CD7-targeting nanobody and use thereof

Abstract

The present invention relates to the field of nanobodies, and specifically relates to a CD7-targeting nanobody and a use thereof. Disclosed is a CD7-targeting nanobody. The nanobody has a small molecular weight and low immunogenicity, and is more suitable for clinical applications such as cell therapy and gene therapy.

Description

Nanobody targeting CD7 and use thereof TECHNICAL FIELD

[0001] The present application relates to the field of nanobodies, in particular to a nanobody targeting CD7 and use thereof. BACKGROUND

[0002] CD7 antigen is a single-chain glycoprotein, which is a marker antigen molecule in the process of T cell development. In addition to healthy human thymocytes, T cells and natural killer cells, hematopoietic stem and progenitor cells such as myeloid lineage precursor cells also express CD7. A large number of studies have shown that CD7 molecules are expressed on T lymphocyte leukemia and lymphoma in most people, and about 10% of cancer cells of acute myeloid leukemia (AML) patients also express CD7 antigen.

[0003] In addition, CD7 is an endocytic receptor, which rapidly triggers endocytosis after binding to anti-CD7 antibody. This property makes CD7 an ideal target for targeted delivery of various functional molecules to CD7 + cells, such as T cells.

[0004] However, traditional four-chain antibodies have the disadvantages of large molecular weight, weak binding force for binding to antigens, low affinity, difficulty in modification, and poor stability. Therefore, the demand for an anti-CD7 antibody with small molecular weight, high specificity and stability has not been met. SUMMARY

[0005] Therefore, in order to at least solve one of the above technical problems, the first aspect of the present application provides an anti-CD7 nanobody, which comprises a CDR-H1 region, a CDR-H2 region and a CDR-H3 region.

[0006] The CDR-H1 region is selected from an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30 or 34;

[0007] The CDR-H2 region is selected from an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31 or 35; and

[0008] the CDR-H3 region is selected from an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, or 36.

[0009] In some embodiments of the application,

[0010] the CDR-H1 region, CDR-H2 region, CDR-H3 region have amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, respectively;

[0011] the CDR-H1 region, CDR-H2 region, CDR-H3 region have amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, respectively;

[0012] the CDR-H1 region, CDR-H2 region, CDR-H3 region have amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, respectively;

[0013] the CDR-H1 region, CDR-H2 region, CDR-H3 region have amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, respectively;

[0014] the CDR-H1 region, CDR-H2 region, CDR-H3 region have amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, respectively;

[0015] the CDR-H1 region, CDR-H2 region, and CDR-H3 region are determined according to the IMGT numbering scheme, the Kabat numbering scheme, the Martin numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, or the Contact numbering scheme.

[0016] the CDR-H1 region, CDR-H2 region, and CDR-H3 region are determined according to the IMGT numbering scheme, the Kabat numbering scheme, the Martin numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, or the Contact numbering scheme.

[0017] the CDR-H1 region, CDR-H2 region, and CDR-H3 region are determined according to the IMGT numbering scheme, the Kabat numbering scheme, the Martin numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, or the Contact numbering scheme.

[0018] the CDR-H1 region, CDR-H2 region, and CDR-H3 region are determined according to the IMGT numbering scheme, the Kabat numbering scheme, the Martin numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, or the Contact numbering scheme.

[0019] In some embodiments of the application, the CDR-H1 region, CDR-H2 region, and CDR-H3 region are determined according to the IMGT numbering scheme, the Kabat numbering scheme, the Martin numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, or the Contact numbering scheme.

[0020] In some embodiments of the application, the CDR-H1 region, CDR-H2 region, and CDR-H3 region are determined according to the Martin (Martin numbering scheme) numbering scheme.

[0021] In some embodiments of the application, the amino acid sequence of the anti-CD7 Nanobody is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, or SEQ ID NO: 33.

[0022] In some embodiments of the application, the anti-CD7 Nanobody can bind to human CD7 (UniProt KB No. P09564).

[0023] In some embodiments of the application, any of the aforementioned amino acid sequences further comprises a derivative sequence which optionally has at least one amino acid added, deleted, modified and / or substituted, and which is capable of retaining CD7 binding affinity.

[0024] In some embodiments of the application, the number of amino acids added, deleted, modified and / or substituted is 1-5 (e.g., 1-3, preferably 1-2, more preferably 1).

[0025] In some embodiments of the application, the derivative sequence which has at least one amino acid added, deleted, modified and / or substituted, and which is capable of retaining CD7 binding affinity, is an amino acid sequence which has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or sequence identity to any of the aforementioned amino acid sequences.

[0026] The anti-CD7 Nanobody disclosed in the present application also includes conservative variants thereof, wherein the conservative variants are polypeptides in which up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by similar or analogous amino acids, while retaining CD7 binding affinity, as compared to the amino acid sequence of the anti-CD7 Nanobody disclosed in the present application.

[0027] In a second aspect, the present application provides an isolated polynucleotide encoding the anti-CD7 Nanobody provided in the first aspect of the present application.

[0028] In a third aspect, the present application provides a chimeric antigen receptor (CAR) comprising:

[0029] (a) an extracellular antigen binding region;

[0030] (b) a transmembrane region; and

[0031] (c) an intracellular signaling domain;

[0032] wherein the extracellular antigen binding region comprises the anti-CD7 Nanobody provided in the first aspect of the application;

[0033] Preferably, the chimeric antigen receptor further comprises a hinge region and a costimulatory signaling domain.

[0034] In some embodiments of the application, the transmembrane region of the CAR is selected from the transmembrane region of CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8a, CD8b, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIy, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.

[0035] In some embodiments of the application, the transmembrane region of the CAR comprises the transmembrane region of CD8a; preferably the transmembrane region of human CD8a.

[0036] In some embodiments of the application, the transmembrane region of human CD8a has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39.

[0037] In some embodiments of the application, the intracellular signaling domain of the CAR is selected from the intracellular signaling domain of CD3e, CD3y, CD35, CD3zeta, CD79a, CD79B, FceRly, FceRb, FcyRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12, and other intracellular signaling domains of proteins comprising at least one ITAM.

[0038] In some embodiments of the application, the intracellular signaling domain of the CAR comprises an intracellular signaling domain of CD3 zeta; preferably an intracellular signaling domain of human CD3 zeta.

[0039] In some embodiments of the application, the intracellular signaling domain of the CAR comprises an intracellular signaling domain of CD3 zeta; preferably an intracellular signaling domain of human CD3 zeta.

[0040] In some embodiments of the application, the CAR further comprises a hinge region connecting the extracellular antigen-binding region and the transmembrane region.

[0041] In some embodiments of the application, the hinge region of the CAR is connected to the extracellular antigen-binding region and the transmembrane region in sequence, the hinge region being located between the C-terminus of the extracellular antigen-binding region and the N-terminus of the transmembrane region.

[0042] In some embodiments of the application, the hinge region of the CAR is selected from the hinge region of the following proteins: CD28, CD8, CD8a, CD8b, CD3, CD45, Ig4, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

[0043] In some embodiments of the application, the hinge region of the CAR comprises a hinge region of CD8a; preferably a hinge region of human CD8a.

[0044] In some embodiments of the application, the hinge region of the CAR comprises a hinge region of CD8a; preferably a hinge region of human CD8a.

[0045] In some embodiments of the application, the CAR further comprises a co-stimulatory signaling domain.

[0046] In some embodiments of the application, the co-stimulatory signaling domain of the CAR comprises a co-stimulatory signaling domain of at least one of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8a, CD8b, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcaRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-l, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, and MyD88.

[0047] In some embodiments of the application, the co-stimulatory signaling domain comprises a co-stimulatory signaling domain of 4-1BB and / or a co-stimulatory signaling domain of CD28; preferably a co-stimulatory signaling domain of human 4-1BB and / or human CD28.

[0048] In some embodiments of the application, the co-stimulatory signaling domain of human 4-1BB has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42.

[0049] In some embodiments of the application, the CAR further comprises a signal peptide located at the N-terminus of the CAR.

[0050] In some embodiments of the application, the signal peptide of the CAR is selected from the group consisting of a signal peptide of HLA-A, CD8a, CD28, CD33, Igkappa, IL-2, and GM-CSFRa.

[0051] In some embodiments of the application, the signal peptide of the CAR comprises a signal peptide of CD8a; preferably a signal peptide of human CD8a.

[0052] In some embodiments of the application, the signal peptide of human CD8a has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37.

[0053] In a fourth aspect, the present application provides an isolated polynucleotide encoding the chimeric antigen receptor provided in the third aspect of the present application.

[0054] In a fifth aspect, the present application provides a vector, wherein the surface of the vector comprises a targeting molecule, wherein the targeting molecule comprises the anti-CD7 Nanobody provided in the first aspect of the present application;

[0055] Preferably, the vector is selected from at least one of the group consisting of a lipid nanoparticle, a virus-like particle, an extracellular vesicle, an adenovirus, an adeno-associated virus, a lentiviral vector (LVV) and a retroviral vector (RVV).

[0056] More preferably, the vector is a LVV or a RVV.

[0057] In some embodiments of the present application, the targeting molecule further comprises a transmembrane region, wherein the anti-CD7 Nanobody is directly or indirectly linked to the transmembrane region, exposed on the surface of the vector.

[0058] Preferably, the transmembrane region is selected from the transmembrane region of the following proteins:

[0059] CD28, CD2, CD4, CD8a, CD5, CD3e, CD3d, CD3z, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD154 (CD40L), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRa, FcRp, FcRy, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCRp, TIM3, TRIM, LPA5, and Zap70.

[0060] More preferably, the transmembrane region comprises the transmembrane region of CD8a.

[0061] In some embodiments of the present application, the transmembrane region of the targeting molecule is the transmembrane region of any one of the aforementioned CD8a.

[0062] In some embodiments of the present application, the targeting molecule further comprises a linker domain, and the anti-CD7 nanobody is indirectly linked to the transmembrane domain via the linker domain;

[0063] Preferably, the linker domain is selected from the group consisting of:

[0064] (a) an immunoglobulin hinge region selected from the group consisting of wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA, and IgD hinge regions;

[0065] (b) a hinge region selected from the group consisting of wild-type or modified hinge regions of CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

[0066] (c) all or a portion of an Fc domain selected from the group consisting of one or more of a CHI domain, a CH2 domain, and a CH3 domain; and

[0067] (d) a stalk region of a type II C-lectin selected from the group consisting of stalk regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D;

[0068] More preferably, the linker domain comprises a hinge region of CD8a.

[0069] In some embodiments of the present application, the linker domain of the targeting molecule is a hinge region of CD8a.

[0070] In some embodiments of the present application, the targeting molecule comprises, in order from N-terminus to C-terminus, the anti-CD7 nanobody, the linker domain, and the transmembrane domain.

[0071] In some embodiments of the present application, a polynucleotide encoding the targeting molecule comprises, in order from 5’ end to 3’ end, any of the aforementioned signal peptides, any of the aforementioned anti-CD7 nanobodies, any of the aforementioned linker domains, and any of the aforementioned transmembrane domains.

[0072] In some embodiments of the present application, the vector is an LVV or an RVV, the targeting molecule comprises, in order from N-terminus to C-terminus, the anti-CD7 nanobody, the linker domain, and the transmembrane domain, and the viral envelope of the LVV or the RVV comprises the targeting molecule.

[0073] In some embodiments of the present application, the viral envelope of the LVV or the RVV comprises a viral glycoprotein.

[0074] In some embodiments of the application, the viral glycoprotein is selected from the group consisting of a Vesiculovirus strain glycoprotein, a NiV glycoprotein G, a Measles virus glycoprotein H, a Lentivirus glycoprotein, a Rabies virus glycoprotein (RVG), a GaLV glycoprotein, a MLV-A glycoprotein, a RD114 glycoprotein, a FPV glycoprotein, an EboV glycoprotein, and an LCMV glycoprotein, or any combination thereof.

[0075] In some embodiments of the application, the Vesiculovirus strain glycoprotein is selected from the group consisting of a Vesiculovirus Indiana strain glycoprotein, a Vesiculovirus Cocal strain glycoprotein, a Vesiculovirus Maraba strain glycoprotein, a Vesiculovirus Morreton strain glycoprotein, a Vesiculovirus Alagoas strain glycoprotein, a Vesiculovirus New Jersey strain glycoprotein, a Vesiculovirus Carajas strain glycoprotein, a Vesiculovirus Chandipura strain glycoprotein, a Vesiculovirus Eptesicus strain glycoprotein, a Vesiculovirus Isfahan strain glycoprotein, a Vesiculovirus Jurona strain glycoprotein, a Vesiculovirus Malpais strain glycoprotein, a Vesiculovirus Perinet strain glycoprotein, a Vesiculovirus Piry strain glycoprotein, a Vesiculovirus Radi strain glycoprotein, a Vesiculovirus Rhinolopus strain glycoprotein, and a Vesiculovirus Yug Bogdanovac strain glycoprotein, or any combination thereof.

[0076] In some embodiments of the application, the viral glycoprotein is:

[0077] (a) a Vesiculovirus Indiana strain glycoprotein (VSV-G); or

[0078] (b) a Vesiculovirus Cocal strain glycoprotein (Cocal-G).

[0079] In some embodiments of the application, the viral glycoprotein comprises a first mutation that inhibits its ability to specifically bind to a viral glycoprotein receptor.

[0080] In some embodiments of the application, the viral glycoprotein comprises a second mutation that enhances its ability to antagonize complement inactivation.

[0081] In some embodiments of the application, the VSV-G or Cocal-G comprises a first mutation that inhibits its ability to specifically bind to its receptor, LDL-R.

[0082] In some embodiments of the application, the VSV-G or Cocal-G comprises a second mutation that enhances its ability to antagonize complement inactivation.

[0083] In some embodiments of the application, the viral glycoprotein comprising the first mutation and / or the second mutation retains the ability to mediate membrane fusion and mediate endosomal / lysosomal escape.

[0084] In some embodiments of the application, the first mutation and the second mutation can be referred to the published patents WO2024067870A1 and WO2024213019A1, the contents of which are incorporated herein by reference in their entirety.

[0085] In a sixth aspect, the present application provides an engineered immune effector cell comprising the CAR provided in the third aspect of the present application or the isolated polynucleotide provided in the fourth aspect of the present application;

[0086] Preferably, the immune effector cell is selected from at least one of T cells, NK cells, NK / T cells, neutrophils, monocytes, dendritic cells and macrophages.

[0087] More preferably, the immune effector cell is a T cell.

[0088] In some embodiments of the application, the immune effector cell membrane expresses the CAR provided in the third aspect of the present application.

[0089] In some embodiments of the application, the engineered immune effector cell is a CAR-T cell that membrane expresses the CAR provided in the third aspect of the present application.

[0090] In a seventh aspect, the present application provides a composition comprising the anti-CD7 Nanobody provided in the first aspect of the present application or the engineered immune effector cell provided in the sixth aspect of the present application.

[0091] Preferably, the composition can further comprise a pharmaceutically acceptable excipient or carrier.

[0092] In an eighth aspect, the present application provides use of the anti-CD7 Nanobody provided in the first aspect of the present application, the chimeric antigen receptor provided in the third aspect of the present application, the vector provided in the fifth aspect of the present application, the engineered immune effector cell provided in the sixth aspect of the present application or the composition provided in the seventh aspect of the present application in the preparation of a medicament for treating a T cell-related and / or CD7-related disease.

[0093] Preferably, the T cell-related and / or CD7-related disease comprises a T cell-related and / or CD7-related tumor, a T cell-related and / or CD7-related autoimmune disease, and a T cell-related and / or CD7-related inflammatory disease.

[0094] More preferably, the T cell and / or CD7-related tumor comprises peripheral T cell lymphoma (NHL), NK / T cell lymphoma, anaplastic large cell lymphoma (ALCL), enteropathy T cell lymphoma, acute myeloid leukemia (AML), large granular lymphocytic leukemia (T-LGL), and fetal center T cell lymphoma (FTCL).

[0095] In some embodiments of the application, the CD7 is human CD7 (UniProt KB No. P09564).

[0096] In a ninth aspect, the present application provides a reagent for detecting a CD7 protein or a functional domain comprising an Epitope thereof, the reagent comprising the anti-CD7 Nanobody according to the first aspect of the present application.

[0097] Preferably, the reagent further comprises a modifying moiety linked to the anti-CD7 Nanobody, the modifying moiety comprising a detectable label or a therapeutic agent.

[0098] More preferably, the detectable label comprises at least one of an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, and biotin.

[0099] In some embodiments of the application, the CD7 is human CD7 (UniProt KB No. P09564).

[0100] In a tenth aspect, the present application provides a method for detecting a CD7 protein or a functional domain comprising an Epitope thereof, not for diagnostic purposes, the method comprising obtaining a sample suspected of containing a CD7 protein or a functional domain comprising an Epitope thereof, contacting the sample with the anti-CD7 Nanobody according to the first aspect of the present application or the reagent according to the ninth aspect of the present application to form an antibody-antigen complex, and detecting the presence of the antibody-antigen complex.

[0101] In an eleventh aspect, the present application provides a method for treating a T cell-related and / or CD7-related disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the anti-CD7 Nanobody according to the first aspect of the present application, the engineered immune effector cell according to the sixth aspect of the present application, or the composition according to the seventh aspect of the present application.

[0102] Preferably, the T cell-related and / or CD7-related disease is selected from the group consisting of a T cell-related and / or CD7-related tumor, a T cell-related and / or CD7-related autoimmune disease, and a T cell-related and / or CD7-related inflammatory disease.

[0103] More preferably, the T cell and / or CD7-related tumor comprises peripheral T cell lymphoma (NHL), NK / T cell lymphoma, anaplastic large cell lymphoma (ALCL), intestinal T cell lymphoma, acute myeloid leukemia (AML), large granular lymphocytic leukemia (T-LGL), and fetal center T cell lymphoma (FTCL).

[0104] In some embodiments of the present application, the CD7 is human CD7 (UniProt KB No. P09564).

[0105] In some embodiments of the present application, the subject is a human.

[0106] In a twelfth aspect, the present application provides an expression vector comprising the isolated polynucleotide according to the second aspect of the present application or the isolated polynucleotide according to the fourth aspect of the present application.

[0107] Preferably, the expression vector is selected from at least one of the group consisting of a plasmid, a lipid nanoparticle, a virus-like particle, an extracellular vesicle, an adenovirus, an adeno-associated virus, a lentivirus vector, and a retrovirus vector.

[0108] The beneficial effects of the present application include:

[0109] Compared with antibodies such as four-chain antibodies (full-length antibodies) and scFv single-chain antibodies, which have larger molecular weights, are less stable, and have lower specificity, the anti-CD7 nanobodies provided by the present application have the advantages of small molecular weight, high solubility, high stability, low immunogenicity, high tissue penetration, no need for additional folding and assembly steps or linker optimization, and are more suitable for clinical applications such as cell therapy and gene therapy.

[0110] Definitions:

[0111] “Nanobody”: As used herein, “nanobody”, “heavy chain variable region of heavy chain antibody”, and “VHH” are used interchangeably. Nanobody or VHH is the antigen binding region of heavy chain only antibody (“HcAb”). Heavy chain antibody is naturally produced by Camelidae and Loriciridae organisms (Bever CS, et al., VHH antibodies: emerging reagents for the analysis of environmental chemicals. Anal Bioanal Chem. 2016 Sep;408(22):5985-6002.).

[0112] Unlike traditional four-chain antibodies, nanobodies have a single variable domain of single-chain antibody. The antigen binding fragment in each heavy chain of heavy chain antibody of Camelidae has a single heavy chain variable domain (VHH) which can have high affinity to antigen without the help of light chain. VHH antibody of Camelidae is called the smallest functional antigen binding fragment with a molecular weight of only about 15 kD, and is also called nanobody.

[0113] Heavy chain antibody lacks light chain and heavy chain constant region 1 (CH1), and only contains 2 heavy chains composed of variable region (VHH) and other constant regions, and the variable region is connected to the constant region through a hinge-like region structure. Each heavy chain of heavy chain antibody of Camelidae contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of heavy chain antibody of Loriciridae contains 1 variable region and 5 constant regions (CH1-CH5).

[0114] Generally, after obtaining heavy chain antibody naturally lacking light chain and heavy chain constant region 1 (CH1), the heavy chain variable region of the heavy chain antibody is cloned to construct a nanobody composed of only one heavy chain variable region. The nanobody contains three CDR-H regions (CDR-H1 region, CDR-H2 region and CDR-H3 region) and four FR regions (FR1 region, FR2 region, FR3 region and FR4 region).

[0115] Nanobody / VHH antibody has natural advantages of good solubility, high stability, strong penetration and wide epitope binding. In terms of application, research on nanobody for autoimmune diseases, blood diseases, viral infections and orthopedic diseases has gradually entered the clinical research stage; in terms of anti-infection, anti-inflammatory diseases and neurodegenerative diseases, nanobody has also shown great advantages.

[0116] The anti-CD7 Nanobodies of the application can be prepared using methods conventional in the art, such as phage display technology well known in the art; alternatively, the various antibodies of the application can be expressed in other cell lines. A suitable mammalian host cell can be transformed with a polynucleotide encoding a Nanobody provided by the application. Transformation can be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and using the virus (or vector) to transduce the host cell. The transformation procedure used will depend on the host to be transformed.

[0117] Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene- mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of the DNA into nuclei, etc. Host mammalian cell lines useful for expression are well known in the art, such as the various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, HEK-293T cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with the essential CD7 binding properties.

[0118] Without materially affecting the activity of the antibodies, one skilled in the art can make one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid alterations to the sequences of the application, particularly the sequences of the anti-CD7 Nanobodies, to obtain variants of the anti-CD7 Nanobodies. These variants include, but are not limited to, deletions, insertions and / or substitutions of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and additions of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids to the C-terminus and / or the N-terminus. In the art, conservative substitutions of amino acids with similar properties are commonly made to preserve the functional properties of the protein. For example, substitutions in the FR regions and / or the CDR regions are made with amino acids having similar properties. The amino acid residues which can be conservatively substituted are well known in the art, and the amino acid residues substituted by the foregoing methods can or can not be encoded by the genetic code. For another example, additions of one or several amino acids to the C-terminus and / or the N-terminus also usually do not change the functional properties of the protein. They are all considered to be within the scope of the protection of the present application.

[0119] "Inhibit" : When referring to the ability of a viral glycoprotein to bind to its receptor (binding ability) being "inhibited", the term "inhibit" includes completely eliminating the binding ability, as well as significantly reducing the binding ability. In particular embodiments, "significantly reducing" means a reduction selected from the group consisting of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, at least 4%, at least 3%, at least 2%, and at least 1%, relative to (a) a wild-type viral glycoprotein or (b) a viral glycoprotein that does not comprise the first mutation provided by the application.

[0120] "Sequence identity": In general, "sequence identity" or "sequence homology" refers to the exact correspondence of nucleotides and nucleotides or amino acids and amino acids, respectively, of two polynucleotide or polypeptide sequences. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity". Whether nucleic acid or amino acid sequences, the percent identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Sequence information can also be compared using the Advanced BLAST computer program, available from the National Institutes of Health, to determine percent identity. The BLAST program is based on the following alignment method: Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (typically nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine percent identity over the entire length of the proteins being compared.

[0121] The sequence identity described herein can be measured using sequence analysis software, for example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The present application also includes molecules having antibody heavy chain variable regions with CDR regions that have at least 80% and more (preferably 95% and more, most preferably 98% and more) homology to the CDR regions identified herein.

[0122] “Specific binding”: As used herein, the term “specific binding” refers to binding that occurs between pairs of molecular species (e.g., a receptor and a ligand). Binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions when the interaction of the two species results in a non-covalently bound complex. In various embodiments, specific binding between one or more species is direct. In some embodiments of the application, the affinity of specific binding is about 2-fold greater than background binding, about 5-fold greater than background binding, about 10-fold greater than background binding, about 20-fold greater than background binding, about 50-fold greater than background binding, about 100-fold greater than background binding, or about 1000-fold or more greater than background binding.

[0123] “Chimeric antigen receptor”: i.e., CAR, modified T cells (CAR-T cells) as an immunotherapy strategy, has been widely valued and applied in tumor treatment, especially in hematological malignancies. The principle of preparing CAR-T cells is to make T cells express receptor structures that can specifically recognize tumor cell surface antigens through genetic modification, and after the specific binding of the receptor and the tumor cell surface antigen, activate its downstream immune costimulatory factors and T cells, thereby activating T cells to secrete related cytokines and specifically kill tumor cells. The structure of CAR is generally composed of four parts: an extracellular antigen binding region (commonly an antibody with antigen recognition function such as nanobodies and single-chain antibodies (scFv) and the like), a hinge region, a transmembrane region and an intracellular signal transduction domain. At present, according to the number of costimulatory molecules added to the intracellular signal transduction domain, CAR structure is usually divided into first generation (without costimulatory molecules), second generation (containing one costimulatory molecule) and third generation (containing two costimulatory molecules), and the second generation CAR structure is currently the most widely used in marketed products and clinical research.

[0124] In some embodiments of the application, the extracellular antigen binding region of the CAR comprises at least one binding domain, and the at least one binding domain comprises an anti-CD7 nanobody disclosed herein.

[0125] "T cell": T cells are one of several white blood cells important in the human immune system and play a major role in the adaptive immune response. One of the main functions of T cells is immune-mediated cell death, which is accomplished primarily by two T cell subtypes: CD8 + T cells (Cytotoxic T Cells) and CD4 + T cells (Helper T Cells).

[0126] In some embodiments of the present application, the T cells are CD4 + / CD8 - , CD4 - / CD8 + , CD4 + / CD8 + , CD4 - / CD8 - T cells or a combination thereof. In some embodiments of the present application, the CD4 + T cells produce IL-2, TFN, TNF or a combination thereof upon expression of a CAR and binding to a target cell, such as a cancer cell. In some embodiments of the present application, the CD8 + T cells lyse an antigen-specific target cell upon expression of a CAR and binding to a target cell, such as a cancer cell.

[0127] "Nucleic acid": refers to any chemical and / or substance including a polymer comprising nucleotides, such as a polynucleotide. As used herein, "nucleic acid", "polynucleotide", and "gene" are used synonymously. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by the sequence of bases, which represents the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5' to 3'. As used herein, the term "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. A "nucleic acid" can be linear or circular. Furthermore, a "nucleic acid" includes both the sense (coding) strand and the antisense (template) strand, as well as single- and double-stranded forms. Moreover, a "nucleic acid" as described herein can contain naturally-occurring or non-naturally-occurring nucleotides. Examples of non-naturally-occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues.

[0128] “Signal Peptide”: Signal Peptide, sometimes also referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).) that is present at the N-terminus of most newly synthesized proteins that are destined for the secretory pathway (occasionally found at the C-terminus or internally) (Owji, et al., A comprehensive review of signal peptides: Structure, roles, and applications, European Journal of Cell Biology. 97(6):422-441. (2018)).

[0129] “Retrovirus” and “Retroviral Vector”: i.e. Retrovirus and Retroviral Vector. A retrovirus is a virus that can integrate a copy of the DNA contained in its RNA genome into the DNA of the host cell it infects, thereby altering the host cell’s genome. An overview of available packaging lines is provided in Cold Spring Harbour Laboratory Press, 1997, page 447 by JM Coffin, SM Hughes et al., which is incorporated herein in its entirety by reference.

[0130] “Lentivirus”: Lentiviruses are complex retroviruses that contain additional genes with regulatory or structural functions in addition to the usual retroviral genes gag, pol, and env. The higher complexity enables the virus to regulate its life cycle, as it does during latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIV).

[0131] “Lentiviral Vector”: i.e. Lentiviral Vector, which is generated by multiple attenuations of HIV virulence genes through gene editing, genetic engineering, etc. techniques, for example, deleting genes env, vif, vpr, vpu, and nef, so that the lentiviral vector has biosafety.

[0132] Lentiviral vectors can stably integrate exogenous payload genes such as target genes into the chromosomes of target cells, allowing the target cells to express the delivered target genes for a long time, providing a great advantage for gene therapy. In addition, they do not transfer viral genes, thus avoiding the problem of transduced cells being destroyed by cytotoxic T cells.

[0133] Packaging plasmids for packaging lentiviral vectors are known in the art, see Naldini, et al., (1996) Science 272:263-7; Zufferey, et al., (1998) J. Virol. 72:9873-9880; Dull, et al., (1998) J. Virol. 72:8463-8471, U.S. Patent No. 6,013,516 and U.S. Patent No. 5,994,136, each of which is incorporated by reference in its entirety.

[0134] Lentiviral vectors or retroviral vectors are generally packaged in packaging cells by lentiviral vector packaging systems or retroviral vector packaging systems. Exemplary, processes and methods for packaging lentiviral vectors see Merten OW, et al., Production of lentiviral vectors. Mol Ther Methods Clin Dev. 2016, which is incorporated by reference in its entirety.

[0135] Packaging cells can be genetically engineered to otherwise improve the immunological properties of lentiviral vectors and retroviral vectors disclosed herein and / or to increase the efficiency of lentiviral vectors and retroviral vectors to transduce target cells; such otherwise including but not limited to adding genes, deleting genes, and introducing point mutations into genes.

[0136] In some embodiments of the present application, the packaging cells include but are not limited to at least one of the following cells: NS0 cells, Vero cells, HeLa cells, COS cells, CHO cells, HEK cells, BHK cells, and MDCK II cells.

[0137] In some embodiments of the present application, the packaging cells are HEK-293T cells.

[0138] “MOI”: i.e., “Mutiplicity of Infection (MOI)”, refers to the number of virions added to each cell during infection. For example, when one million virions are added to one million cells, MOI = 1.

[0139] “Immune effector cell”: refers to an immune cell that can perform an immune effector function. In some embodiments of the present application, the immune effector cell is a T cell. In some embodiments of the present application, the T cell is a CD4 + / CD8 - , CD4 - / CD8 + , CD4 + / CD8 + , CD4 - / CD8 - T cells or combinations thereof. In some embodiments of the application, the T cells produce IL-2, TFN and / or TNF upon expression of the CAR and binding to a tumor cell. In some embodiments of the application, the CD8 + T cells lyse an antigen-specific target cell upon expression of the CAR and binding to the target cell.

[0140] The engineered immune effector cells of the application are prepared by introducing a CAR into an immune effector cell, such as a T cell. In some embodiments of the application, the CAR is introduced into the immune effector cell by transfection of any one of the isolated nucleic acids.

[0141] Methods of introducing a vector or isolated nucleic acid into a mammalian cell are known in the art. The described vectors can be transferred into an immune effector cell by physical, chemical, or biological methods.

[0142] Physical methods of introducing a vector into an immune effector cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, E.F. and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). In some embodiments of the application, the vector is introduced into the cell by electroporation. In some embodiments of the application, the vector is introduced into the cell by PEI (Polyethylenimine, a polyethylenimine transfection reagent) transfection reagent.

[0143] Biological methods of introducing a vector into an immune effector cell include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, such as human, cells.

[0144] Chemical methods of introducing a vector into an immune effector cell include colloidal dispersion systems, such as including macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome.

[0145] "Pharmaceutically acceptable excipient or carrier": Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants. Preferably, the compositions disclosed herein also include an adjuvant that is nontoxic to recipients at the dosages contemplated. Such adjuvants include, but are not limited to, saline, buffered solutions, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the compositions can contain substances that improve, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. The optimal composition can be determined in view of the intended route of administration, mode of delivery, and desired dosage.

[0146] "Subject": The terms "patient," "subject," "individual," or "object" are used interchangeably herein in the present application and include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human.

[0147] "Treat" or "treatment" refers to the administration of a therapeutic method described herein to a subject to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of cancer cell infiltration into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth). The therapeutic method effective to treat a patient can vary depending on a variety of factors such as the disease state, age, body weight, and the ability of the therapy to elicit an anti-cancer response in the subject.

[0148] "Therapeutically effective amount": The therapeutically effective amount of a composition containing an anti-CD7 Nanobody or engineered immune effector cell of the present application will depend, for example, on the severity of the treatment and the target. Those of ordinary skill in the art will

[0149] The frequency of dosing will depend on the pharmacokinetic parameters of the anti-CD7 Nanobody or engineered immune effector cell in the formulation being used. Clinicians will determine appropriate dosing schedules, including frequency of dosing and repeated dosages, to achieve the desired effect. The compositions can therefore be administered as a single dose, or as two or more doses (which can or can not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implanted pump or catheter.

[0150] "Administering": The route of administration of the compositions is routine in the art, e.g., oral, nasal, by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal, in situ, or intralesional injection, can also be by sustained release systems or by implantation devices.

[0151] "and / or": Is to be taken as specific alternatives, that is, "one or the other, or both".

[0152] "comprising": As used herein, the term "comprising" is to be taken in its broadest possible sense, that is, to mean including, among other things, but not necessarily limited to, the stated steps, or elements, or steps or elements thereof. In some embodiments of the application, the terms "including," "has," "have," and "contains" are synonymous with "comprising" and are used in their broadest sense.

[0153] "embodiment": Reference throughout this specification to "some embodiments" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. Thus, appearances of the phrases "in some embodiments" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0154] "operably linked": Refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence wherein the linkage results in expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is associated with the second nucleic acid sequence in a functional relationship as described herein. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. DNA sequences that are operably linked can be contiguous and, as is often the case, in the same reading frame.

[0155] All publications, literature citations, and patents mentioned herein are hereby incorporated by reference in their entirety, as if each individual publication, literature citation, or patent was specifically and individually indicated to be incorporated by reference. In case of conflict between the present application (including any definitions herein) and any incorporated literature reference, the present application controls. However, any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be, construed as being prior art to the present application.

[0156] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. BRIEF DESCRIPTION OF DRAWINGS

[0157] Figure 1: This figure shows the detection results of the ability of the anti-CD7 nanobodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92 or JY2-105 to specifically bind to human CD7 antigen in Example 1.

[0158] Figure 2: The plasmid map of the main plasmid in Example 2;

[0159] Figure 3: A schematic diagram of the plasmid maps of mutant envelope plasmids 1, 2, 3, 4, 5, 6, 7, 8 or 9 in Example 2;

[0160] Figure 4: In Example 2, the viral envelope contains LVVs expressing anti-CD7 nanoantibodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92, or JY2-105, which respectively transduce CD7. + Flow cytometry results of Jurkat cell efficiency;

[0161] Figure 5: In Example 2, the viral envelope contains LVVs expressing anti-CD7 nanoantibodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92, or JY2-105, which transduce CD7. - The flow cytometry results of Nalm6 cell efficiency are shown in the figure. Detailed Implementation

[0162] The following detailed description of the invention's concept and resulting technical effects, in conjunction with specific implementation schemes, provides a clear and complete understanding of the invention's technical problems, solutions, and effects. Clearly, the described implementation schemes are only some, not all, of the invention's embodiments; other implementation schemes obtained by those skilled in the art based on these schemes without inventive effort are all within the scope of protection of this invention.

[0163] For experimental methods in the following embodiments where specific conditions are not specified, conventional methods and conditions known in the art should be followed, or the product instructions should be selected accordingly. All reagents and raw materials not specifically specified in this invention are commercially available.

[0164] Example 1

[0165] Preparation of anti-CD7 nanobodies.

[0166] A. Animal immunization and immune response tests

[0167] (1) Select healthy alpacas as immunization objects, collect 10 mL of blood before immunization, separate the serum as negative serum.

[0168] (2) Week 1, first immunization: mix complete Freund's adjuvant with CD7 antigen (0.4 mg) 1:1 by volume, emulsify and inject subcutaneously in multiple points, and then use incomplete Freund's adjuvant mixed with the CD7 antigen (0.4 mg) 1:1 for subsequent booster immunization, with an immunization interval of 2 weeks, a total of 6 times; collect 5 mL of peripheral blood before immunization and after each immunization, separate the serum, and monitor the immune response by ELISA method to confirm the serum titer.

[0169] (3) After the sixth immunization, use ELISA method to detect the total IgG titer in the serum of the animals, reaching 10000 level, collect 50 mL of peripheral blood, separate the lymphocytes and store in TRIzol reagent for subsequent antibody phage library construction.

[0170] CD7 antigen used in immunized alpacas:

[0171] Product name: Human CD7 Protein, Fc Tag (MALS verified), brand: ACRO, catalog number: CD7-H5253.

[0172] B. Construction of antibody phage library

[0173] (1) Reverse transcription

[0174] After the animal immunization is completed, the separated lymphocytes are used to extract RNA and the total RNA obtained is reverse transcribed using the Takara reverse transcription kit; the above total RNA sample is divided into two parts, one uses Oligo dT Primer in the kit as primer, and the other uses Random 6-mers in the kit as primer, and the total RNA obtained in the above step is reverse transcribed into cDNA according to the reverse transcription kit instructions, and is saved into 2 centrifuge tubes respectively.

[0175] (2) PCR amplification

[0176] a. PCR amplification of specific antibody fragments from reverse-transcribed cDNA, PCR amplification is performed using Taq DNA Polymerase Hot Start enzyme; all PCR products are subjected to 1% agarose gel electrophoresis, and the gel is cut to recover the band of the desired fragment size of about 600-700 bp, which is the first round of PCR amplification product, stored at -20°C;

[0177] b. The first round of PCR amplification product was used as a template for the second round of PCR reaction. After the reaction, 1% agarose gel electrophoresis was performed, and the target band was recovered by gel extraction. The DNA purification kit was used for DNA purification of the PCR reaction solution.

[0178] (3) Enzymatic digestion and ligation

[0179] The second round of PCR amplified target gene fragments and pComb3XSS phage plasmid vector were digested with restriction enzymes Spe I and Sac I, respectively. After digestion, the VHH target gene fragments were ligated to the pComb3XSS phage plasmid vector using ligase to construct a recombinant plasmid.

[0180] (4) Bacterial library construction

[0181] a. Take a 50 μL of TG1 competent cells and melt on ice for 5-10 min;

[0182] b. Add 100 ng of ligation product to the pre-cooled 1 mm spaced electric transformation cup, and set the parameters in the electric transformation instrument to 1800 V and 1 mm. Then click the button to transform;

[0183] c. Immediately after the electric transformation is completed, add 1 mL of 37°C preheated SOC culture solution, mix well, and then incubate at 37°C and 200 rpm for 1 h;

[0184] d. Take 20 or more 100 ng ligation systems and use the competent cells for electric transformation reaction according to the above method;

[0185] e. Take 100 μL from the recovered bacterial solution, perform 10-fold gradient dilution, and then plate it. Incubate at 37°C overnight;

[0186] f. Collect all the remaining bacterial solutions and evenly spread them on 20 or more 15 cm culture plates (2 x YT containing 100 μg / mL Amp, 2% agarose). Incubate at 37°C overnight;

[0187] g. According to the dilution ratio and the number of single colonies, calculate the number of transformed colonies that can be obtained from all reactions, which is the library capacity of the bacterial library. At the same time, randomly select several single colonies from the gradient dilution plate for colony PCR. If the PCR product has a single band of about 400 bp, it is considered a positive clone. In this way, the positive rate of the bacterial library is estimated;

[0188] h. The overnight cultured plate colonies were scraped with 2 x YT liquid medium, placed in a 50 mL centrifuge tube, and the OD600 value was measured. Add 20% glycerol to a final concentration and store at -80°C.

[0189] (5) Phage library construction

[0190] a. Inoculate the bacterial library into 100 mL of 2xYT liquid medium (containing 100 μg / mL Amp) with an initial OD600 value of 0.1, and incubate at 37°C, 250 rpm until OD600 is 0.5-0.55;

[0191] b. Add helper phage at a ratio of 1:20 (number of bacteria: number of phages), and incubate at 37°C, 250 rpm for 30 min;

[0192] c. Add Kana at a final concentration of 50 μg / mL, and incubate at 30°C, 250 rpm overnight, centrifuge, and collect the supernatant;

[0193] d. Add 1 / 4 volume of pre-cooled PEG / NaCl, mix well, and incubate on ice for at least 30 min, centrifuge at 4°C, 4000 rpm for 20 min, remove the supernatant, and add 1 mL of PBS buffer to dissolve the precipitate; add 1 / 4 volume of pre-cooled PEG / NaCl again, and incubate on ice for 10 min, centrifuge at 4°C, 12000 g for 10 min, remove the supernatant, and dissolve the precipitate in 1 mL of PBS, and store at -80°C, to obtain the purified phage library.

[0194] C. Phage screening

[0195] (1) First round of screening

[0196] a. Place the magnetic bead bottle on a vortex shaker for 20 s to resuspend the magnetic beads. Take 100 μL of magnetic beads into a 1.5 mL centrifuge tube, and place it on a magnetic stand for 1 min, magnetically separate, and remove the supernatant;

[0197] b. Add 1 mL of Buffer 2 (1xPBS plus 0.05% Tween-20) to the centrifuge tube, cover the tube, resuspend the magnetic beads well, magnetically separate, and remove the supernatant;

[0198] c. Repeat step b twice, for a total of three washes;

[0199] d. Add 1 mL of Buffer 2 and 40 μg of screening antigen (product name: Human CD7 Protein, His Tag (MALS verified), brand: ACRO, item number: CD7-H82E4), resuspend the magnetic beads well, and place the centrifuge tube on a rotary mixer, and rotate at room temperature for 60 min;

[0200] e. Magnetically separate, remove the supernatant, and wash the magnetic beads 5 times according to the method of step C(1)(b);

[0201] f. Discard the wash solution in the centrifuge tube, add 1 mL of blocking solution (3% BSA) solution, resuspend the magnetic beads by shaking thoroughly, and place the centrifuge tube in a rotary mixer for 60 min at room temperature;

[0202] g. Discard the wash solution in the centrifuge tube, add 1 mL of Buffer 2, and add 50 μL of the prepared phage library as the input phage library for the first round of screening, and rotate for 60 min at room temperature;

[0203] h. Place the centrifuge tube in a magnetic stand, magnetically remove the supernatant, add 1 mL of Buffer 2 for washing, magnetically separate, and remove the wash solution, for a total of 10 washes;

[0204] i. Magnetically remove the supernatant, remove the residual liquid as much as possible, add 1 mL of 0.25 mg / mL Trypsin solution, and rotate for elution for 30 min at room temperature;

[0205] j. Add 10 μL of 10% AEBSF to terminate the elution, place the centrifuge tube in a magnetic stand, magnetically separate, and transfer the supernatant to a new 1.5 mL centrifuge tube, which is the phage eluate for the first round of screening.

[0206] (2) Detection of the titer of the phage eluate for the first round of screening

[0207] a. Perform single colony streaking of the TG1 strain stored in a -80°C freezer on 2xYT solid medium, and incubate overnight at 37°C (store at 4°C for one week), and pick a single colony from the single colony plate into 5 mL of 2xYT medium, and incubate overnight at 37°C;

[0208] b. Take 500 μL of the overnight culture and transfer it into 5 mL of 2xYT liquid medium, and incubate at 37°C, 250 rpm for about 45-60 min until the OD600 is 0.5-0.55;

[0209] c. Take 10 μL of the phage eluate for the first round of screening, and dilute it by 10-fold in a 1.5 mL centrifuge tube, for a total of 12 gradients, i.e., dilute 10 μL of the phage eluate for the first round of screening to 100 μL, then take 10 μL from the 100 μL and dilute it to 100 μL, and so on, for a total of 12 gradients to 10-12, and shake to mix;

[0210] d. Add 90 μL of the TG1 bacterial solution to each dilution centrifuge tube, mix, and incubate at 37°C for 30 min;

[0211] e. Take 5 μL from each dilution centrifuge tube and add it to 2xYT solid medium (Amp), and incubate overnight at 37°C with inversion;

[0212] f. Count the number of colonies on the plate that can be distinguished clearly, and calculate the number of phage particles per milliliter of phage solution according to the following formula, i.e. the titer of the phage library: T (pfu / ml) = N x D x 400

[0213] wherein T is the titer of the phage (unit pfu / mL), D is the dilution factor, and N is the number of single colonies on the corresponding dilution factor.

[0214] (3) Amplification of the first round of phage eluate

[0215] a. The TG1 strain stored in a -80°C refrigerator was pre-streaked on a 2 x YT solid medium and incubated overnight at 37°C (stored at 4°C for one week), and a single colony was picked from the single colony plate to 5 mL of 2 x YT medium and incubated overnight at 37°C;

[0216] b. 500 μL of the overnight culture was transferred to 5 mL of 2 x YT liquid medium, and incubated at 37°C, 250 rpm for about 45-60 min until the OD600 value was 0.5-0.55;

[0217] c. 500 μL of the phage eluate obtained after the first round of screening was added to the bacterial solution with an OD600 value of 0.5-0.55 (the remaining eluate was stored at 4°C);

[0218] d. Continue to incubate at 37°C, 250 rpm for 30 min;

[0219] e. The entire bacterial solution was evenly spread on a 245 mm square plate containing 2% agarose with 100 μg / mL Amp, and incubated overnight at 37°C;

[0220] f. The square plate was taken from the overnight culture, 6 mL of 2 x YT liquid medium (containing 100 μg / mL Amp) was added to the surface of the culture plate, and the colonies on the square plate were gently scraped off with a spreader and collected in a 15 mL centrifuge tube, i.e. the amplified bacterial sub-library, and the OD600 value of the bacterial solution was measured using a spectrophotometer, i.e. the OD600 value of the eluate bacterial library, and glycerol was added to a final concentration of 20%, i.e. the first round of bacterial library.

[0221] g. The eluate bacterial library was calculated according to the following formula, and transferred to 100 mL of 2 x YT liquid medium (containing 100 μg / mL Amp) to make the initial OD600 value 0.1:

[0222] wherein V is the volume of the transferred bacterial solution (unit μL), and OD600 is the OD600 value of the constructed eluate bacterial library.

[0223] h.37°C, 250 rpm culture until the OD600 of the bacterial solution reaches 0.5-0.55;

[0224] i. Calculate and add helper phage M13K07 to the number of bacteria according to the following formula: phage number = 1:20:

[0225] Wherein V is the volume of added helper phage (unit mL), T helper-phage is the titer of helper phage used, OD600 is the OD600 value of the bacterial solution;

[0226] j. Continue to culture at 37°C, 250 rpm for 30 min;

[0227] k. Add 50 μg / mL Kana with final concentration respectively, and culture overnight at 30°C, 250 rpm.

[0228] (4) First round of phage purification

[0229] a. Transfer the overnight culture of the bacterial solution to a new 50 mL centrifuge tube, centrifuge at 4000 rpm, 4°C for 20 min;

[0230] b. Transfer the supernatant after centrifugation to a new 50 mL centrifuge tube, add 1 / 4 volume of 4°C pre-cooled 20% PEG / 2.5M NaCl, mix thoroughly, and then place on ice for 30 min;

[0231] c. Centrifuge at 4000 rpm, 4°C for 20 min, discard the supernatant, and invert on paper for 2 min;

[0232] d. Add 1 mL of PBS to resuspend the precipitate, and transfer the resuspension to a new 1.5 mL centrifuge tube, centrifuge at 12000 rpm, 4°C for 5 min;

[0233] e. Transfer the supernatant after centrifugation to a new 1.5 mL centrifuge tube, add 1 / 4 volume of pre-cooled 20% PEG / 2.5M NaCl solution, mix thoroughly, and then place on ice for 10 min;

[0234] f. Centrifuge at 12000 rpm, 4°C for 10 min, discard the supernatant, and add 1 mL of PBS to resuspend the precipitate;

[0235] g. Centrifuge at 12000 rpm, 4°C for 2 min, and transfer the supernatant to a new 1.5 mL centrifuge tube, which is the first round of phage sub-library screening, store at -80°C for long-term preservation, and at -20°C for short-term (1-2 weeks) preservation

[0236] h. Detect the titer of the first round of phage sub-library screening, the method is the same as described in C(2) above.

[0237] (5) Second round of screening

[0238] The screening method is the same as step C(1) above, and the input phage is the 0.25 mL phage sub-library obtained in the first round of screening, which is used as the input phage library for the second round of screening, to obtain the second round of phage eluate.

[0239] (6) Detection of the titer of the second round of phage eluate

[0240] The method is the same as step C(2) above.

[0241] (7) Amplification and purification of the second round of eluate

[0242] The method is the same as C(2)-(3) above, to obtain the second round of phage sub-library.

[0243] (8) Detection of the titer of the second round of phage sub-library

[0244] The method is the same as step C(2) above.

[0245] (9) Single clone ELISA detection

[0246] a. The TG1 strain stored in a -80°C refrigerator was pre-streaked on a 2xYT solid medium and incubated overnight at 37°C (stored at 4°C for one week), and a single colony was picked from the single colony plate and inoculated into 5 mL of 2xYT medium and incubated overnight at 37°C;

[0247] b. 500 μL of the overnight culture was transferred into 5 mL of 2xYT liquid medium, and incubated at 37°C, 250 rpm for about 45-60 min until the OD600 value was 0.5-0.55;

[0248] c. 10 μL of the third round of phage eluate was diluted 10-fold in a 1.5 mL centrifuge tube, for a total of 12 gradients, i.e. 10 μL of the phage library was diluted to 100 μL, and then 10 μL of the diluted solution was further diluted to 100 μL, and so on, for a total of 12 gradients, and mixed well;

[0249] d. 90 μL of the bacterial solution with an OD600 value of 0.5-0.55 was added to each dilution centrifuge tube and mixed well;

[0250] e. Continue to incubate at 37°C, 250 rpm for 30 min;

[0251] f. The bacterial solution was evenly spread on a solid medium plate containing 100 μg / mL Amp and incubated overnight at 37°C;

[0252] g. Randomly pick single colonies from the overnight culture plates into sterile 96-well cell culture plates (P1-P3) with 200 μL 2xYT medium (containing 100 μg / mL Amp) per well, and incubate at 37°C overnight;

[0253] h. Take 2 μL of the overnight culture and transfer into a new 96-well cell culture plate with 200 μL 2xYT liquid medium (containing 100 μg / mL Amp) per well, and incubate at 37°C for 3-5 h. Store the overnight culture before transfer at 4°C;

[0254] i. Calculate and add helper phage M13K07 to each well to achieve a ratio of 1:20 of phage to bacteria according to the following formula:

[0255] Where V is the volume (in mL) of helper phage added, T helper-phage is the titer of the helper phage used;

[0256] j. Incubate at 37°C for 30 min, and then add Kana at a final concentration of 50 μg / mL, and incubate at 30°C overnight;

[0257] k. Centrifuge the 96-well plates at 4°C and 4000 rpm for 10 min after overnight incubation, and store at 4°C for later use;

[0258] l. Biotinylate the screening antigen (CD7-B), and coat the enzyme-labeled plate (1 ng / μL, coating solution: 1xPBS, 100 μL / well) with the screening antigen (CD7-His) and BSA as a control, and incubate at 4°C overnight;

[0259] m. Discard the liquid in the enzyme-labeled plate after overnight coating, and add 200 μL PBS buffer per well, and wash the enzyme-labeled plate at room temperature for 3 times, 10 min each time;

[0260] n. Add 200 μL blocking solution (3% BSA) per well to block the enzyme-labeled plate, and incubate at room temperature for 1 h;

[0261] o. Discard the blocking solution, and add 200 μL PBST (1xPBS plus 0.1% Tween 20, same below) buffer per well, and wash the enzyme-labeled plate at room temperature for 3 times, 10 min each time;

[0262] p. Add 140 μL 3% BSA per well, and then add 60 μL supernatant after centrifugation in step C(9)(k), and incubate at room temperature for 2 h;

[0263] q. Discard the liquid in the enzyme-labeled plate, and wash with 200 μL PBST buffer per well for 3 times, 10 min each time;

[0264] r. Add M13 Bacteriophage Antibody (HRP), Mouse Mab, 1:60000 diluted in PBS, 100 μL / well, incubate at room temperature for 1 h;

[0265] s. Discard the liquid in the ELISA plate, add 200 μL of PBST buffer to each well, wash 6 times, 5 min each time;

[0266] t. Add 100 μL of TMB single-component color developing solution to each well, develop color in the dark for 2-3 min, add 100 μL of 1 M HCl to each well to stop the reaction, and read OD450nm value with an enzyme-labeled instrument, record and save.

[0267] (10) Secondary verification of positive clones by ELISA

[0268] In order to exclude false positive results, secondary verification by ELISA was carried out, and the method was the same as C(9) above.

[0269] (11) Sequencing of positive clones

[0270] Positive monoclonal antibodies were selected according to the ELISA detection data and secondary verification data.

[0271] 5 μL of positive clone bacterial liquid from the monoclonal ELISA detection C(9) (g) plate was inoculated into 1 mL of 2xYT medium (containing 100 μg / mL Amp), and cultured at 37°C, 250 rpm until OD600 reached 0.8-1.0 (about 6-8 h). 0.5 mL of bacterial liquid was used for sequencing, and the remaining bacterial liquid was added with 20% glycerol to a final concentration, and stored at -20°C.

[0272] (12) Sequence analysis

[0273] The sequenced sequence was analyzed by sequence comparison using GENtle software, and the antibody sequence was translated into amino acid using GENtle software.

[0274] D. Experimental results

[0275] (1) The results of two rounds of screening are shown in Table 1 below.

[0276] Table 1

[0277] (2) ELISA detection results of monoclonal antibodies

[0278] After the immunopipette screening, 288 single clones were randomly selected for ELISA detection. After the positive single clone ELISA secondary verification, 285 positive single clones were selected for sequencing, and 272 normal nucleic acid sequences were obtained (excluding 10 double peaks and 3 no 3' end primer sequences). After translating the 272 nucleic acid sequences into amino acid sequences and sorting, 164 different nanobody sequences were obtained. Sequence-specific clones were screened by Sanger sequencing single clone and DANMAN sequence alignment, and the sequence-specific nanobodies screened were numbered as JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92 and JY2-105, respectively.

[0279] The gradient dilution ELISA method was used to detect the ability of each anti-CD7 nanobody to specifically bind to the screening antigen and biotinylated screening antigen (BSA as negative control), respectively. The detection results are shown in Tables 2-10, respectively.

[0280] Table 2

[0281] Table 3

[0282] Table 4

[0283] Table 5

[0284] Table 6

[0285] Table 7

[0286] Table 8

[0287] Table 9

[0288] Table 10

[0289] From Tables 2-10, it can be seen that the anti-CD7 nanobodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92 and JY2-105 can effectively bind to the screening antigen and biotinylated screening antigen.

[0290] The amino acid sequence of the JY2-07 is shown as SEQ ID NO:1; the amino acid sequences of the CDR-H1 region, CDR-H2 region, CDR-H3 region of the JY2-07 are shown as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 respectively.

[0291] The amino acid sequence of the JY2-11 is shown as SEQ ID NO:5; the amino acid sequences of the CDR-H1 region, CDR-H2 region, CDR-H3 region of the JY2-11 are shown as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 respectively.

[0292] The amino acid sequence of the JY2-27 is shown as SEQ ID NO:9; the amino acid sequences of the CDR-H1 region, CDR-H2 region, CDR-H3 region of the JY2-27 are shown as SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 respectively.

[0293] The amino acid sequence of the JY2-28 is shown as SEQ ID NO:13; the amino acid sequences of the CDR-H1 region, CDR-H2 region, CDR-H3 region of the JY2-28 are shown as SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 respectively.

[0294] The amino acid sequence of the JY2-44 is shown as SEQ ID NO:17; the amino acid sequences of the CDR-H1 region, CDR-H2 region, CDR-H3 region of the JY2-44 are shown as SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 respectively.

[0295] The amino acid sequence of the JY2-53 is shown as SEQ ID NO:21; the amino acid sequences of the CDR-H1 region, CDR-H2 region, CDR-H3 region of the JY2-53 are shown as SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 respectively.

[0296] The amino acid sequence of the JY2-61 is shown as SEQ ID NO:25; the amino acid sequences of the CDR-H1 region, CDR-H2 region, CDR-H3 region of the JY2-61 are shown as SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 respectively.

[0297] The amino acid sequence of the JY2-92 is shown as SEQ ID NO: 29; the amino acid sequences of the CDR-H1 region, CDR-H2 region, and CDR-H3 region of the JY2-92 are shown as SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively.

[0298] The amino acid sequence of the JY2-92 is shown as SEQ ID NO: 29; the amino acid sequences of the CDR-H1 region, CDR-H2 region, and CDR-H3 region of the JY2-92 are shown as SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively.

[0299] SEQ ID NO: 1-36 are shown in Table 12 below.

[0300] Table 12

[0301] E. Flow detection

[0302] His Tag tag is connected to the C-terminal end of each anti-CD7 nanobody: when synthesizing the anti-CD7 nanobodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92, and JY2-105, a His-tag protein is connected to the tail / C-terminal end of each anti-CD7 nanobody, and the connection method is known to those skilled in the art.

[0303] Day 0, prepare 9 groups of Jurkat cell culture systems: resuspend 1 x 10 5 Jurkat cells in 0.5 mL of Jurkat cell culture medium (1640 culture medium and 10% FBS); +

[0304] Incubate 9 groups of the Jurkat cell culture systems with the anti-CD7 nanobodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92, or JY2-105 (antibody concentration is 0.1 mg / mL) connected to the His Tag tag at 4°C for 30 min, and then incubate the APC-His tag antibody (use amount according to the instruction manual) at 4°C for 30 min; the control group antibody is a commercialized anti-CD7 monoclonal antibody with high specificity and strong binding capacity, and the CD7 + Jurkat cells are co-incubated; ​

[0305] Day 2, the flow cytometry detection was performed on each group of Jurkat cell culture system respectively, and the results were shown in Figure 1.

[0306] As shown in Figure 1, the anti-CD7 nanobodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92 and JY2-105 had the same ability of specific binding to CD7 antigen as the commercial anti-CD7 monoclonal antibody, and the binding of CD7 + The efficiency of Jurkat cells was close to 100%.

[0307] Flow cytometry used antibody: APC anti-His Tag Antibody, brand: BIOLEGEND, item number: #362605;

[0308] Commercial anti-CD7 monoclonal antibody: brand: BIOLEGEND, item number: #343114;

[0309] APC-His tag antibody: brand: BIOLEGEND, item number: #362605.

[0310] Example 2

[0311] The membrane expression anti-CD7 nanobodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92 or JY2-105 were constructed on the viral envelope of lentiviral vector (LVV) as membrane expression targeting molecules to improve the ability of LVV to target and transduce CD7 + cells.

[0312] A. LVV packaging

[0313] 1. Designing membrane expression anti-CD7 nanobodies

[0314] The polynucleotides encoding each of the membrane expression anti-CD7 nanobodies sequentially encode from 5' end to 3' end: human CD8a signal peptide, anti-CD7 nanobody (JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92 or JY2-105), human CD8a hinge region, human CD8a transmembrane region;

[0315] (1) The amino acid sequence of the human CD8a signal peptide is shown in SEQ ID NO: 37;

[0316] (2) The amino acid sequences of the CD7 nanobodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92 and JY2-105 are as shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29 or SEQ ID NO:33, respectively;

[0317] (3) The amino acid sequence of the hinge region of the human CD8α is shown in SEQ ID NO:38;

[0318] (4) The amino acid sequence of the transmembrane region of the human CD8α is shown in SEQ ID NO:39.

[0319] 2. Prepare the LVV packaging system:

[0320] (1) Prepare the following 4 types of plasmids:

[0321] Contains polynucleotides encoding mutant VSV-G and membrane-expressed anti-CD7 nanobodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, and JY2-24.

[0322] Mutant envelope plasmids 1, 2, 3, 4, 5, 6, 7, 8, or 9 of polynucleotides 2-92 or JY2-105, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and lentiviral GFP (GFP fluorescent protein) master plasmid (plasmid map shown in Figure 2); the mutant envelope plasmids and the master plasmids were synthesized using conventional molecular cloning methods;

[0323] Figure 3 shows a schematic diagram of the plasmid maps of the mutant envelope plasmids 1-9; where “anti-CD7nanoAb gene” means the polynucleotide encoding the anti-CD7 nanoantibodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92 or JY2-105.

[0324] The amino acid sequence of the wild-type VSV-G is shown in SEQ ID NO: 40, and the mutant VSV-G comprises a K47 deletion mutation relative to the wild-type VSV-G, and the amino acid sequence of the mutant VSV-G is shown in SEQ ID NO: 41; the K47 deletion weakens, loses, or inhibits the ability of the mutant VSV-G to specifically bind to LDL-R, but at the same time retains the ability to mediate membrane fusion and endosome / lysosome escape;

[0325] The LVV surface comprising the mutant VSV-G in the viral envelope constructs the membrane-expressed anti-CD7 nanobody JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92, or JY2-105 as a targeting molecule, which can target the LVV to transduce CD7 + The ability of the cells is significantly enhanced.

[0326] (2) Transfect the packaging cells:

[0327] The four plasmids are transfected into the packaging cells HEK-293T cells by PEI reagent, and the virus envelope comprising different targeting molecules, i.e., the membrane-expressed anti-CD7 nanobody JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92, or JY2-105, is packaged into the LVV targeting CD7 + The cells; the specific steps are as follows:

[0328] Add 9 μg of the master plasmid, 4 μg of the pMDLg / pRRE packaging plasmid, 2 μg of the pRSV-REV packaging plasmid, and 2 μg of the mutant envelope plasmid 1, 2, 3, 4, 5, 6, 7, 8, or 9 to 1 mL of Opti-MEM medium, shake well, add 64 μL of PEI reagent, blow evenly, stand for 10 minutes, then add to the culture medium of HEK-293T cells, update the culture medium after 6 hours, collect the culture medium supernatant after 48 hours of transfection, filter with a 0.45 um filter, centrifuge at 50000g for 2.5h, discard the supernatant, resuspend each group of LVV with 200 μL of F12 medium and freeze at -80℃.

[0329] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: brand: GIBCO, catalog number: #C12430500BT; FBS: brand: EXCELL, catalog number: #FSP500;

[0330] Opti-MEM alpha reduced serum medium: brand: GIBCO, catalog number: #SP0272;

[0331] F12 medium: brand: GIBCO, item number: #C11330500BT;

[0332] Needle filter: brand: SORFA, item number: #622120.

[0333] B. Transduction of CD7 + Jurkat cells and CD7 - Nalm6 cells

[0334] Day 0, 20 μL of virus supernatant of each group of LVV containing different said membrane expressed anti-CD7 nanobodies as targeting molecules were respectively taken to transduce 1 x 10 5 CD7 + Jurkat cells and 1 x 10 5 CD7 - Nalm6 cells, respectively.

[0335] Day 2, the expression of GFP molecules in each group of cells was detected, and the results are shown in Figure 4 (transduction of Jurkat cells) and Figure 5 (transduction of Nalm6 cells), respectively;

[0336] As can be seen from Figure 4 and Figure 5, the said LVV containing different targeting molecules, membrane expressed anti-CD7 nanobodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92 or JY2-105 can effectively transduce CD7 + and LDL-R + Jurkat cells (the positive rates are 13.51%, 7.40%, 6.95%, 12.86%, 12.96%, 4.12%, 17.45%, 4.34%, 49.57%, respectively), and it is difficult to effectively transduce CD7 - and LDL-R + Nalm6 cells (the positive rates are 0.83%, 0.78%, 0.69%, 0.73%, 0.73%, 0.76%, 0.77%, 0.98%, 0.66%, respectively).

[0337] This proves that: the anti-CD7 nanobodies JY2-07, JY2-11, JY2-27, JY2-28, JY2-44, JY2-53, JY2-61, JY2-92 or JY2-105 can all be used as targeting molecules expressed on the viral envelope of LVV, which can significantly improve the ability of LVV to target transduce CD7 + cells.

Claims

1. An anti-CD7 Nanobody, characterized in that, The anti-CD7 Nanobody comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the amino acid sequence of the CDR-H1 region, the CDR-H2 region, and the CDR-H3 region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; the amino acid sequence of the CDR-H1 region, the CDR-H2 region, and the CDR-H3 region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; the amino acid sequence of the CDR-H1 region, the CDR-H2 region, and the CDR-H3 region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; the amino acid sequence of the CDR-H1 region, the CDR-H2 region, and the CDR-H3 region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; the amino acid sequence of the CDR-H1 region, the CDR-H2 region, and the CDR-H3 region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; the amino acid sequence of the CDR-H1 region, the CDR-H2 region, and the CDR-H3 region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; the amino acid sequence of the CDR-H1 region, the CDR-H2 region, and the CDR-H3 region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively; the amino acid sequences of the CDR-H1 region, CDR-H2 region, and CDR-H3 region are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively; or the amino acid sequences of the CDR-H1 region, CDR-H2 region, and CDR-H3 region are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively.

2. The anti-CD7 Nanobody according to claim 1, characterized in that, the amino acid sequence of the anti-CD7 Nanobody is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, or SEQ ID NO:

33.

3. An isolated polynucleotide, comprising, The isolated polynucleotide encodes the anti-CD7 Nanobody of claim 1 or 2.

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises: (a) an extracellular antigen binding region; (b) a transmembrane region; and (c) an intracellular signaling domain; wherein the extracellular antigen binding region comprises the anti-CD7 Nanobody of claim 1 or 2. Preferably, the chimeric antigen receptor further comprises a hinge region and a costimulatory signaling domain.

5. An isolated polynucleotide, comprising, The isolated polynucleotide encodes the chimeric antigen receptor of claim 4.

6. A vector, characterized in that, The surface of the carrier comprises a targeting molecule, the targeting molecule comprising the anti-CD7 Nanobody of claim 1 or 2. Preferably, the carrier is selected from at least one of a lipid nanoparticle, a virus-like particle, an extracellular vesicle, an adenovirus, an adeno-associated virus, a lentiviral vector, and a retroviral vector. More preferably, the carrier is a lentiviral vector (LVV) or a retroviral vector (RVV).

7. The carrier of claim 6, wherein, The targeting molecule further comprises a transmembrane region, the anti-CD7 Nanobody being directly or indirectly linked to the transmembrane region, exposed on the surface of the carrier; Preferably, the transmembrane region is selected from the transmembrane region of the following proteins: CD28, CD2, CD4, CD8a, CD5, CD3e, CD3d, CD3z, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD154 (CD40L), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRa, FcRp, FcRy, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPa, pTa, TCRa, TCRp, TIM3, TRIM, LPA5, and Zap70; More preferably, the transmembrane region comprises a transmembrane region of CD8a.

8. The carrier of claim 7, wherein, The targeting molecule further comprises a linker domain, the anti-CD7 nanobody is indirectly linked to the transmembrane region via the linker domain; Preferably, the linker domain is selected from the group consisting of: (a) an immunoglobulin hinge region, the immunoglobulin hinge region is selected from the group consisting of wild-type or modified IgGl, IgG2, IgG3, IgG4, IgA, and IgD hinge regions; (b) a hinge region, the hinge region is selected from the group consisting of wild-type or modified hinge regions of CD28, CD7, CD8, CD8a, CD8b, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (c) all or a portion of an Fc domain, the Fc domain is selected from the group consisting of one or more of a CH1 domain, a CH2 domain, and a CH3 domain; and (d) a stalk region of a type II C-lectin, the type II C-lectin is selected from the group consisting of stalk regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; More preferably, the linker domain comprises a hinge region of CD8a.

9. An engineered immune effector cell, characterized in that, The engineered immune effector cell comprises the chimeric antigen receptor of claim 4 or the polynucleotide of claim 5; Preferably, the immune effector cell is selected from the group consisting of at least one of a T cell, an NK cell, an NK / T cell, a neutrophil, a monocyte, a dendritic cell, and a macrophage; More preferably, the immune effector cell is a T cell.

10. A composition characterized in that, The composition comprises the anti-CD7 Nanobody of claim 1 or 2 or the engineered immune effector cell of claim 9; Preferably, the composition can further comprise a pharmaceutically acceptable excipient or carrier.

11. Use of the anti-CD7 Nanobody of claim 1 or 2, the chimeric antigen receptor of claim 4, the vector of any one of claims 6-8, the engineered immune effector cell of claim 9 or the composition of claim 10 for the manufacture of a medicament for the treatment of a T cell-related and / or CD7-related disease; Preferably, the T cell-related and / or CD7-related disease comprises a T cell-related and / or CD7-related tumor, a T cell-related and / or CD7-related autoimmune disease and a T cell-related and / or CD7-related inflammatory disease; More preferably, the T cell-related and / or CD7-related disease comprises a T cell and / or CD7-related tumor, which comprises peripheral T-cell lymphoma (NHL), NK / T-cell lymphoma, anaplastic large cell lymphoma (ALCL), intestinal T-cell lymphoma, acute myeloid leukemia (AML), large granular lymphocytic leukemia (T-LGL) and fetal center T-cell lymphoma (FTCL).

12. A reagent for detecting a CD7 protein or a functional domain comprising an epitope thereof, characterized in that, The agent comprises the anti-CD7 Nanobody of claim 1 or 2; Preferably, the agent further comprises a modifying moiety linked to the anti-CD7 Nanobody, which modifying moiety comprises a detectable label or a therapeutic agent; More preferably, the detectable label comprises at least one of an enzyme, a radionuclide, a fluorescent dye, a luminescent substance and biotin.

13. A method for detecting CD7 protein or a functional domain comprising an antigenic determinant thereof for non-diagnostic purposes, characterized in that, The method comprises obtaining a sample suspected of containing a CD7 protein or a functional domain comprising an antigenic determinant thereof, contacting the sample with the anti-CD7 Nanobody of claim 1 or 2 or the agent of claim 12 to form an antibody-antigen complex, and detecting the presence of the antibody-antigen complex; preferably, the CD7 is human CD7 (UniProt KB No: P09564).

14. A method of treating a T cell-related and / or CD7-related disease in a subject, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-13, or a pharmaceutically acceptable salt thereof. The method comprises administering to a subject a therapeutically effective amount of the anti-CD7 Nanobody of claim 1 or 2, the engineered immune effector cell of claim 9 or the composition of claim 10; Preferably, the T cell-related and / or CD7-related disease is selected from a T cell-related and / or CD7-related tumor, a T cell-related and / or CD7-related autoimmune disease and a T cell-related and / or CD7-related inflammatory disease; More preferably, the T cell and / or CD7-related tumor comprises peripheral T-cell lymphoma (NHL), NK / T-cell lymphoma, anaplastic large cell lymphoma (ALCL), intestinal T-cell lymphoma, acute myeloid leukemia (AML), large granular lymphocytic leukemia (T-LGL) and fetal center T-cell lymphoma (FTCL).

15. An expression vector comprising the nucleic acid of claim 14. The expression vector comprises the isolated polynucleotide of claim 4 or claim 6; Preferably, the expression vector is selected from at least one of a plasmid, a lipid nanoparticle, a virus-like particle, an extracellular vesicle, an adenovirus, an adeno-associated virus, a lentivirus vector, and a retrovirus vector. Preferably, the expression vector is selected from at least one of a plasmid, a lipid nanoparticle, a virus-like particle, an extracellular vesicle, an adenovirus, an adeno-associated virus, a lentivirus vector, and a retrovirus vector.

Images