Antigen binding molecule for recognizing CD5 and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an antigen binding molecule for recognizing a CD5 molecule and application of the antigen binding molecule. The antigen binding molecule for identifying the CD5 molecule obtained by screening is a brand new human antibody sequence, so that the immunogenicity of a mouse antibody is avoided; experiments show that the antigen-binding molecule for identifying the CD5 molecule can identify the CD5 molecule with high affinity, can be used as a detection reagent for detecting the expression condition of the CD5 molecule on the surface of a target cell, can also be used as a detection reagent for identifying the expression condition of the CD5 in a target sample such as a tumor sample, and can also be used as an in-vivo tracing molecule in some embodiments, so that the antigen-binding molecule can be used for identifying the CD5 molecule. And the CD5 molecule is subjected to tracing marking. The invention further provides a CD5 chimeric antigen receptor CAR-CD5 containing the single-chain antibody, and after the CAR-CD5 is combined with CD5 on tumor cells, the obvious anti-tumor activity is shown.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to single-chain antibodies targeting CD5 and their applications. Background Art

[0002] Adoptive cell therapy (ACT) refers to separating immune active cells from tumor patients, amplifying and functionally identifying them in vitro, and then re-infusing them into the patients to directly kill tumors or stimulate the body's immune response to kill tumor cells.

[0003] Immunotherapy is the fourth tumor treatment method after surgery, radiotherapy, and chemotherapy. Chimeric antigen receptor (CAR) T cell technology is a newly rapidly developed cell immunotherapy technology. CAR is an artificially synthesized fusion receptor, which structurally includes an extracellular antigen-binding region, a transmembrane region, an intracellular signal transduction region, and a co-stimulatory signal region. The extracellular region is a monoclonal antibody sequence (single-chain variable fragment, scFv) that recognizes tumor-associated antigens. The transmembrane region connects the extracellular region and the intracellular region, and the commonly used transmembrane region molecules are selected from the transmembrane functional domains of genes such as CD3, CD4, CD8, and CD28.

[0004] T-ALL (acute T lymphoblastic leukemia) is a highly aggressive hematological T cell malignancy disease, accounting for about 25% of adult ALL and 15% of childhood ALL. Approximately 64,000 patients are diagnosed with ALL globally every year. The recurrence rate and mortality rate of T cell malignancy patients are usually very high. The prognosis of recurrent patients is poor, the treatment options are extremely limited, and the survival rate over 5 years is less than 10%.

[0005] CD5 is a T cell surface glycoprotein, also known as lymphocyte antigen T1 / LEU1 and LEU1. It is phosphorylated by LYN at tyrosine residues, so CD5 can create a binding site for PTPN6 / SHP-1. CD5 may be a receptor that regulates T cell proliferation and is expressed at different developmental and activation stages of human B cells. CD5 is often expressed in normal T cells, approximately 85% of T cell malignancies, and some B cell malignancies, and it acts as an inhibitory receptor for the T cell receptor (TCR) and B cell receptor (BCR).

[0006] The extracellular antigen recognition domain, which can also be referred to as the extracellular antigen-binding domain, can specifically recognize tumor surface antigens. The CAR molecule transmits immune cell activation signals through the hinge region and transmembrane region by virtue of the characteristic of specifically recognizing tumor antigens through the antigen-binding domain. The selection of the extracellular antigen recognition domain is crucial for the exertion of CAR function, and it is impossible to simply infer whether an antigen-binding molecule is suitable for CAR molecule construction based on data such as antibody affinity. Therefore, for immunotherapy targeting CD5, it is very necessary to screen antigen-binding molecules that recognize CD5. Summary of the Invention

[0007] In view of this, the present invention provides an antigen-binding molecule that recognizes CD5 and its applications. The antigen-binding molecule described in the present invention can be scFv, which is a brand-new human antibody sequence, avoiding the immunogenicity of murine antibodies, and can all recognize CD5 molecules with high affinity. The CD5 antigen-binding molecule provided by the present invention can also be used to construct a protein composition such as a chimeric antigen receptor. After the chimeric antigen receptor binds to CD5 on tumor cells through the CD5 antigen-binding molecule, it shows obvious anti-tumor activity. The CD5 antigen-binding molecule therein is a fully humanized antibody, CD5-scFv, which has a more suitable affinity for human CD5 antigen.

[0008] An antigen-binding molecule that recognizes a CD5 molecule, wherein the three CDRs of its light chain respectively have the amino acid sequences shown in any one of SEQ ID NO: 1 to 12, or amino acid sequences having at least 80% homology therewith; the three CDRs of its heavy chain respectively have the amino acid sequences shown in any one of SEQ ID NO: 13 to 24, or amino acid sequences having at least 80% homology therewith.

[0009] In some embodiments, the CDR sequences of the antigen-binding molecule are selected from any one of the following a) to d):

[0010] a) The three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 1, 5, and 9, or amino acid sequences having at least 80% homology therewith; the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 13, 17, and 21, or amino acid sequences having at least 80% homology therewith;

[0011] b) The three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 2, 6, and 10, or amino acid sequences having at least 80% homology therewith; the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 14, 18, and 22, or amino acid sequences having at least 80% homology therewith;

[0012] c), the three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 3, 7 and 11, or amino acid sequences having at least 80% homology therewith; the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 15, 19 and 23, or amino acid sequences having at least 80% homology therewith;

[0013] d), the three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 4, 8 and 12, or amino acid sequences having at least 80% homology therewith; the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 16, 20 and 24, or amino acid sequences having at least 80% homology therewith.

[0014] In some specific embodiments, the present invention provides four antigen-binding molecules, respectively labeled as CD5(10), CD5(13), CD5(15) and CD5(16), including the following CDR region sequences:

[0015] CD5(10): The three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 1, 5 and 9, and the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 13, 17 and 21;

[0016] CD5(13): The three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 2, 6 and 10, and the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 14, 18 and 22;

[0017] CD5(15): The three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 3, 7 and 11, or amino acid sequences having at least 80% homology therewith; the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 15, 19 and 23, or amino acid sequences having at least 80% homology therewith;

[0018] CD5(16): The three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 4, 8 and 12, and the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 16, 20 and 24.

[0019] In some embodiments, among the antigen-binding molecules of the present invention, the 4 FRs of the light chain are selected from the amino acid sequences of any of the following or amino acid sequences having at least 80% homology therewith:

[0020] The amino acid sequences at positions 1-26, 33-49, 53-88, or 98-108 of the amino acids shown in SEQ ID NO:25;

[0021] Or, the amino acid sequences at positions 1-26, 33-49, 53-88, and 98-108 of the amino acids shown in SEQ ID NO:26;

[0022] Or, the amino acid sequences at positions 1-26, 33-49, 53-88, 97-107 of the amino acids shown in SEQ ID NO:27;

[0023] Or, the amino acid sequences at positions 1-26, 33-49, 53-88, 98-108 of the amino acids shown in SEQ ID NO:28.

[0024] The 4 FRs of the heavy chain are selected from any one of the following amino acid sequences or an amino acid sequence having at least 80% homology thereto:

[0025] The amino acid sequences at positions 1-25, 34-50, 59-96, or 109-119 of the amino acids shown in SEQ ID NO:29;

[0026] Or, the amino acid sequences at positions 1-25, 34-50, 59-96, or 109-119 of the amino acids shown in SEQ ID NO:30;

[0027] Or, the amino acid sequences at positions 1-25, 34-50, 59-96, or 113-123 of the amino acids shown in SEQ ID NO:31;

[0028] Or, the amino acid sequences at positions 1-25, 34-50, 59-96, 116-126 of the amino acids shown in SEQ ID NO:32.

[0029] In some specific embodiments, the antigen-binding molecules of the present invention are respectively labeled as CD5(10), CD5(13), CD5(15), CD5(16), and respectively contain the FR region sequences shown in the following ①-④:

[0030] CD5(10): ① The 4 FRs of its light chain respectively have the amino acid sequences at positions 1-26, 33-49, 53-88, and 98-108 of the amino acids shown in SEQ ID NO:25; the 4 FRs of its heavy chain respectively have the amino acid sequences at positions 1-25, 34-50, 59-96, 109-119 of the amino acids shown in SEQ ID NO:29;

[0031] CD5(13): ② The four FRs of its light chain respectively have the amino acid sequences at positions 1 - 26, 33 - 49, 53 - 88, and 98 - 108 of the amino acids shown in SEQ ID NO:26; the four FRs of its heavy chain respectively have the amino acid sequences at positions 1 - 25, 34 - 50, 59 - 96, and 109 - 119 of the amino acids shown in SEQ ID NO:30;

[0032] CD5(15): ③ The four FRs of its light chain respectively have the amino acid sequences at positions 1 - 26, 33 - 49, 53 - 88, and 97 - 107 of the amino acids shown in SEQ ID NO:27; the four FRs of its heavy chain respectively have the amino acid sequences at positions 1 - 25, 34 - 50, 59 - 96, and 113 - 123 of the amino acids shown in SEQ ID NO:31;

[0033] CD5(16): ④ The four FRs of its light chain respectively have the amino acid sequences at positions 1 - 26, 33 - 49, 53 - 88, and 98 - 108 of the amino acids shown in SEQ ID NO:28; the four FRs of its heavy chain respectively have the amino acid sequences at positions 1 - 25, 34 - 50, 59 - 96, and 116 - 126 of the amino acids shown in SEQ ID NO:32.

[0034] In some embodiments, the light chain of the antigen - binding molecule has the amino acid sequence shown in any one of SEQ ID NO:25 - 28, or an amino acid sequence having at least 80% homology therewith; its heavy chain has the amino acid sequence shown in any one of SEQ ID NO:29 - 32, or an amino acid sequence having at least 80% homology therewith.

[0035] In the antigen - binding molecule of the present invention, the light chain and the heavy chain are connected by a linker; in some embodiments, the linker has the amino acid sequence shown in SEQ ID No.72; or an amino acid sequence having at least 80% homology therewith. The sequence of the linker is not limited thereto, and those common in the art are acceptable. In some examples, the sequence of the linker can also be (GGGGS)n, where n = 1 - 5.

[0036] In some specific examples, four antigen - binding molecules that can recognize the CD5 molecule with high affinity were screened and obtained in the present invention, which are respectively labeled as CD5(10), CD5(13), CD5(15), and CD5(16), and successively contain the light chains and heavy chains shown in A) - D):

[0037] A) Its light chain has the amino acid sequence shown in SEQ ID NO: 25; its heavy chain has the amino acid sequence shown in SEQ ID NO: 29; labeled as CD5(10).

[0038] B) Its light chain has the amino acid sequence shown in SEQ ID NO: 26; its heavy chain has the amino acid sequence shown in SEQ ID NO: 30, labeled as CD5(13);

[0039] C) Its light chain has the amino acid sequence shown in SEQ ID NO: 27; its heavy chain has the amino acid sequence shown in SEQ ID NO: 31, labeled as CD5(15);

[0040] D) Its light chain has the amino acid sequence shown in SEQ ID NO: 28; its heavy chain has the amino acid sequence shown in SEQ ID NO: 32, labeled as CD5(16).

[0041] In some specific embodiments, the antigen-binding molecules CD5(10), CD5(13), CD5(15), and CD5(16) of the present invention have the amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 37, respectively.

[0042] The present invention also provides nucleic acids encoding the single-chain antibody CD5-scFv of the claims.

[0043] In some embodiments, the nucleic acid encoding the light chain of the antigen molecule has the nucleotide sequence shown in SEQ ID NO: 64, 66, 68, or 70, or has a nucleotide sequence with at least 80% homology to the sequences shown in SEQ ID NO: 64, 66, 68, 70;

[0044] The nucleic acid encoding the heavy chain of the antigen molecule has the nucleotide sequence shown in SEQ ID NO: 65, 67, 69, or 71, or has a nucleotide sequence with at least 80% homology to the sequences shown in SEQ ID NO: 65, 67, 69, or 71.

[0045] The present invention also provides an expression vector comprising the nucleic acid encoding the antigen-binding molecule described above.

[0046] The present invention also provides a protein combination comprising the antigen-binding molecule of the present invention.

[0047] In some embodiments, the protein combination further comprises a transmembrane region, a hinge region, and an intracellular signaling functional domain.

[0048] In some embodiments, the transmembrane region includes, but is not limited to, the transmembrane regions of DAP10, DAP12, NKG2D, CD4, CD8α, or CD28;

[0049] The hinge region includes, but is not limited to, the hinge regions of IgG, IgD, CD7, or CD8α / CD28;

[0050] The intracellular signaling functional domain includes a primary signal transduction domain and / or a costimulatory domain.

[0051] The primary signal transduction domain includes, but is not limited to, the signal transduction domains of one or more of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa, etc.;

[0052] The costimulatory domain includes the signal transduction domains selected from one or more of the following molecules: DAP10, CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-l, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-l, ITGB7, TNFR2, TRANCE / RANKL, DNAMl, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and the ligands specifically binding to CD83.

[0053] In some embodiments, the protein combination of the present invention is a chimeric antigen receptor, and the chimeric antigen receptor includes, in sequence from the N-terminus to the C-terminus: the antigen-binding molecule of the present invention, a hinge region, a transmembrane region, and an intracellular signaling region.

[0054] In some embodiments, the hinge region is selected from the 8h hinge region derived from CD8a, the 8h(dc) hinge region, or the 7h hinge region derived from CD7, or the G4h hinge structure derived from IgG, including but not limited to these; in some specific embodiments, the amino acid sequences of the 8h hinge region, 8h(dc) hinge region, and 7h hinge region derived from CD8a are shown in SEQ ID NO: 45, 54, and 53 respectively, or amino acid sequences having at least 80% homology therewith.

[0055] In some embodiments, the transmembrane region is selected from at least one of CD8a, NKp44, and PDGFRβ, including but not limited to these; in some specific embodiments, the structure of the transmembrane region is at least one of CD8TM, PDGFRβTM, and NKp44TM. Specifically, the amino acid sequence of CD8TM is shown in SEQ ID NO: 46, the amino acid sequence of PDGFRβTM is shown in SEQ ID NO: 55; the amino acid sequence of NKp44TM is shown in SEQ ID NO: 56.

[0056] In some embodiments, the intracellular signaling region is selected from CD137-CD3ζ (BBz) chain or CD137 (or also referred to as BB)-DAP10, including but not limited to these. In some specific embodiments, the amino acid sequence of CD137 ICD is shown in SEQ ID NO: 47, the amino acid sequence of CD3ζ (which can be represented by CD3z) is shown in SEQ ID NO: 48, and the amino acid sequence of DAP10 is shown in SEQ ID NO: 57.

[0057] In the present invention, the protein combination may also be a chimeric antigen receptor, and the chimeric antigen receptor further includes a cytokine, and the cytokine includes but is not limited to at least one of IL-15, IL15-Ra, IL-2 (H9), IL12, IL21, IL-2, IL-7, IL-4, IL-17 or a fusion protein formed by fusing two of them. In some embodiments, the cytokine is a fusion protein of IL-15 and IL15-Ra, abbreviated as mbIL15. In some specific embodiments, the amino acid sequence of mbIL15 is shown in SEQ ID NO: 58.

[0058] In the present invention, the position of the cytokine can be the N-terminus or the C-terminus of the chimeric antigen receptor, and is connected to other elements in the chimeric antigen receptor through a linker peptide. The linker peptide is at least one of self-cleaving 2A peptides such as P2A, T2A, E2A, and F2A; in some specific embodiments, the linker peptide is 2A. In some embodiments, the linker peptide can also be an IRES (Internal Ribosome Entry Site); in some embodiments, the linker peptide can be any peptide sequence that can be cleaved in cells.

[0059] In some specific embodiments, the structure of the protein combination of the present invention is selected from any of the following:

[0060] CAR-1: CD5(JK-02-B10 / CD5(10))-scFv-8h-8TM-BBz-2A-mbIL15 (SEQ ID NO: 38);

[0061] CAR-2: CD5(JK-11-C08 / CD5(13))-scFv-8h-8TM-BBz-2A-mbIL15 (SEQ ID NO: 39);

[0062] CAR-4: CD5(JK-14-H12 / CD5(15))-scFv-8h-8TM-BBz-2A-mbIL15 (SEQ ID NO: 40);

[0063] CAR-5: CD5(JK-12-C05 / CD5(16))-scFv-8h-8TM-BBz-2A-mbIL15 (SEQ ID NO: 41);

[0064] CAR-6: IL2(H9)-PDGFRβTM-2A-CD5(JK-11-C08 / CD5(13))-scFv-8h-8TM-BBz (SEQID NO: 42);

[0065] CAR-7: IL2(H9)-PDGFRβTM-2A-CD5(JK-11-C08 / CD5(13))-scFv-8h-NKp44TM

[0066] -BB-DAP10 (SEQ ID NO: 43).

[0067] The present invention also provides nucleic acids encoding the protein combination.

[0068] In some embodiments, the nucleic acids of the present invention include at least one of the following gene structures:

[0069] CD5-scFv-8h-8TM-BBz-2A-mbIL15;

[0070] IL2(H9)-PDGFRβTM-2A-CD-scFv-8h-8TM-BBz;

[0071] IL2(H9)-PDGFRβTM-2A-CD5-scFv-8h-NKp44TM-BB-DAP10;

[0072] Among them, scFv is the coding nucleic acid of the antigen-binding molecule described in the present invention.

[0073] In some embodiments, the nucleic acid includes the nucleic acid encoding any one of the structures of CAR-1, CAR-2, CAR-4, and CAR-5; the nucleic acid has a nucleotide sequence shown in any one of SEQ ID NOs: 59-60, SEQ ID NOs: 62-63, or a nucleotide sequence having at least 80% homology thereto. In some specific embodiments, the sequence of the nucleic acid is shown in any one of SEQ ID NOs: 59-60, SEQ ID NOs: 62-63.

[0074] In some embodiments, the nucleic acid encoding the protein combination described in the present invention further includes a leader peptide. The present invention has no special limitation on the sequence of the leader peptide, and common types in the art can be used. In some specific embodiments, the sequence of the leader peptide is shown in SEQ ID NO: 44.

[0075] The present invention also provides an expression vector containing the nucleic acid.

[0076] The present invention also provides engineered cells that express at least one of ①-④:

[0077] ① The antigen-binding molecule described in the present invention;

[0078] ② The nucleic acid encoding the antigen-binding molecule described in the present invention, the nucleic acid encoding the protein combination described in the present invention;

[0079] ③ The protein combination described in the present invention;

[0080] ④ The expression vector containing the nucleic acid described in ②.

[0081] In some embodiments, the engineered cells express the protein combination described in the present invention and express at least one cytokine selected from IL-15, IL15-Ra, IL-2(H9) or a fusion protein formed by fusing two or more of these cytokines.

[0082] In some embodiments, the engineered cells are immune cells, including but not limited to T cells, B cells, NK cells, DC cells or macrophages.

[0083] The present invention also provides the use of any one of the following I) - V) in the preparation of anti-tumor drugs;

[0084] I) The antigen-binding molecule of the present invention;

[0085] II) The nucleic acid encoding the antigen-binding molecule of the present invention, the nucleic acid encoding the protein combination of the present invention;

[0086] III) The protein combination of the present invention;

[0087] IV) The expression vector containing the nucleic acid described in ②.

[0088] V) The engineered cells of the present invention.

[0089] The present invention also provides a drug, comprising any one of the following I) - V) and a pharmaceutically acceptable excipient:

[0090] I) The antigen-binding molecule of the present invention;

[0091] II) The nucleic acid encoding the antigen-binding molecule of the present invention, the nucleic acid encoding the protein combination of the present invention;

[0092] III) The protein combination of the present invention;

[0093] IV) The expression vector containing the nucleic acid described in II);

[0094] V) The engineered cells of the present invention.

[0095] The present invention also provides the use of any one of the following 1) - 4) as a screening marker or as a marker in the detection of CD5;

[0096] 1) The antigen-binding molecule of the present invention;

[0097] 2) The nucleic acid encoding the antigen-binding molecule of the present invention, the nucleic acid encoding the protein combination of the present invention;

[0098] 3) The protein combination of the present invention;

[0099] 4) The expression vector containing the nucleic acid described in 2).

[0100] The present invention also provides a screening marker or a detection marker, comprising any one of the following 1) - 4):

[0101] 1) The antigen-binding molecule of the present invention;

[0102] 2) Nucleic acids encoding the antigen-binding molecules of the present invention, nucleic acids encoding the protein combinations of the present invention;

[0103] 3) The protein combinations of the present invention;

[0104] 4) An expression vector containing the nucleic acid of 2).

[0105] The label may be one or more selected from the following for labeling: enzymes, biotin, fluorescent indicators, chemiluminescent indicators, isotopes, colloidal indicators, latex microspheres, magnetic bead indicators; wherein, the enzymes are selected from one or more of the following: horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-anti-peroxidase conjugate, alkaline phosphatase-anti-alkaline phosphatase conjugate, β-galactosidase-anti-β-galactosidase conjugate; fluorescent indicators are selected from one or more of the following: AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red or PerCP dye; chemiluminescent indicators are selected from one or more of the following: acridinium ester, acridinium sulfonamide and its derivatives, luminol (3-aminophthalhydrazide), isoluminol (4-aminophthalhydrazide), isoluminol isothiocyanate (ILITC) and its derivatives, N-(4-aminobutyl)-N-ethylisoluminol (ABEI), 4,5-diaminophthalhydrazide (DPH), aminobutylethyl phthalhydrazide (ABENH); isotopes are selected from one or more of the following: 125I / 131I / 124I, 3H, 14C, 111In, 89Zr, 32P; colloidal indicators are selected from one or more of the following: colloidal gold, colloidal carbon, colloidal selenium. The labeled antibody can be applied to flow cytometry, ELISA, and immunohistochemistry.

[0106] The beneficial effects of the present invention are as follows:

[0107] 1) The scFv in the CD5 chimeric antigen receptor provided by the present invention is a brand-new human antibody sequence, avoiding the immunogenicity of murine antibodies.

[0108] 2) After the CD5 chimeric antigen receptor provided by the present invention binds to CD5 on tumor cells, it shows obvious anti-tumor activity, and the CD5-scFv therein is a fully humanized antibody with a more appropriate affinity for the human CD5 antigen. Brief Description of the Drawings

[0109] Figure 1 Show the recognition result of JK-02-B10 protein to CD5 antigen;

[0110] Figure 2 Show the recognition result of JK-11-C08 protein to CD5 antigen;

[0111] Figure 3 Show the recognition result of JK-12-C05 protein to CD5 antigen;

[0112] Figure 4 Show the recognition result of JK-14-E05 protein to CD5 antigen;

[0113] Figure 5 Show the recognition result of JK-14-H12 protein to CD5 antigen;

[0114] Figure 6 Show the in vitro killing results of CAR-1, CAR-2, CAR-4, and CAR-5;

[0115] Figure 7 Show the in vivo pharmacodynamic experiment results of CAR-1, CAR-2, CAR-4, and CAR-5;

[0116] Figure 8 Show the in vivo expansion results of CAR-1, CAR-2, CAR-4, and CAR-5;

[0117] Figure 9 Show the in vivo pharmacodynamic experiment results of CAR-2;

[0118] Figure 10 Show the in vivo survival experiment results of CAR-2;

[0119] Figure 11 Show the in vitro killing experiment results of CAR-7;

[0120] Figure 12 Show the positive cell ratio results of CAR-2;

[0121] Figure 13 Show: the in vitro killing results of T cells modified by CAR-1 and CAR-2;

[0122] Figure 14 Show the killing results of multiple batches of CAR-2. The abscissa represents CD5 CAR-T and Control-T, and the ordinate represents the tumor killing ratio;

[0123] Figure 15 Show the continuous detection results of CAR-2 multi-round stimulated CAR-T. Specific implementation manners

[0124] The present invention provides a single-chain antibody targeting CD5, a CD5-CAR structure, CAR cells and their applications. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0125] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0126] Glossary:

[0127] 1. Expression: The expression described in the present invention includes both the direct expression of cellular proteins and the expression of genes or vectors containing the target gene after transcription and translation in cells to form proteins.

[0128] 2. 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 linker molecules, cells expressing antigen conjugates (TAC), etc. The CAR-T cells are T cells expressing chimeric antigen receptors or CARs.

[0129] 3. Protein combination: refers to a natural or artificially modified / simulated protein structure formed by multiple proteins connected by chemical bonds or linkers, 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 or multiple proteins, polypeptides or protein functional domains, and can also be a chimeric antigen receptor or a co-stimulatory receptor (CCR, chimeric costimulatory receptor). In some embodiments, the protein combination can be a chimeric antigen receptor (CAR), and the chimeric antigen receptor contains an antigen recognition region.

[0130] 4. 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. A CAR generally includes at least one extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain contains a primary signaling domain and / or a co-stimulatory signal; that is, in some embodiments, the intracellular signaling domain has only a primary signaling domain; in some embodiments, the intracellular signaling domain has only a co-stimulatory signaling domain; in some embodiments, the intracellular signaling domain has both a co-stimulatory and a primary signaling domain; in some embodiments, the intracellular signaling domain has multiple co-stimulatory and primary signaling domains.

[0131] 5. In some embodiments, the extracellular antigen-binding region of the "chimeric antigen receptor" or "CAR" structure may further comprise other non-CD5 target molecules that can recognize entities expressed on the surface of solid tumors, hematological tumor cells / tissues. The extracellular antigen-binding domain can specifically recognize antigens. Non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen-binding regions (Fab) selected from libraries, single-domain fragments, or natural ligands that bind to their homologous receptors, and artificially designed target-specific recognition domains that recognize specific targets, such as combinations with fibronectin type III (FN3) domains, designed ankyrin repeat proteins (DARPins) with target-specific recognition of specific targets. In some embodiments, the extracellular antigen-binding region may comprise scFv, Fab, or natural ligands, and any derivatives thereof. The extracellular antigen-binding region may refer to molecules other than intact antibodies, which may comprise a part of an intact antibody and can bind to the antigen bound by the intact antibody. Examples of antibody fragments may 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), where the scFv can be a murine antibody or a fully human or 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. The formation of recognition of multiple targets or different sites of the CD5 molecule, and the non-CD5 target molecules include, but are not limited to: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, 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. In some embodiments, the extracellular antigen-binding region of the "chimeric antigen receptor" or "CAR" structure may further comprise a binding molecule that can recognize a site of CD5 not recognized by the antigen-binding molecules described in the present invention.

[0132] 6. 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 hinge of human CD8), a transmembrane structure (such as the transmembrane region of human CD8, CD8TM), and an intracellular signaling domain. The intracellular signaling domain can be a primary intracellular signaling domain having only 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, etc. It can also contain, in addition to the primary intracellular signaling domain, one or two or more co-stimulatory signaling domains, or can contain only co-stimulatory signaling domains. 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 lymphocyte activation molecule (SLAM protein), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP10, 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.

[0133] 7. "Chimeric antigen receptor" or "CAR" can have various structures, such as including cytokine, antibody gene sequences that can be secreted or membrane-expressed; such as including structures that can be regulated to be activated or inactivated, and the structures for regulated 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.

[0134] 8. 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 it can be a fusion form of the expressed molecule and other polypeptides such as polypeptides derived from human CD8 and CD4.

[0135] 9. 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 of the present invention by adopting any publicly known or unknown cell preparation protocols based on the content disclosed in this specification. Therefore, regardless of the above-mentioned CAR structure, as long as it contains the antigen-binding molecule (CD5-ScFv) that recognizes CD5 of the present invention, it can be applied to construct engineered immune cells such as T cells, NK cells, macrophages, etc. described in this article.

[0136] 10. Those skilled in the art should be aware that there are many ways to define available CDRs, such as the Kabat system, the Chothia system, and the IMGT system. The amino acid numbering in the Kabat system is based on the Kabat numbering system (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991));

[0137] The amino acid numbering in the Chothia system is based on the Chothia numbering system (see, for example, Chothia and Lesk J. Mol. Biol. 196:901-917 (1987));

[0138] The amino acid numbering in the MacCallum system is based on the MacCallum numbering system (see MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008));

[0139] The amino acid numbering in the IMGT system is based on the IMGT numbering (see, for example, Lefranc MP. (2013) IMGT Unique Numbering. In: Dubitzky W., Wolkenhauer O., Cho KH., Yokota H. (eds) Encyclopedia of Systems Biology. Springer, New York, NY; https: / / doi.org / 10.1007 / 978-1-4419-9863-7_127) system;

[0140] The amino acid numbering in the AHo system (see, for example, Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001)) is based on the AHo numbering system.

[0141] Since CDRs can be defined in different ways, those skilled in the art can define CDRs in different ways from those used for LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 in this application, so as to obtain CDRs that are somewhat different in sequence from the combination of the heavy chain variable region and the light chain variable region identical to this application. Those skilled in the art should be aware that, for the CDR combinations obtained from the antibodies in this application through different definition methods, even if the sequences are different from LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 in this application, the antibodies containing the said CDR combinations are still undoubtedly covered by the scope of this application.

[0142] 11. The terms "heavy chain" ("HC"), "light chain" ("LC"), "light chain variable region" ("VL"), "heavy chain variable region" ("VH"), "framework region" ("FR") refer to the domains in naturally occurring immunoglobulins and the corresponding domains of synthetic (e.g., recombinant) binding proteins (e.g., humanized antibodies). The basic structural unit of a naturally occurring immunoglobulin (e.g., IgG) is a tetramer having two light chains and two heavy chains. The amino-terminal ("N") portion of each chain includes a variable region of about 100 to 110 or more amino acids, which is mainly responsible for antigen recognition. The carboxyl-terminal ("C") portion of each chain is defined as the constant region. The light chain has a single constant domain, and the heavy chain usually has three constant domains and a hinge region. Thus, the structure of the light chain of a naturally occurring IgG molecule is VL-CL from the N (nitrogen) terminus to the C (carbon) terminus, and the structure of the IgG heavy chain is VH-CH1-H-CH2-CH3 from the N terminus to the C terminus (where H is the hinge region). The variable region of an IgG molecule consists of complementarity-determining regions (CDRs) (containing residues that contact the antigen) and non-CDR segments (referred to as framework segments or framework regions, which maintain the structure and determine the positions of the CDR loops). Thus, the VL and VH domains have the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure from the N terminus to the C terminus.

[0143] 12. In natural antibodies, the variability is not evenly distributed in the variable regions of the antibodies. It is concentrated in three segments in the variable regions of the light and heavy chains, which are called complementarity-determining regions (CDRs) or hypervariable regions. The CDRs on the heavy chain can be referred to as HCDRn, where "n" is an integer and does not represent the order of the CDRs on the heavy chain. Similarly, the CDRs on the light chain can be referred to as LCDRn, where "n" is an integer that labels the CDR and does not represent the order of the CDRs on the light chain. The more highly conserved parts in the variable regions are called frameworks (FRs). The variable regions of natural heavy and light chains each contain four FR regions connected by three CDRs. The CDRs in each chain are held together by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody [see Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)].

[0144] The sequences involved in the present invention are shown in Table 1:

[0145] Table 1

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] The present invention will be further described below in conjunction with embodiments:

[0157] Example 1 Preparation of CD5 scFv

[0158] (1) Construction of a fully human phage antibody library

[0159] PBMC was separated using Ficoll separation solution. The Ficoll separation solution was slowly added to normal human blood so that a clear separation interface was maintained between the Ficoll separation solution and the normal human blood. A 50 mL centrifuge tube containing the blood and the separation solution was centrifuged at about 15 °C for 20 min. After centrifugation, the entire liquid surface was divided into four layers. The upper layer was the plasma mixture, the lower layer was red blood cells and granulocytes, the middle layer was the Ficoll liquid, and there was a narrow white cloudy layer mainly composed of PBMCs at the junction of the upper and middle layers, that is, the PBMC cell layer. The plasma mixture in the upper layer was carefully aspirated with a sterile Pasteur pipette, and then the PBMCs were aspirated with a new sterile Pasteur pipette to obtain the separated PBMCs.

[0160] Total RNA was extracted by a conventional method and reverse transcribed into cDNA. Then, according to the method in the article by Sblattero, D., Bradbury, A. (1998) A definitive set of oligonucleotide primers for amplifying human V regions. Immunotechnology 3, 271 - 278, primer design was carried out, where VL was in the front or the back, VH was in the back or the front, and they were connected by a flexible Linker in the middle. The heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody were obtained by PCR, and the scFv nucleic acid fragment was amplified by the conventional overlapping PCR method (the PCR method refers to "Molecular Cloning: A Laboratory Manual" (Third Edition), USA, Joe Sambrook, David Russell. Science Press). The scFv nucleic acid fragment was ligated with the phagemid vector pComb3XSS, and the product was transformed into the TGI strain by an electroporator to obtain a fully human single-chain antibody library.

[0161] The library bacterial solution was added to fresh LB liquid medium for resuscitation and cultured until OD600 ≈ 0.5. Then, VSCM13 helper phage was added at a multiplicity of infection of VCSM13:bacteria = 50:1, mixed well, and allowed to stand before continuing to culture in a shaker. The culture was centrifuged to discard the supernatant, and the precipitate was resuspended in SOB medium with ampicillin and kanamycin double resistance and cultured overnight. The bacterial solution was centrifuged for 20 min, the supernatant was collected and PEG 8000 2.5 mol / L NaCl solution was added, incubated on ice for 1 hour, and then centrifuged for 30 min. The precipitated phage was resuspended in PBS and filtered through a 0.22 μm filter membrane.

[0162] (2) Antigen panning

[0163] Patent 202211370708.7 introduces the steps of antigen panning into this application. Antibodies against the antigens described in this application:

[0164] Co-incubate CD5 protein with His-Fc tag and protein G magnetic beads to prepare CD5-protein G conjugated magnetic beads, and draw the conjugated magnetic beads into the phage panning of the prepared fully human single-chain antibody library phage. After 3-4 rounds of panning processes of co-incubation, washing and elution, specific monoclonal antibodies against the antigen can be enriched.

[0165] After panning, pick monoclonal colonies and culture them in SOB-AG medium. Shake at 37°C and 250 rpm until the OD600 is about 0.5. Add helper phage (helper phage:bacteria = 50:1), let it stand and infect at 37°C for 30 min, shake on a shaker at 37°C and 250 rpm for 45 min, and finally resuspend with SOB-AK medium and incubate overnight at 30°C and 250 rpm. The next day, centrifuge to obtain the supernatant, prepare phage, and use chemiluminescence detection or ELISA method to screen monoclonal colonies (theoretically, clones with P / N (signal-to-background ratio) > 3 by chemiluminescence method and P / N > 2.1 by ELISA method can be identified as suspected positive clones. The results of this panning are shown in Table 2). Five clones numbered JK-02-B10, JK-11-C08, JK-12-C05, JK-14-E05, and JK-14-H12 were screened out and specifically bound to Jurkat cells endogenously expressing CD5. The specific data are as follows:

[0166] Table 2: Binding of CD5 scFv phage to Jurkat cells endogenously expressing CD5 detected by chemiluminescence method

[0167]

[0168]

[0169] Express and purify the clones screened above with Jurkat cells endogenously expressing CD5 to obtain ScFv polypeptide protein. After normal identification by SDS-PAGE electrophoresis, perform concentration detection and store at -80°C.

[0170] (3) Perform flow cytometry staining specificity detection after SDS-PAGE identification of scFv purification effect.

[0171] Aliquot Jurkat or C8166 cells that endogenously express the CD5 antigen into 1.5 mL Ep tubes, 5e5 - 1e6 cells / tube. Centrifuge at 300 g for 5 min at 4°C, discard the supernatant, resuspend with 50 μL of 30 μg / ml positive clone scFv solution respectively, incubate at 4°C for 1 h, then add 1 ml of PBS to resuspend the cells, centrifuge at 300 g for 5 min at 4°C, discard the supernatant, add 30 μl of Anti-His-647 fluorescent secondary antibody and resuspend the cells, then incubate at 4°C in the dark for 30 min. Finally, resuspend with 1 ml of PBS and wash the cells twice at 300 g for 5 min at 4°C, discard the supernatant and resuspend with 100 μL of PBS. Detect the positive rate of flow cytometry staining and the mean fluorescence intensity (MFI) for all cell tubes on the machine. The specific flow cytometry staining data can be seen in Figures 1 - 5 。

[0172] The results showed that JK-02-B10, JK-11-C08, JK-12-C05, JK-14-E05, and JK-14-H12 could label His and recognize and detect the expression of CD5 molecules on the cell surface. Therefore, JK-02-B10, JK-11-C08, JK-12-C05, JK-14-E05, and JK-14-H12 could be used as CD5 labeling reagents in applications including but not limited to detection kits, detection antibodies, CD5 molecule tracer reagents, etc.

[0173] Example 2 Construction of CAR Plasmids Targeting CD5

[0174] The scFv sequence was obtained by PCR amplification and then ligated into lentiviral vectors containing different co-stimulatory signals by restriction enzyme digestion, thus obtaining CAR plasmids targeting CD5 with different structures as follows (in the following structures, 8h represents the extracellular hinge domain of CD8a as the hinge region, 8TM represents the transmembrane domain of CD8a as the transmembrane region, BB represents the intracellular signaling domain of CD137, Z represents the CD3ζ chain, and the intracellular signal with the structure of CD137-CD3ζ is abbreviated as BBz), mbIL-15 is a fusion protein of IL-15 and IL15-Ra, IL-2(H9) is super IL-2, PDGFRβTM is the transmembrane domain of PDGFRβ, NKp44TM is the transmembrane domain of NKp44, and DAP10 is the intracellular domain of DAP10: Among them, the corresponding scFv numbers and CAR structures in CAR-1 to 7 are respectively:

[0175] CAR-1: CD5(JK-02-B10 / CD5(10))-scFv-8h-8TM-BBz-2A-mbIL15;

[0176] CAR-2: CD5(JK-11-C08 / CD5(13))-scFv-8h-8TM-BBz-2A-mbIL15;

[0177] CAR-3: CD5(JK-14-E05 / CD5(14))-scFv-8h-8TM-BBz-2A-mbIL15;

[0178] CAR-4: CD5(JK-14-H12 / CD5(15))-scFv-8h-8TM-BBz-2A-mbIL15;

[0179] CAR-5: CD5(JK-12-C05 / CD5(16))-scFv-8h-8TM-BBz-2A-mbIL15;

[0180] CAR-6: IL2(H9)-PDGFRβTM-2A-CD5(JK-11-C08 / CD5(13))-scFv-

[0181] 8h-8TM-BBz;

[0182] CAR-7: IL2(H9)-PDGFRβTM-2A-CD5(JK-11-C08 / CD5(13))-scFv-8h-NKp44TM-BB-DAP10.

[0183] Example 3 Preparation of Lentivirus and Infection of NK Cells

[0184] In this example, CAR-1 to CAR-7 were transfected into NK cells by lentivirus.

[0185] In this example, the lentivirus was packaged by the calcium phosphate method. Specifically, 293T cells were cultured in DMEM medium containing 10% FBS (w / v) until in a better state. The packaging plasmid (RRE:REV:2G) and the expression plasmid were added to a 1.5 mL centrifuge tube in a certain proportion. CaCl2 and 2×HBS were added, and after mixing, it was left standing at room temperature and then added to the treated 293T cell culture medium. After 3 - 5 h, the medium was changed again to 10 mL of DMEM medium containing 10% FBS. After 48 h or 72 h, the cell supernatant was collected, the virus was purified, and the titer was measured.

[0186] When detecting the titer of the prepared lentivirus, CHO cells were used for detection. CHO cells at a density of 1e5 per well were infected with the virus to be tested. After 48 hours, the total CAR expression was detected using Protein-L, and the positive rate was calculated. The formula for calculating the positive rate is: Titer (Tu / ml) = 1e5 × positive rate × dilution factor × 1000 ÷ virus volume (uL). The titers of the above CAR-structured viruses are shown in Table 3. The virus titer of CAR-3 could not be detected, which did not meet the actual usage requirements.

[0187] Table 3 List of CD5-CAR Lentivirus Titers

[0188]

[0189] Application of the screened ScFv against CD5 in CAR-NK:

[0190] Lymphocytes were separated using gradient centrifugation. After centrifugation, the second white lymphocyte layer was taken, washed with physiological saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. After obtaining PBMC, magnetic sorting was performed using CD56 antibody-labeled Microbeads to obtain CD56-positive NK cells. The CAR-NK positive rate was detected using Protein-L 3 days after transduction. The results of detecting the CAR-NK positive rate by labeling NK cells with CD3-FITC and CD56-BV510 (BioLegend) are shown in Table 4. CAR1, CAR-2, CAR4, and CAR-5 all had relatively high CAR positive rates and were able to infect NK cells better.

[0191] Table 4 Results of Detecting the CD5-CAR-NK Positive Rate

[0192]

[0193] The CAR structure described in Example 2 is a nucleotide sequence structure contained in a vector. After transduction into immune cells such as NK cells and T cells, engineered immune cells are formed. During the process of transcription and translation, the self-cleaving peptide in the structure (in some embodiments, the self-cleaving peptide can be replaced by a linker such as IRES (Internal Ribosome Entry Site) that can independently express multiple proteins. Self-cleaving peptides such as P2A / T2A / E2A / F2A, and P2A is used in this embodiment) is cleaved, and the genes before and after the self-cleaving peptide are independently expressed in the cell. Taking the structure of CAR1's CD5(JK-02-B10)-scFv-8h-8TM-BBz-2A-mbIL15 as an example, the engineered immune cells finally formed by expressing CAR1 in immune cells are engineered cells that independently express CD5(JK-02-B10)-scFv-8h-8TM-BBz protein and mbIL15 protein at the same time. In some embodiments, engineered immune cells can also be prepared by separately transducing related protein genes, such as separately transducing CAR (CD5(JK-02-B10)-scFv-8h-8TM-BBz) and mbIL15 genes. Any of the above preparation schemes can obtain the CAR-T and CAR-NK cells of Example 3 and subsequent examples. Different preparation schemes do not affect the application and function of the antigen-binding molecule that recognizes CD5 described in this article in constructing the CAR structure.

[0194] Example 4 In vitro killing experiments of NK cells modified with CAR-1, CAR-2, CAR-4, and CAR-5

[0195] Using NALM6-CD5-Luc-GFP cells (NALM6 cells that externally express the CD5 antigen) as positive target cells, the CAR-NK cells and target cells are seeded in the target cells at the corresponding ratio. After 24 hours, the killing is detected by luciferase. Luciferase principle: 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-NK cell killing rate = 1 - (fluorescence value of the experimental group ÷ fluorescence value of the blank control group) × 100%.

[0196] The results are shown in the figure and Figure 6 as well as Table 5. The NK cells modified with CAR1, CAR-2, CAR4, and CAR-5 showed good effects in the killing experiment against NALM6-CD5-Luc-GFP.

[0197] Table 5 In vitro killing data of CAR-1, CAR-2, CAR-4, and CAR-5

[0198] Structure Antigen-binding molecule included Killing rate (%) CNK / 5.40 CAR-1 CD5(10) 87.66 CAR-2 CD5(13) 95.20 CAR-4 CD5(15) 63.32 CAR-5 CD5(16) 89.22

[0199] Note: " / " indicates that the antigen-binding molecule of the present invention is not included.

[0200] The above results indicate that the antigen-binding molecules JK-02-B10, JK-11-C08, JK-12-C05, and JK-14-H12 that recognize CD5 of the present invention can be adapted to the CAR structure combination and all can play an excellent role in killing tumors.

[0201] Example 5 In Vivo Pharmacodynamic Evaluation of NK Cells Modified with CAR-1, CAR-2, CAR-4, and CAR-5

[0202] NALM6-CD5-Luc-GFP cells were used to pre-form tumors in NCG mice by tail vein injection at a dose of 1e6 cells / mouse, and the pre-forming tumor time was 3 days. NK cells modified with CAR-1, CAR-2, CAR-4, and CAR-5 were cryopreserved after 13 days of in vitro culture; after the pre-forming tumors were completed, the CAR-NK cells were resuscitated and injected into the pre-formed NCG mice by tail vein injection at a dose of 3e6 cells / mouse. NK cells without CAR modification were injected as a control group at the same dose and in the same manner. On the 7th and 13th days, the luciferase fluorescent substrate was intraperitoneally injected once, and in vivo imaging was performed using a small animal in vivo imager to detect the tumor load.

[0203] The results are as Figure 7 、 Figure 8 shown. CAR-1 and CAR-2 have the ability to kill tumors compared with NK cells without CAR modification. Among them, NK cells modified with CAR-2 have relatively the best in vivo pharmacodynamics and amplification. It shows that the CAR-modified CAR-NK engineered immune cells constructed with the antigen-binding molecules JK-02-B10 and JK-11-C08 that recognize CD5 of the present invention have an excellent effect of inhibiting or even eliminating tumor cells.

[0204] Example 6 In Vivo Persistence Experiment of NK Cells Modified with CAR-2

[0205] Pre-tumor formation was carried out by injecting NCG mice via the tail vein with Jurkat-Luc-GFP cells (endogenously expressing the CD5 antigen) at a dose of 3.5e6 cells per mouse for 3 days. The CAR-2 modified NK cells were cryopreserved after 13 days of in vitro culture; after pre-tumor formation was completed, the CAR-NK cells were thawed and injected into the pre-tumor formed NCG mice via the tail vein at a dose of 3e6 cells per mouse. The unmodified NK cells were injected in the same dose and manner as a control group. The luciferase fluorescent substrate was intraperitoneally injected once every 7 days, and in vivo imaging was performed using a small animal in vivo imager to detect the tumor load. Blood was collected from the orbital cavity, and the copy number of BBz in the blood was detected by RT-PCR to confirm the in vivo persistence of CAR-NK cells.

[0206] The results are as Figure 9 , Figure 10 shown, where each of the multiple curves in Figure 10 represents the detection curve of the CAR copy number in each mouse. The CAR-2 modified NK cells have good in vivo persistence and can survive in mice for up to 49 days. This indicates that the CAR-modified CAR-NK engineered immune cells constructed with the antigen-binding molecule JK-11-C08 that recognizes CD5 of the present invention have excellent in vivo persistence ability in the tumor environment.

[0207] Example 7 Evaluation of the in vitro function of CAR7 structure-modified NK cells containing the CD5 ScFv of the present invention

[0208] Using NALM6-CD5-Luc-GFP cells (NALM6 cells exogenously expressing the CD5 antigen) as positive target cells and NALM6-Luc-GFP cells as negative target cells, the CAR-NK cells and target cells were plated in the target cells at the corresponding ratio. After 24 hours, killing was 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-NK cell killing rate = 1 - (fluorescence value of the experimental group ÷ fluorescence value of the blank control group) × 100%. The results are as Figure 11 shown.

[0209] The results showed that, compared with negative target cells, the CAR-7 modified NK cells with the structure of IL2(H9)-PDGFRβTM-2A-CD5(JK-11-C08)-scFv-8h-NKp44TM-BB-DAP10 also had good in vitro killing effects. It was shown that the antigen-binding molecule JK-11-C08 that recognizes CD5 described in the present invention could be adapted to different structures of CARs, and CAR-NK engineered immune cells modified with different structures of CARs containing JK-11-C08 all had excellent effects of inhibiting and even clearing tumor cells.

[0210] Example 8 Application of CAR-2 in CAR-T

[0211] Lymphocytes were isolated by gradient centrifugation; after centrifugation, the second layer of white lymphocyte layer was taken, washed with physiological saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 h, and then the activated PBMC was infected at a certain multiplicity of infection (MOI). On the 7th day after virus infection, the positive rate of CAR-T was detected by flow cytometry. The antibody used was: Protein-L-PE. 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 positive rate of CAR and the expression intensity of CAR. For the expression of CAR2 containing the CD5 ScFv in T cells, Protein L was used to label CAR, and the proportion of CAR-positive cells was detected by flow cytometry. The results were as Figure 12 shown.

[0212] It can be Figure 12 seen that CAR2 containing the CD5 ScFv can be highly expressed in T cells.

[0213] Using NALM6-CD5-Luc-GFP cells (NALM6 cells that exogenously express the CD5 antigen) as positive target cells and NALM6-Luc-GFP cells as negative target cells, the CAR-T cells and target cells were seeded in the target cells at corresponding ratios, and the killing 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%.

[0214] The results were as Figure 13 and Figure 14As shown, T and C represent T cells from two different donors. T-Con and C-Con represent control T cells that do not express the CAR-2 vector. T-CAR-2 and C-CAR-2 represent CAR-T cells derived from two different donors after expressing the CAR-2 vector. It can be seen that CAR-T cells modified with CAR-2 (such as T-CAR-2 and C-CAR-2) have significant in vitro killing effects.

[0215] The results were analyzed using the Paired-T test, showing that CD5 CAR-T cells have obvious specific killing effects on tumor cells expressing the CD5 antigen (P = 0.0286).

[0216] The CAR-T (CAR-2) cells targeting CD5 and Control-T cells were co-cultured with Nalm6-CD5-Luc-GFP tumor cells at an effector-to-target ratio of 2:1. The proportion of GFP+ tumor cells in the cell population was detected by flow cytometry. When the proportion of GFP+ cells was less than 10%, the cells were collected and counted, and tumor cells were replenished again according to the corresponding effector-to-target ratio for repeated stimulation and killing. When the proportion of GFP+ tumor cells in the cell population exceeded 90%, it was used as the killing test endpoint. The number of rounds of stimulation of CD5 CAR-T cells and Control-T cells against tumor cell stimulation reflects the function and persistence of CAR-T cells in response to multiple rounds of target cell stimulation. As Figure 15 shown, the abscissa represents the time of continuous killing of tumor cells, and the ordinate represents different cell types. The results prove that CAR-2 modified T cells have good continuous killing ability in vitro.

[0217] In summary, JK-02-B10, JK-11-C08, JK-12-C05, JK-14-E05, and JK-14-H12 screened by us can all recognize the CD5 molecule with high affinity. They can be used as detection reagents to detect the expression of the CD5 molecule on the surface of target cells, and can also be used as detection reagents to recognize target samples, such as the CD5 expression in tumor samples. In some embodiments, they can also be used as in vivo tracer molecules to trace and label the CD5 molecule; among them, JK-02-B10, JK-11-C08, JK-12-C05, and JK-14-H12 can be used as antigen recognition domains in engineered immune cells, such as CAR-NK cells and CAR-T cells expressing polypeptides containing JK-02-B10 / CD5(10), JK-11-C08 / CD5(13), JK-12-C05 / CD5(16), and JK-14-H12 / CD5(15).

[0218] 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 antigen-binding molecule that recognizes the CD5 molecule, characterized in that, The three CDRs of its light chain respectively have the amino acid sequences shown in any one of SEQ ID NO: 1 to 12, or amino acid sequences having at least 80% homology therewith; The three CDRs of its heavy chain respectively have the amino acid sequences shown in any one of SEQ ID NO: 13 to 24, or amino acid sequences having at least 80% homology therewith.

2. The antigen-binding molecule according to claim 1, characterized in that, It is an antigen-binding molecule as shown in any one of a) to e): a), The three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 1, 5 and 9, or amino acid sequences having at least 80% homology therewith; the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 13, 17 and 21, or amino acid sequences having at least 80% homology therewith; b), The three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 2, 6 and 10, or amino acid sequences having at least 80% homology therewith; the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 14, 18 and 22, or amino acid sequences having at least 80% homology therewith; c), The three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 3, 7 and 11, or amino acid sequences having at least 80% homology therewith; the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 15, 19 and 23, or amino acid sequences having at least 80% homology therewith; d), The three CDRs of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 4, 8 and 12, or amino acid sequences having at least 80% homology therewith; the three CDRs of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 16, 20 and 24, or amino acid sequences having at least 80% homology therewith.

3. The antigen-binding molecule according to claim 1, characterized in that, Its light chain has the amino acid sequence shown in any one of SEQ ID NO: 25 to 28, or an amino acid sequence having at least 80% homology therewith; Its heavy chain has the amino acid sequence shown in any one of SEQ ID NO: 29 to 32, or an amino acid sequence having at least 80% homology therewith.

4. The antigen-binding molecule according to any one of claims 1 to 3, characterized in that, It is an antigen-binding molecule as shown in any one of A) - D): A) Its light chain has the amino acid sequence shown in SEQ ID NO: 25; its heavy chain has the amino acid sequence shown in SEQ ID NO: 29; B) Its light chain has the amino acid sequence shown in SEQ ID NO: 26; its heavy chain has the amino acid sequence shown in SEQ ID NO: 30; C) Its light chain has the amino acid sequence shown in SEQ ID NO: 27; its heavy chain has the amino acid sequence shown in SEQ ID NO: 31; D) Its light chain has the amino acid sequence shown in SEQ ID NO: 28; its heavy chain has the amino acid sequence shown in SEQ ID NO:

32.

5. The antigen-binding molecule according to any one of claims 1 to 4, characterized in that, It has the amino acid sequence shown in any one of SEQ ID NO: 33-34 or SEQ ID NO: 36-37.

6. A nucleic acid encoding the antigen-binding molecule according to any one of claims 1 to 5.

7. The nucleic acid according to claim 6, characterized in that, The nucleic acid encoding the light chain of the antigen molecule has the nucleotide sequence shown in SEQ ID NO: 64, 66, 68 or 70, or has a nucleotide sequence with at least 80% homology to the sequence shown in SEQ ID NO: 64, 66, 68, 70; The nucleic acid encoding the heavy chain of the antigen molecule has the nucleotide sequence shown in SEQ ID NO: 65, 67, 69 or 71, or has a nucleotide sequence with at least 80% homology to the sequence shown in SEQ ID NO: 65, 67, 69 or 71.

8. An expression vector comprising the nucleic acid according to claim 6 or 7.

9. A protein combination, characterized in that, It includes the antigen-binding molecule according to any one of claims 1-5.

10. The protein combination according to claim 9, characterized in that, It further contains a transmembrane region, a hinge region and an intracellular signaling functional domain.

11. The protein combination according to claims 9 - 10, characterized in that, The transmembrane region includes but is not limited to the transmembrane regions of DAP10, DAP12, NKG2D, CD4, CD8α or CD28; The hinge region includes but is not limited to the hinge regions of IgG, IgD, CD7 or CD8α / CD28; The intracellular signaling functional domain includes a primary signal transduction domain and / or a co-stimulatory domain, The primary signal transduction domain includes but is not limited to the signal transduction domains of one or more of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa; The co-stimulatory domain includes but is not limited to the signal transduction domains of one or more of the following molecules: A ligand that specifically binds to DAP10, CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-l, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-l, ITGB7, TNFR2, TRANCE / RANKL, DNAMl, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D or CD83.

12. The protein combination according to claim 11, characterized in that, The hinge region is selected from the 8h hinge region derived from CD8a, the 8h(dc) hinge region, or the 7h hinge region derived from CD7, or the G4h hinge structure derived from IgG; The transmembrane region is optionally selected from CD8a, NKp44 or PDGFRβ; The intracellular co-stimulatory signal is optionally selected from CD137 or DAP10; The intracellular primary stimulation signal region is selected from the CD3ζ chain.

13. The protein combination according to claim 12, wherein, The amino acid sequences of the 8h hinge region, 8h(dc) hinge region, and 7h hinge region derived from CD8a are shown in SEQ ID NO: 45, 54, and 53 respectively, or amino acid sequences having at least 80% homology thereto; The amino acid sequences of the 8TM derived from CD8a, the CD28TM of CD28 transmembrane, the transmembrane domain β of PDGFR, and the transmembrane domain of NKp44 are shown in SEQ ID NO: 46, 49, 55, and 56 respectively, or amino acid sequences having at least 80% homology thereto; The amino acids of the intracellular signal functional domains derived from CD137 and DAP10 are shown in SEQ ID NO: 47 and 57 respectively, or amino acid sequences having at least 80% homology thereto; The amino acid sequence of the CD3ζ chain is shown in SEQ ID NO: 48, or an amino acid sequence having at least 80% homology thereto.

14. A nucleic acid encoding the protein combination according to any one of claims 9 to 13.

15. The nucleic acid according to claim 14, wherein, Comprising at least one of the following gene structures: CD5-scFv-8h-8TM-BBz-2A-mbIL15; IL2(H9)-PDGFRβTM-2A-CD5-scFv-8h-8TM-BBz; IL2(H9)-PDGFRβTM-2A-CD5-scFv-8h-NKp44TM-BB-DAP10; The scFv is the nucleic acid described in claim 6 or 7.

16. The nucleic acid according to claim 15, wherein, It has a nucleotide sequence shown in any one of SEQ ID NO: 59 - 63, or a nucleotide sequence having at least 80% homology thereto.

17. An expression vector comprising the nucleic acid according to any one of claims 14 to 16.

18. An engineered cell, wherein, It expresses at least one of ① - ④: ① The antigen-binding molecule described in any one of claims 1 - 5; ② The nucleic acid described in claim 6 or 7, or the nucleic acid described in any one of claims 14 - 16; ③ The protein combination described in any one of claims 9 - 13; ④ The expression vector described in claim 8 or the expression vector described in claim 17.

19. The engineered cell according to claim 17, wherein, It expresses the protein combination described in any one of claims 7 - 13, and expresses at least one cytokine selected from IL-15, IL15-Ra, IL-2(H9) or a fusion protein formed by fusing two or more of them.

20. The engineered cell according to claim 18, wherein, The cell is an immune cell, including but not limited to T cells, B cells, NK cells, DC cells or macrophages.

21. Use of any one of (I) to (V) in the preparation of an anti-tumor drug; I) The antigen-binding molecule according to any one of claims 1 to 5; II) The nucleic acid according to claim 6 or 7, or the nucleic acid according to any one of claims 14 to 16; III) The protein combination according to any one of claims 9 to 13; IV) The expression vector according to claim 8 or 17; V) The engineered cell according to any one of claims 18 to 20.

22. A drug, characterized in that, It includes any one of I) - V) and a pharmaceutically acceptable excipient: I) The antigen-binding molecule described in any one of claims 1 - 5; II) The nucleic acid described in claim 6 or 7, or the nucleic acid described in any one of claims 14 - 16; III) The protein combination described in any one of claims 9 - 13; IV) The expression vector described in claim 8 or 17; V) The engineered cell described in any one of claims 18 - 20.

23. Use of any one of 1) to 5) as a screening marker or as a marker in detecting CD5; 1) The antigen-binding molecule according to any one of claims 1 to 5; 2) The nucleic acid according to claim 6 or 7, or the nucleic acid according to any one of claims 14 to 16; 3) The protein combination according to any one of claims 9 to 13; 4) The expression vector according to claim 8 or 17; 5) The engineered cell according to any one of claims 18 to 20.

24. A screening marker or a detection marker, characterized in that, It includes any one of 1) - 5): 1) The antigen-binding molecule described in any one of claims 1 - 5; 2) The nucleic acid described in claim 6 or 7, or the nucleic acid described in any one of claims 14 - 16; 3) The protein combination described in any one of claims 9 - 13; 4) The expression vector described in claim 8 or 17; 5) The engineered cell described in any one of claims 18 - 20.

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