CN117343177A - Camel heavy chain antibody or antigen binding fragment and application thereof - Google Patents
Camel heavy chain antibody or antigen binding fragment and application thereof Download PDFInfo
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- CN117343177A CN117343177A CN202210779773.9A CN202210779773A CN117343177A CN 117343177 A CN117343177 A CN 117343177A CN 202210779773 A CN202210779773 A CN 202210779773A CN 117343177 A CN117343177 A CN 117343177A
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Classifications
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001129—Molecules with a "CD" designation not provided for elsewhere
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Landscapes
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Abstract
The invention discloses a camel heavy chain antibody or antigen binding fragment and application thereof. The antibody or antigen binding fragment comprises one or more of the 5 amino acid sequences; these 5 amino acid sequences are camelid nanobody VHH that specifically bind CD 38. The invention constructs an anti-CD 38 CAR by using the 5 amino acid sequences obtained by screening, constructs the anti-CD 38 CAR on immune cells by using a slow virus transduction technology, and performs in vitro function verification and clinical function verification by taking the anti-CD 38 CAR-T as an example. The in vitro experiment result shows that the anti-CD 38 CAR-T has good target cell killing capability and can stimulate secretion of a large amount of cytokines related to anti-tumor activity; clinical results show that anti-CD 38 CAR-T cells that use nanobodies to perform targeting functions can be specifically expanded in patients after stimulation in vivo with CD38 positive tumor cells.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a camel heavy chain antibody or antigen binding fragment and application thereof.
Background
1. Characteristics of alpaca nano antibody
Nanobodies are naturally heavy chain-deficient single domain antibodies containing only the heavy chain variable region, classical antibody molecules (IgG) are composed of two heavy chains comprising 1 VL region and 1 CL region, the heavy chains comprising 1 VH region and 3 CH regions (CH 1, CH2 and CH 3), the VH and VL regions of the light and heavy chains constituting the variable regions of antibody recognition antigens (Fv), the CL and CH regions being termed the constant regions of the antibody, and are critical for the development of ADCC and CDC against recruited immune cells. As shown in fig. 1, a small antibody consisting of only the heavy chain variable region (variable domain of heavy chain of heavy-chain, VHH) of the heavy chain, which recognizes a binding antigen, or which can exist alone stably in vitro, is called a single domain antibody (single domain antibody, sdAb) or a nanobody (Nb) or VHH antibody. Nanobodies have complete antigen recognition capability.
Alpaca nanobodies were first reported in 1993 by immunologists of the university of brussel freedom, hamers-masterman, at belgium, and this novel antibody was found not only in dromedaries, but also in other camelids, dromedaries, llamas, alpacas, llamas, and alpacas. Such antibodies naturally lack a light chain, also known as single domain heavy chain antibodies, and in comparison to conventional antibodies, camel heavy chain antibodies lack a CH1 (conventional region of heavy chain, CH) region between the heavy chain variable region and the hinge region, except for the light chain. The average length of CDR3 of human and mouse VH is 12 and 9 amino acids respectively, while the CDR3 of nanometer antibody is 13-18 amino acids, which compensates the decrease of antigen binding force caused by light chain deletion to a certain extent; in FR2 of the traditional antibody, 4 amino acid residues V37, G44, L45 and W47 participate in the interaction of VL, while in Nb, the 4 amino acids are mutated into F/Y37, E/Q44, R/C45, G/F/S/L47, and the hydrophobic amino acid is mutated into hydrophilic amino acid, so that the nanobody is easier to dissolve.
Compared with the traditional antibody, the alpaca nano antibody has good stability, small molecular weight, which is only 1/10 (about 15 kD) of the traditional whole antibody, and the crystal diameter is only 2.5nm and the length is 4nm, so that the alpaca nano antibody has strong tissue penetrating power and can even pass through the blood brain barrier; nanobodies have antigen specificity and high affinity by virtue of only 3 complementarity-determining region (CDRs), whereas common antibodies require 6 CDRs; the binding specificity of the nano antibody and an antigen target is stronger, and the nano antibody can reach an epitope which cannot be reached by the traditional antibody, and the long CDR3 region can extend into the crack of the surface receptor of bacteria or viruses due to the neutral activity of the enzyme; the modified polypropylene composite material also has higher stability, can resist more severe conditions such as high temperature resistance, and is easier to store and transport; the nano antibody is suitable for industrialized mass production by utilizing phage screening and other technologies, the period of the traditional monoclonal antibody prepared by cell fusion is about 6 months long, and the monoclonal monomer can be prepared more rapidly by bypassing cell fusion by phage display technology; the transformation and the humanization are easier, the nano antibody has more than 80 percent of sequence homology with the heavy chain variable region of the human antibody, and the 3D structures of the nano antibody and the human antibody can be overlapped, so the humanization is simple; in addition, nanobodies are easier to construct as multivalent and multifunctional antibodies.
Application of CD38 antigen
The CD38 antigen is a type II transmembrane glycoprotein of 46kDa in size. Its ligand is CD31, also known as PECAM-1. In addition to expression on endothelial cells, expression is also in lymphoid cells (follicular B cells and plasma cells), lung (alveolar ducts, alveoli and lymphatic ducts) and kidney (glomerular cells). In general, CD38 is expressed at low levels on normal lymphoid and myeloid cells, but the level of CD38 expression in plasma cells (also known as effector B cells) is particularly high.
CD38 interacts with the ligand CD31 and plays an important role in regulating cell migration, receptor-mediated adhesion, and signaling. In addition, the CD38 protein is also a bifunctional extracellular enzyme having both cyclase and hydrolase activities, involved in nucleotide metabolism.
CD38 expression is associated with a variety of diseases including aids, autoimmune diseases (e.g., systemic lupus erythematosus), type 2 diabetes, osteoporosis, and cancer. The research shows that CD38 is highly expressed in a large number of malignant blood cancers, especially in cancers such as Multiple Myeloma (MM), acute myelogenous leukemia (ALL), acute B-lymphocytic leukemia (B-ALL) and the like, so that the CD38 becomes a development target point of a drug for treating the multiple myeloma, and a plurality of drug enterprises are developing at home and abroad at present.
The prednisone up to Lei Tuoyou mab (Daratumumab) was the first humanized anti-CD 38 mab available on the market worldwide, the sarcolisa of Sanofi (Sanofi) was the FDA approved CD38 antibody drug of clause 2. Currently, only up to Lei Tuoyou monoclonal antibody, one CD38 monoclonal antibody, is commercially available in 2021 in China, and has remarkable effect of treating MM. At present, most of the research medicines taking CD38 as a target point are monoclonal antibodies, and no anti-CD 38 CAR-T product exists on the market.
3. Development of anti-CD 38 CAR-T technology
The CAR-T therapy, namely chimeric antigen receptor T cell immunotherapy, expresses Chimeric Antigen Receptor (CAR) comprising scFv single-chain antibody, co-stimulatory domain and other elements on the surface of T cells through genetic engineering technology, so that the CAR-T therapy can recognize specific tumor antigens, and has good curative effects for treating malignant hematological cancers, including acute B-lymphocyte leukemia, large B-cell non-Hodgkin lymphoma, recurrent refractory CD19 positive malignant lymphoma and the like. The CAR structure is formed by embedding a single-chain variable region scFv (antigen receptor) capable of directly recognizing a tumor antigen and a co-stimulatory signal in an antibody into the activation signal pathway cd3ζ chain of T cells. CD38 antigen is expressed on most AML and myeloma cells and is hardly expressed on Hematopoietic Stem Cells (HSCs), making it a potential target for CAR-T therapy for the treatment of hematological tumor disease patients. The traditional construction of the CAR-T cells comprises the steps of connecting an antibody heavy chain variable region (VH) and a light chain variable region (VL) gene into scFv (single-chain variable fragment) through a flexible Linker (such as (G4S) 3), constructing corresponding CAR target plasmids according to sequences, producing viruses by combining a virus packaging system, transfecting T cells with the viruses to prepare CAR-T cells of corresponding targets, and expressing the scFv segments on the surfaces of the T cells so as to perform the function of targeting the antibodies to tumor antigens. However, the conventional scFv forms have large molecular weight, large steric hindrance, instability and difficulty in successful transduction by lentivirus, so as to be used as an alternative to scFv (conventional VH and VL in series), nanobody VHH is widely used as an antigen binding domain of CAR-T due to its small size, small molecular weight, low immunogenicity, good stability, strong antigen recognition ability, strong penetration ability, and the like. However, the production of CAR-T cells in combination with nanobodies differs in phage library construction.
4. Prior art and disadvantages associated with the creation of the present invention
Antibody genetic engineering techniques, such as phage display techniques, are continually evolving to provide a means for the mass, rapid production of antibodies. Phage display is an emerging expression technology, which is established and developed in 1990, and exogenous antibody fragments are fused with capsid proteins of phage, so that the exogenous antibody fragments are displayed on the surface of phage, antibodies of specific antigens are screened, and the antibodies can be produced in a large scale through prokaryotic or eukaryotic cell expression. After the camel is immunized by antigen, the phage display technology is used for screening the corresponding nano antibody, after the camel is immunized, the camel blood is collected, lymphocytes are separated, total RNA is extracted, cDNA can be obtained through reverse transcription, and the cDNA is subcloned into a phagemid vector to be combined with an auxiliary phage M13K07 for preparing a monovalent nano antibody display library. The size and diversity of the constructed nanobody library are the precondition for obtaining high affinity nanobody, and also, collection of fresh blood and preparation of mRNA and cDNA are key to constructing high quality library.
Phage display technology is the method of choice for antibody screening because it is mature and stable and suitable for large-scale production of antibodies. In addition to phage display technology, other related technologies have also been successfully applied to antibody production, including ribosome or mRNA display technology, yeast or bacterial surface display technology, and yeast two-hybrid screening technology, among others. Multivalent display libraries of nanobodies can be rapidly screened from libraries to high quality nanobodies in yeast and bacterial systems by using flow cell sorting (FACS). Ribosome and mRNA display technology was performed in vitro, eliminating the transformation step. In addition, after each round of reverse transcription polymerase chain reaction (RT-PCR), minor mutations in the sequence may be introduced, which may facilitate screening for more binding antibodies. Compared with other screening methods, the method has the advantages of simple library construction, large library capacity and simple and convenient screening, and is also an ideal screening technology.
Currently, anti-CD 38 monoclonal antibodies are mainly used for the treatment of malignant hematological cancers positively expressed by CD38, and anti-CD 38 antibodies target CD38 cancer cells mainly through antibody-dependent cytotoxicity and antibody-dependent phagocytosis. Currently, 2 CD38 monoclonal therapeutic antibodies (isatuximab) are marketed in batches and show a certain therapeutic effect clinically. There remains a need to develop more effective treatments for CD38 positive hematological malignancies.
CAR-T therapy has shown good therapeutic effects on hematological cancers including acute B-cell leukemia, large B-cell non-Hodgkin lymphoma and the like, but currently research drugs taking CD38 as targets are mostly monoclonal antibodies, and no anti-CD 38 CAR-T product exists on the market. Moreover, the traditional CD38 monoclonal antibody drugs mainly have two main problems:
a. the structure is complex, the molecular weight is large, and the blood brain barrier is not easy to penetrate; while nanobodies have only the variable region (VHH) of heavy chain antibodies, have small molecular weight, good solubility, strong penetrability, and low immunogenicity.
b. The core technology of traditional monoclonal antibody medicines is iterated from an ascites method/hybridoma cell in-vitro culture technology (murine monoclonal antibody) to a chimeric antibody technology (murine chimeric monoclonal antibody), from an antibody humanization technology (humanized antibody) to a phage display antibody library technology/humanized antibody transgenic mouse technology (fully humanized antibody), and the immunogenicity problem is a difficulty. In contrast, nanobodies are prepared by immunizing alpaca to obtain antibody genes, and then screening the alpaca antibody library by phage display screening technology to obtain the most suitable antibody sequences, wherein the steps comprise alpaca immunization, phage library construction and antibody screening are more advantageous in preparation.
Disclosure of Invention
In order to obtain a novel camel heavy chain antibody or antigen binding fragment which specifically binds to CD38, particularly a VHH antibody which specifically binds to CD38 antigen, the invention discloses 5 amino acid sequences by utilizing the advantages of small molecular weight, good solubility, strong penetrability and low immunogenicity of the VHH antibody. Although these 5 amino acid sequences are screened as nanobodies, it will be apparent to those skilled in the art that these 5 amino acid sequences may be part of the amino acid sequence of a camelid heavy chain antibody that binds CD38, or of an antigen binding fragment, by virtue of their property of specifically binding to CD38 antigen. Therefore, the specific scheme of the invention is as follows:
a camelid heavy chain antibody or antigen binding fragment comprising:
a1 Amino acid sequence shown as SEQ ID NO. 6; or (b)
a2 Amino acid sequence shown as SEQ ID NO. 7; or (b)
a3 Amino acid sequence shown in SEQ ID NO. 8; or (b)
a4 Amino acid sequence shown as SEQ ID NO. 9; or (b)
a5 Amino acid sequence shown as SEQ ID NO. 10;
the camelid heavy chain antibody is a polyclonal antibody or an anti-CD 38 monoclonal antibody;
the camelid heavy chain antibody or antigen binding fragment is capable of specifically binding to CD38 antigen.
In some embodiments, the antigen binding fragment is a VHH antibody; the amino acid sequence of the VHH antibody is selected from:
a1 Amino acid sequence shown as SEQ ID NO. 6;
a2 Amino acid sequence shown as SEQ ID NO. 7;
a3 Amino acid sequence shown in SEQ ID NO. 8;
a4 Amino acid sequence shown as SEQ ID NO. 9;
a5 Amino acid sequence shown in SEQ ID NO. 10.
In another aspect, the invention discloses a nucleic acid encoding a camelid heavy chain antibody or antigen binding fragment as described above.
In some embodiments, the nucleic acid comprises:
c1 A nucleotide sequence shown as SEQ ID NO. 1; or (b)
c2 A nucleotide sequence shown as SEQ ID NO. 2; or (b)
c3 A nucleotide sequence shown as SEQ ID NO. 3; or (b)
c4 A nucleotide sequence shown as SEQ ID NO. 4; or (b)
c5 A nucleotide sequence shown as SEQ ID NO. 5.
In some embodiments, the nucleic acid is selected from the group consisting of:
c1 A nucleotide sequence shown as SEQ ID NO. 1;
c2 A nucleotide sequence shown as SEQ ID NO. 2;
c3 A nucleotide sequence shown as SEQ ID NO. 3;
c4 A nucleotide sequence shown as SEQ ID NO. 4;
c5 A nucleotide sequence shown as SEQ ID NO. 5.
In a third aspect, the invention also discloses a vector comprising a nucleic acid as described above. In some embodiments, the vector is a recombinant lentiviral vector; the recombinant lentiviral vector contains a nucleic acid as described above.
In a fourth aspect, the invention also discloses a host cell comprising a nucleic acid as described above or a vector as described above.
In a fifth aspect, the invention also discloses a method for producing a camelid heavy chain antibody or antigen binding fragment, which cultures the host cells as described above and recovering the camelid heavy chain antibody or antigen binding fragment from the culture.
In a sixth aspect, the invention also discloses an anti-CD 38 CAR comprising an anti-CD 38 nanobody; the anti-CD 38 nanobody is an antigen-binding fragment as described above.
In some embodiments, the anti-CD 38 CAR is structured as a CD8 Leader-anti-CD 38 nanobody-CD 8 Hinge-CD8 TM-co-stimulatory domain-intracellular signal peptide, comprising a CD8 Leader membrane receptor signal peptide (CD 8 Leader), an anti-CD 38 nanobody, a CD8 Hinge chimeric receptor Hinge region (CD 8 Hinge), a CD8 TM chimeric receptor transmembrane region (CD 8 TM), a co-stimulatory domain, and an intracellular signal peptide, in series. The anti-CD 38 CAR in this embodiment replaces the anti-CD 38 scFv in a traditional anti-CD 38 CAR with an anti-CD 38 nanobody.
In some embodiments, the costimulatory domain comprises, but is not limited to, one or more selected from the group consisting of CD28, OX40, and 4-1 BB. Further, the intracellular signal peptide is cd3ζ.
In some embodiments, the CAR structure includes, but is not limited to, a CD8 leader-anti-CD 38 nanobody-CD 8 finger-CD 8 TM-co-stimulatory domain-intracellular signal peptide structure.
In a seventh aspect, the invention also discloses an anti-CD 38 CAR-T or anti-CD 38 CAR-NK or anti-CD 38 CAR-M, having modified thereon a Chimeric Antigen Receptor (CAR) which is an anti-CD 38 CAR as described above. In some embodiments, the T cells in the anti-CD 38 CAR-T include, but are not limited to, one or more of an αβ T cell, γδ T cell, NKT cell, MAIT cell, CIK cell.
In an eighth aspect, the invention also discloses the use of a camelid heavy chain antibody or antigen binding fragment as described above, or a nucleic acid as described above, or a vector as described above, or a host cell as described above, or an anti-CD 38 CAR-T or anti-CD 38 CAR-NK or anti-CD 38 CAR-M as described above, in the manufacture of an anti-tumour medicament.
In some embodiments, the tumor is a CD 38-positive-expressed tumor or a BMCA-positive-expressed tumor.
In some embodiments, the tumor is multiple myeloma, chronic Lymphocytic Leukemia (CLL), mantle cell lymphoma MCL, acute myeloid leukemia AML, acute gonococcal leukemia ALL.
As used herein, T cells in the term "CAR-T" are understood to be T cells in the broad sense, i.e., including but not limited to αβ T cells and/or γδ T cells, but also including some atypical T cells, such as Natural Killer T (NKT) cells, mucosa-associated invariant T (Mucosal AssociatedInvariant T, MAIT) cells, cytokine-Induced Killer (CIK) cells, and the like, unless specifically indicated as being T cells of a certain class when embodied. These atypical T cells also act as effector types for T lymphocytes. The source of T cells is also not limited to peripheral blood, including cord blood, stem cells, IPSCs, cell lines, and other cell types from which T cells differentiate. αβt and γδ T cells are a classification of typical T cells according to TCR type.
As used herein, the term "host cell" is to be understood in a broad sense and may be a prokaryotic cell or a eukaryotic cell; may be any suitable host cell known in the art, e.g., mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.
As used herein, the term "single-chain variable fragment" (scFv) consists of a variable region of the light chain (VL), a variable region of the heavy chain (VH) and a linker, the heavy and light chain variable regions each comprising 3 complementarity determining regions (CDR 1, CDR2, CDR 3) and 4 framework regions (FR 1, FR2, FR3, FR 4). Can bind to antigen.
Cancer is an important disease threatening human health, and the proportion of people dying from various cancers is on an increasing trend year by year. With the continuous development of science and technology, the diagnosis and treatment conditions of cancer are greatly improved, but the defects still exist, the treatment of cancer is mainly carried out by means of chemotherapy or operation, and the like, by means of the means, side effects are often great, and irreversible damage is caused to the health of patients during the treatment. CD38 is a transmembrane glycoprotein molecule expressed on the surface of tumor cells of various blood systems, and can promote proliferation, infiltration and metastasis of tumor cells, and the higher the malignancy of the tumor, the higher the expression level of CD38 is, which is considered as a biomarker of malignant blood tumor. The polyclonal and monoclonal antibodies of the CD38 are developed successively by taking the CD38 as a target protein, and have the advantages of short production period and the like, but the traditional IgG form has large molecular weight, is not easy to clear by organisms, and has poor tissue permeability, such as the monoclonal antibody (mAb) cannot penetrate through the blood brain barrier. As a preferred scheme for scFv (conventional VH and VL), nanobodies are widely used as antigen binding domains for CAR-T due to their small size, low molecular weight, low immunogenicity, good stability, strong antigen recognition and penetration. The present invention results clearly demonstrate the killing ability of cd38+ tumor cells using VHH-directed CD38 CAR-T cells with few applications of nanobodies of CD38, supporting the further development of such CD38 CAR-T for clinical trials and applications.
The invention discloses a camel heavy chain antibody or antigen binding fragment and application thereof. The antibody or antigen binding fragment comprises one or more of the 5 amino acid sequences; these 5 amino acid sequences are camelid nanobody VHH that specifically bind CD 38. The invention constructs an anti-CD 38 CAR by using the 5 amino acid sequences obtained by screening, constructs the anti-CD 38 CAR on immune cells by using a slow virus transduction technology, and performs in vitro function verification and clinical function verification by taking the anti-CD 38 CAR-T as an example. The in vitro experiment result shows that the anti-CD 38 CAR-T has good target cell killing capability and can stimulate the secretion of a large amount of cytokines (such as IFN-gamma and TNF alpha) related to the anti-tumor activity; clinical results show that anti-CD 38 CAR-T cells that use nanobodies to perform targeting functions can be specifically expanded in patients after stimulation in vivo with CD38 positive tumor cells. The invention discloses a group of anti-CD 38 VHH antibodies for the first time, and the anti-CD 38 VHH antibodies are used in a CAR to identify antigens, and have good technical effects, thereby being beneficial to improving the treatment of the CAR-T, CAR-NK and the CAR-M.
The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.
Drawings
FIG. 1 is a schematic diagram of a conventional antibody and nanobody.
FIG. 2 is a5 VHH based anti-CD 38 CAR structure.
FIG. 3 is a graph of the percentage of anti-CD 38 CAR-T cells detected that bind to the antigen CD 38-HIS.
FIG. 4 is a comparison of the killing function of anti-CD 38 CAR-T cells prepared from 551-555 together with 5 sets of anti-CD 38 nanobodies against target cells.
FIG. 5 is a comparison of anti-CD 38 CAR-T cells prepared from 551-555 together with 5 sets of anti-CD 38 nanobodies at IFN-gamma and TNF alpha cytokine release levels.
FIG. 6 is the expansion of anti-CD 38 CAR-T (pUT 553) cells after 8 patients were infused.
Detailed Description
The invention is further described with reference to the following detailed description in order to make the technical means, the inventive features, the achieved objects and the effects of the invention easy to understand. The present invention is not limited to the following examples.
The attached table:
table 1, 5 anti-CD 38 VHH nucleotide and amino acid sequence numbering
| Name of the name | Nucleotide sequence numbering | Amino acid sequence numbering |
| FA-2E VHH | SEQ ID NO:1 | SEQ ID NO:6 |
| FA-7G VHH | SEQ ID NO:2 | SEQ ID NO:7 |
| FB-1E VHH | SEQ ID NO:3 | SEQ ID NO:8 |
| FB-5G VHH | SEQ ID NO:4 | SEQ ID NO:9 |
| FB-8A VHH | SEQ ID NO:5 | SEQ ID NO:10 |
TABLE 2 nucleotide sequence and amino acid sequence numbering of anti-CD 38 CAR partially related elements
| Name of the name | Nucleotide sequence numbering | Amino acid sequence numbering |
| CD8 leader | SEQ ID NO:11 | SEQ ID NO:17 |
| CD8 Hinge | SEQ ID NO:12 | SEQ ID NO:18 |
| CD8 TM transmembrane region | SEQ ID NO:13 | SEQ ID NO:19 |
| CD28 co-stimulatory domain | SEQ ID NO:14 | SEQ ID NO:20 |
| OX40 co-stimulatory domains | SEQ ID NO:15 | SEQ ID NO:21 |
| CD3ζ | SEQ ID NO:16 | SEQ ID NO:22 |
TABLE 3 anti-CD 38 CAR nucleotide sequences
| Name of the name | Nucleotide sequence numbering | Amino acid sequence numbering |
| FA-2E CAR | SEQ ID NO:23 | SEQ ID NO:28 |
| FA-7G CAR | SEQ ID NO:24 | SEQ ID NO:29 |
| FB-1E CAR | SEQ ID NO:25 | SEQ ID NO:30 |
| FB-5G CAR | SEQ ID NO:26 | SEQ ID NO:31 |
| FB-8A CAR | SEQ ID NO:27 | SEQ ID NO:32 |
TABLE 4 PCR amplification of VHH Gene primer sequence numbering
| Name of the name | Numbering device |
| CS-F | SEQ ID NO:33 |
| CS-R | SEQ ID NO:34 |
| VHH-F | SEQ ID NO:35 |
| VHH-R | SEQ ID NO:36 |
Example 1 screening of anti-CD 38 nanobodies
The experimental animal Bactrian camels are fed into inner Mongolia. Helper phage M13KO7 was purchased from NEB under accession number N0315S. Plasmid minipump kit and DNA gel recovery kit were purchased from Tiangen Biochemical technologies Co. The antigen Human CD38 Protein, his Tag (MALS verify) was purchased from Baipase under the designation CD8-H5224.HRP anti-His Tag anti-ibody was purchased from Biolegend and the required primers were purchased from Biobioengineering Co. SfiI was purchased from NEB. T4 DNA ligase was purchased from NEB. Tg1 E.coli was from Ubachel.
Selecting alpaca over two years old as an immunized animal, wherein the alpaca immunization steps are as follows: selecting the back of the alpaca neck, and shaving the back hair close to the area of the arced lymph node; iodophor was used for disinfection and disinfectant cleaning. 1mg of CD38-HIS antigen protein was diluted to 1mg/ml with 1 XPBS, thoroughly mixed with 1ml of complete Freund's adjuvant, and then immunized with incomplete adjuvant. Multipoint immunization was performed by subcutaneous injection, and blood was collected from the jugular vein of alpaca using a vacuum blood collection tube prior to immunization as a negative serum control. Boosting is carried out after each immunization for one week, the total immunization is carried out for 4 times, and the alpaca peripheral blood is collected after immunization. The lymphocytes are separated by using a peripheral blood lymphocyte separation solution, and the RNA is extracted and reverse transcribed into cDNA. PCR reactions were performed using the primer sequences shown in Table 4. The primer pairs for the first round of PCR were CS-F and CS-R, and the procedure was set as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s for 35 cycles; finally, the extension is carried out at 72 ℃ for 5 minutes. The first round PCR products were identified by agarose gel electrophoresis and the PCR products were recovered by gel. And carrying out a second round of PCR reaction by taking the gel recovery product as a template. The second round PCR primer pairs were VHH-F and VHH-R, and the procedure was set to: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s for 35 cycles; finally, the extension is carried out at 72 ℃ for 5 minutes. After purifying the PCR product, the PCR product is digested with SfiI, and the digested product is purified and then connected with a linearization vector through T4 DNA ligase. The purified ligation product was electrotransformed into Tg1 E.coli competent. After the electric transferred Tg1 escherichia coli is shake-flask cultured to the logarithmic phase, the phage is infected by using helper phage M13KO7, and phage are collected the next day, so as to obtain a nanobody phage library. Phagemid vector PA008 was from eucadi and was cut by SfiI to form the linearized vector described above.
Phage were eluted with pancreatin solution using CD38-HIS antigen as target, and 4 rounds of affinity panning were performed on nanobody phage library. Positive screening is carried out by phage enzyme-linked immunosorbent assay (phageELISA), and finally 5 positive clones (namely 5 nanobodies or 5 VHHs) are obtained, wherein the nucleotide sequence and the amino acid sequence numbers of the positive clones are shown in Table 1, and the positive clones are FA-2E, FA-7G, FB-1E, FB-5G, FB-8A respectively.
EXAMPLE 2 construction of anti-CD 38-CAR and lentiviral packaging
Lentiviral backbone plasmid vector pUT-Bacbone, from Ucat; both pPac-R, pPac-GP and pEnv-G are from Ubacodi; DMEM medium was from Gibco and 293T cells were from ATCC.
5 sets of anti-CD 38 nanobodies (FA-2E, FA-7G, FB-1E, FB-5G, FB-8A) obtained in example 1 were respectively constructed into 5 sets of recombinant lentiviral vectors, which were designated as PUT551, PUT552, PUT553, PUT554, and PUT555, respectively.
The method comprises the following specific steps: mixing a lentiviral backbone plasmid vector pUT-Bacbone with EcoR I and BamH I restriction endonucleases for enzyme digestion; after gel electrophoresis (120V, 20 min), the linear plasmid of 7000bp size was recovered using an agarose gel DNA recovery kit; designing corresponding primers to synthesize a target gene, namely an anti-CD 38-CAR plasmid gene, by Polymerase Chain Reaction (PCR), wherein the target gene comprises a CD8 leader membrane receptor signal peptide, an anti-CD 38 VHH, a CD8 alpha chimeric receptor Hinge region Hinge, a CD8 alpha chimeric receptor transmembrane region TM, a CD28 co-stimulatory domain, an OX40 co-stimulatory domain and a CD3 zeta intracellular signal peptide which are sequentially connected in series; the PCR reaction product is recovered by agarose gel electrophoresis (120V, 20 min) and purified by a purification kit, and the obtained vector linear plasmid and the target gene fragment are subjected to homologous recombination according to a seamless cloning kit to obtain the target plasmid embedded with the target gene (CD 38 CAR). The 5 anti-CD 38 CARs were structured as shown in figure 2.
The 293T cells were transfected with the appropriate amount of the plasmid of interest as follows: 2500ul CaCl was added to a 15mL centrifuge tube 2 3 packaging plasmids (80 ug pPac-R),100G of pPac-GP, 60G of pEnv-G) and 220ug of the target plasmid, then the bacterial endotoxin test water is added to 5000ul, 5000ul of HBS is added, and the shaking is continued for about 20s after the addition is completed. 293T cells were taken, 1mL of the mixed transfection reagent was added, mixed well with gentle shaking, and placed in an incubator. After 6h of transfection, the culture supernatant was discarded and 10mL of 4% DMEM complete medium was added. After 24h of transfection, 10mL of 4% DMEM complete medium was continued to be added. Collecting virus supernatant at 4 ℃ after 24 hours, 48 hours and 72 hours of culture, preserving, filtering the virus supernatant by using a 0.45um filter after the virus supernatant is collected, adding polyethylene glycol 8000 (PEG 8000) after filtering residues of cells, concentrating the virus, incubating at 4 ℃ for 12 hours, centrifuging at 3000rpm for 15 minutes, discarding the supernatant after centrifuging, adding a proper amount of sterile PBS to dissolve virus precipitate, subpackaging and preserving the dissolved virus in a refrigerator at-80 ℃ to obtain the recombinant lentiviral vector. The recombinant lentiviral vector contains an anti-CD 38 CAR gene.
Example 3 preparation of anti-CD 38 CAR-T
The recombinant lentiviral vectors pUT 551-pUT 555 obtained in example 2 are respectively transduced into primary T cells, CAR-T cells are prepared, and a flow cytometer detects the CAR positive rate; CAR positive rate is defined as the percentage of cells that are able to bind to CD38 antigen. The primary antibody for flow detection is Human CD38 Protein, his Tag and the secondary antibody is anti-His fluorescence. The method comprises the following specific steps: separating Peripheral Blood Mononuclear Cells (PBMCs) with a mononuclear cell separation fluid; CD 4-separating magnetic beads and CD 8-separating magnetic beads for sorting lymphocyte T cells; CD3 and CD28 antibodies activate T cells; adding slow virus to respectively transduce T cells, placing into 37 ℃ and 5% CO 2 Transduction in incubator for 48h; changing the culture medium after 48h of transduction, and counting and supplementing liquid every 1-2 days; the CAR positive rate was examined by flow cytometry about 7 days after transduction, with the primary antibody being the Human CD38 Protein, his Tag, and the secondary antibody being anti-His fluorescent. In addition to the 5 CAR-T cells (551, 552, 553, 554, 555) constructed using nanobodies, 2354CAR-T cells (using conventional anti-CD 38 scFv to recognize antigens) were constructed by prior art techniques (such as CN107337736 a). As shown in fig. 3, the CAR positive rate in each of these 6 CAR-T cells was 20% or more.
Example 4 in vitro functional validation of anti-CD 38 CAR-T
CD4/CD8 magnetic beads, CD3 antibody and CD28 antibody are all from meitian and Tsaoko; IL-2 from Peprotech; the A-IMV medium was from Gibco; FITC-Protein L Protein was from Biolegend. LDH killing kit source: cytoTox 96Non-Radioactive Cytotoxicity Assay, promega, cat No. REF: g1782; the A-IMV medium was from Gibco; k562 (CD 38 negative) from Shanghai national academy of sciences cell Bank; the myeloid leukemia cell line K562-CD38 (abbreviated as K562-CD 38) overexpressing CD38 is from Shanghai Ubachel.
Effector NC (T cells without a transvirus, as negative control, i.e. control in fig. 4 and 5), 2354 (conventional scFv control CAR-T cells), 551, 552, 553, 554, 555 (CAR-T cells constructed with VHH nanobodies) total 7 groups of cells were cultured separately; culturing target cells K562, CD38 (PSE 2348) -K562 (i.e., K562-CD38 as described above) respectively; performing cytotoxicity experiments by grouping and incubating effector cells and target cells, wherein the experimental effect target ratio is set to be 5:1 and 2.5:1 respectively; after overnight incubation, the killing efficiency was measured using Lactate Dehydrogenase (LDH) method, while effector cells were co-incubated with target cells and the supernatant was taken to measure cytokine levels.
The specific experimental steps are as follows: 1) Collecting a suspension of target cells: adherent cells were discarded, 10mL of physiological saline was used to infiltrate the bottom of the dish, then the physiological saline was discarded, 1mL of pancreatin was added to digest the cells per dish, the digestion time was determined according to the cell line characteristics, the cells were observed under a microscope without adherent, suspended and morphologically rounded, and digestion was terminated by adding 3mL of complete medium. Centrifugation at 1500rpm for 5min, discarding supernatant, adding 5mL PBS respectively, resuspending, centrifugation at 1500rpm for 5min, discarding supernatant. 2) Target cell plating: target cells were resuspended individually with 1mL AIM-V killer medium (AIM-V+4% FBS). Counting was performed using a hemocytometer. 96. Adding 1x104 holes of target cells into each hole of a hole plate, preparing target cell suspension by using a killing culture medium according to the required cell quantity, spreading the target cells into a 96-hole cell culture plate after preparing, centrifuging for 5 minutes, and placing into a 37 ℃ carbon dioxide incubator for culturing for 2 hours to promote the adherence of the target cells. 3) Effector cell suspensions were collected, T cells were mixed well and counted using the hemocytometer. Taking according to the dosage of effector cells required for killingEffector cells were centrifuged at 1500rpm for 5min and resuspended in 1mL AIM-V killer medium (AIM-V+4% FBS) individually, trypan blue staining counts. 4) Target cells have been added 1X10 per well of a 96-well plate 4 When E: T (effector cells: target cells) was set to 2.5:1, 2.5X10 cells were added per well 4 The method comprises the steps of carrying out a first treatment on the surface of the E, t=5: 1. effector cells 5X 10 4 Holes; e, t=10: 1, effector cells 1×10 5 Each well had a volume of 50 ul/well. Formulation E, t=10: 1, effector cells 1×10 5 Well, prepared with 1-fold volume of cell mass, 2-fold gradient diluted with killing medium, formulated E: t=5: 1 and E, t=2.5: 1. is described. 5) After the plate is paved, sealing the 96-well plate by using a sealing film, putting the sealing film into a centrifuge, centrifuging for 5min after the sealing film is 250g and 3 g, and putting the sealing film into a constant temperature incubator at 37 ℃ for incubation for 24h. 6) The detection was performed according to the kit instructions and finally absorbance was measured at 490nm using an enzyme-labeled instrument. 7) The killing efficiency calculation formula: cytotoxicity% = experimental group kill/maximum kill x 100%.
And (3) a step of detecting cytokines: after co-incubation of effector T cells with target cells, the supernatants were collected for cytokine detection in 1.5mL EP tubes. a sample treatment: after centrifugation of the cell suspension at 1500rpm for 3min, the supernatant was collected in a fresh 1.5mL EP tube. b, preparing a standard product: and adding 2mL of standard substance diluent to dilute the standard substance freeze-dried powder, and standing for 15min at room temperature. The standard was then diluted 2-fold gradient (10 gradients total from the highest concentration to the blank dilution). c 50. Mu.L of standard or sample was added to the new EP tube, followed by 50. Mu.L of magnetic beads and 50. Mu.L of detection antibody, vortexing well, and incubation at room temperature for 3h. d washing twice with washing Buffer (Wash Buffer), discarding the supernatant, adding 200. Mu.L Wash Buffer, and re-suspending on-machine to detect cytokine IL-2/IL-4/IL-6/IL-10/IFNgamma/TNF alpha.
As shown in FIG. 4, the LDH killing results showed that the killing efficiency of the pUT551 and pUT552 groups on CD38+K562 target cells was low, and the killing efficiency of the pUT553 and pUT554 groups on CD38+K562 target cells was within the normal range, and the pUT555 groups on both K562 (CD 38 negative cells) and K562-CD38 (CD 38 positive cells) showed that the off-target effect was possible.
The results of cytokine release level detection are shown in FIG. 5, and are substantially consistent with LDH results, and IFN gamma/TNF alpha is almost negative after the groups 551 and 552 are incubated with CD38+K562 target cells, and IFN gamma/TNF alpha is expressed in the group 553 and 554 except for the group CD38+K562 on non-target cells K562.
In summary, pUT553 (FB-1E) group was the most potent in killing and did not have off-target effects, and thus was selected as the final anti-CD 38 CAR-T cell (pUT 553) based on nanobody FB-1E design for subsequent experiments.
Example 5 in vivo amplification and therapeutic Effect of anti-CD 38 CAR-T
After screening for appropriate patients with Multiple Myeloma (MM), acute Myeloid Leukemia (AML), acute Lymphoblastic Leukemia (ALL) (requisite inclusion group condition: positive expression of tumor cells CD 38), the clinical therapeutic efficacy and safety of anti-CD 38 CAR-T (CAR number pUT553, FB-1E-VHH performs the targeting function) cell therapy for MM, AML, ALL patients was evaluated. The anti-CD 38 CAR-T cells were infused with Fludarabine and Cyclophosphamide (FC) (fludarabine 30 mg/m) 2 Per day, 3 consecutive days, cyclophosphamide 300mg/m 2 Per day) for 3 consecutive days to reduce the number of lymphocytes and tumor cells in the body, to the extent of "clear stranguria", while improving the efficacy of CAR-T cells. Then according to the dosage of 1×10 6 The CAR-T cells/kg pass through the venous CAR-T cells singly or in batches, and are checked at a specified time evaluation point after the reinfusion is finished to evaluate the curative effect and toxic and side effects. The qPCR method was used to detect CAR copy number in blood after patient reinfusion of CAR-T cells at a given time point.
Clinical results as shown in FIG. 6, the CAR-T cells were all able to expand in vivo after infusion of anti-CD 38 CAR-T in 8 MM, AML, ALL patients, suggesting that anti-CD 38 CAR-T cells targeting with nanobody VHH (FB-1E) could specifically expand in patients after stimulation with CD38 positive tumor cells in vivo.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (11)
1. A camelid heavy chain antibody or antigen binding fragment comprising:
a1 Amino acid sequence shown as SEQ ID NO. 6; or (b)
a2 Amino acid sequence shown as SEQ ID NO. 7; or (b)
a3 Amino acid sequence shown in SEQ ID NO. 8; or (b)
a4 Amino acid sequence shown as SEQ ID NO. 9; or (b)
a5 Amino acid sequence shown as SEQ ID NO. 10;
the camelid heavy chain antibody is a polyclonal antibody or an anti-CD 38 monoclonal antibody;
the camelid heavy chain antibody or antigen binding fragment is capable of specifically binding to CD38 antigen.
2. The camelid heavy chain antibody or antigen binding fragment of claim 1, wherein the antigen binding fragment is a VHH antibody; the amino acid sequence of the VHH antibody is selected from:
a1 Amino acid sequence shown as SEQ ID NO. 6;
a2 Amino acid sequence shown as SEQ ID NO. 7;
a3 Amino acid sequence shown in SEQ ID NO. 8;
a4 Amino acid sequence shown as SEQ ID NO. 9;
a5 Amino acid sequence shown in SEQ ID NO. 10.
3. Nucleic acid encoding a camelid heavy chain antibody or antigen binding fragment according to claim 1 or 2.
4. A nucleic acid according to claim 3, comprising:
c1 A nucleotide sequence shown as SEQ ID NO. 1; or (b)
c2 A nucleotide sequence shown as SEQ ID NO. 2; or (b)
c3 A nucleotide sequence shown as SEQ ID NO. 3; or (b)
c4 A nucleotide sequence shown as SEQ ID NO. 4; or (b)
c5 A nucleotide sequence shown as SEQ ID NO. 5.
5. Vector, characterized in that it comprises a nucleic acid according to claim 3 or 4.
6. Host cell, characterized in that it comprises a nucleic acid according to claim 3 or 4 or a vector according to claim 5.
7. A method for producing a camelid heavy chain antibody or antigen binding fragment, characterized in that a host cell according to claim 6 is cultured and the camelid heavy chain antibody or antigen binding fragment is recovered from the culture.
8. An anti-CD 38 CAR comprising an anti-CD 38 nanobody; the anti-CD 38 nanobody is an antigen-binding fragment according to claim 1 or 2.
9. An anti-CD 38 CAR-T or anti-CD 38 CAR-NK or anti-CD 38 CAR-M, having modified thereon a chimeric antigen receptor which is an anti-CD 38 CAR according to claim 8.
10. Use of a camelid heavy chain antibody or antigen binding fragment according to claim 1 or 2, or a nucleic acid according to claim 3, or a vector according to claim 5, or a host cell according to claim 6, or an anti-CD 38 CAR according to claim 8, or an anti-CD 38 CAR-T or anti-CD 38 CAR-NK or anti-CD 38 CAR-M according to claim 9, in the manufacture of an anti-tumour medicament or a medicament for the treatment of a B cell derived autoimmune disease.
11. Use of a camelid antibody or CD38 antigen binding fragment that binds CD38 according to claim 1 or 2 to reduce the level of cell surface expression of CD38 or to reduce the level of intracellular expression of CD 38.
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