Bispecific antigen-binding construct and preparation method and use thereof

Abstract

Disclosed are a bispecific antigen-binding construct and the preparation method and use thereof, wherein the construct comprises a first antigen binding unit and a second antigen binding unit, the first antigen binding unit is a single chain variable region antibody fragment ScFV which specifically binds to the surface antigen of immune cells, and the second antigen binding unit is a Slit2D2 protein fragment which specifically binds to the surface antigen Robo1 of tumour cells. That is to say, the construct can bind to the surface antigen of immune cells and the surface Robo1 molecule of tumour cells at the same time, so that as the distance between tumour cells and immune cells get smaller, the quiescent immune cells are effectively activated, and the effect of killing and wounding tumours is produced. The construct has advantages of small molecular weight and good tissue penetrability, has significant killing and wounding effects on the tumour cells which express a large amount of Robo1, and can be used in the development of anti-tumour drugs.

Claims

1. A nucleotide encoding: a bispecific antigen-binding construct comprising a first antigen-binding unit and a second antigen-binding unit, wherein the first antigen-binding unit is an antibody or an antibody fragment that specifically binds to an immune cell surface antigen CD3; and the second antigen-binding unit is a Slit2 or a Slit2 fragment that specifically binds to a tumor cell surface antigen RoBo1; wherein the first antigen-binding unit is an antibody or an antibody fragment comprising a VH and a VL of SEQ ID NOs: 2 and 3, respectively; wherein the second antigen-binding unit is a Slit2 or Slit2 fragment that comprises SEQ ID NO: 1; wherein the construct comprises the amino acid sequence SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

2. A vector comprising the nucleotide of claim 1.

3. A method of treating a tumor disease by administering: a bispecific antigen-binding construct comprising a first antigen-binding unit and a second antigen-binding unit, wherein the first-antigen binding unit is an antibody or an antibody fragment that specifically binds to an immune cell surface antigen CD3; the second antigen-binding unit is a Slit2 or a Slit2 fragment that specifically binds to a tumor cell surface antigen RoBo1; wherein the first antigen-binding unit is an antibody or an antibody fragment comprising a VH and a VL of SEQ ID NOs: 2 and 3, respectively; wherein the second antigen-binding unit is a Slit2 or Slit2 fragment that comprises SEQ ID NO: 1; wherein the construct comprises the amino acid sequence SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

4. The method of claim 3, wherein the tumor disease is characterized by increased expression of RoBo1 in the tumor cells as compared to normal cells.

5. The method of claim 4, wherein the tumor disease is lung cancer or breast cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a construction scheme of the bispecific antigen binding construct of the invention.

(2) FIG. 2 illustrates an expression vector map of the bispecific antigen binding construct of the present invention.

(3) FIG. 3 illustrates a SDS-PAGE electropherogram of the bispecific antigen binding construct protein in Example 1, wherein M is the marker lane, and 6 and 7 are the lanes of the bispecific antigen binding construct protein.

(4) FIG. 4 illustrates a result of tumor killing experiments for detecting the drug activity in Example 2.

(5) FIG. 5 illustrates a growth change result of the tumor volume of the treatment group and the control group mice of the MHCC97H+PBMC tumor model in Example 3.

(6) FIG. 6 illustrates the change of body weight of experimental mice over treating time, wherein the mice is from the MHCC97H+PBMC tumor model in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

(7) The technical solutions in the embodiments of the present invention are clearly and completely described in the following context with reference to the drawings in the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by a person skilled in the art without any creative work are within the scope of the protection of the present invention.

Example 1: Preparation of the Bispecific Antigen Binding Construct Slit2D2-ScFV

(8) The bispecific antigen binding construct prepared in this example is Slit2D2-VH-VL, and its construction scheme is as shown in FIG. 1.

(9) 1. Protein Expression

(10) 1.1 Experimental Materials:

(11) Cell line: ExpiCHO-S™ cells (Gibco Catalog No. A29127);

(12) Transfection kit: ExpiFectamine™ CHO Transfection Kit (Gibco Catalog No. A29129) OptiPRO™ SFM (Gibco Catalog No. 12309-050);

(13) Culturing Medium: ExpiCHO™ Expression Medium (Gibco Catalog No. A29100-01);

(14) pCDNA3.4 plasmid vector (Invitron).

(15) 1.2 Experiment Procedure:

(16) According to the designed Slit2D2-ScFV gene sequence (as shown in SEQ ID NO: 10), the Slit2D2-VH-VL gene fragment was obtained by total gene synthesis, and then PCR product was obtained by PCR amplifying by the Slit2D2-VH-VL gene fragment as a template. The aforesaid product was purified and recovered with a gel recovery kit. According to the theory of T-A cloning, the PCR product was cloned into a pCDNA3.4 vector. The recombinant plasmid map is as shown in FIG. 2. The recombinant plasmid was transformed into E. coli TOP10, and then it was screened with ampicillin. The positive clones were picked and the completion of the construction of the vector was confirmed by sequencing. Plasmid DNA was extracted by using endo-free DNAextraction kit, which was used for the transfection of ExpiCHO-S™ cells. The ExpiCHO-S™ cells were cultured by shaking at 37° C. in an incubator containing 8% CO.sub.2. The plasmid DNA was transfected into the ExpiCHO-S™ cells when the density reached 2.5×10.sup.6 cells/ml. The transfected ExpiCHO-S™ cells were cultured in the ExpiCHO™ Expression Medium, and the supernatant of the culturing fluid was collected 5 days after transfection and then centrifuged at a high speed. The supernatant was kept for subsequent purification.

(17) 2. Protein Purification

(18) 2.1 Experimental Instruments and Materials:

(19) AKTA protein purification system;

(20) Binding Buffer: 20 mM phosphate, 0.5 M NaCl, 20 mM imidazole, adjusted to pH 7.4;

(21) Elution Buffer: 20 mM phosphate, 0.5 M NaCl, 500 mM imidazole, adjusted to pH 7.4; deionized water;

(22) 20% Ethanol solution.

(23) 2.2 Experiment Procedure:

(24) (1) Starting the AKTA device, and connecting a His column to the AKTA;

(25) (2) Washing the His column with 3-5 column volumes of deionized water;

(26) (3) Equilibrating the His column with 5 column volumes of the Binding Buffer;

(27) (4) The filtered cell supernatant was taken out and flowed through the His column in turn;

(28) (5) Washing off the protein impurities with the Binding Buffer until the absorbance approaches zero at UV280;

(29) (6) Eluting the protein of interest using the Elution Buffer, and starting collecting the eluent while absorbance was greater than 400 at UV280;

(30) (7) Dialyzing the collected protein eluent with PBS at 4° C., and the dialyzed protein was stored at −80° C.;

(31) (8) After the collection of protein, washing the His column using 10 column volumes of Binding Buffer;

(32) (9) Washing the His column using 10 column volumes of deionized water;

(33) (10) Washing the His column using 10 column volumes of 20% ethanol, and the His column was placed in 20% ethanol and stored at 4° C.;

(34) (11) Dialyzing the collected protein eluent with a dialysis bag to remove the salt ions, and the eluent is converted into a PBS solution for preserving the protein.

(35) Note: a. Use a 0.45 μm filtering membrane to filter all the reagents for use in the purification process.

(36) b. During the purification process, it is prohibited to allow air to enter the His column.

(37) c. The entire process should be performed on ice to prevent protein inactivation.

(38) 2.3 Experimental Result

(39) The target protein was obtained by the aforesaid purification procedure, and its SDS-PAGE electrophores is as shown in FIG. 3. As shown in FIG. 3, the molecular weight of the target protein was about 55 KD with extremely high purity. No protein impurity was detected, and it was indicated that the recombinant expression vector of Slit2D2-VH-VL had been successfully constructed, and the expression of Slit2D2-VH-VL in the host cell had been achieved.

Example 2 Tumor Killing Test for Detecting the Drug Activity

(40) The killing activity of the drug on tumor cells was determined by lactate dehydrogenase (LDH) releasing method.

(41) 1.1 Experimental Materials:

(42) Test kit: CytoTox 96@ Non-Radioactive Cytotoxicity Assay kit (promega);

(43) Kit components: 5 vials of Substrate Mix, 60 ml Assay Buffer, 25 μl LDH Positive Control, 5 ml Lysis Solution (10×), 65 ml Stop Solution;

(44) Target cells: breast cancer cells MDA-MB-231, liver cancer cells SMCC7721, PBMC;

(45) Drug: bispecific antigen binding construct protein Slit2D2-VH-VL (obtained from the preparing and purifying in Example 1);

(46) Medium: DMEM (10% FBS, 5% double antibody) (Gibco), T cell medium (Gibco);

(47) Reagent preparation: Assay Buffer was incubated at 37° C. 12 ml Assay Buffer is taken into a Substrate Mix bottle to prepare the CytoTox96® Reagent.

(48) 1.2 Experiment Procedure:

(49) (1) Target cells (MDA-MB-231/SMCC7721) is taken and added into a 96-well plate at 100 μl/4×10.sup.3/well;

(50) Effector cells PBMC is taken and added into a 96-well plate at 100 μl/2×10.sup.5/well (i.e., effector-target ratio is 50:1);

(51) The drug was prepared in 3 ladder gradients: 1.2 μg/ml, 0.12 μg/ml, and 0.012 μg/ml;

(52) The Design of LDH release control group (6 replicate wells was set up in each group):

(53) (a) Culture solution control group: pure serum-free 1640 culture solution;

(54) (b) LDH low release group: target cells 4×10.sup.3/well;

(55) (c) LDH high release group: target cells 4×10.sup.3/well (treated after culturing);

(56) (d) Effector cell control group: PBMC 2×10.sup.5/well;

(57) (2) Putting the 96-well plate with cells into an incubator set at 37° C. and a volume fraction of 5% CO.sub.2 to culture overnight (18-24 hours);

(58) (3) After the culture is ended, adding lysate (Lysis Solution 10×) to the LDH high release group at 1l/O well, and incubating at 37′C for 45-60 min;

(59) (4) Transferring cells of each well to an EP tube, and centrifuging at 1000 rpm for 5 min;

(60) (5) 50 μl of supernatant obtained by the aforesaid centrifugation was taken and added into an ELISA plate and added with 50 μl of CytoTox® Reagent to each well of ELISA plate, and then incubated at room temperature for 30 min;

(61) (6) Adding 50 μl of Stop Solution to each well to terminate the reaction;

(62) (7) Using a microplate reader to detect the absorbance value of the sample obtained in the step (6) at 492 nm.

(63) 1.3 Experimental Results:

(64) Calculation formula of killing activity of tumor cells is:
% cytotoxicity=(experimental group−LDH low release group−effector cell control group)/(LDH low release group−LDH low release group)×100%

(65) The experimental result is as shown in FIG. 4. As shown in FIG. 4, the drug (bispecific antigen binding construct protein Slit2D2-VH-VL prepared and purified in Example 1) show significant killing activity in both liver cancer cell SMCC-7721 and breast cancer cell MDA-MB-231 cells, and the killing activity of the drug was increased with the drug concentration.

Example 3

(66) 1.1 Experimental Design

(67) The experiment was divided into three groups: 1. blank control group (a group free from PBMCinoculation), 2. solvent control group, 3. sample group. The design of the experiment is as shown in Table 1.

(68) TABLE-US-00001 TABLE 1 Experimental Design Number Testing Method Drug of Testing sample of delivery Groups animals sample dosage(mg/kg) administering cycle 1 4 — — — 2 8 Solvent 0 i.v qd × 7 day control 3 8 ZD016 1 i.v qd × 7 day Note: i.v: tail vein administration; qd: one administering per day; ZD016: Slit2D2-VH-VL, which was prepared and purified in Example 1.

(69) 1.2 Experimental Materials

(70) 1.2.1 Experimental Animals: 20 NOD/SCID mice, male, 5-6 weeks (the age at which tumor cell inoculation is carried out, in weeks), weighed 19.3-21.7 g. The mice are purchased from Beijing Huafukang Biotechnology Co., Ltd., and the animal certificate number is 11401300037778. The mice are maintained under SPF level environment.

(71) 1.2.2 Experimental Sample: ZD016, packaging specification: 0.65 mg/ml×0.5 ml×3 vials, sealed preservation at −80° C.

(72) 1.2.3 Cells: PBMC, packaging specification: 5 ml×1 vial, sealed preservation at −80° C., 9×10.sup.7 cells are obtained upon recovery, and the cell survival rate is 94.2%.

(73) 1.3 Experimental Methods and Steps

(74) 1.3.1 Preparation of Test Drug

(75) Solvent control group: PBS, stored at 4° C.;

(76) Sample group: 0.554 ml of ZD016 was taken and added with 3.046 ml of PBS, and the mixture was vortexed evenly to give 3.6 ml of 0.1 mg/ml solution. The solution was filtered and ready for use.

(77) All operations were performed on ice and stored at −80° C.

(78) 1.3.2 Cells: MHCC97H cells were cultured in the DMEM medium containing 10% fetal bovine serum. MHCC97H cells in the exponential growth phase were collected, and resuspended in PBS to a suitable concentration and then mixed with the PBMCat 1:1, and then matrigel was added at a same volume for injecting tumors subcutaneously into mice.

(79) 1.3.3 Animal Modeling and Grouping: 20 male mice were intraperitoneally injected with CD122 (0.2 mg/mouse) on the day before inoculation, and 5×10.sup.6MHCC97H+5×10.sup.6 PBMC cells were inoculated subcutaneously on the right side on the day of inoculation. The animals were randomly grouped in the dosing on the day of inoculation (see Table 1).

(80) 1.3.4 Experimental Observation: After tumor inoculation, routine monitoring includes the effects of the tumor growth and the treatment on normal behavior of animals, and Specifically, the contents of which include the activity of the experimental animals, eating and drinking, weight gain or loss (the weight was measured 3 times per week), eyes, coat and other abnormal conditions. The clinical symptoms observed during the experiment were all recorded in the raw data.

(81) The experimental protocols for animal experiments in this experiment were reviewed and approved by Animal Ethics Committee. During the experiment, the animal experiments were all carried out according to the requirements of AAALAC.

(82) 1.3.5 Determination of Treatment Based on Experimental Results

(83) Relative tumor inhibition rate TGI (%): TGI=1−T/C (%). T/C % is the relative tumor growth rate, i.e., the percentage of the relative tumor volumes of the treatment group and the control group at a certain time point, wherein T and C are the relative tumor volumes (RTV) of the treatment group and the control group at a certain time point, respectively.

(84) The calculation formula is as follows: T/C %=T.sub.RTV/C.sub.RTV*100% (T.sub.RTV: mean RTV of the treatment group; C.sub.RTV: mean RTV of the solvent control group; RTV=V.sub.t/V.sub.0, V.sub.0 is tumor volume of the animal at the time of grouping, and V.sub.t is tumor volume of the animal after treatment).

(85) 1.3.6 Experimental Termination Point

(86) When a single tumor volume exceeds 3000 mm.sup.3 in one single animal, or the average tumor volume exceeds 2000 mm.sup.3 in a group of animals, or when the animals were at agonal stage, the single or whole group of animals was euthanized.

(87) In the experiment, on the 25th day after grouped administration (on the 25th day after inoculation), the mean tumor value of the non-PBMC-inoculated group reached 1464 mm.sup.3, and the mean tumor value of the solvent control group reached 776 mm.sup.3. After the tumor volume was recorded, the mice were euthanized, and then the experiment was terminated. No tissue was collected.

(88) 1.4 Experimental Result

(89) 1.4.1 Result of Anti-tumor Effect of Test Drug ZD016 in the MHCC97H+PBMC Tumor Model

(90) The Tumor growth of the treatment group and the control group is as shown in Table 2 and FIG. 5.

(91) The average tumor volume of the completely blank control group (the group free of PBMCinoculation) was 1464 mm.sup.3 25 days after grouped administration, and the average tumor volume of the solvent control group was 776 mm.sup.3 25 days after grouped administration. The average tumor volume of the test group ZD016 (1 mg/kg, qd×7 days) was 421 mm.sup.3 25 days after grouped administration, and the relative tumor inhibition rate TGI (%) was 45.76%.

(92) TABLE-US-00002 TABLE 2 TGI and T/C values of each experimental group in the MHCC97H + PBMC tumor model 19 days after grouped administration Tumor P Vlaue Experimental volume (compared with group (χ ± S, mm.sup.3) TGI T/C(%) the control group) The first group: 1464 ± 127  — — — completely blank control group The second 776 ± 375 — — — group: solvent control group The third group 421 ± 243 45.76 54.24 0.4400 ZD016(1 mg/kg)

(93) 1.4.2 Safety Study Results of the Test Drug ZD016 in the MHCC97H+PBMC Tumor Model

(94) The test drug ZD016 (1 mg/kg, qd×7 days) treatment group showed no animal death, and no obvious drug toxicity, and said drug was well tolerated during the treatment.

(95) The weight changes in the treatment group and the control group upon administration are as shown in FIG. 6.

(96) The above only shows the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modifications, equivalents, and the like made within the spirit and principles of the present invention should be included in the scope of the present invention.