CN113045675A

CN113045675A - Antibody for resisting CD22 protein molecule and application thereof

Info

Publication number: CN113045675A
Application number: CN202110367389.3A
Authority: CN
Inventors: 赵振东; 张重阳; 黄前荣; 刘佳; 吴维海; 牛璘灿
Original assignee: Beijing Rongyi Medical Biotechnology Co ltd
Current assignee: Beijing Rongyi Medical Biotechnology Co ltd
Priority date: 2020-04-07
Filing date: 2021-04-06
Publication date: 2021-06-29
Anticipated expiration: 2041-04-06
Also published as: CN111518217A; CN113045675B

Abstract

The invention discloses an antibody for resisting a CD22 protein molecule and application thereof, belonging to the technical field of immunity. The antibody for resisting the CD22 protein molecule comprises a heavy chain variable region, wherein the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The invention also discloses a chimeric antigen receptor, a fusion protein and a carrier. The anti-CD22 protein molecule antibody is a small molecular weight antibody derived from alpaca, adopts a nano antibody targeting a CD22 molecule as a specific recognition structural component of a receptor, can specifically recognize and kill cells taking a CD22 molecule as a marker, and treats diseases taking CD22+ as the marker.

Description

Antibody for resisting CD22 protein molecule and application thereof

Technical Field

The invention relates to an antibody for resisting a CD22 protein molecule and application thereof, belonging to the technical field of immunity.

Background

In recent years, the field of immunology has increasingly studied for transforming medicine, in particular tumor immunity. The CAR-T cell immunotherapy strategy is widely used in tumor immunotherapy. This immunotherapy strategy achieved good results in transformation medicine early in the 90 s. CAR-T cells undergo a first, second to third generation transition. Through the innovation of the technology and design, the activation of cytotoxic action and the lasting of killing signals in the treatment process are gradually optimized. CAR-T cells targeting CD22 molecules are widely applied, especially for treating blood tumors such as B-lymphocytoma. The regulation of multiple signals is required for effective stimulation and immunological function of T cells in normal organisms, mainly comprises the recognition of T Cell Receptor (TCR) and MHC-antigen peptide complex as a first signal, the recognition and activation of costimulatory molecules on the surface of T cells as a second signal, and even requires cytokines to participate in forming a third signal. The chimeric antigen receptor mainly performs artificial tandem integration on protein molecules required by T cell stimulation and activation processes, so as to promote the activation and specific killing of T cells. Specific killing of CAR-T cells is primarily recognition and binding of the antibody molecule part of the front end. Currently, the use of monoclonal antibody Single chain variable domains (ScFv) based on human or other species for specific recognition of target proteins is widespread. But has the following defects: on the basis that the affinity of the ScFv is equivalent to that of the nanobody, the ScFv has a large molecular weight compared to the nanobody, and has certain limitations on molecular expression and function development. Nanobodies are the smallest naturally occurring antibody fragments. ScFv is derived from its parent monoclonal antibody, and may have some disadvantages in terms of activity and stability.

In view of the above, there is a need to provide an antibody against CD22 protein molecule for constructing a novel chimeric antigen receptor and/or fusion protein to overcome the deficiencies of the prior art.

Disclosure of Invention

One of the purposes of the present invention is to provide an antibody against CD22 protein molecule. The anti-CD22 protein molecule antibody is a small molecular weight antibody derived from alpaca, adopts a nano antibody targeting CD22 molecule as a specific recognition structural component of a receptor, can specifically recognize and kill cells expressing CD22, such as CD22+ cells in adaptation diseases caused by B cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute myelogenous leukemia and a large amount of autoantibodies expressed by plasma cells, such as lupus erythematosus, rheumatoid arthritis, autoimmune purpura and the like, and inhibits and treats diseases taking CD22 molecule as a marker.

The technical scheme for solving the technical problems is as follows: an antibody for resisting a CD22 protein molecule is derived from alpaca and comprises a heavy chain variable region, wherein the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

The principle of the antibody against the CD22 protein molecule of the present invention is:

the invention adopts CD22 single-chain antibody as the specific recognition structural component of the receptor. The CD22 single-chain antibody is derived from alpaca, and is an alpaca naturally-occurring antibody molecule with a deleted light chain. The antibody only comprises a heavy chain Variable region (VHH) and two conventional CH2 and CH3 regions, and the structure of the heavy chain Variable region (VHH) which is independently cloned and expressed is equivalent to the structure stability and the binding activity with an antigen in an intact heavy chain antibody, and is the antibody structure with the minimum protein molecular weight which is known at present, so the structure of the heavy chain Variable region (VHH) is called as a nanobody (Nb). Compared with the Single chain variable region (ScFv) of the monoclonal antibody in the prior art, the heavy chain variable region of the CD22 Single chain antibody has the advantage of smaller structure, and has advantages in expression of chimeric antigen receptors and modification of T cells.

The anti-CD22 protein molecule antibody has the following beneficial effects:

the anti-CD22 protein molecule antibody is a small molecular weight antibody derived from alpaca, adopts a nano antibody targeting CD22 molecule as a specific recognition structural component of a receptor, can specifically recognize and kill cells expressing CD22 molecule, and treats diseases taking CD22 molecule as a marker, such as B cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute myelogenous leukemia and adaptation diseases caused by a large amount of autoantibodies expressed by plasma cells, such as lupus erythematosus, rheumatoid arthritis, autoimmune purpura and the like.

Another object of the present invention is to provide a chimeric antigen receptor. The chimeric antigen receptor contains a nano antibody of an anti-human CD22 protein molecule, targets and identifies cells expressing the human CD22 protein molecule, can cause the identified target cells to die, and simultaneously causes host cells to release cytokines and the like, and the host cells expressing the chimeric antigen receptor can play an anti-tumor effect or an anti-autoimmune disease effect.

The technical scheme for solving the technical problems is as follows: a chimeric antigen receptor having the structure: SP-VHH-HINGE-TM-CD-SD, wherein SP is a signal peptide or leader peptide, VHH is an antibody against the CD22 protein molecule of claim 1, is an antigen binding domain, HINGE is a HINGE region, TM is a transmembrane region, CD is a costimulatory region, SD is a signal transduction region, and "-" is a linker peptide or peptide bond.

The chimeric antigen receptor of the invention has the following beneficial effects:

1. the chimeric antigen receptor contains a nano antibody of an anti-human CD22 protein molecule, targets and identifies cells expressing the human CD22 protein molecule, can cause the identified target cells to die, and simultaneously causes host cells to release cytokines and the like, and the host cells expressing the chimeric antigen receptor can play an anti-tumor effect or an anti-autoimmune disease effect.

2. The chimeric antigen receptor of the invention can be used for detecting cells or other biological tissues or cells expressing human CD22 protein molecules, such as bacteriophage, viruses, bacteria and the like; can also be used for identifying and detecting free human CD22 protein molecules.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the SP is selected from any one of signal peptides of CD8 alpha, GM-CSFR and Ig kappa, wherein the nucleotide sequence of the CD8 alpha signal peptide is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4; the nucleotide sequence of the GM-CSFR signal peptide is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 6; the nucleotide sequence of the signal peptide of the Ig kappa is shown as SEQ ID NO.7, and the amino acid sequence is shown as SEQ ID NO. 8.

The adoption of the further beneficial effects is as follows: the signal peptide can mediate the localization distribution of the CAR protein expression outside the cell membrane.

Further, the HINGE is selected from a CD8 alpha HINGE region, the nucleotide sequence of which is shown as SEQ ID NO.9, and the amino acid sequence of which is shown as SEQ ID NO. 10.

The adoption of the further beneficial effects is as follows: the CD8 a chain hinge region can promote stabilization of the extracellular domain of the CAR structure and promote the formation of dimers of the extracellular domain CAR structure.

Further, the TM is selected from a transmembrane region of CD8 alpha or CD28, wherein the nucleotide sequence of the transmembrane region of CD8 alpha is shown as SEQ ID NO.11, and the amino acid sequence is shown as SEQ ID NO. 12; the nucleotide sequence of the CD28 transmembrane region is shown as SEQ ID NO.13, and the amino acid sequence is shown as SEQ ID NO. 14.

The adoption of the further beneficial effects is as follows: the CD8 a chain transmembrane region can facilitate localization of CAR structures on cell membranes.

Further, the CD is selected from a co-stimulation region of CD28 and/or 4-1BB, wherein the nucleotide sequence of the co-stimulation region of CD28 is shown as SEQ ID NO.15, and the amino acid sequence is shown as SEQ ID NO. 16; the nucleotide sequence of the 4-1BB costimulatory region is shown as SEQ ID NO.17, and the amino acid sequence is shown as SEQ ID NO. 18.

The adoption of the further beneficial effects is as follows: the costimulatory region of the CD28 molecule can act as a costimulatory molecule to activate T cells. The co-stimulation region of the 4-1BB molecule can be used as a co-stimulation molecule to activate T cells.

Furthermore, the SD is a CD3 zeta intracellular signal transduction zone, the nucleotide sequence of the SD is shown as SEQ ID NO.19, and the amino acid sequence of the SD is shown as SEQ ID NO. 20.

The adoption of the further beneficial effects is as follows: the CD3 ζ intracellular signaling region can transduce a T cell activation signal to an intracellular signaling pathway.

The third object of the present invention is to provide a fusion protein. The fusion protein can realize the functions of target recognition of antigen and playing the role of cytotoxicity; can play a role of specifically marking target antigens; can also be used for detecting cells expressing human CD22 protein molecules or other biological tissues or cells, such as bacteriophage, virus, bacteria, etc.; can also be used for identifying and detecting free human CD22 protein molecules.

The technical scheme for solving the technical problems is as follows: a fusion protein comprises the anti-CD22 protein molecule antibody.

The fusion protein has the beneficial effects that:

the fusion protein can realize the functions of target recognition of antigen and playing the role of cytotoxicity; can play a role of specifically marking target antigens; can also be used for detecting cells expressing human CD22 protein molecules or other biological tissues or cells, such as bacteriophage, virus, bacteria, etc.; can also be used for identifying and detecting free human CD22 protein molecules.

The fourth object of the present invention is to provide a vector. The vector of the present invention may be a plasmid vector, and may carry the nucleotide sequence of the antibody against the CD22 protein molecule, or the nucleotide sequence of the chimeric antigen receptor, or the nucleotide sequence of the fusion protein. The vector of the present invention may be one which expresses either the nucleotide sequence of the antibody against the CD22 protein molecule or the nucleotide sequence of the chimeric antigen receptor or the nucleotide sequence of the fusion protein alone, or may be one which is co-expressed with a helper plasmid to synthesize a viral vector, which may be transfected into a host cell, and the nucleotide sequence of the antibody against the CD22 protein molecule or the chimeric antigen receptor or the nucleotide sequence of the fusion protein may be transferred into the host cell, and the antibody against the CD22 protein molecule, the chimeric antigen receptor or the fusion protein may be transiently expressed in the host cell, or the nucleotide sequence may be integrated into the chromosome of the host cell, and then the antibody against the CD22 protein molecule, the chimeric antigen receptor or the fusion protein may be expressed.

The technical scheme for solving the technical problems is as follows: a vector comprising any one of the nucleotide sequence of the antibody against the CD22 protein molecule, the nucleotide sequence of the chimeric antigen receptor and the nucleotide sequence of the fusion protein.

The carrier of the invention has the following beneficial effects:

1. the vector of the invention can stably store, replicate and amplify the nucleotide sequence of the antibody, the chimeric antigen receptor or the fusion protein, more conveniently transfer the nucleotide of the antibody, the chimeric antigen receptor or the fusion protein into host cells, and more efficiently express the antibody, the chimeric antigen receptor or the fusion protein.

2. The vector of the invention carries a nucleotide sequence containing a nano antibody with smaller molecular weight, can be stored, copied and amplified more stably, and can also be transduced into host cells more efficiently and express the nano antibody, and the nano antibody has stability superior to that of the traditional antibody.

The fifth object of the present invention is to provide a host cell. The host cell of the present invention belongs to an immune cell, or a stem cell or a progenitor cell capable of differentiating into an immune cell. The host cells of the invention are immune cells of a class including T cells, NK cells, NKT cells, mononuclear macrophages and dendritic cells, and have the functions of killing or phagocytosis, immunoregulation and cytokine release. T cells in the host cell of the present invention include various subtypes such as α β T cells, γ δ T cells, CD4+ T cells, CD8+ T cells, regulatory T cells, and killer T cells. Another class of host cells of the invention are stem cells, which may be induced pluripotent or hematopoietic stem cells, capable of differentiating into one or more of the above-described immune cells.

The technical scheme for solving the technical problems is as follows: a host cell comprising any one of the nucleotide sequence of the anti-CD22 protein antibody, the chimeric antigen receptor, the fusion protein nucleotide sequence and the vector, or any one of the nucleotide sequence of the anti-CD22 protein antibody, the chimeric antigen receptor and the fusion protein nucleotide sequence integrated with a foreign gene in a chromosome.

The host cell of the invention has the following beneficial effects:

1. the host cell of the invention, through expressing the anti-CD22 protein molecule antibody, the chimeric antigen receptor and the fusion protein, plays a role in targeting CD22 molecules and recognizing and killing CD22+ cells, and simultaneously combines the targeting of the anti-CD22 protein molecule antibody and the functions of killing, regulating and secreting cytokines of the host cell.

2. The host cell of the invention can target and recognize and kill CD22+ cells and play a role in resisting tumors or eliminating cells.

Drawings

FIG. 1 shows the detection of the affinity of the VHH region of the BA125 nano antibody of the present invention and the extracellular domain protein of CD 22.

FIG. 2 shows the expression of the chimeric antigen receptor comprising the VHH region of BA125 nanobody of the present invention in 293T and the binding of CD22 protein molecule. Wherein, A picture and C picture are 293T cell after lentivirus infection and do not use CD22 antigen for immunofluorescence staining; panel B shows staining of 293T cells with anti-VHH f (ab)2 antibody following lentivirus infection, showing that 293T cells express a23 chimeric antigen receptor; and D, a graph shows that 293T cells are detected by CD22 protein after being infected by lentivirus, and the BA125 chimeric antigen receptor expressed by the 293T cells can be targeted and identified with CD22 protein.

FIG. 3 is a flow cytometry assay for CAR-T cells expressing a chimeric antigen receptor in an embodiment of the invention. Wherein, A is that T cells are not infected by lentivirus carrying the chimeric antigen receptor gene of the invention, and the T cells can not be detected to be combined with CD22 antigen; and B, the picture B shows that when T cells are infected by 1000 mu L of lentivirus stock solution carrying the chimeric antigen receptor gene disclosed by the invention, the T cells can be detected to express the chimeric antigen receptor, and the CAR-T cells expressing the chimeric antigen receptor are detected to be positive by about 76.5 percent through F (ab)2 antibody staining of anti-VHH.

FIG. 4 shows the results of the present invention, in which CD22-K562 stimulates the activation of CAR-T cells to produce TNF α cytokines. In the figure, Effector cells refer to Effector cells; PMA/iono refers to phorbol ester and ionomycin; TNF α refers to tumor necrosis factor α.

FIG. 5 shows the results of the present invention, in which CD22-K562 stimulates the activation of CAR-T cells to produce IFN γ cytokines. In the figure, Effector cells refer to Effector cells; PMA/iono refers to phorbol ester and ionomycin; IFN γ refers to interferon γ.

FIG. 6 is a graph showing the detection of killing of CD22-K562 target cells by CAR-T cells in an example of the invention. In the figure, E: t ratio, meaning effective target ratio; % cytoxicity, means percent cytotoxic effect.

Detailed Description

The principles and features of this invention are described below in conjunction with the following detailed drawings, which are given by way of illustration only and are not intended to limit the scope of the invention.

CAR-T Cell Immunotherapy, collectively known as Chimeric Antigen Receptor T-Cell Immunotherapy.

CAR, known collectively as the Chimeric Antigen Receptor, Chimeric Antigen Receptor.

Example 1: preparation of Nanobody targeting CD22 molecule

Step 1: expressing CD22 protein extracellular segment in 293T cell by eukaryotic expression system, adding His tag at C terminal, purifying protein with nickel column, purifying protein with molecular sieve, detecting protein concentration, packaging, and storing at-80 deg.C.

Step 2: the purified CD22 molecule is used as antigen to immunize alpaca, and the antibody titer of alpaca peripheral blood serum is detected after four times of antigen immunizations, wherein the intervals are 20 days.

And step 3: collecting alpaca peripheral blood, extracting Peripheral Blood Mononuclear Cells (PBMC), extracting RNA, carrying out reverse transcription, carrying out PCR (polymerase chain reaction) amplification on a heavy chain variable region, and constructing a library after connecting a vector.

And 4, step 4: positive clones (OD value greater than 1) were screened by phage display using indirect ELISA, and antibody clone sequences capable of binding antigen were screened.

And 5: the heavy chain variable region sequence of the nanobody targeting the CD22 molecule was confirmed by sequencing.

Example 2: affinity analysis of Nanobodies targeting CD22 molecules

Step 1: the heavy chain variable region sequence of the nanobody targeting CD22 molecule obtained in example 1 was cloned into a Mycoplasma Hominis (MH) antibody expression vector, and a His tag was added to the C-terminus. The vector transfects 293T cells to express the antibody in eukaryotic cells, antibody protein is purified through a nickel column, protein concentration is detected after protein is purified through a molecular sieve, and the protein is stored at 4 ℃.

Step 2: antigen-antibody binding affinity (kinetic) analysis was performed using a Biacore T200 instrument (available from GE Healthcare), amino-coupling of CD22 protein molecules using a CM5 chip, while cyclically binding the coupled antigen protein using different concentrations of antibody protein, analyzing the binding kinetic curve and calculating affinity.

And step 3: through affinity verification, the KD value is screened to reach 10^-8The antibody clone of M is named BA125 nanometer antibody.

Example 3: construction of antibody plasmid vector against CD22 protein molecule

Step 1: the BA125 nano antibody sequence obtained in the embodiment 2 is amplified by PCR and spliced with other structural component sequences of the chimeric antigen receptor synthesized by the gene by an overlapping PCR method. The complete anti-CD22 protein molecule antibody comprises a nano antibody heavy chain variable region targeting a CD22 molecule, a CD8 alpha chain hinge region, a CD8 alpha chain transmembrane region, a CD28 molecule intracellular segment, a 4-1BB molecule intracellular segment and a CD3 zeta chain which are connected in sequence.

Step 2: inserting the constructed DNA fragment into a PLVX lentiviral expression vector (the original vector is PLVX-IRES-ZsGreen1 after being modified, replacing the CMV promoter with a PGK promoter, and removing the IRES-ZsGreen1 structure), and verifying the accuracy of plasmid construction through sequencing to obtain the anti-CD22 protein molecule antibody lentiviral expression plasmid vector.

Example 4: packaging and preparation of lentiviruses

Step 1: lentiviral packaging was performed in 293T cells and the desired plasmids included PLVX-BA125-28BBz, dR8.74 and MD2.G. The transfection is carried out by using a Polyethyleneimine (PEI) transfection reagent, and the specific method is described in the specification of the polyethyleneimine transfection reagent. The polyethyleneimine transfection reagent is commercially available, e.g., from Sigma under the reference number 408727.

Step 2: passage 293T cells to a new 10cm culture dish, and inoculation density is 70-80%;

and step 3: transfection was carried out 12h after passage, according to the mass ratio of PLVX-BA125-28 BBz: dR8.74: md2.g ═ 6:5:1, plasmid mass to volume ratio: and 2.5ul of polyethyleneimine, proportionally mixing plasmids in a serum-free and antibiotic-free DMEM basal medium, proportionally preparing the polyethyleneimine, standing at room temperature for 5min, mixing plasmid suspension and polyethyleneimine suspension in equal volume, standing at room temperature for 20min, carefully adding transfection suspension into 293T cell culture supernatant, and adding 1mL of transfection suspension into each dish.

And 4, step 4: and (3) carrying out total liquid change 12h after transfection, and continuing to culture after changing a complete DMEM culture medium.

And 5: after the total transfection time was 36h, the culture supernatant was collected, centrifuged at 2600g for 10min, transferred to a new centrifuge tube and stored.

Example 5: 293T cell expresses chimeric antigen receptor of anti-CD22 molecule and recognition of CD22 protein molecule by chimeric antigen receptor

Step 1: 293T cells were cultured adherent to the wall, 80% confluency.

Step 2: take 1000. mu.L of lentivirus stock solution, add polybrene (polybrene) with final concentration of 6. mu.g/mL, mix well, add into 10cm petri dish.

And step 3: the cells are normally cultured in an incubator, and after the lentivirus is infected for 6 hours, the cells are replaced by a fresh complete culture medium and are continuously cultured for 24 to 48 hours.

And 4, step 4: the transduced 293T cells were harvested by digestion, collected and divided into three tubes, 1X 10 per tube⁶Cells were washed with PBS and centrifuged at 300g for 3 min.

And 5: discard the supernatant, resuspend the cells in 500ul PBS, three tubes of cells respectively: 1) adding no antibody, 2) adding Biotin Goat Anti-Alpaca antibody (Biotin-coat Anti-Alpaca IgG) and specific recognition heavy chain variable region (VHH region) antibody (purchased from Jackson Immuno company of America) according to the volume ratio of 1:500, 3) adding FITC fluorescence labeled CD22 protein, shaking and mixing uniformly, and incubating for 20min at room temperature.

Step 6: cells were washed with 3mL PBS, shaken well and centrifuged at 300g for 3 min. This was repeated twice.

And 7: discard the supernatant, resuspend the cells in 300ul PBS, 2) add 1ug of PE-labeled Streptavidin (PE-Streptavidin) antibody (purchased from BD Co., Becton, Dickinson and Company) to the tube cells, mix well with shaking, and incubate at room temperature for 20 min.

And 8: cells were washed with 3mL PBS, shaken well and centrifuged at 300g for 3 min.

And 7: discard the supernatant, resuspend the cells in 300ul PBS, mix well by shaking, and test on the machine.

The results obtained from the flow cytometry examination demonstrated that, in addition to the detection of the expression of the anti-CD22 chimeric antigen receptor on the surface of 293T cells by antibodies specifically recognizing the heavy chain variable region (VHH region), the expression of the anti-CD22 chimeric antigen receptor on the surface of 293T cells was also detected specifically by the human CD229 protein, indicating that the anti-CD22 chimeric antigen receptor expressed on the surface of 293T cells specifically recognizes the human CD22 protein.

Example 6: isolation and culture of PBMC (peripheral blood mononuclear cells)

Step 1: peripheral blood samples of 50mL healthy persons were taken and diluted with an equal volume of PBS.

Step 2: prepare a new centrifuge tube and add an equal volume of lymphocyte spacer, and the pasteur tube carefully pipettes the diluted sample into the upper layer of the spacer.

And step 3: centrifuging at room temperature of 600g for 20min, and rotating the centrifuge in a slowly rising and slowly falling mode.

And 4, step 4: the pasteur tubes carefully pipetted the middle buffy coat cells into a new centrifuge tube.

And 5: adding PBS 3-5 times the volume of the white blood cell layer to dilute the white blood cell layer, washing, mixing well, and centrifuging for 10min at 300 g.

Step 6: discard the supernatant, resuspend the cell pellet with a small volume of PBS, add 40mL PBS to wash again, mix well, centrifuge for 5min at 300 g.

And 7: the supernatant was discarded, the CELLs were resuspended in a T-CELL serum-free Medium (STEM CELL), and activated for 48h by adding IL2, 50ng/mL CD3 activating antibody and 50ng/mL CD28 activating antibody at a final concentration of 1000 IU/mL.

And 8: the cell suspension was collected, centrifuged at 300g for 3min, the supernatant discarded, the medium was replaced with fresh one and IL2 was added to the medium to a final concentration of 1000IU/mL and the culture was continued.

Example 7: transduction preparation of CAR-T cells

Step 1: t cells cultured for 7 days are taken for transduction experiment, and 1.2 multiplied by 10 are counted and collected⁷Cells were resuspended using a complete T cell culture medium and seeded in 24-well plates, 500ul of cell suspension per well, 5X 10 per well⁵A cell.

Step 2: 12mL of the packaged lentivirus suspension is taken, polybrene (polybrene) with the final concentration of 5ug/mL is added, the mixture is fully and evenly mixed, and the mixture is added into a 24-well plate, wherein each well contains 500ul of virus liquid.

And step 3: and (3) placing the cells in an incubator for normal culture, after infecting the lentiviruses for 12 hours, resuspending all the cells, centrifuging, replacing the cells with a fresh complete culture medium in a full-volume manner, and continuing to culture the cells.

Example 8: positive rate detection of CAR-T cell CAR structural expression

Step 1: transduced T cells were cultured for 4-6 days for positive rate detection of CAR expression and flow cytometry was used for detection.

Step 2: collecting 1X 10⁶Cells were washed with PBS and centrifuged at 300g for 3 min.

And step 3: the supernatant was discarded, 500ul of PBS was used to resuspend the cells, Biotin Goat Anti-Alpaca antibody (Biotin-coat Anti-Alpaca IgG) and antibody specifically recognizing the heavy chain variable region (VHH region) (purchased from Jackson Immuno, USA) were added at a volume ratio of 1:500, shaken and mixed well, and incubated at room temperature for 30 min.

And 4, step 4: the cells were washed with 1mL PBS, shaken well and centrifuged at 300g for 3 min.

And 5: the supernatant was discarded, 100ul of PBS was used to resuspend the cells, 1test dose of anti-CD3-APC-Cy7 antibody (purchased from BD, Becton, Dickinson and Company) was added, along with 1ug of PE-labeled Streptavidin (PE-Streptavidin) antibody (purchased from BD, Becton, Dickinson and Company), vortexed, and incubated at room temperature for 30 min.

Step 6: the cells were washed with 1mL PBS, shaken well and centrifuged at 300g for 3 min.

Example 9: CAR-T cell activation function assay

Step 1: CAR-T release of cytokines after co-incubation with CD22-K562(K562 cell line stably expressing CD22 molecule) target cells was tested using untransduced T cells as controls.

Step 2: the CAR T cells in culture were collected and harvested by centrifugation by cell counting to obtain the cells required for the activation experiment.

And step 3: similarly, CD22-K562 cells were collected to give the desired number of cells.

And 4, step 4: the cells were seeded according to the protocol of Table 1, in V-bottom 96-well plates,

TABLE 1 cell seeding protocol

Note: in the table, E refers to effector cells; t refers to target cells, target cells; effect cell only means that there are only Effector cells. PMA/iono refers to phorbol ester and ionomycin. Two secondary wells were tested simultaneously per group.

And 5: the cells are placed in an incubator for normal culture and incubation for 24 h.

Step 6: carefully pipette cell supernatants into new EP tubes, and detect the expression levels of TNF-alpha and IFN-gamma in cell culture supernatants by ELISA method using a doctor's biological product kit, the specific operation is performed according to the instruction.

Example 10: CAR-T cell in vitro killing activity detection

Step 1: CAR-T was tested for cytotoxicity against target cells after co-incubation with CD22-K562(K562 cell line stably expressing CD22 molecule) target cells using untransduced T cells as controls.

Step 2: by using

An experiment is carried out by using an EuTDA cytoxicity Reagents kit (purchased from Perkin Elmer company in the United states), CD22-K562 target cells are pre-marked according to the kit operation instructions, T cell culture media are used for being inoculated in a 96-well plate with a V bottom, 5000 cells are arranged in each well, and a spontaneous release hole and a maximum release hole are simultaneously arranged;

and step 3: the collected effector cells are inoculated into a pore plate according to the proportion, and the ratio of the effector cells to the effector cells is determined according to the proportion of E: t is inoculated in a gradient of 1:1, 5:1, 10:1, 30:1 and 60:1, and the volume of the culture medium in each well is 200 ul.

And 4, step 4: after the cells were normally cultured in an incubator at 37 ℃ for 2 hours, the target cells in the maximum release pores were lysed using a lysine buffer.

And 5: after further incubation for 2h, i.e. after co-incubation for 4h, the V-well plate was placed on a plate centrifuge for centrifugation at 500g for 5min according to the specification.

Step 6: carefully sucking 20ul of supernatant per well into a 96-well plate (white board) special for fluorescence measurement, adding 200ul of Eu-Solution into each well, shaking the table, shaking and incubating at room temperature in a dark place for 15 min.

And 7: the fluorescence signal from each well was detected using time-resolved fluorescence (time-resolved fluorescence).

And 8: percent cell killing was calculated according to the following equation.

And (4) conclusion:

the invention adopts the self-screened nano antibody to replace the traditional scFv as the main strategy, and the chimeric antigen receptor designed and established has uniqueness. We successfully screened a nanobody clone capable of targeting CD22 protein, and verified that the heavy chain variable region cloned by nanobody BA125 can be well combined with CD22, and has better affinity (fig. 1). The experiment sets 5 different concentration gradients of the nanobody, which are 2nM, 4nM, 8nM, 16nM and 32nM respectively, and reports the affinity KD (M) value of the nanobody BA125 clone protein and the CD22 protein to be 9.47E-09M through curve fitting.

We successfully constructed a chimeric antigen receptor structure lentivirus expression vector targeting CD22 molecule and derived from the heavy chain variable region of a nano antibody, namely BA125-28 BBz. We successfully generated a lentivirus for chimeric antigen receptor transduction, with which we successfully infected 293T cells and detected that CAR-293T cells were able to specifically bind to human CD22 antigen (fig. 2D), and that 293T cells expressed proteins containing VHH structures by indirect methods (fig. 2B). Meanwhile, the T cells are infected by the lentivirus and are amplified to obtain the CAR-T cells. The prepared CAR-T cell can well express the designed chimeric antigen receptor structure through experimental verification (FIG. 3B).

The CAR-T cells prepared by us were verified by cell activation functional experiments to be able to perform efficient activation and secrete corresponding cytokines when stimulated by specific target cells (fig. 4 and 5).

It was verified by cytotoxic experiments that the CAR-T cells we produced were able to specifically kill target cells (fig. 6). The CAR-T cell function detection process is added with a chimeric antigen receptor structure constructed by anti-CD22 scFv submitted in geneBank as a comparison. The experiments prove that the chimeric antigen receptor structure and the CAR-T cell which are designed by the inventor can well kill target cells in vitro. The chimeric antigen receptor from the nanobody source designed by the inventor has certain effectiveness and uniqueness. Therefore, the chimeric antigen receptor BA125-CD8 alpha-CD 28-41BB-CD3 zeta in one of the preferred embodiments of the invention can be applied to the treatment of tumors (such as B cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute myelogenous leukemia and indications caused by the expression of a large amount of autoantibodies by plasma cells, such as lupus erythematosus, rheumatoid arthritis, autoimmune purpura and the like).

The plasmid vector and the lentiviral vector for constructing the chimeric antigen receptor are not limited to the vectors used in the illustrated examples of the present invention, and the plasmid vector can be pCDH series vectors and other vectors suitable for cloning, expressing proteins and packaging viruses besides pLVX series vectors. The viral vector is not limited to a lentiviral vector, and may be other viral vectors of other retroviruses, adenoviruses, adeno-associated viruses, oncolytic viruses, and the like, and plasmid vectors adapted thereto.

Based on the anti-CD22 protein molecule antibody, in addition to the construction of the chimeric antigen receptor, the nucleotide and/or amino acid sequence of the anti-human CD22 protein molecule nanobody can be used as a part of a target sequence, and other fusion proteins can be constructed, including but not limited to bispecific antibody, linked/fused IgG1, IgG2a, IgG2b, IgG3, IgG4 heavy chain constant region, other receptor molecule fusion proteins, and the like.

Based on the antibodies against the CD22 protein molecule of the invention, the nucleotide and/or amino acid sequence of the antibodies against the CD22 protein molecule can be part of a sequence of interest, in addition to being used for the study, preparation and production of CAR-T cells, also for the study, preparation and production of CAR- γ δ T cells, CAR-NK cells, CAR-NKT cells, CAR-Mo/M Φ cells, and as modified molecules of induced pluripotent stem cells (iPS) and their induced cells (such as T cells, NK cells, NKT cells, Mo or M Φ cells derived from iPS induction).

The anti-CD22 protein molecule antibody of the invention can be applied to the field of tumor immunotherapy and can also be used for detection, and can be used as a part of a human CD22 protein molecule detection reagent or a kit.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Beijing Rong good jade medical Biotechnology Limited liability company

<120> antibody against CD22 protein molecule and application thereof

<150> 202010264776.X

<151> 2020-04-07

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 417

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttggtggtcc tggctgctct actacaaggt gtccaggctc aggtgcagct ggtggagtct 60

gggggaggct tggtgcagcc tggggggtct ctgagactct cctgtgcagc ctctggaagc 120

atcttcggta gcaataccat gggctggttc cgccaggctc cgggacagca gcgcgagttg 180

gtcgcagcta tgtctactct tggtagaaca agttatgcag tctccgtgaa gggccgattc 240

accatctcca gagacaacgc caagaacacg gtgtacctgc aaatgaacag cctgaaacct 300

gaggacacgg ccgtctatta ttgcaaatat actgggacca cgccacggga catctactgg 360

ggcctgggga cccaggtcac cgtctcctca gaacccaaga caccaaaacc acaagac 417

<210> 2

<211> 139

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Leu Val Val Leu Ala Ala Leu Leu Gln Gly Val Gln Ala Gln Val Gln

1 5 10 15

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

20 25 30

Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Gly Ser Asn Thr Met Gly

35 40 45

Trp Phe Arg Gln Ala Pro Gly Gln Gln Arg Glu Leu Val Ala Ala Met

50 55 60

Ser Thr Leu Gly Arg Thr Ser Tyr Ala Val Ser Val Lys Gly Arg Phe

65 70 75 80

Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn

85 90 95

Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys Tyr Thr Gly

100 105 110

Thr Thr Pro Arg Asp Ile Tyr Trp Gly Leu Gly Thr Gln Val Thr Val

115 120 125

Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Asp

130 135

<210> 3

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 4

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 5

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atccca 66

<210> 6

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 7

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggagacag acacactcct gttatgggta ctgctgctct gggttccagg ttccactggt 60

<210> 8

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<210> 9

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 10

<211> 45

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 11

<211> 84

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gcaaccacag gaac 84

<210> 12

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 13

<211> 81

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 14

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 15

<211> 123

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 16

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 17

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aaacggggca gaaagaagct cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 18

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 19

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 20

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

Claims

1. An antibody for resisting a CD22 protein molecule, which is derived from alpaca and comprises a heavy chain variable region, wherein the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

2. A chimeric antigen receptor having the structure: SP-VHH-HINGE-TM-CD-SD wherein SP is a signal peptide, VHH is an antibody against the CD22 protein molecule of claim 1, is an antigen binding domain, HINGE is a HINGE region, TM is a transmembrane region, CD is a costimulatory region, SD is a signal transduction region, and "-" is a linker peptide or peptide bond.

3. A fusion protein comprising an antibody against the CD22 protein molecule of claim 1.

4. A vector comprising any one of the nucleotide sequence of an antibody against the CD22 protein molecule of claim 1, the nucleotide sequence of a chimeric antigen receptor of claim 2, and the nucleotide sequence of a fusion protein of claim 3.

5. A host cell comprising any one of the nucleotide sequence of the antibody against the CD22 protein molecule of claim 1, the chimeric antigen receptor of claim 2, the nucleotide sequence of the fusion protein of claim 3, and the vector of claim 4, or any one of the nucleotide sequence of the antibody against the CD22 protein molecule of claim 1, the nucleotide sequence of the chimeric antigen receptor of claim 2, and the nucleotide sequence of the fusion protein of claim 3, integrated into the chromosome.