CN115925948A

CN115925948A - anti-CD 22 nano antibody and application thereof

Info

Publication number: CN115925948A
Application number: CN202211203949.2A
Authority: CN
Inventors: 狄升蒙; 石磊; 余学军
Original assignee: Huadao Shanghai Biopharma Co ltd
Current assignee: Huadao Shanghai Biopharma Co ltd
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2023-04-07
Anticipated expiration: 2041-12-16
Also published as: CN114149506B; CN114149506A; CN115925948B

Abstract

The invention discloses an anti-CD 22 nano antibody and application thereof. The anti-CD 22 nano antibody comprises a heavy chain variable region, wherein the amino acid sequence of CDR1 of the heavy chain variable region comprises the sequence shown by SEQ ID No.2, the amino acid sequence of CDR2 of the heavy chain variable region comprises the sequence shown by SEQ ID No.4, and the amino acid sequence of CDR3 of the heavy chain variable region comprises the sequence shown by SEQ ID No. 6. The anti-CD 22 antibody has high affinity and specificity, can effectively target a tumor antigen CD22, prepares a chimeric antigen receptor by using the anti-CD 22 antibody, and further prepares chimeric antigen receptor cells, and the chimeric antigen receptor cells can kill tumor cells efficiently and have high specificity.

Description

anti-CD 22 nano antibody and application thereof

The application is a divisional application of patent application No. 202111545594.0 (application date of the original application is 2021, 12 and 16, and the name of the invention is anti-CD 22 antibody and application thereof).

Technical Field

The invention belongs to the technical field of biological medicines, and relates to an anti-CD 22 nano antibody and application thereof.

Background

B-cell acute lymphoblastic leukemia (B-ALL) is an invasive hematological malignancy characterized by CD19 ⁺ Clonal expansion of B cell precursors. B-ALL is one of the most common malignancies in children, with a poor prognosis in adults, although less common. Although more than 90% of patients get complete remission after first-line treatment, the prognosis for refractory/relapsed (R/R) B-ALL patients is poor.

Adoptive transfer of artificial Chimeric antigen receptor T cells (CAR-T) engineered to express antigens associated with the surface of targeted tumor cells is a revolutionary cancer immune cell therapy. At present, CD19 CAR-T therapy is one of the ideal cell therapies for B-ALL, CD19 is uniformly expressed on malignant cells, whereas off-target expression is limited to normal B cells, and CD19 CAR-T induced B-cell dysgenesis is easily controlled clinically by using γ -immunoglobulin. CD19 CAR-T thoroughly changes the treatment of R/R B-ALL, and the complete remission rate reaches 80-90 percent; however, 40% to 60% of patients treated with CD19 targeted immunotherapy still relapse after 1 year. The types of recurrence are mainly divided into two categories: one still being CD19 ⁺ Recurrence of (1), in generalIs poor T cell function or CAR-T cells are short in duration; another is the recurrence of CD19, whose disease is relapsed with loss of cell surface CD19 expression, representing a novel "stem cell origin-related" tumor escape mechanism.

Another promising target for CAR-T treatment of B-ALL is CD22.CD22 is a sialic acid binding adhesion molecule, the expression of which is primarily restricted to B cells and is highly expressed in most B cell malignancies. CD22 targeted immunotherapy has been developed and tested in several studies, for example CN111320703A discloses a CD22 targeted chimeric antigen receptor and its use, the chimeric antigen receptor sequence is selected from one of the following two structures, the first structure: the CD22 antigen binding domain and the constant domain of the T Cell Receptor (TCR); the second structure is as follows: a transmembrane protein signal peptide, a CD22 antigen binding domain, a hinge region of a CD8 protein molecule, a transmembrane region, a 4-1BB costimulatory domain and a CD3 zeta intracellular signaling domain in serial order; furthermore, in a study of B-ALL patients in children and young adults with disease progression after CD19 CAR-T treatment, CD22 CAR-T induced a complete remission rate of 73%, which was specific for CD19 ⁺ And CD19 ^- The effectiveness of B-ALL was the same. However, recurrence is also common, and a subset of patients have recurrence associated with down-regulation of CD22 expression. Thus, R/R B-ALL remains clinically challenging.

In summary, the development of efficient CAR-T therapy for R/R B-ALL is of great significance in the field of B-ALL therapy.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides an anti-CD 22 nano antibody and an application thereof, wherein the anti-CD 22 nano antibody has high affinity and specificity and can efficiently target CD22, and CAR-T cells constructed by taking the anti-CD 22 antibody as an antigen binding domain can efficiently target tumor cells CD22 so as to mediate immune cells to kill the tumor cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an anti-CD 22 antibody, wherein the anti-CD 22 antibody comprises a heavy chain variable region, the amino acid sequence of CDR1 of the heavy chain variable region comprises the sequence shown in SEQ ID No.1 or SEQ ID No.2, the amino acid sequence of CDR2 of the heavy chain variable region comprises the sequence shown in SEQ ID No.3 or SEQ ID No.4, and the amino acid sequence of CDR3 of the heavy chain variable region comprises the sequence shown in SEQ ID No.5 or SEQ ID No. 6.

The anti-CD 22 antibody is screened, and the anti-CD 22 antibody only comprises a heavy chain variable region, has high affinity and specificity, can efficiently target a CD22 antigen, has a simple structure, is easy to prepare, and has important application value in the field of preparing medicaments taking CD22 as a target spot.

SEQ ID No.1：GDTHSSYC。

SEQ ID No.2：GYTNSRRY。

SEQ ID No.3：VDSDGKQ。

SEQ ID No.4：IYTGDGDSRT。

SEQ ID No.5：AAAPWCYREAEDFTI。

SEQ ID No.6：AADVWYHGDWNDPKLYPY。

Preferably, the amino acid sequence of CDR1 of the heavy chain variable region comprises the sequence shown in SEQ ID No.1, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.3, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 5.

Preferably, the amino acid sequence of CDR1 of the heavy chain variable region comprises the sequence shown in SEQ ID No.2, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.4, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 6.

Preferably, the heavy chain variable region further comprises framework regions FR1-FR4.

Preferably, the amino acid sequence of FR1 includes the sequence shown in SEQ ID No.7 or SEQ ID No. 8.

Preferably, the amino acid sequence of FR2 comprises the sequence shown in SEQ ID No.9 or SEQ ID No. 10.

Preferably, the amino acid sequence of FR3 includes the sequence shown in SEQ ID No.11 or SEQ ID No. 12.

Preferably, the amino acid sequence of FR4 comprises the sequence shown in SEQ ID No. 13.

Preferably, the amino acid sequence of the heavy chain variable region comprises the sequence shown in SEQ ID No.14 or SEQ ID No. 15.

SEQ ID No.7：EVQLVESGGDSVQPGGSLRLSCAVS。

SEQ ID No.8：EVQLVESGGASVQAGGSLTLSCAAS。

SEQ ID No.9：LAWFRQAPGKEREGVAF。

SEQ ID No.10：MAWFRQTPGKEREGVAY。

SEQ ID No.11：

IHADSVKGRFTGSRDNTKNTLFLQMDSLQLEDTAMYYC。

SEQ ID No.12：

YYADSVKGRFTISRDNAKGTVNLQMNSLQPGDSAMYYC。

SEQ ID No.13：WGQGTQVTVSS。

SEQ ID No.14：

EVQLVESGGDSVQPGGSLRLSCAVSGDTHSSYCLAWFRQAPGKEREGVAFVDSDGKQIHADSVKGRFTGSRDNTKNTLFLQMDSLQLEDTAMYYCAAAPWCYREAEDFTIWGQGTQVTVSS。

SEQ ID No.15：

EVQLVESGGASVQAGGSLTLSCAASGYTNSRRYMAWFRQTPGKEREGVAYIYTGDGDSRTYYADSVKGRFTISRDNAKGTVNLQMNSLQPGDSAMYYCAADVWYHGDWNDPKLYPYWGQGTQVTVSS。

The coding gene of the anti-CD 22 antibody is inserted into an expression vector to obtain a recombinant expression vector, and the recombinant expression vector is introduced into cells and cultured, and then separated and purified to obtain the anti-CD 22 antibody.

In a second aspect, the present invention provides a nucleic acid molecule comprising a gene encoding the anti-CD 22 antibody of the first aspect.

Preferably, the nucleic acid molecule comprises the sequence shown as SEQ ID No.16 or SEQ ID No. 17.

SEQ ID No.16：

gaagtgcagctggtggaatctggcggcgatagcgtgcagcctggcggcagcctgagactgagctgtgccgtgagcggcgatacccatagcagctattgtctggcctggtttagacaggcccctggcaaagaaagagaaggcgtggcctttgtggattccgatggaaaacagattcatgccgatagcgtgaaaggccggtttaccggctccagagataataccaaaaataccctgtttctgcagatggatagcctgcagctggaagataccgccatgtattattgtgccgccgccccttggtgttatagagaagccgaagattttaccatttggggccagggcacccaggtgaccgtgagcagc。

SEQ ID No.17：

gaggtgcagctggtggagagcggcggcgccagcgtgcaggccggcggcagcctgaccctgagctgtgctgcctctggatacacaaattctagaagatatatggcctggtttagacagaccccaggaaaagagagagaaggcgtggcttatatctatacaggcgatggagattctagaacatattatgccgatagcgtgaagggcagattcacaattagcagagacaacgccaagggcaccgtgaacctgcagatgaacagcctgcagcccggcgacagcgccatgtactactgcgccgccgacgtgtggtaccacggcgactggaacgacccaaagctgtacccctactgggggcagggcacccaggtgaccgtgagcagc。

The nucleic acid molecule of the invention is capable of accurately encoding the anti-CD 22 antibody of the first aspect.

In a third aspect, the present invention provides a chimeric antigen receptor comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, and a signaling domain, the antigen binding domain being the anti-CD 22 antibody of the first aspect.

In the invention, the anti-CD 22 antibody is used for constructing a chimeric antigen receptor which can efficiently target CD22.

Preferably, the signal peptide comprises a CD8 α signal peptide.

Preferably, the antigen binding domain also includes an anti-CD19 antibody.

Preferably, the antigen binding domain comprises an antigen binding domain formed by connecting an anti-CD19 antibody and an anti-CD 22 antibody in series in sequence or an antigen binding domain formed by connecting an anti-CD 22 antibody and an anti-CD19 antibody in series in sequence.

Preferably, the hinge region comprises a CD8a hinge region;

preferably, the transmembrane region comprises any one of or a combination of at least two of a CD8a transmembrane region, a CD28 transmembrane region, or a DAP10 transmembrane region;

preferably, the signal transduction domain comprises an immunoreceptor tyrosine-activation motif;

preferably, the signal transduction domain further comprises a co-stimulatory molecule comprising any one of the 4-1BB, CD28 intracellular domain, OX40, ICOS or DAP10 intracellular domain or a combination of at least two thereof

Preferably, the chimeric antigen receptor further comprises CD3 epsilon or CD3 gamma.

Preferably, the amino acid sequence of the signal peptide comprises the sequence shown in SEQ ID No. 18.

Preferably, the amino acid sequences of the CD8a hinge region and the transmembrane region comprise the sequence shown in SEQ ID No. 19.

Preferably, the amino acid sequence of the immunoreceptor tyrosine activation motif comprises the sequence shown in SEQ ID No. 20.

Preferably, the amino acid sequence of CD3 epsilon comprises the sequence shown in SEQ ID No. 21.

Preferably, the amino acid sequence of the CD3 gamma comprises the sequence shown in SEQ ID No. 22.

Preferably, the chimeric antigen receptor comprises a first peptide chain obtained by fusing an anti-CD19 antibody and a CD3 epsilon and a second peptide chain obtained by fusing an anti-CD 22 antibody and a CD3 gamma, or the chimeric antigen receptor comprises a first peptide chain obtained by fusing an anti-CD 22 antibody and a CD3 epsilon and a second peptide chain obtained by fusing an anti-CD19 antibody and a CD3 gamma.

In a fourth aspect, the present invention provides an expression vector comprising a gene encoding the chimeric antigen receptor of the third aspect.

Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to the third aspect, preferably a lentiviral vector.

In a fifth aspect, the present invention provides a recombinant lentivirus comprising the expression vector of the fourth aspect.

In the present invention, the recombinant lentivirus is prepared from mammalian cells transfected with the expression vector and helper plasmid of the fourth aspect.

In a sixth aspect, the present invention provides a chimeric antigen receptor immune cell expressing the chimeric antigen receptor of the third aspect.

The invention utilizes the anti-CD 22 antibody to prepare a chimeric antigen receptor and further prepare a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell can specifically identify CD22 and/or CD19 positive tumor cells and kill the cells efficiently, and simultaneously can release various cell factors including IL-2, TNF-alpha, IFN-gamma factors and the like to play a role in killing the cells.

Preferably, the chimeric antigen receptor immune cell comprises the expression vector of the fourth aspect and/or the recombinant lentivirus of the fifth aspect.

Preferably, the immune cells comprise any one of T lymphocytes, B lymphocytes, NK cells, mast cells or macrophages or a combination of at least two thereof.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor immune cell of the sixth aspect.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

In an eighth aspect, the present invention provides the use of the anti-CD 22 antibody of the first aspect, the nucleic acid molecule of the second aspect, the chimeric antigen receptor of the third aspect, the expression vector of the fourth aspect, the recombinant lentivirus of the fifth aspect, the chimeric antigen receptor immune cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect, in the preparation of a medicament for treating a tumor.

Preferably, the tumor comprises B-cell acute lymphoblastic leukemia.

Compared with the prior art, the invention has the following beneficial effects:

(1) The anti-CD 22 antibody only comprises a heavy chain variable region, has high affinity and specificity, can efficiently target a CD22 antigen, and has a simple structure and easy preparation;

(2) In the invention, the anti-CD 22 antibody is utilized to construct a chimeric antigen receptor, the chimeric antigen receptor can efficiently target CD22, and in addition, an anti-CD 22 antibody and an anti-CD19 antibody can be simultaneously adopted as an antigen binding domain, so that the chimeric antigen receptor capable of simultaneously targeting CD22 and CD19 is prepared;

(3) The chimeric antigen receptor cell can specifically recognize CD22 and/or CD19 positive tumor cells, kill the cells efficiently, and release various cytokines including IL-2, TNF-alpha, IFN-gamma factors and the like to play a role in killing the cells.

Drawings

FIG. 1A is a graph showing the results of Biacore's affinity assay for anti-CD 22 antibody (CD 22-28);

FIG. 1B is a graph showing the result of the affinity of Biacore for detecting anti-CD 22 antibody (CD 22-29);

FIG. 2 is a graph showing the results of FACS detection of CD22 antigen on the cell surface recognized by anti-CD 22 antibody;

FIG. 3A is a plasmid map of a lentiviral vector expressing a chimeric CD22 antigen receptor (HD SIN03 CD22 (V28) 41BBz (ka));

FIG. 3B is a plasmid map of a lentiviral vector expressing a chimeric CD22 antigen receptor (HD SIN03 CD22 (V29) 41BBz (ka));

FIG. 3C is a plasmid map of a lentiviral vector expressing a chimeric CD22 antigen receptor (HD SIN03 CD19-CD22 (V28) 41BBz (ka));

FIG. 3D is a plasmid map of a lentiviral vector expressing a chimeric CD22 antigen receptor (HD SIN03 CD19-CD22 (V29) 41BBz (ka));

FIG. 3E is a plasmid map of a lentiviral vector expressing a chimeric CD22 antigen receptor (HD SIN03 CD22 (V28) -CD19-41BBz (ka));

FIG. 3F is a plasmid map of a lentiviral vector expressing a chimeric CD22 antigen receptor (HD SIN03 CD22 (V29) -CD19-41BBz (ka));

FIG. 3G is a plasmid map of a lentiviral vector expressing a chimeric CD22 antigen receptor (HD SIN03 CD19-CD3 ε -T2A-CD22 (V28) -CD3 γ (ka));

FIG. 3H is a plasmid map of a lentiviral vector expressing a chimeric CD22 antigen receptor (HD SIN03 CD19-CD3 ε -T2A-CD22 (V29) -CD3 γ (ka));

FIG. 4A is a schematic diagram of the structure of a CD22 chimeric antigen receptor (antigen binding domain containing only CD22 antibody);

FIG. 4B is a schematic diagram of the structure of the CD22 chimeric antigen receptor (CD 19 antibody before CD22 antibody);

FIG. 4C is a schematic diagram of the structure of the CD22 chimeric antigen receptor (CD 19 antibody after CD22 antibody);

FIG. 4D is a schematic representation of the structure of the CD22 chimeric antigen receptor (CD 19 antibody before CD22 antibody with CD3 ε -T2A attached between them);

FIG. 5 is a graph of chimeric antigen receptor expression rate in CAR-T cells;

FIG. 6A is a graph showing the killing effect of CAR-T cells of the present invention on K562-luci cells;

FIG. 6B is a graph showing the killing effect of CAR-T cells of the present invention on K562-CD19-luci cells;

FIG. 6C is a graph showing the killing effect of CAR-T cells of the present invention on K562-CD22-luci cells;

FIG. 6D is a graph showing the killing effect of CAR-T cells of the present invention on K562-CD19-CD22-luci cells;

FIG. 7 is a graph of IL-2 cytokine secretion levels by CAR-T cells;

FIG. 8 is a graph of TNF- α cytokine secretion levels by CAR-T cells;

FIG. 9 is a graph of the IFN- γ cytokine secretion levels of CAR-T cells.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

In the invention, a phage display technology is utilized to screen a VHH immune library of the alpaca immunized by the CD22, so that the high-affinity nano antibody for resisting the CD22 is obtained.

Example 1

In this example, phage nanobody library was constructed and panning and ELISA primary screening were performed.

1. Construction of phage Nanobody libraries

(1) Adopting CD22-Fc expressing an extracellular region to immunize bactrian camel, and extracting 200mL of peripheral blood after the titer is verified by ELISA;

(2) Sorting lymphocytes to obtain peripheral blood mononuclear lymphocyte sediment and extracting RNA;

(3) By using

III, synthesizing first-strand cDNA (complementary deoxyribonucleic acid) by using reverse transcriptase by taking RNA as a template, and then amplifying a VHH (very high frequency) gene by using nested PCR (polymerase chain reaction);

(4) Inserting VHH gene into pMECS phage display carrier, after electrotransformation of TG1 competent cell, taking bacterial liquid to perform library identification, uniformly coating the rest culture on LB/AMPGLU flat plate, collecting bacterial lawn after bacterial growth, adding 50% glycerol of 1/3 volume, uniformly mixing and subpackaging, storing at-80 ℃, successfully constructing the product with the storage capacity of more than 10 ⁹ The phage display camelid VHH immune library of (a).

2. Panning of phage Nanobody libraries

Diluting the purified CD22-His recombinant protein to 4 mu g/mL by using a PBS buffer solution, taking a 96-well enzyme label plate, selecting 3 wells, adding 100 mu L (400 ng/well) into each well, coating overnight at 4 ℃, and using the PBS as a negative control; discarding the coating solution, adding 150 μ L of 2% defatted inner powder into each well, and sealing at 25 deg.C for 1 hr; washing with PBST for 4 times, taking the prepared phage solution, diluting to 5 × 1011pfu/ml with 2% milk powder, adding an enzyme-labeled plate, 100 μ L/hole, and incubating at 25 deg.C for 2h; discarding the phage sample, washing with PBST for 10 times, then washing with PBS for 5 times, adding 100 μ L of freshly prepared 0.1M triethylamine to each well, standing at 25 deg.C for 10min, sucking out the eluate, and rapidly neutralizing with equal volume of 1M Tris-HCl (pH7.4); taking part of the eluent to determine the titer of the phage; taking 400 μ L of eluate, infecting 4mL of fresh logarithmic phase TG1 bacterial solution (OD 600 about 0.6), incubating at 37 deg.C for 30min, adding 16mL of 2 XYT/AMP-GLU, culturing at 37 deg.C and 200r/min to OD ₆₀₀ Up to 0.7. Taking 100 mu L of bacterial suspension, performing gradient dilution, and uniformly smearing the bacterial suspension on a2 XYT/ampicillin/glucose agar plate so as to perform library capacity and diversity determination; inoculating 100 μ L bacterial suspension, i.e. phage display carrier library, into 2 XYT/AMP-GLU culture medium, culturing to logarithmic phase, adding adjuvantAssisting phage, performing library rescue, determining the titer of phage required for obtaining phage particles, and concentrating and purifying to obtain phage particles for the next round of screening; the rest bacteria liquid is centrifuged and then resuspended by 2 XYT culture solution with proper volume, smeared on a plate with screening resistance for overnight culture, scraped from the plate by proper liquid culture solution, added with 2 XYT culture solution containing 50% glycerol with 1/3 volume for resuspension and subpackaged, and all the bacteria are preserved at-80 ℃.

The above screening operation was repeated 3 times.

3 rounds of solid phase screening are carried out on the immune nano antibody library in vitro, so that phage clones with binding activity are effectively enriched. After prokaryotic induction expression is carried out on the monoclonal phage, phage clones capable of combining with the extracellular region of the antigen are further screened out by ELISA.

3. Phage packaging

100 μ L of the frozen stock solution from the previous round was added to 100mL of 2 XYT/AMPGL culture solution and cultured at 37 deg.C with shaking (200 rpm) until logarithmic phase (OD) ₆₀₀ Value 0.6), 90. Mu.L of the helper phage M13K07 (1.7X 10) was added ¹³ PFU/mL), the mixture is firstly kept still at 37 ℃ for 30min,2800 Xg is centrifuged for 10min to collect thalli, then is resuspended by 200mL 2 XYT/AMP-KAN culture medium, is shaken at 37 ℃ for 12h,4 ℃ and 3800 Xg are centrifuged for 30min to remove thalli collection supernatant, 1/5 volume of precooled PEG/NaCl is added for mixing, after phage is precipitated at 2h,4 ℃ and 3800 Xg are centrifuged for 30min to collect phage, 2mL final volume of PBS solution is used for resuspension and is transferred to a 15mL centrifuge tube, 4 ℃ and 12000 Xg is centrifuged for 15min to collect supernatant, 1/5 volume of precooled PEG/NaCl solution is added, the mixture is inverted and mixed up and down, and is kept still on ice for 2h; centrifuging at 4 deg.C and 10000 Xg for 10min, discarding supernatant, resuspending phage precipitate with 1mL PBS, incubating overnight at 4 deg.C with shaking table to dissolve phage particles completely, mixing phage solution with equal volume of 60% glycerol, packaging into 1.5mL EP tube, and storing at-80 deg.C.

And 3 rounds of panning on the phage library by using CD22 antigen, in order to avoid losing sequence diversity, primary ELISA screening is carried out on panning products of 2 nd round and 3 rd round, positive clones are randomly selected from the panning products and are induced to express, an expression supernatant is a crude VHH antibody, and the VHH antibody sequence of a monoclonal strain is determined by sequencing.

Example 2

This example was performed on Fluorescence Activated Cell Sorting (FACS) candidate clones.

Nalm6 (CD 22) was cultured according to standard cell culture protocols ⁺ )、Raji(CD22 ⁺ )、K562(CD22 ^- ) (both available from ATCC) and SK-hep-1 (CD 22) ^- Purchased from shanghai cell bank of chinese academy of sciences), CD22 positive and negative cell suspensions were prepared using trypsinized cells, centrifuged (300 × g,5 min) to remove the culture medium and then resuspended with Flow Buffer to 2 × 10 ⁶ cell/mL, V-bottom 96-well plates 2X 10 additions per well ⁵ Cell suspension of individual cells, centrifugation at 300 Xg for 5min to remove supernatant, adding VHH antibody crude extract to resuspend cells, and incubation at 4 ℃ for 1h, centrifugation at 300 Xg for 5min to remove supernatant, flow Buffer to resuspend cells, using Flow Buffer to dilute APC anti-his antibody to 2. Mu.g/mL, resuspend cells at 100. Mu.L per well, incubation at 4 ℃ for 1h, flow Buffer to wash cells for 3 times, then using 200. Mu.L Flow Buffer to resuspend cells and detecting by up-Flow cytometry, and screening two candidate antibodies, named CD22-28 and CD22-29.

Example 3

This example performs VHH-mIgG2a Fc nanobody expression, purification, and antibody affinity determination.

In order to further identify the antibodies screened in example 2, it was necessary to express the antibodies in mammalian cells, and therefore, a plasmid vector C-4pCP.Stuffer-mCg a-FC expressing VHH with a mouse Fc tag was first constructed, which included the following steps:

(1) PCR amplification of CD22 VHH: CD22-28 and CD22-29, the reagents used are shown in Table 1, the primers are shown in Table 2, and the system and PCR reaction conditions are shown in Table 3;

TABLE 1

TABLE 2

TABLE 3

(2) The digestion system and reaction conditions are shown in Table 4, respectively, for the digested vector

Purifying by using a PCR purification kit, dissolving the air-dried DNA into 20 mu L of water, and detecting the concentration of the DNA;

TABLE 4

(3) The homologous recombination reaction system was 10. Mu.L, as shown in Table 5;

TABLE 5

(4) Adding all homologous recombination reaction systems into DH5 alpha competent cells, and transforming the DH5 alpha competent cells under the transformation conditions shown in Table 6;

TABLE 6

(5) Selecting monoclonal PCR for pre-identification by the transformation plate, wherein the condition of a PCR identification system is shown in Table 7; sending the vector to a sequencing company for sequencing identification, wherein the sequencing result is in line with expectation, and successfully constructing a plasmid vector with a mouse Fc tag expression VHH.

TABLE 7

293E cells were passaged to a cell density of about 2.6X 10 approximately 24h prior to plasmid transfection ⁶ cells/mL, 0.15mg scFV-mIgG1/100mL 293E was transfected into 293E cells by PEI method, DNA: PEI = 1:2. 37 ℃, 130rpm, 8% CO ₂ The cell culture supernatant was collected by shaking culture for 6 days at 3000rpm,30min, and the collected supernatant containing the target antibody was filtered by 0.45 μm Sterile Millex-GP Filter Unit, and then subjected to MabSelect ^TM SuRe ^TM After concentration by centrifugation, the column was washed with 1 XPBS, the protein eluted with 0.1M (mol/L) Gly-HCl and neutralized with 1/10 volume of Tris-HCl pH 8.5, and dialyzed overnight at 4 ℃ for protein, quantified by the method of A280 assay by NanoDrop 2000, and the antibody purity was determined by SEC-HPLC.

In addition, affinity determination was performed by Biacore on the purified 2 CD22 VHH antibodies (CD 22-28 and CD 22-29). Biacore is a bioanalytical sensing technology developed based on Surface Plasmon Resonance (SPR), and can detect and track the whole process of the binding and dissociation of molecules in a solution to and from a molecule immobilized on a chip surface, record the whole process in the form of a sensorgram, and provide kinetic and affinity data, during the measurement, antibodies are immobilized on the chip surface, a mobile phase is a solution containing antigens, and the measurement results are shown in table 8 and fig. 1A and 1B, and it is known from the measurement results that these 2 CD22 VHH antibodies have high affinity.

TABLE 8

Stationary phase	Mobile phase	ka(1/Ms)	kd(1/s)	KD(M)
					0.5μg/mL CD22-28	CD22	2.79E+05	1.44E-04	5.14E-10
0.5μg/mL CD22-29	CD22	2.16E+05	1.88E-05	8.68E-11

Example 4

This example was performed by flow assay of anti-CD 22 single chain antibody.

Nalm6 tumor cells and purified 2 strains of recombinant anti-CD 22 VHH-mIgG2 antibodies (CD 22-28 and CD 22-29) are incubated for 30min in ice bath, a blank control group is not added with the anti-CD 22 VHH-mIgG2 antibodies, then the cells are incubated for 30min with APC-labeled goat anti-mouse IgG antibodies, and the detection is carried out by a flow cytometer, and the result is shown in FIG. 2, which indicates that the anti-CD 22 antibodies prepared by screening can recognize CD22 antigens on the cell surfaces.

Example 5

This example prepares lentiviral vectors expressing a chimeric antigen receptor for an anti-CD 22 antibody (CD 22 VHH) and a chimeric antigen receptor co-expressing CD22 VHH and CD19 scFv.

Construction of lentiviral vectors HD SIN03 CD22 (V28) 41BBz (ka) and HD SIN03 CD22 (V29) 41BBz (ka) carrying the chimeric antigen receptors for CD22 VHH (CD 22-28 and CD 22-29), and 6 lentiviral vectors also carrying the CD19 antibody simultaneously:

HD SIN03 CD19-CD22 (V28) 41BBz (ka) and HD SIN03 CD19-CD22 (V29) 41BBz (ka), with the CD19 antibody preceding the CD20 antibody;

HD SIN03 CD22 (V28) -CD19-41BBz (ka) and HD SIN03 CD22 (V29) -CD19-41BBz (ka), CD19 antibody follows CD20 antibody;

HD SIN03 CD19-CD3 ε -T2A-CD22 (V28) -CD3 γ (ka) and HD SIN03 CD19-CD3 ε -T2A-CD22 (V29) -CD3 γ (ka), the CD19 antibody preceding the CD20 antibody with a CD3 ε -T2A intermediate;

the vector maps are shown in FIGS. 3A-3H, and the chimeric antigen receptor containing a signal peptide, CD22 VHH, CD19 scFv, CD8 alpha hinge region, transmembrane region, immunoreceptor tyrosine activation motif, CD3 epsilon or CD3 gamma is expressed, and the structural schematic diagram of the chimeric antigen receptor is shown in FIGS. 4A-4D.

1. Construction of HD SIN03 CD22 (V28) 41BBz (ka) and HD SIN03 CD22 (V29) 41BBz (ka) Lentiviral vectors

1. A PCR reaction system was prepared according to Table 10, and the CD22 VHH fragment was amplified using the primers shown in Table 9.

TABLE 9

Note: CD22 (28) -R may be substituted for CD22 (29) -R.

Watch 10

Reagent	Volume (μ L)
		10 Xbuffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10μM primer F	1
		10μM primer R	1
Template DNA(cDNA clone)	1
		PCR-grade pure water	33
KOD-Plus-Neo	1

The above reagent is from TOYOBO Inc.

After preparation, the reaction was performed according to the PCR procedure shown in Table 11.

TABLE 11

2. A PCR reaction system was prepared according to Table 13, and a signal peptide was added to the antibody fragment, and the amino acid sequence of the signal peptide is shown in Table 12 using primers: MALPVTALLLPLALLLHAARP (SEQ ID No. 18).

TABLE 12

Note: CD22 (28) -R may be substituted for CD22 (29) -R.

Watch 13

After the preparation, PCR reaction was carried out according to the PCR procedure shown in Table 11, after the reaction was completed, the PCR product was subjected to 1% agarose gel electrophoresis, and fragments of about 500bp were recovered and quantified by the UV absorption method.

3. A PCR reaction system was prepared according to Table 14, and the CD8a hinge-TM-41BB-CD3Z fragment was amplified using the following primers:

CD8aH-F：accacgacgccagcgccgcgac。

Vector-R：tcgataagcttgatatcg。

after the preparation, PCR reaction was carried out according to the PCR procedure shown in Table 11, after the PCR was finished, 1% agarose gel electrophoresis was carried out, and fragments of about 780bp were recovered and quantified by UV absorption.

TABLE 14

Reagent	Volume (μ L)
		10 Xbuffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10μM primer F CD8aH-F	1
		10μM primer R Vector-R	1
Template DNA (HD CD19 CAR)	1
		PCR-grade pure water	33
KOD-Plus-Neo	1

The reagent is from TOYOBO Inc.

4. Mu.g of the constructed HD SIN03 CD19 BBz (ka) plasmid was digested with BamHI and EcoRI, reacted in a water bath at 37 ℃ for 2 hours, and then the vector was recovered.

5. Connecting the 2 fragments recovered in the

steps

2 and 3 with the vector obtained in the step 4 by using recombinase, wherein a recombination reaction system is shown in table 15, reacting in water bath at 37 ℃ for 0.5h after the preparation is finished, transforming to escherichia coli stbl3 competent cells by a conventional method, selecting monoclone from a solid culture medium, culturing overnight, carrying out PCR identification, preparing a PCR reactant as shown in a graph 16, carrying out PCR program as shown in table 17, selecting positive clone after the PCR is finished for further sequencing identification, and obtaining a sequencing result which is in line with the expectation.

Watch 15

Reagent	Dosage of
		HD SIN03 CD19 41BBz(ka)	150ng
CD8a signal CD22 VHH	15ng
		CD8a hinge-TM-41BB-CD3Z	15ng
5x CE MultiS buffer	2μL
		Exnase MultiS	1μL
PCR-grade pure water	Up to 10μL
		Total volume	10μL

TABLE 16

TABLE 17

The amino acid sequences of the CD22 VHH 2 clones (CD 22-28, CD22-29) are shown as SEQ ID No.14 and SEQ ID No. 15.

The amino acid sequences of the CD8 α hinge and transmembrane regions are:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID No.19)。

the amino acid sequence of the immunoreceptor tyrosine activation motif is:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID No.20)。

the nucleotide sequence of the CD22 VHH is shown in SEQ ID No.16 and SEQ ID No. 17.

2. Construction of HD SIN03 CD19-CD22 (V28) 41BBz (ka) and HD SIN03 CD19-CD22 (V29) 41BBz (ka) Lentiviral vectors

1. The PCR reaction system shown in Table 18 was prepared using HD SIN03 CD1941BBz (ka) as a template, and CD8a leader-CD19 scFv fragments were amplified. The primers used were as follows:

BamH-CD8a sig-F：

GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC。

Linker(75)-CD19VH-R：

CACCCGATCCGCCGCCCCCAGATCCGCCCCCACCGGACCCTCCACCGCCTGAACCGCCCCCTCCTGAGGAGACGGTGACTGAG。

after the preparation, PCR reaction was carried out according to the PCR procedure shown in Table 19, after the PCR was finished, 1% agarose gel electrophoresis was carried out, and fragments of about 800bp were recovered and quantified by an ultraviolet absorption method.

Watch 18

Reagent	Volume (μ L)
		10x buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10μM primer F	1
		10μM primer R	1
Template DNA	1
		PCR-grade pure water	33
KOD-Plus-Neo	1

Watch 19

2. The PCR reaction systems of Table 18 were prepared using HD SIN03 CD22 (V28) 41BBz (ka) and HD SIN03 CD22 (V29) 41BBz (ka) as templates, respectively, to amplify CD22 (V28) -CD8ahinge-TM-41BB-CD3Z and CD22 (V29) -CD8a finger-TM-41 BB-CD3Z fragments using the following primers:

Linker(75)-CD22(V28)-F：

GGCGGCGGATCGGGTGGTGGTGGTAGTGAAGTGCAGCTGGTGGAATC；

Vector-R：TCGATAAGCTTGATATCG；

Linker(75)-CD22(V29)-F：

GGCGGCGGATCGGGTGGTGGTGGTAGTGAGGTGCAGCTGGTGGAG；

Vector-R：TCGATAAGCTTGATATCG。

after the preparation, PCR reaction was carried out according to the PCR procedure shown in Table 19, after the PCR was completed, 1% agarose gel electrophoresis was carried out, and fragments of about 1100bp were recovered and quantified by an ultraviolet absorption method.

3. Connecting the 2 fragments with the vector obtained in the first step and the second step by using recombinase, wherein a recombination reaction system is shown in table 20, reacting in water bath at 37 ℃ for 0.5h after the preparation is finished, transforming the recombinant vector into escherichia coli stbl3 competent cells by a conventional method, selecting monoclone from a solid culture medium, culturing overnight, carrying out PCR identification, selecting positive clone after the PCR is finished, further sequencing and identifying, wherein the sequencing result is in line with expectation, the PCR reactant preparation is shown in table 16, and the PCR program is shown in table 17.

Watch 20

3. Construction of HD SIN03 CD22 (V28) -CD1941BBz (ka) and HD SIN03 CD22 (V29) -CD1941BBz (ka) Lentiviral vectors

1. The PCR reaction system of Table 18 was prepared using HD SIN03 CD1941BBz (ka) as a template to amplify a CD19 scFv-CD8ahinge-TM-41BB-CD3Z fragment using the following primers:

Linker(75)-CD19VL-F：

GGCGGCGGATCGGGTGGTGGTGGTAGTGACATCCAGATGACACAG；

Vector-R：TCGATAAGCTTGATATCG；

after the preparation, PCR reaction was carried out according to the PCR procedure shown in Table 19, after the PCR was completed, 1% agarose gel electrophoresis was carried out, and a fragment of about 1400bp was recovered and quantified by an ultraviolet absorption method.

2. The PCR reaction system of Table 18 was prepared using HD SIN03 CD22 (V28) 41BBz (ka) and HD SIN03 CD22 (V29) 41BBz (ka) as templates, respectively, to amplify the CD8a leader-CD22 (V28) VHH and CD8a leader-CD22 (V29) VHH fragments using the primers as follows:

BamH-CD8a sig-F：

GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC；

Linker(75)-CD22(V28)-R：

CACCCGATCCGCCGCCCCCAGATCCGCCCCCACCGGACCCTCCACCGCCTGAACCGCCCCCTCCGCTGCTCACGGTCACCTG；

BamH-CD8a sig-F：

GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC；

Linker(75)-CD22(V29)-R：

after the preparation, PCR reaction was carried out according to the PCR procedure shown in Table 19, after the PCR was completed, 1% agarose gel electrophoresis was carried out, and fragments of about 500bp were recovered and quantified by an ultraviolet absorption method.

3. Connecting the 2 fragments with the vector obtained in the first step and the first step by using recombinase, wherein a recombination reaction system is shown in table 21, reacting in water bath at 37 ℃ for 0.5h after the preparation is finished, transforming the mixture into escherichia coli stbl3 competent cells by a conventional method, selecting monoclone from a solid culture medium, culturing overnight, carrying out PCR identification, preparing a PCR reactant as shown in a graph 16, carrying out PCR program as shown in table 17, selecting positive clone after the PCR is finished, further sequencing and identifying, and the sequencing result is in line with the expectation.

TABLE 21

Reagent	Amount of the composition
		HD SIN03 CD19 41BBz(ka)	150ng
CD19 scFv-CD8a hinge-TM-41BB-CD3Z	28ng
		CD8a leader-CD22 (V28 or V29)	10ng
5x CE MultiS buffer	2μL
		Exnase MultiS	1μL
PCR-grade pure water	Adding to 10 μ L
		Total volume	10μL

4. Construction of HD SIN03 CD19-CD3 epsilon-T2A-CD 22 (V28) -CD3 gamma (ka) and HD SIN03 CD19-CD3 epsilon-T2A-CD 22 (V29) -CD3 gamma (ka) Lentiviral vectors

1. The PCR reaction system of Table 18 was prepared using HD SIN03 CD1941BBz (ka) as a template to amplify the CD8a leader-CD19 scFv fragment using the following primers:

BamH-CD8a sig-F：

GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC；

Linker3-CD19VH-R：

AGATCCGCCCCCACCGGACCCTCCACCGCCTGAACCGCCCCCTCCTGAGGAGACGGTGACTGAG；

after the preparation, PCR reaction was carried out according to the PCR procedure shown in Table 19, after the PCR was completed, 1% agarose gel electrophoresis was carried out, and fragments of about 800bp were recovered and quantified by ultraviolet absorption.

2. A PCR reaction system of Table 18 was prepared using pUC57-CD 3. Epsilon. Expression vector biosynthesized in the Venetian institute of technology as a template to amplify the CD 3. Epsilon. Fragment using the following primers:

Linker3-CD3ε-ECD-F：

CGGTGGGGGCGGATCTGATGGTAATGAAGAAATGG；

T2A-CD3ε-ICD-R：

AGGGCCGGGATTCTCCTCCACGTCACCGCATGTTAGAAGACTTCCTCTGCCCTCGATGCGTCTCTGATTCAG；

after the preparation, PCR reaction was carried out according to the PCR procedure shown in Table 19, after the PCR was completed, 1% agarose gel electrophoresis was carried out, and a fragment of about 600bp was recovered and quantified by an ultraviolet absorption method.

3. The PCR reaction systems of Table 18 were prepared using HD SIN03 CD22 (V28) 41BBz (ka) and HD SIN03 CD22 (V29) 41BBz (ka) as templates, respectively, to amplify CD8a leader-CD22 (V28) and CD8a leader-CD22 (V29) fragments using the primers as follows:

T2A-CD8a sig-F：GGAGAATCCCGGCCCTATGGCCTTACCAGTGACC；

Linker2-CD22(V28)-R：

AGAGCCACCACCTCCGGAGCCGCCACCTCCGGACCCTCCGCCACCGCTGCTCACGGTCACCTG；

T2A-CD8a sig-F：GGAGAATCCCGGCCCTATGGCCTTACCAGTGACC。

Linker2-CD22(V29)-R：

AGAGCCACCACCTCCGGAGCCGCCACCTCCGGACCCTCCGCCACCGCTGCTCACGGTCACCTG；

after the preparation, PCR reaction was carried out according to the PCR procedure shown in Table 19, after the completion of PCR, 1% agarose gel electrophoresis was carried out, and fragments of about 500bp were recovered and quantified by an ultraviolet absorption method.

4. The PCR reaction system of Table 18 was prepared using pUC57-CD 3. Gamma. Expression vector biosynthesized in the Compton corporation as a template, and CD 3. Gamma. Fragments were amplified using the following primers:

Linker2-CD3γ-ECD-F：

CGGAGGTGGTGGCTCTCAGTCAATCAAAGGAAACC；

EcoRI-CD3γ-ICD-R：

TCGATAAGCTTGATATCGAATTCTCAATTCCTCCTCAACTGG；

5. The 4 fragments and the vector obtained in the first step.4 are connected by recombinase, the recombination reaction system is shown in Table 22, the prepared vector is reacted in water bath at 37 ℃ for 0.5h, and the prepared vector is transformed into escherichia coli stbl3 competent cells according to a conventional method. Selecting single clone from a solid culture medium, culturing overnight, performing PCR identification, preparing PCR reactants as shown in a table 16, performing PCR program as shown in a table 17, selecting positive clone after PCR is finished, further sequencing identification, and ensuring that the sequencing result is in line with expectation.

TABLE 22

Reagent	Dosage of
		HD SIN03 CD19 41BBz(ka)	150ng
CD8a leader-CD19scFv	16ng
		CD3ε	12ng
CD8a leader-CD22 (V28 or V29)	10ng
		CD3γ	10ng
5x CE MultiS buffer	2μL
		Exnase MultiS	1μL
PCR-grade pure water	Adding to 10 μ L
		Total volume	10μL

6. Separating the obtained signal peptide-VHH, CD19 scFv-CD8a hinge-TM-41BB-CD3Z, CD (V28 or V29) -CD8 hinge-TM-41BB-CD3Z, signal peptide-CD 19 scFv, CD3 epsilon and CD3 gamma fragments by agarose gel electrophoresis, and then recovering, purifying and quantifying by using an agarose gel DNA fragment recovery kit; the lentiviral expression vector HD SIN03 CD1941BBz (ka) was cut with the restriction enzymes BamHI and EcoRI (from NEB) and the procedure was performed as described. Separating the enzyme digestion product by agarose gel electrophoresis, and then recovering, purifying and quantifying by using an agarose gel DNA fragment recovery kit; and then cloning the corresponding target fragment and the vector into a lentiviral vector by using a recombinase, and carrying out sequencing verification, wherein the sequencing result is in line with expectation.

The amino acid sequence of the CD3 epsilon fragment is (SEQ ID No. 21):

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI。

the amino acid sequence of the CD3 gamma fragment is (SEQ ID No. 22):

QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN。

example 6

This example performed lentiviral packaging, comprising the steps of:

(1) At 1.6X 10 ⁷ Cell number 293T cells plated in 15cm dishes at 37 ℃ C. And 5% CO ₂ Culturing overnightPreparing a packaging virus, wherein a culture medium is DMEM, and 10% Fetal Bovine Serum (FBS) is added;

(2) Dissolving 30 μ g of the 8 lentiviral vectors constructed in example 5, 12.5 μ g of helper plasmid gag/pol and 10 μ g of envelope plasmid VSVg in 2000 μ L of serum-free DMEM culture solution, and mixing;

(3) Mu.g PEI (1. Mu.g/. Mu.L) was dissolved in 2000. Mu.L serum-free DMEM medium, vortexed at 1000rpm for 5 seconds, and incubated at 25 ℃ for 5min;

(4) Formation of transfection complexes: adding the PEI mixed solution into the DNA mixed solution, immediately mixing by vortex or mixing lightly, and incubating for 20min at 25 ℃;

(5) Dripping 4mL of the transfection compound into a 15cm culture dish containing 25mL of DMEM medium, and replacing the fresh medium after 4 hours;

(6) After 48h, collecting the virus liquid supernatant to obtain 8 lentiviruses expressing chimeric antigen receptors with different structures.

Example 7

This example was performed for lentivirus concentration.

Filtering the virus supernatant prepared in example 6 by using a 0.45-micron filter membrane, collecting the filtered virus supernatant into a 50mL centrifuge tube, adding 1/4 of PEG-NaCl virus concentrated solution, reversing the mixture up and down, uniformly mixing the mixture, and standing the mixture at 4 ℃ overnight; centrifuging at 4 deg.C and 3500rpm for 30min; removing supernatant, adding RPMI 1640 medium (containing 10% FBS), and dissolving the heavy suspension virus precipitate; the concentrated lentiviral suspension was aliquoted into 50 μ L aliquots, stored in finished tubes and stored at-80 ℃.

Example 8

This example performs lentivirus titer detection.

500 μ L K cells (1 × 10) ⁵ Individual cells) were seeded into 24-well culture plates; the concentrated lentivirus of example 7 was added to the cell suspension at 1. Mu.L, 0.2. Mu.L and 0.04. Mu.L, respectively, and polybrene was added to a final concentration of 5. Mu.g/mL; 37 ℃,5% of CO ₂ After overnight culture, the fresh medium was replaced; after 72h of infection, 400 Xg centrifugation for 5min, abandoning supernatant collected cells, adding 100. Mu.L PBS +2% FBS heavy suspension cells, adding 1. Mu.g AF488-anti-CD19 scFv and/or PE-anti-VHH antibody, ice incubation for 30min; PBS +2% FBS Wash 2 timesThereafter, 300. Mu.L of PBS +2% FBS was added to resuspend the cells, and the infection efficiency was measured by flow cytometry; taking a cell sample with a positive rate of 15%, calculating the titer (TU/mL) = cell number (10) ⁵ ) X positive rate/virus volume (mL).

Example 9

This example utilizes lentiviruses to transduce T lymphocytes.

Diluting anti-human CD3 antibody and anti-human CD28 antibody with PBS to final concentrations of 1 μ g/mL and 0.5 μ g/mL respectively, coating the well plate, and standing overnight in a refrigerator at 4 deg.C; discarding the antibody coating solution in the pore plate, and washing twice with 1mL of PBS; human PBMC are adjusted to a density of 1X 10 with T cell medium (X-VIVO +10% FBS + IL-2 (300U/mL)) ⁶ mL, then inoculated into CD3 and CD28 antibody coated well plates for activation for 48h; collecting activated T cells, adjusting cell density to 1 × 10 ⁶ Per mL, lentivirus prepared in example 7 was added at multiplicity of infection (MOI) =10, polybrene was added to a final concentration of 5 μ g/mL; at 37 ℃ C, 5% CO ₂ After overnight incubation in the environment, the medium was replaced with fresh medium and passaged every 2 days.

Example 10

This example performed T lymphocyte chimeric antigen receptor expression, comprising the steps of:

(1) After 5 days of infection, 3X 10 of the total amount of the mixture was collected ⁵ Centrifuging the T cells at 4 ℃ at 400 Xg for 5min, discarding the supernatant, and washing the cells once with PBS +2% FBS;

(2) Add 100. Mu.L PBS +2% FBS resuspended cells, add 1. Mu.g AF488-anti-CD19 scFv and PE-anti-VHH antibody, incubate on ice for 30min; after 2 times of PBS + 2-FBS washing, 300. Mu.L of PBS + 2-FBS-resuspended cells were added, and the infection efficiency was examined by flow cytometry using uninfected T cells as a control, and the results are shown in FIG. 5, where the infected CAR-T cells had a clear positive cell population, indicating that the present invention successfully constructed 8 CAR-T cells expressing chimeric antigen receptors of different structures, in which the antigen-binding domain of the chimeric antigen receptor contained only CD22-28 antibody (corresponding to the cell marker CD22 (V28)) or CD22-29 antibody (corresponding to the cell marker CD22 (V29)), and the antigen-binding domain of the chimeric antigen receptor contained both CD22 and CD19 antibodies, but were ranked differently and labeled CD22 (V28) -CD19, CD22 (V29) -CD19, CD19-CD22 (V28), CD19-CD22 (V29), CD19- ε -CD22 (V28) - γ or CD19- ε -CD22 (V29) - γ, respectively.

Example 11

This example used 8 CAR-T cells prepared in example 9 to perform an in vitro toxicity assay comprising the following steps:

(1) Target cell inoculation:

tumor cells K562-luci (CD 19) preserved in this laboratory ^- CD22 ^- )、K562-CD19-luci(CD19 ⁺ CD22 ^- )、K562-CD22-luci(CD19 ^- CD22 ⁺ )、K562-CD19-CD22-luci(CD19 ⁺ CD22 ⁺ ) As the target cells, the concentration of the target cells was adjusted to 2X 10 ⁵ mL, inoculate 50 μ L to 96well plates;

(2) Effector cell inoculation:

the CAR-T cells prepared in example 9 and containing both CD19 and CD22 antibodies and control T (not lentivirus infected) cells were used as effector cells; adding CAR-T cells and control T cells to a 96-well plate at an effective target ratio of 0.3;

each group was set with 2 replicate wells, the average of the 2 replicate wells was taken, where the experimental and control groups were as follows:

experimental groups: CAR-T + target cells;

control group: control T cells + target cells;

(3) After the effector cells and the target cells are co-cultured for 18h, the cells are used

Detecting by the luciferase detection kit, wherein the specific detection step is based on the reference>

The results of the luciferase assay kit instructions are shown in fig. 6A-6D, and the CAR-T cells constructed by the invention have high killing activity on CD22 and CD19 positive tumor cells, and have no killing effect on CD22 and CD19 negative cells, which indicates that the CAR-T cells constructed by the invention also have high specificity.

Example 12

This example detects CAR-T cytokine secretion.

1. Cell culture supernatant

The cell culture of 1:1, the effective target ratio of example 11, was centrifuged at 400 Xg for 10min to remove the precipitate, and the supernatant was stored at-80 ℃ for examination.

2. Reagent preparation

The combined biological ELISA kit (the product numbers are respectively: human gamma interferon ELISA kit: EK180-96, human interleukin 2ELISA kit: EK102-96 and human tumor necrosis factor alpha (TNF-alpha) ELISA kit: EK 182-96) is used for detection, all reagents and samples are recovered to 25 ℃ before detection, 1 Xwashing liquid and 1 Xdetection buffer solution are prepared according to the use instructions, and the antibody is detected.

3. Standard and sample preparation

And (3) standard substance: the standard stock was diluted 2-fold using 5%1640 medium for a total of 8 dilution gradients, including zero concentration.

Sample preparation: the samples were diluted proportionally using 5%1640 medium.

4. Detection step

(1) Soaking the enzyme label plate: adding 300 mu L of 1 Xlotion, standing and soaking for 30s, removing the lotion, and patting the microporous plate on absorbent paper to dry;

(2) Adding a standard substance: add 100. Mu.L of 2-fold diluted standards to the standard wells and 100. Mu.L of 5% medium to the blank wells;

(3) Adding a sample: adding 100 mu L of cell culture supernatant into the sample hole;

(4) Adding a detection antibody: add 50 μ L of diluted detection antibody per well (1;

(5) And (3) incubation: incubating for 2h at 25 ℃ by using a sealing plate and a membrane sealing plate and oscillating at 300 rpm;

(6) Washing: discarding the liquid, adding 300 mu L of washing liquid into each hole, washing the plate for 6 times;

(7) Adding enzyme for incubation: add 100 μ L of diluted horseradish peroxidase-labeled streptavidin per well (1;

(8) And (3) incubation: sealing the plate with a new sealing plate membrane, shaking at 300rpm, and incubating at 25 deg.C for 45min;

(9) Washing: repeating the step (6);

(10) Adding a substrate for color development: adding 100 μ L of chromogenic substrate TMB into each hole, keeping out of the sun, and incubating for 15min at 25 ℃;

(11) Adding a stop solution: adding 100 mu L of stop solution into each hole, and fully and uniformly mixing;

(12) And (3) detection reading: the two-wavelength detection is carried out by using a microplate reader, OD values at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm are measured, and the calibrated OD value is obtained by subtracting the measured value of 630nm from the measured value of 450 nm.

The IL-2, TNF-alpha and IFN-gamma factor secretion results are respectively shown in figures 7-9, wherein the spontaneous CAR-T cell independent culture detects trace amounts of TNF-alpha, IL-2 and IFN-gamma factor in the spontaneous and K562-luci and CAR-T cultures, and IL-2, TNF-alpha and IFN-gamma factors are detected in the K562-CD19-luci, K562-CD22-luci and K562-CD19-CD22-luci respectively and in the CAR-T culture, which indicate that the CAR-T cells constructed by the invention can release cytokines to kill CD19 and/or CD22 positive tumor cells, have high specificity and do not obviously secrete cytokines to CD19 and/or CD22 negative cells.

In conclusion, the anti-CD 22 antibody is prepared by screening, has high affinity and specificity, can effectively target a tumor antigen CD22, utilizes the anti-CD 22 anti-chimeric antigen receptor, and further prepares a chimeric antigen receptor cell, and the chimeric antigen receptor cell can efficiently kill tumor cells, can secrete various cytokines to play a killing function, and has high specificity.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. An anti-CD 22 nanobody, wherein the anti-CD 22 nanobody comprises a heavy chain variable region;

the amino acid sequence of CDR1 of the heavy chain variable region comprises a sequence shown in SEQ ID No. 2;

the amino acid sequence of CDR2 of the heavy chain variable region comprises a sequence shown in SEQ ID No. 4;

the amino acid sequence of CDR3 of the heavy chain variable region comprises the sequence shown in SEQ ID No. 6.

2. The anti-CD 22 nanobody of claim 1, wherein the heavy chain variable region further comprises framework regions FR1, FR2, FR3 and FR4;

preferably, the amino acid sequence of FR1 comprises the sequence shown in SEQ ID No.7 or SEQ ID No. 8;

preferably, the amino acid sequence of FR2 comprises the sequence shown in SEQ ID No.9 or SEQ ID No. 10;

preferably, the amino acid sequence of FR3 comprises the sequence shown in SEQ ID No.11 or SEQ ID No. 12;

preferably, the amino acid sequence of FR4 comprises the sequence shown in SEQ ID No. 13;

preferably, the amino acid sequence of said heavy chain variable region comprises the sequence shown in SEQ ID No. 15.

3. A nucleic acid molecule comprising a gene encoding the anti-CD 22 nanobody of claim 1 or 2;

preferably, the nucleic acid molecule comprises the sequence shown as SEQ ID No. 17.

4. A chimeric antigen receptor comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, and a signaling domain;

the antigen binding domain comprises the anti-CD 22 nanobody of claim 1 or 2;

preferably, the signal peptide comprises a CD8a signal peptide;

preferably, the antigen binding domain comprises an antigen binding domain formed by connecting an anti-CD19 antibody and an anti-CD 22 nano antibody in series in sequence or an antigen binding domain formed by connecting an anti-CD 22 nano antibody and an anti-CD19 antibody in series in sequence;

preferably, the hinge region comprises a CD8a hinge region;

preferably, the signaling domain further comprises a co-stimulatory molecule comprising any one of the 4-1BB, CD28 intracellular domain, OX40, ICOS or DAP10 intracellular domain or a combination of at least two thereof;

preferably, the chimeric antigen receptor comprises a first peptide chain obtained by fusing an anti-CD19 antibody and a CD3 epsilon and a second peptide chain obtained by fusing an anti-CD 22 nano antibody and a CD3 gamma, or the chimeric antigen receptor comprises a first peptide chain obtained by fusing an anti-CD 22 nano antibody and a CD3 epsilon and a second peptide chain obtained by fusing an anti-CD19 antibody and a CD3 gamma.

5. An expression vector comprising a gene encoding the chimeric antigen receptor of claim 4;

preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to claim 4, preferably a lentiviral vector.

6. A recombinant lentivirus comprising the expression vector of claim 5.

7. A chimeric antigen receptor immune cell that expresses the chimeric antigen receptor of claim 4;

preferably, the chimeric antigen receptor immune cell comprises the expression vector of claim 5 and/or the recombinant lentivirus of claim 6;

preferably, the chimeric antigen receptor immune cell comprises any one of T lymphocyte, B lymphocyte, NK cell, mast cell or macrophage or a combination of at least two thereof.

8. A pharmaceutical composition comprising the chimeric antigen receptor immune cell of claim 7;

9. Use of the anti-CD 22 nanobody of claim 1 or 2, the nucleic acid molecule of claim 3, the chimeric antigen receptor of claim 4, the expression vector of claim 5, the recombinant lentivirus of claim 6, the chimeric antigen receptor immune cell of claim 7 or the pharmaceutical composition of claim 8 for the preparation of a medicament for the treatment of a tumor.

10. The use of claim 9, wherein the tumor comprises B-cell acute lymphoblastic leukemia.