Antibodies against malignant tumors and uses thereof

Abstract

The invention relates to the technical field of cell engineering, in particular to an antibody aiming at malignant tumors and application thereof. The scFv of the CD7 provided by the invention is a brand new humanized antibody sequence, so that the immunogenicity of a mouse antibody is avoided; after the CD7 chimeric antigen receptor provided by the invention is combined with CD7 on tumor cells, the obvious anti-tumor activity is shown, and the CD7-scFv is a fully humanized antibody with more appropriate affinity to a human CD7 antigen.

Description

Technical Field

[0001] The present invention relates to the technical field of cell engineering, and in particular to antibodies targeting malignant tumors and applications thereof. Background Art

[0002] Adoptive cell therapy (ACT) refers to the separation of immune-competent cells from cancer patients, their expansion and functional identification in vitro, and then their return to the patient, thereby directly killing the tumor or stimulating the body's immune response to kill tumor cells.

[0003] Immunotherapy is the fourth type of cancer treatment after surgery, radiotherapy and chemotherapy. Chimeric antigen receptor (CAR) T cell technology is a rapidly developing cellular 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. Commonly used transmembrane region molecules are selected from the transmembrane functional domains of genes such as CD3, CD4, CD8 and CD28. The intracellular region has an immune receptor tyrosine activation motif (ITAM), the most commonly used of which are the T cell receptor TCR / CD3ζ chain and the immunoglobulin FC receptor FCεRIγ, which are mainly responsible for signal transduction function; people use molecular cloning methods to recombinate the above elements in vitro to form recombinant plasmids, and then transduce the recombinant plasmids into T cells through viral vectors, electroporation and other means, followed by in vitro culture and amplification, and finally back into the patient's body to treat tumors. This genetically modified and transformed T cell is called CAR-T cell. CAR-T cells recognize and bind tumor antigens through scFv segments, and then activate T cells through CD3ζ chains and costimulatory signal regions. In this way, the modified CAR-T cells recognize and kill tumor cells with two major characteristics: ① Targeting; ② Overcoming MHC restriction, that is, no longer relying on MHC, thus breaking through the problem of immune escape by tumor cells by downregulating the expression of MHC.

[0004] T-ALL (acute T-lymphocytic leukemia) is a highly aggressive hematologic T-cell malignancy disease, accounting for about 25% of adult ALL and 15% of childhood ALL. About 64,000 patients are diagnosed with ALL each year worldwide. Standard treatments for T-ALL include radiotherapy / chemotherapy and stem cell transplantation. The standard chemotherapy regimen has a response rate of only 30%-40%, and the median overall survival of responding patients is 6 months. The recurrence rate and mortality rate of patients with T-cell malignancies are usually high. Relapsed patients have a poor prognosis, extremely limited treatment options, and less than 10% survive for more than 5 years. Overall, the survival status of T-ALL has not changed much in recent years, and the treatment success rate for children is relatively good. The biggest treatment progress is immunotherapy. At present, international clinical trials focus on three targets: CD2, CD5, and CD7, but there are only small-scale clinical case reports and lack of large-scale clinical research data support. Summary of the invention

[0005] In view of this, the present invention provides antibodies against malignant tumors and applications thereof.

[0006] The present invention provides antibodies against malignant tumors and their applications. The scFv in the CD7 chimeric antigen receptor provided by the present invention is a completely new humanized antibody sequence, which avoids the immunogenicity of mouse antibodies. After the CD7 chimeric antigen receptor provided by the present invention binds to CD7 on tumor cells, it shows obvious anti-tumor activity, wherein the CD7-scFv of CAR1 is a fully humanized antibody with a more suitable affinity for human CD7 antigen.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a CD7 scFv comprising any of the following CDR sequences:

[0009] (I), CDR1, CDR2 and CDR3 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 7, 8 and 9 respectively; and

[0010] The CDR1, CDR2 and CDR3 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 10, 11 and 12 respectively; or

[0011] (II), CDR1, CDR2 and CDR3 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 13, 14 and 15, respectively; and

[0012] The CDR1, CDR2 and CDR3 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 16, 17 and 18, respectively; or

[0013] (III), CDR1, CDR2 and CDR3 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 19, 20 and 21, respectively; and

[0014] The CDR1, CDR2 and CDR3 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 22, 23 and 24, respectively; or

[0015] (IV) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (I) to (III); or

[0016] (V) A sequence having an identity of 80% or more to the amino acid sequence shown in any one of (I) to (IV).

[0017] In some specific embodiments of the present invention, the CD7 scFv comprises KK-01-A02, KK-01-C11 or KK-01-D01.

[0018] In some specific embodiments of the present invention, the CD7 scFv comprises any of the following FR sequences:

[0019] (VI), FR1, FR2, FR3 and FR4 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 25, 26, 27 and 28, respectively; and

[0020] The FR1, FR2, FR3 and FR4 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 29, 30, 31 and 32, respectively; or

[0021] (VII), FR1, FR2, FR3 and FR4 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 33, 34, 35 and 36, respectively; and

[0022] The FR1, FR2, FR3 and FR4 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No.37, 38, 39 and 40, respectively; or

[0023] (VIII), FR1, FR2, FR3 and FR4 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 41, 42, 43 and 44, respectively; and

[0024] The FR1, FR2, FR3 and FR4 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No.45, 46, 47 and 48, respectively; or

[0025] (VI) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (i) to (iii); or

[0026] (X) A sequence having 80% or more homology with the amino acid sequence shown in any one of (i) to (iv).

[0027] In some specific embodiments of the present invention, the CD7 scFv comprises the variable region sequence of any of the following:

[0028] (i) the variable region of the light chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.50; and

[0029] The variable region of the heavy chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.51; or

[0030] (ii), the variable region of the light chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.53; and

[0031] The variable region of the heavy chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.54; or

[0032] (iii), the variable region of the light chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.56; and

[0033] The variable region of the heavy chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.57; or

[0034] (iv) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (i) to (iii); or

[0035] (v) A sequence having 80% or more homology with the amino acid sequence shown in any one of (i) to (iv).

[0036] In some specific embodiments of the present invention, the variable region of the light chain and the variable region of the heavy chain are connected by a linker peptide;

[0037] The connecting peptide linker has:

[0038] (vi), the amino acid sequence shown in SEQ ID No.49; or

[0039] (vii) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (vi); or

[0040] (viii) A sequence having a homology of 80% or more to the amino acid sequence shown in (vi).

[0041] In some specific embodiments of the present invention, the CD7 scFv comprises any of the following:

[0042] (1) The CD7 scFv has the amino acid sequence shown in SEQ ID No.52; or

[0043] (2) the CD7 scFv has the amino acid sequence shown in SEQ ID No.55; or

[0044] (3) the CD7 scFv has the amino acid sequence shown in SEQ ID No.58; or

[0045] (4) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (1) to (3); or

[0046] (5) A sequence having a homology of 80% or more to the amino acid sequence shown in any one of (1) to (4).

[0047] The present invention also provides a nucleic acid molecule encoding the CD7 scFv.

[0048] In some specific embodiments of the present invention,

[0049] (6) the CD7 scFv nucleic acid molecule has a nucleotide sequence as shown in SEQ ID No. 64, 65 or 66; or

[0050] (7) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (6) but is different from the nucleotide sequence shown in (6) due to the degeneracy of the genetic code; or

[0051] (8) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (6) or (7), and having the same or similar functions as the nucleotide sequence shown in (6) or (7); or

[0052] (9) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (6) to (8).

[0053] Based on the above research, the present invention also provides a protein combination, including the CD7 scFv, a transmembrane region, a hinge region and an intracellular signaling domain.

[0054] In some specific embodiments of the present invention, the transmembrane region is selected from the transmembrane region of DAP10, DAP12, NKG2D, CD4, CD8α or CD28;

[0055] The hinge region includes but is not limited to the hinge region of IgG, IgD, CD7 or CD8α / CD28;

[0056] The intracellular signaling domain may comprise a primary signaling domain and / or a co-stimulatory domain, wherein the primary signaling domain comprises a signaling domain of one or more molecules selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa, CD79α, CD79β, CD66d, DAP10 or DAP12;

[0057] The co-stimulatory domain comprises a signal transduction domain selected from one or more of the following molecules:

[0058] 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), CD 160. CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, 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 a ligand that specifically binds to CD83.

[0059] In some specific embodiments of the present invention,

[0060] (10), the transmembrane region has the amino acid sequence shown in SEQ ID No.59; or

[0061] (11), the hinge region has the amino acid sequence shown in SEQ ID No.60; or

[0062] (12), the intracellular signaling domain has an amino acid sequence as shown in SEQ ID No.61;

[0063] (13), the primary signal transduction domain has the amino acid sequence shown in SEQ ID No.62;

[0064] or

[0065] (14) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (10) to (13); or

[0066] (15) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of (10) to (14).

[0067] In some specific embodiments of the present invention, the primary signal transduction domain is CD3ζ.

[0068] In some specific embodiments of the present invention, the hinge region is derived from the extracellular hinge domain of CD8a; and the transmembrane region is derived from the transmembrane domain of CD8a.

[0069] In some specific embodiments of the present invention, the protein combination includes CAR-1, CAR-2 and / or CAR-3;

[0070] (A), the CAR-1, CAR-2 and CAR-3 have the amino acid sequences shown in SEQ ID No. 4, 5, and 6; or

[0071] (B) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (A); or

[0072] (C) An amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in (A) or (B).

[0073] The present invention also provides genetic elements, including the nucleic acid molecules.

[0074] In some specific embodiments of the present invention, the genetic elements include CAR-1, CAR-2 and CAR-3;

[0075] (D), the CAR-1, CAR-2 and CAR-3 have the nucleotide sequences shown in SEQ ID No. 1, 2 and 3 respectively; or

[0076] (E), a nucleotide sequence obtained by replacing, 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

[0077] (F) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in (D) or (E).

[0078] The present invention also provides an artificial vector, comprising the gene element and an acceptable auxiliary agent or vector.

[0079] The present invention also provides virus particles, including the artificial vector.

[0080] The present invention also provides an engineered cell, comprising any of the following:

[0081] (a), the CD7 scFv; and / or

[0082] (b), the nucleic acid molecule; and / or

[0083] (c), the protein combination; and / or

[0084] (d), the genetic element; and / or

[0085] (e), the artificial carrier; and / or

[0086] (f) the virus particles.

[0087] In some specific embodiments of the present invention, the engineered cells include but are not limited to NK cells, T cells, macrophages, DC cells and related prerequisite cells.

[0088] The present invention also provides the use of any of the following items in detecting CD7 expression:

[0089] Ⅰ), the CD7 scFv; and / or

[0090] II), the nucleic acid molecule; and / or

[0091] III), the protein combination; and / or

[0092] IV), said genetic element; and / or

[0093] V), the artificial carrier.

[0094] The present invention also provides the use of any of the following items in the preparation of a product for detecting CD7 expression:

[0095] Ⅰ), the CD7 scFv; and / or

[0096] II), the nucleic acid molecule; and / or

[0097] III), the protein combination; and / or

[0098] IV), said genetic element; and / or

[0099] V), the artificial carrier.

[0100] In some specific embodiments of the present invention, the product includes a kit, a detection antibody and / or a CD7 molecule tracing reagent.

[0101] The present invention also provides a product, comprising:

[0102] Ⅰ), the CD7 scFv; and / or

[0103] II), the nucleic acid molecule; and / or

[0104] III), the protein combination; and / or

[0105] IV), said genetic element; and / or

[0106] V), the artificial carrier.

[0107] The present invention also provides the use of any of the following items in the preparation of a drug for preventing and / or treating a disease:

[0108] 1) the CD7 scFv; and / or

[0109] 2) the nucleic acid molecule; and / or

[0110] 3) the protein combination; and / or

[0111] 4) the genetic element; and / or

[0112] 5) the artificial carrier; and / or

[0113] 6) the virus particles; and / or

[0114] 7) The engineered cells.

[0115] In some embodiments of the invention, the disease comprises a tumor.

[0116] In some specific embodiments of the present invention, the tumor includes but is not limited to acute T-lymphocytic leukemia.

[0117] The present invention also provides a drug comprising any of the following items and a pharmaceutically acceptable adjuvant or auxiliary agent:

[0118] 1) the CD7 scFv; and / or

[0119] 2) the nucleic acid molecule; and / or

[0120] 3) the protein combination; and / or

[0121] 4) the genetic element; and / or

[0122] 5) the artificial carrier; and / or

[0123] 6) the virus particles; and / or

[0124] 7) The engineered cells.

[0125] The present invention also provides a drug combination, comprising the drug and any other effective ingredients.

[0126] The present invention provides antibodies against malignant tumors and their applications. The scFv in the CD7 chimeric antigen receptor provided by the present invention is a completely new humanized antibody sequence, which avoids the immunogenicity of mouse antibodies. After the CD7 chimeric antigen receptor provided by the present invention binds to CD7 on tumor cells, it shows obvious anti-tumor activity, wherein the CD7-scFv of CAR1 is a fully humanized antibody with a more suitable affinity for human CD7 antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0128] Figure 1 The binding force between CD7 scFv and antigen detected by Elisa in Example 1 is shown;

[0129] Figure 2 The figure shows the flow cytometry staining of Jurkat cells by the prokaryotic expression protein of KK-01-A02 in Example 1; wherein the figure on the left shows the detection of CD7 of Jurkat without specific antibody labeling, and the figure on the right shows the recognition of CD7 of Jurkat by KK-01-A02;

[0130] Figure 3 The figure shows the flow cytometry staining of Jurkat cells by the prokaryotic expression protein of KK-01-C11 in Example 1; wherein the figure on the left shows the detection of CD7 of Jurkat without specific antibody labeling, and the figure on the right shows the recognition of CD7 of Jurkat by KK-01-C11;

[0131] Figure 4 The figure shows the flow cytometry staining of Jurkat cells by the prokaryotic expression protein of KK-01-D01 in Example 1; wherein the figure on the left shows the detection of CD7 of Jurkat without specific antibody labeling, and the figure on the right shows the recognition of CD7 of Jurkat by KK-01-D01;

[0132] Figure 5 The results of three CAR-NK in vitro killing experiments in Example 4 are shown;

[0133] Figure 6 The cytokine secretion test results in Example 4 are shown;

[0134] Figure 7 The results of the in vivo efficacy experiment of CAR-NK in Example 5 are shown. DETAILED DESCRIPTION

[0135] The present invention discloses antibodies against malignant tumors and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve them. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0136] CD7 is a transmembrane glycoprotein that is mainly expressed on T cells and NK cells and cells in the early stages of T, B, and bone marrow cell differentiation. During lymphocyte development, it can act as a co-stimulatory receptor for the interaction between T and B lymphocytes. CD7 is expressed in more than 90% of lymphoblastic T-cell leukemias and lymphomas and about 30% of acute myeloid leukemias. Studies have shown that CD7 has no significant effect on the function of peripheral blood T cells, so CD7-CAR or anti-CD7 ADCs can be used to kill tumors.

[0137] The present invention provides a CD7 antibody and a chimeric antigen receptor targeting CD7;

[0138] The present invention provides a CD7-CAR structure that can efficiently activate NK targeted killing.

[0139] Table 1 Nucleotide sequences

[0140]

[0141]

[0142] Table 2 Amino Acid Sequence Listing

[0143]

[0144]

[0145]

[0146]

[0147]

[0148] The antibody against malignant tumor provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0149] The present invention will be further described below in conjunction with embodiments:

[0150] Example 1 Preparation of CD7 scFv

[0151] Ficoll separation solution is used for PBMC separation, and the Ficoll separation solution is slowly added to normal human blood so that the Ficoll separation solution and normal human blood maintain a clear separation interface. A 50mL centrifuge tube containing the blood and the separation solution is centrifuged at about 15°C for 20min. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll liquid, wherein at the junction of the upper and middle layers, there is a narrow band of white cloud layer mainly composed of PBMC, i.e., the PBMC cell layer. Carefully suck off the upper plasma mixture with a sterile Pasteur pipette, and then suck PBMC with a new sterile Pasteur pipette to obtain separated PBMC.

[0152] Total RNA was extracted by conventional methods and reverse transcribed into cDNA. Primers were then designed according to the method in Sblattero, D., Bradbury, A. (1998) A definitive set of oligonucleotide primers for amplifying human V regions. Immunotechnology 3, 271-278, wherein VL was in front or in the back, VH was in the back or in the front, and the middle was connected by a flexible linker. PCR was used to obtain the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody, and the scFv nucleic acid fragment was amplified by conventional overlapping PCR (PCR method reference "Molecular Cloning: A Laboratory Manual" (third edition), Joe Sambrook, David Russell, Science Press, USA), the scFv nucleic acid fragment was connected to the phagemid vector, and the product was converted by an electroporator to obtain a fully human single-chain antibody library.

[0153] The CD7 protein with the His-Fc tag was co-incubated with proteinG magnetic beads to prepare CD7-proteinG coupled magnetic beads for panning of fully human single-chain antibodies. After 3-4 rounds of co-incubation, washing and elution, specific monoclonal antibodies against the antigen can be enriched.

[0154] Antigen panning steps: co-incubate the CD7 protein with Fc tag with proteinG magnetic beads to prepare CD7-proteinG coupled magnetic beads, and then put the coupled magnetic beads into the prepared fully human single-chain antibody library phage panning. After 3-4 rounds of co-incubation, washing and elution, specific monoclonal antibodies against the antigen can be enriched. Specific panning data are shown in Table 3:

[0155] Table 3 Selection of scFv against CD7 antigen in the fully human scFv library

[0156]

[0157]

[0158] After the selection, the ELISA method was used to screen the monoclones, and finally three clones numbered KK-01-A02, KK-01-C11, and KK-01-D01 were selected, which specifically bound to the human CD7 antigen. The specific data are as follows: Figure 1 :

[0159] The specific sequences of the peptides that recognize CD7 that were screened are shown in Table 4:

[0160] Table 4

[0161]

[0162]

[0163] Among them, the CDR sequences are in bold italics.

[0164] The clones screened above were purified and expressed by Jurkat cells endogenously expressing CD7 to obtain a fully human scFv polypeptide protein targeting CD7, and the recognition ability of the scFv targeting CD7 to the CD7 molecule was verified using flow cytometry.

[0165] Jurkat cells endogenously expressing CD7 antigen were divided into 1.5mL Eppendorf tubes, 5e5-1e6 cells / tube, centrifuged at 4℃300g for 5min, supernatant discarded, 50μL 30ug / ml positive clone scFv solution was added to resuspend, incubated at 4℃ for 1h, then 1ml PBS was added to resuspend the cells, centrifuged at 4℃300g for 5min, supernatant discarded, 30μl Anti-His-647 fluorescent secondary antibody was added and resuspended, incubated at 4℃ in the dark for 30min, finally resuspended with 1ml PBS and washed twice at 4℃300g for 5min, supernatant discarded and resuspended with 100μL PBS. All cell tubes were tested for flow cytometry staining positivity and mean fluorescence intensity (MFI). The specific flow staining data are as follows: Figure 2-4 As shown:

[0166] Figure 2-Figure 4 The graph on the left represents the CD7 detection of Jurkat in the absence of specific antibody labeling, and the graph on the right represents the CD7 recognition of Jurkat by KK-01-A02, KK-01-C11, and KK-01-D01. The results show that the monoclonal antibody polypeptide can recognize CD7 on the surface of Jurkat cells. The 3 clones KK-01-A02, KK-01-C11, and KK-01-D01 screened by the present invention can all be CD7 antibodies expressed on the cell surface, and can be used as CD7 labeling reagents in detection kits, detection antibodies, and CD7 molecular tracing reagents.

[0167] Example 2 Construction of CAR plasmid targeting CD7

[0168] The scFv sequence was obtained by PCR amplification, and then the sequence was connected to a lentiviral vector containing different hinges, different transmembrane domains, and different co-stimulatory signals by restriction endonuclease digestion, thereby obtaining CAR plasmids with different structures targeting CD7. The structures are as follows (8h in the following structure indicates that the hinge region is the extracellular hinge domain derived from CD8a, 8TM indicates that the transmembrane region is the transmembrane domain derived from CD8a, BB indicates the intracellular signal domain derived from CD137, Z indicates the CD3ζ chain, and GMR indicates the signal peptide of the GMCSF receptor). The corresponding scFv numbers in CAR1 to 3 are KK-01-A02 (CD7(3)), KK-01-C11 (CD7(4)), and KK-01-D01 (CD7(5)), respectively:

[0169] CAR-1:GMR-CD7(3)-8h-8TM-BBz

[0170] CAR-2: GMR-CD7(4)-8h-8TM-BBz

[0171] CAR-3: GMR-CD7(5)-8h-8TM-BBz

[0172] Example 3 Preparation of lentivirus and infection of T / NK lymphocytes

[0173] In this example, CAR-1 to CAR-3 are transfected into NK cells via lentivirus.

[0174] In this example, the lentivirus was packaged using the calcium phosphate method, referring to the Molecular Cloning Laboratory Guide (3rd edition, J. Sambrook et al.), specifically: 293T cells were cultured in DMEM medium containing 10% FBS (w / v) to an optimal state, the packaging plasmid (RRE:REV:2G) and the expression plasmid were added to a 1.5 mL centrifuge tube in a certain ratio, and CaCl was added. 2 and 2×HBS, mix well, let stand at room temperature and then add to the treated 293T cell culture medium. After 3-5 hours, change the medium again to 10mL DMEM medium containing 10% FBS. After 48 hours or 72 hours, collect the cell supernatant, purify the virus and determine the titer.

[0175] The prepared lentivirus titer was determined using CHO cells, using 1e 5 After 48 hours of infection of the virus to be tested, the total CAR expression was detected by Protein-L, and the positive rate was calculated. The positive rate calculation formula is: Titer (TU / mL) = 1e 5 × positive rate × dilution factor × 1000 ÷ virus volume (μL). The virus titers of the above CAR structures are shown in Table 5.

[0176] Table 5 CD7-CAR lentivirus titer list

[0177] Plasmid number Virus titer (TU / mL) CAR-1 5.46E+08 CAR-2 6.51E+08 CAR-3 5.37E+08

[0178] Lymphocytes were separated by gradient centrifugation; after centrifugation, the second layer of white lymphocytes was taken, washed with saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. After obtaining PBMC, CD56 antibody-labeled Microbeads were used for magnetic sorting to obtain CD56-positive NK cells. NK cells can be transduced with lentivirus 24 hours after activation. Three days after transduction, the positive rate of CAR-NK needs to be detected using CD7-6×His (ACRO-Biosystems) (primary antibody) and anti-his-AF647 (GenScript) (secondary antibody). The results of the CAR-NK positive rate test using CD3-PE / Cy7 and CD56-BV510 (BioLegend) labeled NK cells are shown in Table 6. All three CARs can efficiently modify NK cells.

[0179] Table 6 CD7-CAR-NK positive rate detection results

[0180] Plasmid number CAR positive rate (%) CAR-1 89.86 CAR-2 88.54 CAR-3 93.14

[0181] Example 4 In vitro killing and cytokine secretion experiments of CAR-1-CAR-3 modified NK cells

[0182] NALM6-CD7-Luc-GFP cells (NALM6-Luc-GFP exogenously expresses CD7) were used as positive target cells, and NALM6-Luc-GFP cells were used as negative target cells. CAR-NK cells and target cells were plated on target cells at a ratio of 1:4, and killing was detected by luciferase after 24 hours. Luciferase principle: When the target cells are lysed with lysis buffer during the detection, the luciferase in them will decompose the substrate to emit fluorescence, and the formula for analyzing the results using fluorescence value is: CAR-NK cell killing rate = 1-(fluorescence value of experimental group ÷ fluorescence value of blank control group).

[0183] The results are as follows Figure 5 As shown in Table 7, there was no significant difference in the in vitro killing function of the three CAR-NKs (CNK represents unmodified NK).

[0184] Table 7 In vitro killing data

[0185] structure NALM6-CD7-Luc-GFP CAR-1 79.77％ CAR-2 79.68％ CAR-3 82.27％ CNK 10.57％

[0186] The cell supernatant was collected 24 hours after killing to detect the IFN-γ secretion ability of CAR-NK cells after being stimulated by target cells. The collected supernatant was tested for IFN-γ secretion using ELISA (enzyme-linked immunosorbent assay). The results are as follows Figure 6 As shown in Table 8, relative to CNK, the secretion of IFN-γ factor by CAR-1 and CAR-3 (7520.36 pg / ml and 7615.91 pg / ml, respectively) after target cell stimulation was significantly higher than that by CAR-2 (5906.66 pg / ml).

[0187] Table 8 IFN-γ secretion data (unit: pg / ml)

[0188] structure CD7-NALM6-Luc-GFP CAR-1 7520.36 CAR-2 5906.66 CAR-3 7615.91 CNK 0

[0189] Example 5 Evaluation of the efficacy of CAR-NK cells in vivo

[0190] Using NALM6-CD7-Luc-GFP as the target cell, NCG mice were injected through the tail vein at a dose of 5e5 / mouse for tumor-bearing for 1 day. CAR-NK was cultured in vitro for 13 days and then frozen; after tumor-bearing, CAR-NK was resuscitated and injected into NCG mice with tumors through the tail vein at a dose of 5e6 / mouse. CNK cells were injected in the same dose and manner as a control group. After that, the fluorescent substrate of luciferase was injected intraperitoneally once every 7 days, and in vivo imaging was performed using a small animal in vivo imaging instrument to detect tumor-bearing conditions ( Figure 7 ). In addition, submandibular blood was collected every 7 days after CAR-NK transfusion, and the copy number of BBz gene in the blood was detected by qPCR.

[0191] The results are as follows Figure 7 As shown, the CAR1-CAR3 constructed by KK-01-A02, KK-01-C11, and KK-01-D01 we screened can all exert good in vivo effectiveness. Among them, NK cells modified by CAR-1 and CAR-3 have relatively good in vivo efficacy advantages over NK cells modified by CAR-2 and unmodified NK.

[0192] It should be noted that although the present invention takes NK cells and CAR-NK cells as examples, based on the content disclosed in the present invention, technicians in the field can obviously infer that other immune cells such as T cells, macrophages, DC cells and related prerequisite cells can also express the engineered immune cells described herein containing CAR for CD7, and specifically recognize CD7, and play a killing role against malignant tumors expressing CD7. The chimeric antigen receptor constructed by using the scFv for CD7 as an extracellular antigen recognition domain or one of the extracellular antigen recognition domains described in this application can be expressed in T cells, NK cells, macrophages, DC cells and related prerequisite cells; it can be co-expressed with fusion proteins of any structure in T cells, NK cells, macrophages, DC cells and related precursor cells, and used for drugs or drug combinations for malignant tumors expressing CD7 for tumor treatment.

[0193] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A CD7 scFv, characterized in that A CDR sequence comprising any of the following: (I), CDR1, CDR2 and CDR3 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 7, 8 and 9 respectively; and The CDR1, CDR2 and CDR3 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 10, 11 and 12 respectively; or (II), CDR1, CDR2 and CDR3 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 13, 14 and 15, respectively; and The CDR1, CDR2 and CDR3 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 16, 17 and 18, respectively; or (III), CDR1, CDR2 and CDR3 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 19, 20 and 21, respectively; and The CDR1, CDR2 and CDR3 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 22, 23 and 24, respectively; or (IV) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (I) to (III); or (V) A sequence having an identity of 80% or more to the amino acid sequence shown in any one of (I) to (IV).

2. The CD7 scFv according to claim 1, characterized in that A FR sequence comprising any of the following: (VI), FR1, FR2, FR3 and FR4 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 25, 26, 27 and 28, respectively; and The FR1, FR2, FR3 and FR4 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 29, 30, 31 and 32, respectively; or (VII), FR1, FR2, FR3 and FR4 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 33, 34, 35 and 36, respectively; and The FR1, FR2, FR3 and FR4 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No.37, 38, 39 and 40, respectively; or (VIII), FR1, FR2, FR3 and FR4 of the light chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No. 41, 42, 43 and 44, respectively; and The FR1, FR2, FR3 and FR4 of the heavy chain of the CD7 scFv have the amino acid sequences shown in SEQ ID No.45, 46, 47 and 48, respectively; or (VI) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (i) to (iii); or (X) A sequence having 80% or more homology with the amino acid sequence shown in any one of (i) to (iv).

3. The CD7 scFv according to claim 1 or 2, characterized in that A variable region sequence comprising any of the following: (i) the variable region of the light chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No. 50; and The variable region of the heavy chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.51; or (ii), the variable region of the light chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.53; and The variable region of the heavy chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.54; or (iii), the variable region of the light chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.56; and The variable region of the heavy chain of the CD7 scFv has the amino acid sequence shown in SEQ ID No.57; or (iv) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (i) to (iii); or (v) A sequence having 80% or more homology with the amino acid sequence shown in any one of (i) to (iv).

4. The CD7 scFv according to any one of claims 1 to 3, characterized in that The variable region of the light chain and the variable region of the heavy chain are connected by a linker peptide; The connecting peptide linker has: (vi), the amino acid sequence shown in SEQ ID No.49; or (vii) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (vi); or (viii) A sequence having a homology of 80% or more to the amino acid sequence shown in (vi).

5. The CD7 scFv according to any one of claims 1 to 4, characterized in that Include any of the following: (1) The CD7 scFv has the amino acid sequence shown in SEQ ID No.52; or (2) the CD7 scFv has the amino acid sequence shown in SEQ ID No.55; or (3) the CD7 scFv has the amino acid sequence shown in SEQ ID No.58; or (4) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (1) to (3); or (5) A sequence having a homology of 80% or more to the amino acid sequence shown in any one of (1) to (4).

6. A nucleic acid molecule encoding the CD7 scFv according to any one of claims 1 to 5.

7. The nucleic acid molecule according to claim 6, wherein (6) the CD7 scFv nucleic acid molecule has a nucleotide sequence as shown in SEQ ID No. 64, 65 or 66; or (7) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (6) but is different from the nucleotide sequence shown in (6) due to the degeneracy of the genetic code; or (8) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (6) or (7), and having the same or similar functions as the nucleotide sequence shown in (6) or (7); or (9) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (6) to (8).

8. A protein combination, characterized in that It comprises the CD7 scFv according to any one of claims 1 to 5, a transmembrane region, a hinge region and an intracellular signaling domain.

9. The protein combination according to claim 8, characterized in that The transmembrane region is selected from the transmembrane region of DAP10, DAP12, NKG2D, CD4, CD8α or CD28; The hinge region includes but is not limited to the hinge region of IgG, IgD, CD7 or CD8α / CD28; The intracellular signaling domain may comprise a primary signaling domain and / or a co-stimulatory domain, wherein the primary signaling domain comprises a signaling domain of one or more molecules selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa, CD79α, CD79β, CD66d, DAP10 or DAP12; The co-stimulatory domain comprises a signal transduction domain selected from one or more of the following molecules: 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), CD 160. CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, 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 a ligand that specifically binds to CD83.

10. The protein combination according to claim 8 or 9, characterized in that (10), the transmembrane region has the amino acid sequence shown in SEQ ID No.59; or (11), the hinge region has the amino acid sequence shown in SEQ ID No.60; or (12), the intracellular signaling domain has an amino acid sequence as shown in SEQ ID No.61; (13), the primary signal transduction domain has the amino acid sequence shown in SEQ ID No.62; or (14) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (10) to (13); or (15) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of (10) to (14).

11. The protein combination according to any one of claims 8 to 10, characterized in that The protein combination includes CAR-1, CAR-2 and / or CAR-3; (A), the CAR-1, CAR-2 and CAR-3 have the amino acid sequences shown in SEQ ID No. 4, 5, and 6; or (B) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (A); or (C) An amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in (A) or (B).

12. A genetic element, characterized in that Comprising the nucleic acid molecule according to claim 6 or 7.

13. The genetic element according to claim 12, wherein The gene elements include CAR-1, CAR-2 and CAR-3; (D), the CAR-1, CAR-2 and CAR-3 have the nucleotide sequences shown in SEQ ID No. 1, 2 and 3 respectively; or (E), a nucleotide sequence obtained by replacing, 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 (F) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in (D) or (E).

14. An artificial carrier, characterized in that The invention comprises the gene element according to claim 12 or 13 and an acceptable auxiliary agent or carrier.

15. A virus particle, characterized in that Comprising the artificial carrier as claimed in claim 14.

16. An engineered cell, characterized in that Include any of the following: (a), the CD7 scFv according to any one of claims 1 to 5; and / or (b), a nucleic acid molecule as described in claim 6 or 7; and / or (c) a protein combination as claimed in any one of claims 8 to 11; and / or (d) a genetic element as claimed in claim 12 or 13; and / or (e), an artificial carrier as claimed in claim 14; and / or (f) transducing the viral particles as described in claim 15.

17. The engineered cell according to claim 16, characterized in that The engineered cells include, but are not limited to, NK cells, T cells, macrophages, DC cells and related prerequisite cells.

18. Application of any of the following in detecting CD7 expression: Ⅰ), the CD7 scFv according to any one of claims 1 to 5; and / or II), a nucleic acid molecule as described in claim 6 or 7; and / or III), a protein combination as claimed in any one of claims 8 to 11; and / or IV), the genetic element according to claim 12 or 13; and / or V) The artificial vector as claimed in claim 14.

19. Use of any of the following in the preparation of a product for detecting CD7 expression: Ⅰ), the CD7 scFv according to any one of claims 1 to 5; and / or II), a nucleic acid molecule as described in claim 6 or 7; and / or III), a protein combination as claimed in any one of claims 8 to 11; and / or IV), the genetic element according to claim 12 or 13; and / or V) The artificial vector as claimed in claim 14.

20. The use according to claim 19, characterized in that The product includes a kit, a detection antibody and / or a CD7 molecule tracing reagent.

21. A product, characterized in that include: Ⅰ), the CD7 scFv according to any one of claims 1 to 5; and / or II), a nucleic acid molecule as described in claim 6 or 7; and / or III), a protein combination as claimed in any one of claims 8 to 11; and / or IV), the genetic element according to claim 12 or 13; and / or V) The artificial vector as claimed in claim 14.

22. Use of any of the following in the preparation of a drug for preventing and / or treating a disease: 1), the CD7 scFv according to any one of claims 1 to 5; and / or 2) A nucleic acid molecule according to claim 6 or 7; and / or 3) A protein combination according to any one of claims 8 to 11; and / or 4) The genetic element according to claim 12 or 13; and / or 5) The artificial carrier according to claim 14; and / or 6) The viral particle according to claim 15; and / or 7) The engineered cell according to claim 16 or 17.

23. The disease according to claim 22, characterized in that The disease includes a tumor.

24. A drug, characterized in that Includes any of the following items and pharmaceutically acceptable excipients or adjuvants: 1), the CD7 scFv according to any one of claims 1 to 5; and / or 2) A nucleic acid molecule according to claim 6 or 7; and / or 3) A protein combination according to any one of claims 8 to 11; and / or 4) The genetic element according to claim 12 or 13; and / or 5) The artificial carrier according to claim 14; and / or 6) The viral particle according to claim 15; and / or 7) The engineered cell according to claim 16 or 17.

25. A pharmaceutical combination, characterized in that Comprising the drug as claimed in claim 24 and any other active ingredients.

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