Bispecific antibody for resisting CD3 and CEA antigen

文档序号：496573 发布日期：2022-01-07 浏览：2次中文

阅读说明：本技术 一种抗cd3和cea抗原的双特异性抗体 (Bispecific antibody for resisting CD3 and CEA antigen ) 是由宋海鹏刘原源于建立王准曹慧古一李飞张霞蒋立仲宋亮于 2021-10-12 设计创作，主要内容包括：本发明公开了一种抗CD3抗原的纳米抗体,所述抗CD3抗原的纳米抗体具有独特的3个互补决定区CDR1、CDR2、CDR3。本发明还公开了一种含有抗CD3的VHH结构域和抗CEA的VHH结构域的双特异性抗体,所述双特异性抗体由两条不同的重链序列融合而成,可以分别识别T细胞表面的CD3抗原和癌胚抗原CEA,且该双特异性抗体能够显著增强抗体介导的T细胞杀伤肿瘤靶细胞的活性。(The invention discloses a nano antibody for resisting a CD3 antigen, which has 3 unique complementarity determining regions (CDR 1, CDR2 and CDR 3). The invention also discloses a bispecific antibody containing the VHH domain of anti-CD 3 and the VHH domain of anti-CEA, which is formed by fusing two different heavy chain sequences, can respectively recognize the CD3 antigen and carcinoembryonic antigen CEA on the surface of a T cell, and can remarkably enhance the activity of the T cell mediated by the antibody to kill a tumor target cell.)

1. A nanobody against the T cell surface antigen CD3, wherein the variable region of the nanobody has 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the sequence of CDR1 is shown by SEQ ID No.1, the sequence of CDR2 is shown by SEQ ID No.2 and the sequence of CDR3 is shown by SEQ ID No. 3.

2. The nanobody of claim 1, wherein the sequence of the variable region of the nanobody is represented by SEQ ID No. 4.

3. A nucleic acid encoding the nanobody of claim 2, wherein the sequence of said nucleic acid is represented by SEQ ID No. 5.

4. An expression vector comprising the nucleic acid of claim 3, wherein said vector is pMES 4.

5. A host cell comprising the expression vector of claim 4, wherein said cell is E.coli BL21(DE 3).

6.A bispecific antibody comprising a first heavy chain which is the variable region of the nanobody of claim 2 and a second heavy chain; the variable region of the second heavy chain recognizes the CEA antigen, and the sequence of the variable region is shown by SEQ ID NO. 6.

7. The bispecific antibody of claim 6, wherein a linker peptide is provided between the first heavy chain and the second heavy chain, wherein n (G4S) are connected in series, wherein n is an integer between 1 and 6.

8. The bispecific antibody of claim 7, wherein n is 4, and the amino acid sequence of the bispecific antibody is represented by SEQ ID No. 7.

9. A nucleic acid encoding the bispecific antibody of claim 8, the sequence of which is represented by SEQ ID No. 9.

10. The bispecific antibody according to claim 8, further comprising an antibody constant region at the carboxy-terminus of said bispecific antibody, said constant region having the sequence shown in SEQ ID No. 8.

11. A nucleic acid encoding the bispecific antibody of claim 10, the sequence of which is represented by SEQ ID No. 10.

12. An expression vector comprising the nucleic acid of claim 11, wherein said vector is pFUSE hIgG1-Fc 2.

13. A host cell comprising the expression vector of claim 12, wherein the host cell is a HEK293 cell.

14. Use of the antibody of claim 1 or 2 for the preparation of a medicament for the treatment of tumors.

15. Use of an antibody according to any one of claims 6 to 11 for the manufacture of a medicament for the treatment of tumours.

Technical Field

The invention discloses a polypeptide, and more particularly discloses an antibody, belonging to the field of immunology.

Background

Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a glycoprotein with a molecular weight of about 180 kDa. CEA is a member of the immunoglobulin superfamily and contains 7 domains linked to the cell membrane via a Glycosylphosphatidylinositol (GPI) anchor. The 7 domains include a single N-terminal Ig variable domain and 6 domains homologous to Ig constant domains (a1-B1-a 2-B2-A3-B3). CEA was originally classified as a protein expressed only in fetal tissues and has now been identified in several normal adult tissues. Overexpression of CEA is observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers. Thus, CEA has been identified as a tumor associated antigen. CEA is readily cleaved from the cell surface and shed from the tumor into the bloodstream, either directly or via the lymphatic system. Because of this property, serum CEA levels have been used as clinical markers to diagnose and screen for cancer. Furthermore, CEA has also been used as a tumor marker, and immunological assays to measure elevated CEA in the blood of cancer patients have been used clinically for the prognosis and control of cancer.

More importantly, CEA has become a potentially useful tumor-associated antigen for targeted therapy. There have been reported 2 major approaches to cancer treatment using CEA-targeted immunotherapy. One method uses an anti-CEA antibody to elicit the lytic activity of immune cells, particularly by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), to eliminate CEA-expressing tumor cells. Another method is to specifically target CEA-expressing tumor cells by conjugating the anti-CEA antibody or antibody fragment with effector molecules such as drugs, toxins, radionucleotides, immunomodulators or cytokines, thereby exerting the therapeutic effect of the effector molecules. It is known that activated effector T cells can directly kill tumor cells, and also can induce apoptosis of tumor cells and other stromal cells or chemotactic macrophages by secreting cytokines to achieve nonspecific immune killing. On the other hand, cancer cells also have immune evasion mechanism to evade the killing of immune system, thereby facilitating the growth and metastasis of cancer cells, wherein immunosuppressive cells and molecules such as Regulatory T cells (Tregs), Myeloid Derived Suppressor Cells (MDSCs), tumor-associated macrophages and Interleukin-6 (Interleukin-6, IL-6) play an important role in this process. Therefore, promotion of Antigen Presenting Cells (APCs) Antigen presenting functions, anti-tumor effects of T cells, and inhibition of tumor-associated immunosuppressive factors are the main strategies for immunotherapy.

Bispecific antibodies are an antibody technology developed in recent years that can simultaneously recognize 2 different antigens. By using the bispecific antibody, it is possible to develop a bispecific antibody against both tumor cells and T cells, which recognizes tumor cell antigens, induces ADCC effect, recognizes effector T cells, directs T cells to the vicinity of tumor cells, and enhances the killing effect of effector T cells against tumor cells, such as CEA antigen and CD3 antigen of T cells.

19In 93 years, the Hamers-Casterman et al study found that a class of heavy chain-only dimers (H) was found in camelids (camels, dromedary and llamas) in vivo₂) Antibodies of the type IgG2 and IgG3, which are predominantly of the IgG2 and IgG3, are also referred to as single domain antibodies or single domain antibodies (sdabs) because they lack a light chain and are thus referred to as Heavy chain-only antibodies (HCAbs), whereas their antigen binding site consists of one domain, referred to as a VHH region. Since this class of antibodies is a variable region sequence after removal of the constant region, the molecular weight is only 15kDa, about 10 nm in diameter, and is therefore also referred to as nanobodies (Nbs). In addition, such single domain antibodies, called VNARs, are also observed in sharks. This heavy chain-only antibody was originally recognized only as a pathological form of a human B-cell proliferative disease (heavy chain disease). This heavy chain-only antibody may be due to genomic level mutations and deletions that result in the inability of the heavy chain CH1 domain to be expressed, such that the expressed heavy chain lacks CH1 and thus lacks the ability to bind to the light chain, thus forming a heavy chain dimer.

Nanobodies are comparable in affinity to their corresponding scFv, but surpass scfvs in solubility, stability, resistance to aggregation, refolding, expression yield, and ease of DNA manipulation, library construction, and 3-D structure determination, relative to scfvs of conventional four-chain antibodies.

Nanobodies have minimal functional antigen-binding fragments derived from HCabs in adult camelids, have high stability and high avidity for antigen binding, and can interact with protein clefts and enzymatic active sites, making their action similar to inhibitors. Therefore, the nano-antibody can provide a new idea for designing small molecule enzyme inhibitors from peptide-mimetic drugs. Due to the heavy chain only, nanobodies are easier to manufacture than monoclonal antibodies. The unique properties of nanobodies, such as stability in extreme temperature and pH environments, allow for large yields to be produced at low cost. Therefore, the nano antibody has great value in the treatment and diagnosis of diseases and has great development prospect in the antibody target diagnosis and treatment of tumors.

Based on the problems that a single recognition site or a traditional antibody molecule in the prior art in the field of tumor treatment is large and difficult to reach an acting cell and the like, the invention aims to provide the bispecific nanobody which simultaneously aims at a tumor cell and a T cell, recognizes the CEA antigen of the cancer cell, induces ADCC effect, recognizes the CD3 antigen of the T cell, guides the T cell to the vicinity of the tumor cell, and enhances the killing effect of the T cell on the tumor cell. And can overcome the inherent defects of poor permeability, low targeting effect and the like of the traditional antibody solid tumor.

Disclosure of Invention

Based on the above objects, the present invention provides a nanobody against the T cell surface antigen CD3, the variable region of which has 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the sequence of CDR1 is represented by SEQ ID No.1, the sequence of CDR2 is represented by SEQ ID No.2, and the sequence of CDR3 is represented by SEQ ID No. 3.

In a preferred technical scheme, the variable region sequence of the nanobody is shown by SEQ ID NO. 4. One preferred example of a nanobody having this variable region sequence in the present invention is nanobody 9H 9.

Secondly, the invention also provides a nucleic acid for coding the nano antibody, and the sequence of the nucleic acid is shown by SEQ ID NO. 5.

Thirdly, the present invention provides an expression vector containing the above nucleic acid, which is pMES 4.

Fourth, the present invention provides a host cell comprising the above expression vector, said host cell being E.coli BL21(DE 3).

Fifth, the present invention also provides a bispecific nanobody comprising a first heavy chain and a second heavy chain, wherein the variable region of the first heavy chain recognizes the CD3 antigen and the sequence of the variable region is represented by SEQ ID No. 4; the variable region of the second heavy chain recognizes the CEA antigen, and the sequence of the variable region is shown by SEQ ID NO. 6.

In a preferred embodiment, a connecting peptide is provided between the first heavy chain and the second heavy chain,the connecting peptide is formed by connecting n (G4S) in series and is represented as (G)₄S)_nWherein n is an integer between 1 and 6.

More preferably, n is 4, and the amino acid sequence of the bispecific antibody is shown in SEQ ID No. 7.

Sixth, the present invention also provides a nucleic acid encoding the bispecific antibody, the sequence of which is shown by SEQ ID NO. 9.

Particularly preferably, the bispecific antibody also has an antibody constant region at the carboxyl terminal, and the sequence of the constant region is shown as SEQ ID NO. 8.

Seventh, the present invention also provides a nucleic acid encoding the bispecific antibody with a constant region as described above, the sequence of which is shown by SEQ ID NO. 10.

Eighth, the invention provides an expression vector containing the nucleic acid, wherein the expression vector is pFUSE hIgG1-Fc 2.

Ninth, the invention provides a host cell containing the expression vector, and the host cell is an HEK293 cell.

Finally, the invention also provides the application of the nano antibody of the anti-T cell surface antigen CD3 and the bispecific antibody in the preparation of tumor treatment drugs.

The anti-CD 3 nanobody provided by the invention has higher affinity, and the bispecific nanobody can respectively recognize the CEA antigen and the CD3 antigen on the surface of a T cell. In an ADCC cytotoxicity experiment aiming at LoVo cells, the bispecific antibody provided by the invention shows excellent cytotoxicity, remarkably enhances the activity of T cells mediated by the antibody to kill tumor target cells, has the killing rate of 85 percent, is higher than the combined application of a CEA antibody and a CD3 antibody, and is more remarkably higher than the single application of the two antibodies. Compared with a single nano antibody, the bispecific antibody provided by the invention has longer in vivo half-life, which shows the application value of the bispecific antibody in the fields of clinical treatment of CEA antigen-positive tumors and drug preparation.

Drawings

FIG. 1 is an electrophoretic identification chart of total RNA extracted;

FIG. 2 is a first round of PCR amplification for identifying variable region gene electrophoresis of antibody;

FIG. 3 is the second round of PCR amplification antibody variable region gene electrophoresis identification picture;

FIG. 4 is the electrophoretic identification chart of the product of the double digestion reaction of pMES4 vector;

FIG. 5 is an electrophoretic identification chart of colony PCR-identified transformants;

FIG. 6 is a SDS-PAGE pattern of the Nanobody 9H9 purification;

FIG. 7 is a Biacore analysis nanobody 9H9 affinity graph;

FIG. 8 is a bispecific antibody purification SDS-PAGE profile;

FIG. 9 is a graph showing the results of detection of bispecific antibody by Western blot;

FIG. 10 is a graph showing the results of ADCC cytotoxicity assays;

figure 11 is a graph of the metabolism of bispecific antibody and nanobody in rabbits.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of protection defined by the claims of the present invention.

Example 1 preparation of monovalent Nanobodies against CD3

1.1 immunization of alpaca

Selecting one healthy adult alpaca, uniformly mixing a recombinant humanized CD3 antigen (a manufacturer: abcam, a product number is ab229518) and Freund's adjuvant according to a ratio of 1:1, immunizing the alpaca by adopting a back subcutaneous multipoint injection mode according to 6-7 mu g/kg for four times, and the immunization interval is 2 weeks. Then 10ml of alpaca peripheral blood is collected for constructing a phage display library.

1.2 isolation of Camel-derived lymphocytes

Separating collected alpaca peripheral blood lymphocytes by using camel peripheral blood lymphocyte separation kit (Tianjin tertiary ocean company, Cat. LTS1076) instruction manual operation, each 2.5 × 10⁷Adding 1ml RNA separating agent into living cells, and extracting RNA from 1mlCollecting, and storing at-80 deg.C.

1.3 Total RNA extraction

Repeatedly blowing 1ml of the Tipure Isolation Reagent containing lymphocytes, and standing for 5 minutes; add 200. mu.l chloroform, vortex for 30 seconds and then place for 5 minutes; centrifuging at 4 ℃ and 12000g for 15 minutes, sucking the water phase and transferring into a new EP tube; adding equal amount of isopropanol, and standing for 10 minutes; centrifuging at 12000g for 10min at 4 ℃, and removing supernatant; washing with 1ml of pre-cooled 70% ethanol, centrifuging at 4 ℃ and 7500g for 5 minutes, discarding the supernatant and drying for 5 minutes; 30 μ l of RNase-free water was added to dissolve the precipitate, and the concentration was adjusted to 1 μ g/. mu.l for detection by gel electrophoresis, and the results are shown in FIG. 1.

1.4 Synthesis of cDNA by reverse transcription

The cDNA was reverse-transcribed using the RNA obtained in step 1.3 as a template according to the reverse transcription KIT (Transcriptor first stand cDNA Synthesis KIT from Roche).

1.5 antibody variable region Gene amplification

And carrying out PCR reaction by using cDNA obtained by reverse transcription as a template. Amplification was performed in two rounds, and the primer sequences for the first round of PCR were as follows:

CALL001：GTCCTGGCTGCTCTTCTACAAGG

CALL002：GGTACGTGCTGTTGAACTGTTCC

the PCR reaction conditions and procedures were: 5 minutes at 95 ℃; 30 cycles of 95 ℃ for 30 seconds, 57 ℃ for 30 seconds, 72 ℃ for 30 seconds; 7 minutes at 72 ℃. The agarose gel recovery kit gel was used to recover a band of about 700bp, and the final nucleic acid concentration was adjusted to 5 ng/. mu.l with water (FIG. 2: M is Trans 2K DNA Marker; 1 is negative control; 2 is first round PCR product). The primer sequences for the second round of PCR were as follows:

VHH-Back：GATGTGCAGCTGCAGGAGTCTGGRGGAGG

VHH-For：CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

the PCR reaction conditions and procedures were: 5 minutes at 95 ℃; 30 seconds at 95 ℃, 30 seconds at 55 ℃, 30 seconds at 72 ℃ and 15 cycles; 7 minutes at 72 ℃. PCR products were purified using a PCR product recovery kit (FIG. 3: M is Trans 2K DNA Marker; 1 is the second round PCR product; 2 is the negative control).

1.6 vector construction

pMES4 (purchased from Biovector) and the second PCR product were subjected to PstI and BstEII double digestion, respectively, and 1.5. mu.g of the digested vector and 450ng of the digested second PCR product were taken, and 15. mu. l T4 DNA ligase was added to the digested vector and water to a total volume of 150. mu.l, ligated overnight at 16 ℃ and the ligated product was recovered. The product was recovered using a PCR product recovery kit and eluted with 20. mu.l of water. The double-restriction enzyme digestion result of the pMES4 vector was detected by 1% agarose electrophoresis gel (FIG. 4: M is Trans 5K plus DNA Marker; 1 is pMES4 vector non-restriction enzyme digestion plasmid; and 2 is pMES4 vector double-restriction enzyme digestion product).

1.7 electrotransformation and determination of the storage volume

Mu.l of the purified ligation product was taken and added to a pre-cooled electric cuvette containing 50. mu.l of E.coli TG1 competent cells and placed in an electric converter (ECM 630 electric converter of BTX, USA) for electric conversion, and the electric cuvette was taken out, and the transformant was recovered and cultured. Clones were randomly selected and colony PCR identified (FIG. 5: M is Trans 2K DNA Marker; 1 is negative control; 2-11 is randomly selected monoclonal PCR identified product). The pool capacity (pool capacity ═ number of clones × dilution × positive rate of PCR identification × 10) was estimated from the PCR positive rate. The primer sequences are as follows:

MP57：TTATGCTTCCGGCTCGTATG

GIII：CCACAGACAGCCCTCATAG

1.8 phage amplification

Inoculating recovered bacteria solution into YT-AG culture medium, culturing at 37 deg.C and 200rpm until culture OD₆₀₀0.5. 10ml of the bacterial suspension was taken out and added to 4X 10¹⁰VCSM13, 30min at 37 ℃ for static infection. At 4000rpm, the mixture was centrifuged at room temperature for 10 minutes, and the supernatant was removed. The cells were resuspended in 2 XYT-AK (ampicillin and kanamycin-containing) medium and cultured overnight at 37 ℃ and 200 rpm. Centrifuging, taking a supernatant in a 40ml tube, adding 10ml of PEG/NaCl (20%/2.5M) solution, mixing thoroughly, centrifuging, discarding the supernatant, washing the precipitate with 1ml of ice PBS, centrifuging, taking 250 μ l of precooled PEG/NaCl from the supernatant, mixing thoroughly, washing and resuspending.

Determining the phage titer: TG1 was cultured to OD₆₀₀Gradient dilution of phage with LB medium (0.4), mixed culture of phage TG1 diluted in multiple proportions, and observation of the culture the next dayPlaque formation in the plate, the number of plaques was counted on a dilution gradient plate of 30-300 and the phage titer (pfu) was calculated according to the following formula.

Phage titer (pfu/ml) dilution times plaque number times 100

1.9 Nanobody screening

Positive clones were screened for antigen by ELISA. ELISA plates were coated with antigen, blocked with 5% BSA, and washed with PBST. Mu.l phage supernatant was added to each well and left at 37 ℃ for 1 hour. The supernatant was discarded, and a secondary HRP-labeled mouse anti-M13 antibody was added thereto and the mixture was left at 37 ℃ for 1 hour. The supernatant was discarded, TMB solution was added, incubation was carried out at room temperature for 5 hours, 2M sulfuric acid stop solution was added to each well, and reading was carried out with a microplate reader at 450 nm.

Expression and purification of 1.10 Nano antibody in Escherichia coli

Selecting a clone with a positive phage ELISA result, extracting a plasmid, transforming the plasmid into a strain BL21 competent cell, inducing the protein expression of the nano antibody by IPTG, collecting a supernatant (periplasmic extract), dialyzing the periplasmic extract into PBS, purifying by using Ni-NTA resin, eluting and collecting by using imidazole with different concentrations, carrying out reduced protein electrophoresis analysis on the collected sample, and finally dialyzing the nano antibody into the PBS.

The nano antibody of anti-CD 3 is screened out through alpaca immunity, cell separation, phage library construction and nano antibody screening. Analysis of the antibody light and heavy chain genes was performed on the sequencing results using Vector NTI software to determine the Framework Regions (FRs) and Complementarity Determining Regions (CDRs) of the variable Regions.

The nanobody of one preferred embodiment screened by the present invention is named "9H 9". Through DNA sequencing, the heavy chain nucleic acid sequence of the nano antibody 9H9 is shown as SEQ ID NO.5, the variable region amino acid sequence is shown as SEQ ID NO.4, wherein the amino acid sequences at the 1 st to 25 th positions are FR1, the amino acid sequences at the 26 th to 33 th positions are CDR1, the amino acid sequences at the 34 th to 50 th positions are FR2, the amino acid sequences at the 51 th to 58 th positions are CDR2, the amino acid sequences at the 59 th to 96 th positions are FR3, the amino acid sequences at the 97 th to 107 th positions are CDR3, and the amino acid sequence at the 108 th position and 118 th positions is FR 4.

Example 2 preparation of Nanobody 9H9

2.1 amplification of original strain TG1 of nano antibody and transformation of Escherichia coli BL21(DE3) by recombinant plasmid of nano antibody

The original strain TG1 glycerol strain containing the nano antibody nucleic acid is inoculated into 5ml of fresh LB-A culture medium according to the proportion of 1:1000, and the culture is carried out overnight at 37 ℃ and 200 rpm. The following day, Plasmid was extracted using a Plasmid mini kit (OMEGA) as per the instructions. After verification, 1. mu.l of the plasmid was transformed into 100. mu.l of competent cells, gently mixed, placed on ice for 30 minutes, heat-shocked in a water bath at 42 ℃ for 90 seconds, and cooled in an ice bath for 3 minutes. 600. mu.l of LB medium was added to the centrifuge tube, and the tube was cultured with shaking at 37 ℃ for 60 minutes. 100. mu.l of the supernatant was applied to an LB-A plate using a triangle spreader and cultured overnight at 37 ℃ in an inverted state.

2.2 inducible expression of Nanobodies

The above monoclonal colonies were picked up in LB-A medium and cultured overnight with shaking at 37 ℃. The next day, adding 100ml fresh LB-A culture medium into the bacterial liquid at a ratio of 1:100, and performing shaking culture at 37 deg.C for 3 hr to obtain bacterial liquid OD₆₀₀After adding IPTG to a final concentration of 1mM, the mixture was induced overnight at 30 ℃. On the third day, 8000rpm, the cells were collected by centrifugation for 10 minutes, and 1.5ml of a precooled TES buffer was added to resuspend the pellet. After 2 minutes in ice bath, gently shake for 30 seconds and repeat this cycle 6 times. 3.0ml TES/4 (TES diluted 4-fold with water) was added, gently shaken for 30 seconds, and then allowed to stand on an ice bath for 2 minutes, and the shaking and standing steps were repeated a total of 6 times. After centrifugation at 9000rpm at 4 ℃ for 10 minutes, about 4.5ml of the supernatant (periplasmic extract) was collected.

2.3 purification and characterization of Nanobodies

After resuspending IMAC Sepharose (GE Co.), 2ml was added to the gravity column, and the column was allowed to stand for 30 minutes to allow Sepharose to naturally settle at the bottom of the gravity column, and the preservation buffer was discharged. Adding 2 column volumes of nickel sulfate solution (0.1M) and flowing out the nickel sulfate solution at a flow rate of about 8 seconds per drop; adding 10 times of column volume of balance buffer solution to balance and wash sepharose, and keeping the flow rate unchanged; diluting the sample by 2 times of a balance buffer solution, adding the diluted sample into a gravity column, adjusting the flow rate to be 6 seconds/drop, and collecting the penetration liquid; adding 10 times of column volume of washing buffer solution to wash sepharose, maintaining the flow rate unchanged, and collecting washing solution; adding elution buffer solution with the volume being 3 times of that of the column, maintaining the flow rate at 6 seconds per drop, and collecting the eluent containing the target protein; finally sepharose was washed by sequentially adding 10 column volumes of equilibration buffer, 10 column volumes of pure water and 10 column volumes of 20% ethanol, and finally 4ml of 20% ethanol was retained to preserve the column. The collected samples were subjected to SDS-PAGE detection (FIG. 6: M is a rainbow 180 broad-spectrum protein Marker; 1 is a purified nanobody 9H9 induced by Escherichia coli).

Example 3 determination of the affinity Activity of Nanobodies with antigens

3.1 chip antigen coupling

Preparing the antigen into working solution of 20 mu g/ml by using sodium acetate buffer solutions (pH 5.5, pH 5.0, pH 4.5 and pH 4.0) with different pH values, preparing 50mM NaOH regeneration solution, analyzing the electrostatic binding between the antigen and the surface of a chip (GE company) under different pH conditions by using a template method in a Biacore T100 protein interaction analysis system instrument, selecting a proper pH system with most neutral pH according to the standard that the signal increase amount reaches 5 times RL, and adjusting the antigen concentration as required to serve as the condition during coupling. Coupling the chip according to a template method carried by the instrument: wherein, the 1 channel selects a blank coupling mode, the 2 channel selects a Target coupling mode, and the Target is set as a designed theoretical coupling quantity. The coupling procedure took approximately 60 minutes.

3.2 analyte concentration setting Condition exploration and regeneration Condition optimization

A manual sample injection mode is adopted, a1, 2-channel 2-1 mode is selected for sample injection, and the flow rate is set to be 30 mu l/min. The injection conditions were 120 seconds, 30. mu.l/min. Regeneration conditions were 30 seconds, 30. mu.l/min. The buffer was run continuously empty first until all baselines were stable. Nanobody solutions with larger concentration spans were prepared in running buffer formulations, suggesting settings of 200. mu.g/ml, 150. mu.g/ml, 100. mu.g/ml, 50. mu.g/ml, 20. mu.g/ml, 10. mu.g/ml, 2. mu.g/ml. Preparing a regeneration solution, selecting the regeneration solution with four pH gradients of a glutamate acid system: 1.5,2.0,2.5,3.0. A200. mu.g/ml sample of analyte was manually injected and the 2-channel observed, regenerating from the most neutral pH regenerating buffer until the line of response after 2-channel regeneration returned to the same height as the baseline. And manually injecting a sample of 200 mu g/ml of analyte once again, observing the signal change of the 2-1 channel and recording the binding capacity, regenerating by using a regeneration solution which finally returns the response line to the base line in the previous step, then manually injecting a sample of 200 mu g/ml of analyte once again, observing the signal change of the 2-1 channel and recording the binding capacity, comparing with the value of the previous binding capacity, if the deviation is less than 5 percent, determining that the regeneration solution with the pH value is the optimal regeneration solution, and if the binding capacity of re-injection is lower, continuing to perform the experiment by using a regeneration buffer solution with lower pH value. And taking the selected optimal regeneration solution as a chip surface regeneration reagent after each sample introduction. And respectively injecting analyte concentration samples arranged on the sample injection device, and analyzing the binding capacity of each concentration to finally determine the concentration gradient required by the affinity test.

3.3 affinity assay

According to the optimized sample concentration gradient, the solution is regenerated, and the affinity between the nano antibody and the antigen is tested by using a template method carried by the instrument (wherein the sample introduction condition is set to be 60 seconds and 30 mul/min; the dissociation time is 600 seconds, and the regeneration condition is set to be 30 seconds and 30 mul/min). The signal condition of the 2-1 channel is observed at any time. The affinity testing process took approximately 200 minutes.

3.4 analysis of results

The binding dissociation curves for several concentration gradients were selected using a 1: the 1binding mode was fitted to all curves to obtain the affinity values and binding and dissociation constants (see FIG. 7). The affinity value of the anti-CD 3 nanobody 9H9 is 2.813E-10.

Example 4 preparation of anti-CEA and CD3 bispecific Nanobodies

The bispecific antibody with a constant region can be obtained by connecting the bispecific antibody into pFUSE hIgG1-Fc2, and the pFUSE hIgG1-Fc2 vector (Qingdao Jieshikang organism, cat JR442) can self-express the Fc fragment of human IgG 1. The specific operation is as follows: the amino acid sequence of the anti-CEA nano antibody refers to the amino acid sequence of the nano antibody 11C12 in Chinese patent CN 106946989A. Selecting anti-CEA nanometer antibody 11C12 andthe anti-CD 3 nano antibody 9H9 is subjected to protein fusion expression, and the variable regions of the heavy chains of the two antibodies utilize flexible polypeptide (GGGGS)₄After ligation, the obtained product was sent to Huada Gene Co for gene synthesis. The two restriction enzyme sites EcoRI and BglII are reserved at both ends of the synthesized gene and are connected on a pUC57 vector to obtain pUC57-11C12- (GGGS)₄-9H 9. Cutting target fragment of fusion nano antibody by using EcoRI and Bgl II, connecting into pFUSE hIgG1-Fc2 vector by T4 ligase, constructing recombinant plasmid pFUSE hIgG1-Fc2-11C12- (GGGS)₄9H9, transforming DH5 alpha to competence, and extracting plasmids by using an endotoxin-free macroextraction kit (Tiangen). Human 293 cells were transfected, bispecific nanobodies were purified from the culture supernatant of 293 cells by Protein A chromatography, and the purified bispecific nanobodies were subjected to SDS-PAGE analysis (FIG. 8: M is rainbow 180 broad-spectrum Protein Marker; 1 is purified bispecific nanobody).

Example 5 Western blot detection of the function of bispecific antibodies

Carrying out SDS-PAGE on the recombinant CEA antigen (Huada gene synthesis, GenBank: CAE75559.1) and the recombinant CD3 antigen (manufacturer: abcam, cat # ab229518), then carrying out membrane transfer, and carrying out constant current electrophoresis for 45min, wherein the current is 23mA (the current is 1 time of the membrane area); sealing 5% skimmed milk powder at room temperature for 5h after electrophoresis; after washing the membrane for three times by TBST, performing primary antibody incubation on the NC membrane, wherein the dilution of the antibody (the initial concentration of the antibody is 1mg/ml) is 1:2000, and incubating overnight at 4 ℃; after washing the membrane for three times, incubating a secondary antibody of the NC membrane, wherein the secondary antibody is incubated for 2 hours at room temperature by using HRP-labeled rabbit anti-alpaca VHH (manufacturer: Kingsler, product number A01861) or HRP-labeled rabbit anti-human IgG (manufacturer: abcam, product number ab 6759); and (3) washing the membrane for three times, carrying out DAB color development on the NC membrane, carrying out color development at room temperature for 10min, and then terminating the reaction by using distilled water. The results are shown in FIG. 9. Bispecific antibodies can bind both CEA and CD3 antigens, whereas 11C12 can only bind CEA antigens.

Example 6 ADCC cytotoxicity assay

6.1 leukocyte isolation

2ml of venous blood is taken and put into a test tube added with anticoagulant, and the venous blood is gently mixed. Standing the tube at room temperature or 37 deg.CAnd (5) naturally settling the red blood cells in the incubator for 30-60 min. At this time, the suspension in the tube was seen to be divided into 3 layers, the upper layer was light yellow plasma, the bottom layer was red blood cells, and a pale white leukocyte layer (normal human peripheral blood leukocytes) was formed on the layer adjacent to the red blood cells. The leukocyte-rich cell suspension located above the red blood cell layer was aspirated by a capillary tube and transferred to another tube. Adding Ca-free²⁺、 Mg²⁺Hank's solution is added to the position 3cm away from the test tube port, mixed evenly, centrifuged for 10min at 2000r/min by a horizontal centrifuge, the supernatant is discarded, and the mixture is washed twice by the same method. The precipitated cells are resuspended and counted in a proper amount of Hank's solution of 10% -20% inactivated calf serum to prepare a suspension with the required cell concentration, and the suspension is usually 2X 10⁶/ml。

6.2 culture of tumor cells

Culturing LoVo cells (human colon cancer cells, donated by Experimental animal center of Hospital medical science institute of Zhejiang province) with high CEA in RPMI-1640 culture medium containing 10% fetal calf serum to 75-80% density, discarding the culture medium, washing cells with PBS preheated to 37 deg.C for 2 times, adding 2ml trypsin-EDTA solution, standing at room temperature for 5min, adding 2ml culture medium containing 10% fetal calf serum to terminate the reaction, repeatedly blowing cells with disposable sterile pipette to single cell suspension, centrifuging with horizontal centrifuge 2000r/min for 10min, discarding supernatant, washing with PBS twice, counting, and re-suspending cells with culture medium to 2 × 10⁶One/ml is ready for use.

6.3 cytotoxicity assays

Take 10. mu.l (2X 10) of resuspended tumor cells⁴Respectively), adding 30 μ l of RPMI-1640 medium containing 0.1% BSA, adding 10 μ l (1mg/ml) of different antibodies, incubating at 37 deg.C for 30min, and adding 5 × 10⁵PMBC (effector/target 25) cells, volume 250 μ l, incubated at 37 ℃ for 4h, centrifuged at 2000r/min for 10min in a horizontal centrifuge, supernatant was taken and LDH activity in the supernatant was detected using Roche cytotoxicity detection kit to convert to the degree of tumor cell death, the kit had the code: 11644793001. the results are shown in FIG. 10.

As shown in FIG. 10, the ordinate represents the killing rate of T cells against tumor target cells, and the abscissa 1-6 samples are PMBC + LoVo + bispecific antibody, PMBC + LoVo +11C12+9H9, PMBC + LoVo +11C12, PMBC + LoVo +9H9, pure PMBC, and pure LoVo cells in this order. The bispecific antibody provided by the invention is the column 1, which remarkably enhances the activity of killing tumor target cells by T cells mediated by the antibody, shows excellent cytotoxicity, has the killing rate of up to 85 percent (the killing rate of 6 samples on the abscissa is 85 percent, 70 percent, 60 percent, 12 percent, 3 percent and 2 percent in sequence), is higher than the combined application of the anti-CEA nano antibody 11C12 and the anti-CD 3 nano antibody 9H9 and is more remarkably higher than the single application of the two antibodies, and shows the application value of the bispecific antibody in the clinical treatment of CEA antigen positive tumors.

Example 7 metabolism of bispecific Nanobodies in Rabbit plasma

In this example, the primary study of pharmacokinetics was performed with respect to new zealand rabbits, each 6 new zealand rabbits were used as a group, and the administration dose of the bispecific nanobody, the anti-CEA nanobody 11C12 and the anti-CD 3 nanobody 9H9 was 1nmol/kg by the back subcutaneous administration. Ear vein blood collection was performed at 0.5h, 1h, 2h, 4h, 8h, 12h, 18h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 120h, and 144h after administration, and serum was isolated for antibody titer determination, and a drug titer time curve was plotted, and the results are shown in fig. 11.

The results show that the nanobody is metabolized to a lower level after 72h, while the bispecific antibody still has a high concentration after 72h, with therapeutic effect. Pharmacokinetic parameters were analyzed using GraphPad Prism software and the results are shown in table 1.

TABLE 1 in vivo pharmacokinetic parameters of Nanobodies and bispecific antibodies in rabbits

In Table 1, t_maxRefers to the time at which the antibody titer reaches a maximum; t is t_1/2Refers to the time at which the antibody is half metabolized after reaching its maximum concentration. As can be seen from the table, the half-life of the bispecific antibody in New Zealand rabbits was as long as 48 hoursIs significantly longer than the 24 hours of nanobodies.

Sequence listing

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