and transmembrane of the CAR structure is feasible. Further evaluate whether the ScFv-EGFR(t)-TM-ICD (intracellular signaling region) (A-B-C-I) constructed with the EGFR mutant peptides ERt-3 and ERt-4 as the hinge region of the CAR structure is feasible. With CD22 as the target, CAR-15 was constructed with ERt-3 as the hinge, and CAR-16 was constructed with ERt-4 as the hinge, to further test whether ERt-3 and ERt-4 can be effectively expressed as the CAR hinge region on the CD22 target.

[0224] The results are as Figure 5 shown in Table 6: Figure 5For the detection of the expression of ERt-3 and ERt-4 as the hinge (A-B-C-I structure) of the CAR structure in T cells by flow cytometry therapy technology, the abscissa represents the expression status.

[0225] Table 6 Detection results of the positive rates of CAR-15 and CAR-16

[0226]

[0227] For CAR-15 and CAR-16 modified T cells, the CARs have relatively high expression efficiency, and are completely consistent with the EGFR expression efficiency, indicating that detecting EGFR in this CAR domain can represent the positive rate of the CAR and may also be used as a general detection index for the CAR.

[0228] In summary, the ScFv-EGFR(t)-TM-ICD (intracellular signaling region) (A-B-C-I) CAR structure constructed with CD22 as the target and the EGFR mutant peptides ERt-3 and ERt-4 as the hinge of the CAR structure is feasible, can be normally expressed on T cells, and can be used for the general detection method of CAR expression in CAR-T.

[0229] Example 6 Function verification of CAR-modified T cells constructed with EGFR mutant peptides

[0230] Using CD123+MOLM13-Luc-GFP and THP1-Luc-GFP cells as positive target cells (the target cells are from the corresponding cells purchased from the ATCC official website), and Raji-Luc-GFP as the negative target cell. The CAR-T cells are seeded into the target cells at a ratio of 1:1, and after 24 hours, the killing is detected by Luciferase. Principle of Luciferase: When detecting, the target cells are lysed with lysis buffer, and the Luciferase in them will decompose the substrate to emit fluorescence. The formula for analyzing the results using the fluorescence value is: CAR-T cell killing rate = 1 - (fluorescence value of the experimental group ÷ fluorescence value of the blank control group) × 100%.

[0231] The results are shown in Table 7. The CAR-3 and CAR-4 modified CAR-T cells have effective killing effects on the positive target cells THP1-Luc-GFP and MOLM13-Luc-GFP, and the killing efficiency on the negative target cell Raji-Luc-GFP is extremely low and has no difference from the control Control T. Therefore, the CAR-3 and CAR-4 CAR structures constructed with the EGFR mutant peptides ERt-3 and ERt-4 of the present invention can be highly expressed in immune cells and play their killing roles, and the engineered immune cells of CAR-3 and CAR-4 constructed with the EGFR mutant peptides ERt-3 and ERt-4 have excellent effectiveness.

[0232] Table 7 In vitro killing data of CAR-3 and CAR-4

[0233] CAR structure THP1-Luc-GFP MOLM13-Luc-GFP Raji-Luc-GFP CAR-3 99.77％ 99.7％ 10.04％ CAR-4 98.56％ 99.17％ 10.16％ ControlT 0.00％ 0.37％ 12.79％

[0234] Using CD123+MOLM13-Luc-GFP and THP1-Luc-GFP cells as positive target cells (the target cells are from the corresponding cells purchased from the ATCC official website), and Raji-Luc-GFP as the negative target cell. The CAR-T cells were seeded in the target cells at a ratio of 1:1. After 24 hours of killing, the cell supernatant was collected to detect the IFN-γ secretion ability of CAR-T cells after being stimulated by target cells. The collected supernatant was used to detect the secretion of IFN-γ by ELISA (enzyme-linked immunosorbent assay). Among them, IFN-γ was purchased from BD company, product number: 555142. The specific detection method was operated according to the instruction manual.

[0235] The results are shown in Table 8. After antigen stimulation, both CAR-3 and CAR-4 have strong IFN-γ secretion ability.

[0236] Table 8 Cytokine secretion data of CAR-3 and CAR-4 (unit: pg / mL)

[0237] CAR structure THP1-Luc-GFP MOLM13-Luc-GFP Raji-Luc-GFP CAR-3 6462.88 4087.16 306.00 CAR-4 5394.223 3077.228 299.878 ControlT 0 0 347.112

[0238] The results show that using the EGFR mutant peptide protected by the present invention as the hinge and transmembrane domain of the CAR structure can exert good anti-tumor effects.

[0239] In summary, in Examples 1-6, the EGFR mutant peptides ERt-3 and ERt-4 of the present invention can serve as the hinge structure of the CAR structure, or as the hinge and transmembrane structure of the CAR structure, and are applicable to chimeric antigen receptors of various structures targeting any one or more targets such as CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PD-L1, 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. In addition to chimeric antigen receptors, the EGFR mutant peptides ERt-3 and ERt-4 of the present invention can serve as the hinge structure or the hinge and transmembrane structure and be applied to costimulatory receptors (CCRs, chimeric costimulatory receptors). In some embodiments, the difference between the costimulatory receptor (CCR, chimeric costimulatory receptor) and the chimeric antigen receptor is that it does not contain a primary signal domain.

[0240] Example 7 Detection of the phenotype of CAR-modified T cells constructed with EGFR mutant peptides

[0241] The hinge domain of the CAR mainly comes from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, CD28, 4-1BB or IgG, or all or part of the constant region of the antibody heavy chain. Currently, the commonly used hinge domains of CARs include subsequences of CD8α, IgG1, IgG4, PD-1 or FcγRIIIα, and the commonly used transmembrane regions can be derived from CD8, CD28, OX-40, 4-1BB / CD137, CD2, CD7. Therefore, next, the effects of CAR-3 with a truncated EGFR mutant peptide as the hinge and transmembrane of the CAR and CAR-6 with a traditional CD8 hinge and CD8 transmembrane domain on the function of CD123 CAR-T cells were compared.

[0242] Lymphocytes were separated by gradient centrifugation to obtain human PBMC cells. The obtained PBMC cells were activated with magnetic beads coated with anti-CD3 and CD28 antibodies for 24 h, and then the activated PBMC were infected at a certain multiplicity of infection (MOI). The phenotypes of CAR-T cells were detected on the 7th - 8th day after virus infection by flow cytometry. The antibodies used were: CD3PE-Cy7 (Biolegend, catalog number: 300420), Protein-L-PE (SinoBiological, catalog number: 11044-H07E-P), CD4-FITC (Biolegend, catalog number: 317408), CD8-BV510 (Biolegend, catalog number: 344732), CD45RA-BV421 (Biolegend, catalog number: 304130), CD45RO PerCP-Cy5.5 (Biolegend, catalog number: 304222), CD197-APC (Biolegend, catalog number: 353214).

[0243] For the control verification of CAR-3 and CAR-6, CAR-3 is a CAR-T cell with ERt-3 as the hinge and transmembrane structure, and CAR-6 is its corresponding traditional 8h-8TM structure CAR-T cell. The results are as Figure 6 shown in Table 9:

[0244] Table 9 Proportion of CAR-3 and CAR-6 Tnaive cells (unit: %)

[0245]

[0246] The experimental results showed that when CAR-3 and CAR-6 CAR-T cells were cultured to the 7th day, among 4 batches of donors, the proportion of naive T cells (Tnaive) in CAR-3 CAR-T cells constructed with the EGFR mutant peptide ERt-3 was significantly higher than that in traditional CD8 hinge and transmembrane CAR-6 CAR-T cells, P = 0.0129 (*). A higher proportion of the Tnaive cell subset indicates stronger in vivo persistence of CAR-T cells, thus exerting a better anti-tumor effect.

[0247] For different ScFvs (extracellular recognition domains) targeting the CD123 target, a control verification was carried out with CAR-T cells CAR-8 prepared with a CAR structure constructed with ERt-3 as the hinge and transmembrane structure and traditional 8h-8TM structure CAR-7. The results are as Figure 7 shown in Table 10:

[0248] Table 10 Proportion of each immune cell subset in CAR-7 and CAR-8 CAR-T cells (unit: %)

[0249]

[0250]

[0251] The experimental results show that when CAR-8 and CAR-7 CAR-T cells are cultured for 7 days, the proportion of naive T cells (Tnaive) in CAR-8 CAR-T cells constructed with the EGFR mutant peptide ERt-3 is significantly higher than that in traditional CAR-7 CAR-T cells with a CD8 hinge and transmembrane domain.

[0252] Further verify the applicability of the EGFR mutant peptide as a CAR hinge and transmembrane domain on the CD19 target. Among them, CAR-9 has a traditional CD8 hinge and transmembrane domain, and CAR-10 has a hinge and transmembrane domain of the EGFR mutant peptide. The results are as Figure 8 shown in Table 11:

[0253] Table 11 Proportions of immune cell subsets in CAR-9 and CAR-10 CAR-T cells (unit: %)

[0254] CAR number Tnaive CAR-9 33.18 CAR-10 47.11

[0255] The experimental results show that when CAR-9 and CAR-10 CAR-T cells are cultured for 7 days, the proportion of naive T cells (Tnaive) in CAR-10 CAR-T cells constructed with the EGFR mutant peptide ERt-3 is significantly higher than that in traditional CAR-9 CAR-T cells with a CD8 hinge and transmembrane domain.

[0256] Further compare the differences between CAR-4 containing the ERt-4 mutant peptide and CAR-6 with a traditional CD8 hinge and transmembrane domain. The results are as Figure 9 shown in Table 12.

[0257] Table 12 Proportions of immune cell subsets in CAR-4 and CAR-6 CAR-T cells (unit: %)

[0258]

[0259] The experimental results show that when CAR-4 and CAR-6 CAR-T cells are cultured for 8 days, the proportion of the Tnaive subset of CAR-4 cells is significantly higher than that of CAR-6 cells both under the CD3 gate and the CAR+ gate.

[0260] The results indicate that: for different targets or different extracellular recognition domains of the same target, using the EGFR mutant peptides ERt-3 and ERt-4 protected by the present invention as the hinge and transmembrane domains of the CAR structure is beneficial to the maintenance of the Tnaive phenotype of T cells and CAR-T cells after expression on T cells compared with the traditional CAR structure.

[0261] Example 8 Detection of CAR-modified T cell exhaustion phenotype constructed with EGFR mutant peptides

[0262] T cell exhaustion is a state of dysfunction that T cells undergo under chronic infection and tumor conditions. The main characteristics of exhausted T cells (Tex) are the progressive loss of effector functions, mainly reflected in the continuous high expression of multiple inhibitory receptors, changes in transcription factor expression, and intracellular metabolism. A key protein on the surface of T cells, Tim-3, PD-1, and LAG-3, can serve as markers of T cell exhaustion. Therefore, next, the effects of CAR-3 with a truncated EGFR mutant peptide ERt-3 as the hinge and transmembrane region of CAR, and CAR-6 with a traditional CD8 hinge and CD8 transmembrane domain on the expression of CD123 CAR-T cell exhaustion markers were compared.

[0263] When CAR-3 and CAR-6 CAR-T cells were cultured for 7 days, the expression levels of CAR-T cell exhaustion markers Tim-3, PD-1, and LAG-3 were detected by flow cytometry. The detection antibodies were: CD3 PE-Cy7 (Biolegend, catalog number: 300420), Tim-3 PerCP-Cy5.5 (Biolegend, catalog number: 345016), PD-1-APC (Biolegend, catalog number: 329908), LAG-3 BV421 (Biolegend, catalog number: 369314).

[0264] The detection results are as Figure 10 and Table 13 shows:

[0265] Table 13 Expression levels of CAR-3 and CAR-6 CAR-T cell exhaustion markers (unit: %)

[0266] Exhaustion marker CAR-3 CAR-6 ControlT Tim-3 0.34 0.49 0.21 PD-1 2.02 4.09 3.08 LAG-3 3.69 11.9 5.18

[0267] Compared with CAR-6, the expression levels of the exhaustion markers Tim-3, PD-1, and LAG-3 of CAR-3 were 69.4%, 49.4%, and 31% of CAR-6 using the wild-type ERt-1 structure, significantly lower than those of CAR-6. Among them, the expression difference of LAG-3 was the largest, suggesting that CAR-3 has lower expression levels of exhaustion markers and better anti-tumor potential.

[0268] The effects of CAR-4 with a truncated EGFR mutant peptide ERt-4 as the hinge and transmembrane region of CAR, and CAR-6 with a traditional CD8 hinge and CD8 transmembrane domain on the expression of CD123 CAR-T cell exhaustion markers were compared. The results are as Figure 11 and Table 14 shows:

[0269] Table 14 Expression levels of CAR-4 and CAR-6 CAR-T cell exhaustion markers (unit: %)

[0270] Exhaustion marker CAR-4 CAR-6 Tim-3 0.25 0.49 PD-1 2.09 4.09 LAG-3 4.48 11.90

[0271] Compared with CAR-6, the expression levels of the exhaustion markers Tim-3, PD-1, and LAG-3 in CAR-4 were 51.0%, 51.1%, and 37.6% of that of CAR-6 using the wild-type ERt-1 structure, significantly lower than that of CAR-6. Among them, the expression difference of LAG-3 was the largest, suggesting that CAR-4 has lower exhaustion marker expression levels and better anti-tumor potential.

[0272] The results showed that using the EGFR mutant peptides ERt-3 and ERt-4 protected by the present invention as the hinge and transmembrane domains of the CAR structure could reduce the expression levels of CAR-T cell exhaustion markers and the proportion of CAR-T cell exhaustion after expression on T cells compared with traditional CAR structures.

[0273] Example 9 Detection of the persistence of CAR-modified T cells constructed with EGFR mutant peptides

[0274] The multi-round tumor re-killing ability is to co-incubate CAR cells and tumor cells expressing CAR antigens at a certain ratio in vitro, and effectively evaluate the in vivo anti-tumor function and long-term survival of CAR cells by monitoring the continuous killing of tumor target cells by effector CAR cells, calculating the number of exposures of target cells, and the proliferation / survival rate of CAR cells exposed to antigens for a long time. Therefore, in vitro continuous multi-round tumor re-killing ability experiments were planned to evaluate the anti-tumor ability of CAR-3 and CAR-6 cells and the survival rate of CAR-T cells under continuous multi-round stimulation of target cells.

[0275] CAR-3 and CAR-6 CAR-T cells were counted separately. CD123+THP1-Luc-GFP was used as the positive target cell (the target cell was derived from the corresponding cell purchased from the ATCC official website), centrifuged, and counted. In the first round of plating CAR-T cells and tumor cells: Add the corresponding volume of tumor cells and CAR-T cells (CAR-T cells: tumor cells = 1:1). Observe the cell killing situation when CAR-T cells and tumor cells were co-cultured for 2-3 days, and detect the GFP expression ratio. When GFP > 10%, the cells were treated with a medium change (centrifuged at 300g for 5-10 min, discard the original medium and resuspend with an equal volume of fresh medium). Subsequently, select an appropriate time to send the sample for GFP detection again according to the observed cell killing situation; when GFP ≤ 10%, the next round of experiment was carried out. When CAR-T cells lost their tumor killing ability and the proportion of tumor cells > 90% (GFP > 90%), the stimulation ended.

[0276] The results are as follows Figure 12 As shown, under the stimulation of positive target cells CD123+THP1-Luc-GFP, CAR-3CAR-T cells can effectively kill target cells in three consecutive rounds of target cell stimulation, while CAR-6CAR-T cells can only maintain the killing ability of target cells for two rounds in multiple rounds of stimulation under the same conditions.

[0277] In the present invention, the effects of different extracellular recognition domains on the persistence ability of CAR constructed with EGFR mutant peptides on T cells were also evaluated respectively. Therefore, the multi-round stimulation abilities of CAR-7 and CAR-8 cells were also evaluated respectively. The results are as follows Figure 13 as shown

[0278] The results show that: under the stimulation of positive target cells CD123+THP1-Luc-GFP, CAR-8CAR-T cells can effectively kill target cells in three consecutive rounds of target cell stimulation, while CAR-7CAR-T cells can only maintain the killing ability of target cells for two rounds in multiple rounds of stimulation under the same conditions.

[0279] The results indicate that: for different targets or different extracellular recognition domains of the same target, using the EGFR mutant peptides protected by the present invention as the hinge and transmembrane domains of the CAR structure shows more persistent anti-tumor effects and long-term survival in the face of consecutive multiple rounds of stimulation tests of target cells compared with the traditional CAR structure.

[0280] Example 10 Verification of the ADCC effect of EGFR mutant peptides

[0281] EGFR mutant peptides ERt-3 and ERt-4 can act as safety switches as CAR structures. Among them, the EGFR mutant peptides contain the binding sites of cetuximab, and the constructed CAR has an antibody-dependent cell-mediated cytotoxicity (ADCC effect) similar to that of the traditional EGFRt safety switch (when the traditional EGFRt exerts the ADCC effect, it exists independently of the CAR structure in the forms of CAR-2A-EGFRt and EGFRt-2A-CAR).

[0282] NK cells were isolated from PBMCs (Miltenyi NK isolation kit) as effector cells and co-cultured with CAR-3 CAR-T cells at a ratio of 1:1, with or without cetuximab (Cetuximab, Erbitux). The final concentrations of cetuximab were 10 μg / mL and 50 μg / mL respectively. After 24 h, CD3 and CAR expression were detected by flow cytometry. ADCC percentage = (1 - positive rate of CAR in the monoclonal antibody group / positive rate of CAR in the antibody-free group) × 100%.

[0283] The results are as Figure 14 shown in

[0284] Table 15 ADCC efficiency of CAR-3 at different cetuximab concentrations and effector-target ratios (unit: %)

[0285] NK / CAR-T = 1:1 NK / CAR-T = 4:1 Erbitux 10μg / mL 31.36 72.75 Erbitux 50μg / mL 53.04 74.67

[0286] When the final concentration of cetuximab was 10 μg / mL, the NK / CAR-T cell ratios were 1:1 and 4:1 respectively, and the action time was 24 h, the ADCC efficiencies reached 31.36% and 72.75% respectively. When the final concentration of cetuximab was 50 μg / mL, the NK / CAR-T cell ratios were 1:1 and 4:1 respectively, and the action time was 24 h, the ADCC efficiencies reached 53.04% and 74.67% respectively.

[0287] CAR structures constructed based on the EGFR mutant peptide ERt-4 and different extracellular recognition domains also had ADCC effects. The results are as Figure 15 shown in

[0288] Table 16 ADCC efficiencies of CAR-4 and CAR-6

[0289] CAR structure ADCC efficiency (%) CAR-4 43.23 CAR-6 1.49

[0290] When the final concentration of cetuximab was 10 μg / mL, the NK / CAR-T cell ratio was 1:1, and the action time was 24 h, the ADCC efficiencies of CAR-4 and CAR-6 were 43.23% and 1.49% respectively, which demonstrated the advantages of CAR-4 as a hinge and transmembrane domain, neither affecting the function of CAR-T cells, having a more primitive phenotype and lower expression of exhaustion markers, and in addition, fully exerting the ADCC effect as a safety switch.

[0291] The test results showed that: using the EGFR mutant peptide protected by the present invention as the hinge and transmembrane domain of the CAR structure can not only normally transmit CAR signals and exert the function of CAR-T cells, but also exert the effect of a safety switch, that is, exhibit excellent ADCC effects.

[0292] Example 11 Verification of CAR Structure with EGFR Mutant Peptide in Solid Tumors

[0293] For the CEA target, based on the EGFR mutant peptide ERt-4, ERt-4 was used as the hinge region of the CAR structure to construct the CAR structure. The construction of the CAR and the preparation of CAR-T refer to Example 2 and Example 3, and the constructed CAR structure is CAR-11. The detection results of the positive rate expression are shown in Table 17:

[0294] Table 17 Detection Results of the Positive Rate of CAR-11 (Unit: %)

[0295] CAR positive rate Batch 1 74.31 Batch 2 78.33 Batch 3 79.64

[0296] The preparation results of 3 batches of CAR-T cells showed that the CAR positive rate of CAR-11 was very stable and had a high consistency among multiple batches.

[0297] Using CEA+DLD1-CEA-Luc-GFP cells as positive target cells (the target cells were obtained by exogenous construction from the corresponding cells purchased from the ATCC official website), and using DLD1-Luc-GFP as the control target cells. The CAR-T cells were seeded in the target cells at a ratio of 4:1, and the killing effect was detected by Luciferase 24 hours later. Principle of Luciferase: When detecting, the target cells were lysed with lysis buffer, and the Luciferase in them would decompose the substrate to emit fluorescence. The formula for analyzing the results using the fluorescence value was: CAR-T cell killing rate = 1 - (fluorescence value of the experimental group ÷ fluorescence value of the blank control group) × 100%. The results are as Figure 16 and Table 18 show:

[0298] Table 18 In Vitro Killing Effect of CAR-11 on Target Cells DLD1-CEA-Luc-GFP (Unit: %)

[0299] DLD1-CEA-Luc-GFP DLD1-Luc-GFP CAR-11 90.84 23.15 ControlT 11.82 6.13

[0300] CAR-11 showed a good killing effect on the positive target cells DLD1-CEA-Luc-GFP.

[0301] Using CEA+ target cell A549-CEA-Luc-GFP as the positive target cell (exogenous overexpression of CEA based on the lung cancer cell line A549), pre-tumor formation was carried out by subcutaneous injection of NCG mice at a dose of 3e6 cells / mouse, and the pre-tumor formation time was 15 days. After 8 days of in vitro culture of CAR-11 modified T cells, the pre-tumor formed NCG mice were injected via the tail vein at a dose of 1.5e7 copies / mouse. T cells without CAR modification were injected in the same dose and manner as the control group. After tumor implantation, the tumor growth was observed. On the day of test article administration, the tumor size was measured with a vernier caliper, measuring the longest diameter (length) and the shortest diameter (width). The tumor volume calculation formula was: volume = length × width × width / 2. Tumor measurements were performed twice a week, the tumor volume was calculated, and a graph was plotted. The results are as Figure 17 shown:

[0302] The results showed that CAR-11 modified T cells could significantly inhibit the proliferation of A549-CEA-Luc-GFP cells, demonstrating good anti-tumor effects.

[0303] The same verification was carried out in the pancreatic cancer indication. Clinical tissue samples from pancreatic cancer were subcutaneously transplanted into 6-8 week old immunodeficient mice through an inoculation needle, and the tumor growth changes were observed weekly. When the tumor volume was about 100 mm3, CAR-T cell reinfusion was performed (recorded as D0, and the measurement cycle and analysis were the same as above). At the same time, HE and IHC staining were used to determine the heterogeneity and antigen expression of the tumor tissue. After 8 days of in vitro culture of CAR-11 modified T cells, the pre-tumor formed NCG mice were injected via the tail vein at a dose of 1.5e7 copies / mouse. T cells without CAR modification were injected in the same dose and manner as the control group. After tumor implantation, the tumor growth was observed. On the day of test article administration, the tumor size was measured with a vernier caliper, measuring the longest diameter (length) and the shortest diameter (width). The tumor volume calculation formula was: volume = length × width × width / 2. Tumor measurements were performed twice a week, the tumor volume was calculated, and a graph was plotted. The results are as Figure 18 shown:

[0304] The results showed that CAR-11 modified T cells could completely eliminate PDX tumors derived from pancreatic cancer and maintain it until 45 days without tumor recurrence, effectively demonstrating the in vivo anti-tumor function and long-term survival of CAR-11 cells.

[0305] Furthermore, the same verification was carried out in the liver cancer indication. Clinical tissue samples from liver cancer were subcutaneously transplanted into 6-8 week old immunodeficient mice through an inoculation needle, and the tumor growth changes were observed weekly. When the tumor volume was 1000 mm 3Around this time, CAR-T cell reinfusion was performed (denoted as the D0 measurement cycle, and the analysis was the same as above). At the same time, HE and IHC staining were used to determine the heterogeneity and antigen expression of tumor tissues. After 8 days of in vitro culture, CAR-11-modified T cells were intraperitoneally injected into NCG mice with pre-established tumors at a dose of 5.00E6 copies / mouse. T cells without CAR modification were injected in the same dose and manner as the control group. After tumor implantation, the tumor growth was observed. On the day of the test article administration, the tumor size was measured with a vernier caliper, measuring the longest diameter (length) and the shortest diameter (width). The tumor volume calculation formula was: volume = length × width × width / 2. Tumor measurements were performed twice a week, and the tumor volume was calculated and plotted. The results are as Figure 19 shown:

[0306] The results showed that: CAR-11-modified T cells in the CEA+ liver cancer PDX model with a tumor volume of about 1000 mm 3 showed a strong anti-tumor ability even when the effective dose of CAR was further reduced, and the tumor was effectively controlled.

[0307] In summary, the EGFR mutant peptide described in the present invention can be used as the hinge of the CAR structure or the hinge and transmembrane for CAR design. The CAR structure can be directed against different targets or different extracellular recognition domains of the same target, and can be any CAR structure or combination of CAR structures (such as the combination of the CEA-targeted CAR of CAR11 and the SIRPγ-28TM-28 fusion protein). The above CAR structure can be applied to T cells and NK cells to construct engineered T cells and NK cells, making them have better in vivo efficacy and safety.

[0308] Example 12 Combined Application of EGFR Mutant Peptide in Cell Therapy

[0309] Using the EGFR mutant peptide as the hinge and / or transmembrane domain of the CAR, the linker of the bispecific antibody can be bridged to achieve the combined application with multi-target antibodies / ADC antibodies. The schematic diagram is as Figure 20 shown:

[0310] PSCA ScFv and the EGFR monoclonal antibody cetuximab were respectively constructed into eukaryotic expression vectors, and the corresponding plasmid numbers were A-20-10 (the specific structure was PSCA(ScFv)-Linker-EGFR(cetuximab ScFv)). The accuracy of the plasmid sequence was verified by sequencing, and the CHO eukaryotic expression system was used to produce the antibody. After purification by Protein A, the antibody was identified by SDS-PAGE and size exclusion chromatography.

[0311] Flow cytometry was used to verify whether the bispecific antibody could bind to PSCA, and the detected antibody was the PSCA-His antigen. The results are asFigure 21 , 22 As shown in 22 , the abscissa is the detection density of the PSCA antigen, which is labeled with a His tag and thus represented by His.

[0312] The results show that the bispecific antibody can effectively recognize the PSCA antigen. Moreover, the results also show that there is a high consistency between the positive rate of antigen detection and Protein L detection.

[0313] Using PSCA+HPAC-Luc-GFP cells as positive target cells (the target cells are derived from the corresponding cells purchased from the ATCC official website), where the HPAC target cells are a positive cell line with low to moderate expression of CEA, and the expression ratio is between 30-40%, the results are as Figure 23As shown (the positive rate of CEA was 39.32%). CAR-11 and Control T cells were seeded into target cells at a ratio of 1:2 respectively. A-20-10 is a PSCA / EGFR bispecific antibody with the amino acid sequence "MALPVTALLLPLALLLHAARPELDIEMTQSPSSLSASVGDRVTITCRASQSISSHLNWYQQKSGKAPKLLIYAASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSPPWTFGQGTKLELKRGSTSGSGKPGSGEGSTKGEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDTMIVANAFDIWGQGTTVTVSSEFEAAAKEAAAKEAAAKLEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRGSTSGSGKPGSGEGSTKGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSATS (SEQ ID No.36)". 2 μg and 5 μg of A-20-10 were added respectively, with the group without bispecific antibody as the control. After 24 hours, the killing effect was detected by Luciferase. Principle of Luciferase: When the target cells are lysed with lysis buffer during detection, the Luciferase in them will decompose the substrate to emit fluorescence. The formula for analyzing the results using the fluorescence value is: CAR-T cell killing rate = 1 - (fluorescence value of the experimental group ÷ fluorescence value of the blank control group) × 100%. The results are shown in Table 19:

[0314] Table 19 Killing detection results of bispecific antibody and CAR-11 cells (unit: %)

[0315] HPAC-Luc-GFP CAR-11 23.51 CAR-11 + 2 μg A-20-10 48.91 ControlT 3.52 ControlT + 2 μg A-20-10 8.66

[0316] The results showed that after treatment with the bispecific antibody A-20-10, the killing function of CAR-11 was significantly enhanced, more than doubling compared with that of CAR-11 alone.

[0317] Using HPAC-Luc-GFP cells as positive target cells (the target cells are obtained from the corresponding cells purchased from the ATCC official website and are obtained through exogenous construction), the CAR-T cells were seeded into the target cells at a ratio of 1:2. A-20-10 is a PSCA / EGFR bispecific antibody. 2 μg and 5 μg of A-20-10 were added respectively, and the group without adding the bispecific antibody was used as a control. After 24 hours of killing, the cell supernatant was collected for the detection of the IFN-γ secretion ability of CAR-T cells after being stimulated by target cells. For the collected supernatant, the ELISA (enzyme-linked immunosorbent assay) method was used to detect the secretion of IFN-γ. Among them, IFN-γ was purchased from BD Company, product number: 555142. The specific detection method was operated according to the instruction manual. The results are shown in Table 20:

[0318] Table 20 Detection results of cytokine secretion of bispecific antibody and CAR-11 cells (unit: pg / mL)

[0319] HPAC-Luc-GFP CAR-11 1961.023 CAR-11 + 2 μg A-20-10 3303.315 ControlT 0 ControlT + 2 μg A-20-10 0

[0320] The results show that the CAR containing ERt-4 of the present invention can be combined with the bispecific antibody recognizing PSCA / EGFR for the treatment of pancreatic cancer (HPAC is a pancreatic cancer cell line), and the effectiveness is higher than that of single CAR-T treatment. Although the tumor cells used in the examples are pancreatic cancer cells, and the CAR-T used is CAR-T cells expressing the CAR-11 structure, combined with the bispecific antibody recognizing PSCA / EGFR, based on the content disclosed in the present invention, those skilled in the art can obtain through simple derivation and trial: Any CAR containing the EGFR mutant peptides (ERt-3, ERt-4) of the present invention can be combined with an ADC drug or a multi-target antibody drug containing an EGFR inhibitor (which can be an antibody or a small molecule inhibitor), which can be a bispecific antibody recognizing PSCA / EGFR, or a bispecific or multi-specific antibody recognizing other non-PSCA / EGFR targets.

[0321] In summary, the engineered immune cells prepared based on the CAR structure of the EGFR mutant peptides (ERt-3, ERt-4) of the present invention, including but not limited to CAR-T and CAR-NK, can be combined with an ADC drug or a multi-target antibody drug containing an EGFR inhibitor (which can be an antibody or a small molecule inhibitor).

[0322] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An EGFR mutant, characterized in that it has: (I) an amino acid sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5; or (II) a sequence with one or more amino acids substituted, deleted, added and / or replaced on the basis of 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).

2. A nucleic acid molecule encoding the EGFR mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that it has: (IV) a nucleotide sequence shown in SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 or SEQ ID No.12; or (V) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (IV) but is different from the nucleotide sequence shown in (IV) due to the degeneracy of the genetic code; or (VI) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (IV) or (V), and having the same or similar function as the nucleotide sequence shown in (IV) or (V); or (VII) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (IV) to (VI).

4. Use of any of the following as a hinge region or simultaneously as a hinge region and a transmembrane region in constructing an engineered receptor: (1) the EGFR mutant according to claim 1; and / or (2) the nucleic acid molecule according to claim 2 or 3.

5. An engineered receptor, characterized in that it comprises any of the following: (3) an antigen recognition region, the EGFR mutant according to claim 1, a transmembrane region and an intracellular signaling region; and / or (4) an antigen recognition region, the EGFR mutant according to claim 1 and an intracellular signaling region.

6. The engineered receptor according to claim 5, characterized in that the engineered receptor comprises a fusion protein, a chimeric antigen receptor or a co-stimulatory receptor.

7. The engineered receptor according to claim 5 or 6, characterized in that the intracellular signaling region comprises a primary signaling domain and / or a co-stimulatory signaling domain.

8. The engineered receptor according to claim 7, characterized in that the intracellular signaling region comprises any of the following: 1) a primary signaling domain; or 2) a co-stimulatory signaling domain; or 3) a co-stimulatory signaling domain and a primary signaling domain; or 4) multiple co-stimulatory domains and one primary signaling domain.

9. The engineered receptor according to any one of claims 5 to 8, characterized in that the engineered receptor is a chimeric antigen receptor; the antigen recognition region of the chimeric antigen receptor comprises an antibody fragment, a single-chain antibody (ScFv), a ligand, a receptor or an artificial structure capable of specifically recognizing and binding to a target antigen.

10. The engineered receptor according to claim 9, characterized in that The antigen recognition region includes, but is not limited to, ScFv against one or more of the targets CD123, CD19, CEA or CD22; The transmembrane region includes CD8TM derived from human CD8 transmembrane or CD28TM derived from CD28 transmembrane; The intracellular signaling region includes the signal transduction structure CD3ζ and / or the co-stimulatory signaling domain 4-1BB.

11. The engineered receptor according to claim 10, wherein, (5), the ScFv targeting CD123 has the amino acid sequence shown in SEQ ID No. 27 or SEQ ID No. 28; or (6), the ScFv targeting CD19 has the amino acid sequence shown in SEQ ID No. 29; or (7), the ScFv targeting CEA has the amino acid sequence shown in SEQ ID No. 30; or (8), the ScFv targeting CD22 has the amino acid sequence shown in SEQ ID No. 38; or (9), the CD8TM or CD28TM has the amino acid sequence shown in SEQ ID No. 21 or SEQ ID No. 25; or (10), the CD3ζ has the amino acid sequence shown in SEQ ID No. 23; or (11), the co-stimulatory signaling domain 4-1BB has the amino acid sequence shown in SEQ ID No. 22; or (12), a sequence with substitution, deletion, addition and / or replacement of one or more amino acids based on the amino acid sequence shown in any one of (5) to (11); or (13), an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of (5) to (12).

12. The engineered receptor according to any one of claims 5 to 11, wherein, the engineered receptor further includes SIRPγ; the SIRPγ has: (14), the amino acid sequence shown in SEQ ID No. 24; or (15), a sequence with substitution, deletion, addition and / or replacement of one or more amino acids based on the amino acid sequence shown in (14); or (16), an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (14).

13. The engineered receptor according to any one of claims 5 to 12, wherein, the engineered receptor includes CAR-3, CAR-4, CAR-8, CAR-10 and CAR-11; (A), the CAR-3, CAR-4, CAR-8, CAR-10 and CAR-11 have the amino acid sequences shown in SEQ ID No. 31, 32, 33, 34 and 35 in sequence; or (B), a sequence with substitution, deletion, addition and / or replacement of one or more amino acids based on the amino acid sequence shown in (A); or (C), an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (A) or (B).

14. A gene element, wherein, Comprising the nucleic acid molecule according to claim 2 or 3.

15. The gene element according to claim 14, wherein, the gene element comprises CAR-3, CAR-4, CAR-8, CAR-10 and CAR-11; (D), the CAR-3, CAR-4, CAR-8, CAR-10 and CAR-11 successively have the nucleotide sequences shown in SEQ ID No. 13, 14, 15, 16 and 17; or (E), a nucleotide sequence obtained by substitution, deletion or addition of one or more nucleotide sequences to the nucleotide sequence shown in (D), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (D); or (F), a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in (D) or (E).

16. An artificial vector, wherein, it comprises the gene element according to claim 14 or 15 and an acceptable vector.

17. A virus particle, wherein, it comprises the artificial vector according to claim 16.

18. An engineered cell, wherein, it comprises or expresses any of the following: (a), the EGFR mutant according to claim 1; and / or (b), the nucleic acid molecule according to claim 2 or 3; and / or (c), the engineered receptor according to any one of claims 5 to 13; and / or (d), the gene element according to claim 14 or 15; and / or (e), the artificial vector according to claim 16; and / or (f), the virus particle transduced as claimed in claim 17.

19. The engineered cell according to claim 18, wherein, the engineered cell includes but is not limited to NK cells and T cells.

20. Use of any of the following in enhancing the in vivo and in vitro survival ability of target cells, bridging bispecific antibodies and / or simultaneously recognizing EGFR antigen and other specific target antigens on target cells: (a), the EGFR mutant according to claim 1; and / or (b), the nucleic acid molecule according to claim 2 or 3; and / or (c), the engineered receptor according to any one of claims 5 to 13; and / or (d), the gene element according to claim 14 or 15; and / or (e), the artificial vector according to claim 16; and / or (f), the virus particle transduced as claimed in claim 17; and / or (g), the engineered cell according to claim 18 or 19.

21. Use of any of the following as a detection and / or screening marker: (a), the EGFR mutant according to claim 1; and / or (b), the nucleic acid molecule according to claim 2 or 3; and / or (c), the engineered receptor according to any one of claims 5 to 13; and / or (d), the gene element according to claim 14 or 15; and / or (e), the artificial vector according to claim 16.

22. A detection marker and / or a screening marker, wherein, it comprises any of the following: (a), the EGFR mutant according to claim 1; and / or (b), a nucleic acid molecule as described in claim 2 or 3; and / or (c), an engineered receptor as described in any one of claims 5 to 13; and / or (d), a gene element as described in claim 14 or 15; and / or (e), an artificial vector as described in claim 16.

23. Use of any of the following in the preparation of a medicament for preventing and / or treating a disease: (a), an EGFR mutant as described in claim 1; and / or (b), a nucleic acid molecule as described in claim 2 or 3; and / or (c), an engineered receptor as described in any one of claims 5 to 13; and / or (d), a gene element as described in claim 14 or 15; and / or (e), an artificial vector as described in claim 16; and / or (f), a virus particle transducing as described in claim 17; and / or (g), an engineered cell as described in claim 18 or 19.

24. The use as described in claim 22, characterized in that the disease includes but is not limited to tumors.

25. A medicament, characterized in that it includes any of the following and a pharmaceutically acceptable excipient or adjuvant: (a), an EGFR mutant as described in claim 1; and / or (b), a nucleic acid molecule as described in claim 2 or 3; and / or (c), an engineered receptor as described in any one of claims 5 to 13; and / or (d), a gene element as described in claim 14 or 15; and / or (e), an artificial vector as described in claim 16; and / or (f), a virus particle transducing as described in claim 17; and / or (g), an engineered cell as described in claim 18 or 19.

26. A drug combination, characterized in that it includes the medicament as described in claim 25 and any other active ingredient.

27. The drug combination as described in claim 26, characterized in that the any other active ingredient includes a multi-target antibody drug or an ADC drug containing an EGFR inhibitor.

28. The drug combination as described in claim 27, characterized in that the multi-target antibody drug includes a bispecific antibody recognizing PSCA / EGFR, a bispecific antibody recognizing non-PSCA / EGFR targets or a multispecific antibody recognizing non-PSCA / EGFR targets; the ADC drug containing an EGFR inhibitor includes an antibody containing EGFR or an inhibitor containing EGFR.

Images