CN116178546B

CN116178546B - Multifunctional recombinant antibody and preparation method and application thereof

Info

Publication number: CN116178546B
Application number: CN202211255697.8A
Authority: CN
Inventors: 娄竞; 陈建鹤; 苏冬梅; 靳征; 吕云英; 张若兰; 裴若辰; 欧艳梅; 曲啸; 谢写; 张静; 曾淋
Original assignee: Shenzhen Bestone Technology Development Co ltd
Current assignee: Shenzhen Bestone Technology Development Co ltd
Priority date: 2022-10-13
Filing date: 2022-10-13
Publication date: 2024-06-18
Anticipated expiration: 2042-10-13
Also published as: CN116178546A; WO2024077774A1

Abstract

The invention discloses a multifunctional recombinant antibody and a preparation method and application thereof, and the invention not only provides a monoclonal antibody capable of specifically recognizing human CD73 protein, but also provides a humanized recombinant antibody thereof, and the multifunctional antibody which can recognize human CD73 and has IL15 function can be obtained by further reconstruction on the basis, so that the killing effect of a primary antibody on tumor cells can be effectively enhanced. Meanwhile, the invention also obtains a modified specific CD73 monoclonal antibody-IL 15 bifunctional molecule by introducing a mutated human IgG1 constant region, which can effectively reduce the toxicity of the antibody and improve the safety. The antibody provided by the invention can identify CD73 protein in various tumor cells, further has a killing effect on various tumor cells, and meanwhile, the reconstructed multifunctional recombinant antibody has the advantages of short medicament metabolism period, low toxicity and high safety, and has a huge application prospect.

Description

Multifunctional recombinant antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, relates to a multifunctional recombinant antibody, a preparation method and application thereof, and in particular relates to a multifunctional recombinant antibody capable of recognizing CD73 and having an IL15 function and application thereof.

Background

Antibody drugs have become a major component of anticancer drugs at present, but in terms of solid tumors, the efficacy of antibody drugs is still limited, so it is important to find more novel solid tumor antigens, but it is also relatively difficult. For solid tumor treatment, it is important to overcome the improved therapeutic effect by relieving the inhibition of immune effector cells by Tumor Microenvironment (TME). CD73 is a 5-primary nucleotide hydrolase that hydrolyzes extracellular Adenosine Monophosphate (AMP) to adenosine, which is a powerful immunosuppressive molecule that inhibits activation of CD8 positive T cells, thereby helping cancer cells escape T cells "chase-killing", which can be said to be one of the key components of immunosuppressive microenvironment formation in cancer. CD73 is reported to be expressed in many different cancers, including colon, lung, pancreas, ovary, bladder, leukemia, glioma, glioblastoma, melanoma, thyroid, esophageal, prostate, and breast cancers, among others. Furthermore, CD73 expression in cancer is associated with cell proliferation, migration, neovascularization, invasiveness, metastasis, and shorter survival in patients. CD73 activity has also been proposed as a prognostic marker for papillary thyroid cancer. While CD73 has been shown to regulate tumor cell-to-cell and cell-to-matrix interactions, CD73 expression and activity has also been associated with reduced T cell responses and has been proposed to be involved in drug resistance. Thus, CD73 can directly and indirectly regulate the development of cancer, which embodies its potential as a new therapeutic target, and thus inhibition of its activity by regulation and the development of related therapeutic agents are highly desirable.

IL15 is a cytokine expressed by a variety of cells, including monocytes, macrophages, epidermal cells, fibroblasts, etc., but not T lymphocytes. Unlike many other cytokines, IL15 generally does not secrete cells to function, but rather, in combination with IL15 ra localizes to specific cell membranes, thereby stimulating nearby effector cells, principally NK and cd8+ T cells. IL15 is closely related to IL2, and IL15Rα can bind to the common beta/gamma receptor of IL2 after forming a complex, mediating the biological activity. One advantage of IL15 over IL2 in terms of anti-tumor effect is that IL 15/ra does not stimulate Treg proliferation. Currently, there are a number of IL 15-related molecules that are used in the research and development stage to treat malignant tumors. Among the fastest-evolving ones is ALT-803, a complex whose molecule is formed by IL15 and IL15Rα sushi-hFc 1. Several clinical studies have shown that ALT-803 is effective against a variety of tumors including melanoma, but at the same time, serious toxic side effects, mainly including liver function damage, hypotension, fever, etc., occur.

The development of a drug which can kill various cancer cells and has small toxic and side effects has important significance.

Disclosure of Invention

In view of the above problems, an object of the present invention is to provide a monoclonal antibody capable of specifically recognizing CD73 protein, a hybridoma cell secreting the monoclonal antibody; the monoclonal antibody is subjected to humanized transformation to obtain a recombinant antibody; and multifunctional recombinant antibodies capable of recognizing human CD73 protein and having IL15 function.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a monoclonal antibody capable of recognizing human CD73-ECD protein; the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO:5, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: shown at 7.

The invention prepares a mouse-derived monoclonal antibody capable of specifically recognizing human CD73 protein by immunizing mice with recombinant expressed human CD73-ECD protein, which is named as 3E10. The amino acid sequences of the heavy chain variable region and the light chain variable region of the murine monoclonal antibody are obtained through biological analysis.

Furthermore, the invention also claims a recombinant antibody which is obtained by humanizing the monoclonal antibody 3E10, and the amino acid sequence of the heavy chain variable region of the recombinant antibody is shown as SEQ ID NO:16, the amino acid sequence of the light chain variable region of the recombinant antibody is shown as SEQ ID NO: shown at 18.

As a preferred embodiment of the present invention, the heavy chain amino acid sequence of the recombinant antibody is shown in SEQ ID NO:17, the light chain amino acid sequence of the recombinant antibody is shown as SEQ ID NO: 19.

The inventor of the present invention further analyzes the obtained murine monoclonal antibody 3E10 sequence, replaces the CDR region of the human template, recombines the heavy chain variable region with the human IgG1 constant region, recombines the light chain variable region with the human kappa chain constant region, and simultaneously, based on the three-dimensional structure of the antibody, carries out back mutation on embedded residues, residues directly interacted with the CDR region and residues having important influence on the conformation of VL and VH of each antibody, and finally obtains the humanized recombinant antibody, which is named hu3E10. The amino acid sequence, heavy chain amino acid sequence and light chain amino acid sequence of the humanized recombinant antibody hu3E10 heavy chain variable region and light chain variable region are shown in the description.

Further, the present invention also claims a multifunctional recombinant antibody whose heavy chain comprises an antibody functional region that recognizes human CD73, a human IgG1 constant region functional region, an IL15 functional region, and a nonfunctional amino acid fragment for linking the functional regions; the amino acid sequence of the antibody functional region that recognizes human CD73 comprises the heavy chain variable region amino acid sequence of the recombinant antibody.

The IL15 domain recognizes the functional domain of the human IL2/IL15 beta/gamma receptor.

The inventors of the present invention further modified the humanized antibody obtained as described above, and connected the heavy chain sequence of the antibody to a sequence having an IL15 function, thereby further obtaining a recombinant antibody capable of recognizing human CD73 protein and having an IL15 function.

As a preferred embodiment of the invention, the human IgG1 constant region functional domain is a human IgG1 constant region and has the amino acid sequence shown in SEQ ID NO: shown at 8.

More preferably, the human IgG1 constant region functional domain is a mutated human IgG1 constant region having an amino acid sequence as set forth in SEQ ID NO: shown at 24.

The inventor of the invention uses the sequence of the mutant human IgG1 constant region to obtain the multifunctional recombinant antibody with shorter metabolism period, lower toxic and side effects and better safety.

As a preferred embodiment of the invention, the amino acid sequence of the human IL15 functional region is shown in SEQ ID NO: 21.

More preferably, the amino acid sequence of the human IL15 functional region is as set forth in SEQ ID NO: shown at 22.

SEQ ID NO:22 is that human IL15Rsushi is linked to human IL15 by (GGGGS) ₆ to form single chain IL15, i.e., IL15sc.

In a preferred embodiment of the present invention, the non-functional amino acid fragment for linking each functional region in the multifunctional recombinant antibody is a GGGGS repeat.

More preferably, the GGGGS repeat is (GGGGS) ₃.

As a preferred embodiment of the invention, the amino acid sequence of the functional domain of the human IgG1 constant region of the multifunctional recombinant antibody is shown in SEQ ID NO:8, 8; the heavy chain amino acid sequence of the multifunctional recombinant antibody is shown as SEQ ID NO: indicated at 23; the amino acid sequence of the light chain of the multifunctional recombinant antibody is shown as SEQ ID NO: 19.

The invention prepares a multifunctional antibody which can recognize CD73 and has IL15 function and is named SPGL003.

More preferably, the human IgG1 constant region functional domain of the multifunctional recombinant antibody is a mutated human IgG1 constant region, and the amino acid sequence of the mutated human IgG1 constant region is as shown in SEQ ID NO: shown at 24; the heavy chain amino acid sequence of the multifunctional recombinant antibody is shown as SEQ ID NO: 26; the amino acid sequence of the light chain of the multifunctional recombinant antibody is shown as SEQ ID NO: 19.

The multifunctional recombinant antibody prepared by utilizing the preferred multifunctional recombinant antibody sequence comprises a sequence of a mutated human IgG1 constant region, and is named SPGL004; the multifunctional recombinant antibody SPGL004 not only can effectively identify CD73 in various tumor cells and effectively inhibit the growth of various cancer cells, but also has shorter half-life, lower toxicity and good safety.

Further, the invention also claims nucleotide sequences encoding said monoclonal antibodies, or said recombinant antibodies, or said multifunctional recombinant antibodies.

According to the amino acid sequence of the monoclonal antibody, the recombinant antibody or the multifunctional recombinant antibody, the nucleotide sequence of the corresponding coding gene can be obtained.

As a preferred embodiment of the present invention, the heavy chain variable region nucleotide sequence of the monoclonal antibody is shown in SEQ ID NO:4, the nucleotide sequence of the light chain variable region is shown as SEQ ID NO: shown at 6.

Further, the present invention also claims an expression vector comprising the nucleotide sequence.

By means of molecular biology, expression vectors containing the nucleotide sequences can be constructed.

Further, the invention also claims host cells comprising said expression vectors.

By means of cell biology, host cells capable of efficiently expressing the antibody proteins can be constructed using the expression vectors.

Further, the invention also claims the monoclonal antibody, or the recombinant antibody, or the multifunctional recombinant antibody; or the nucleotide sequence, or the expression vector, or the host cell, in the preparation of a biological agent for the treatment of tumors.

Further, the invention also claims a biological agent comprising said monoclonal antibody, or said recombinant antibody, or said multifunctional recombinant antibody; or at least one of said nucleotide sequence, or said expression vector, or said host cell.

Further, the invention also provides a preparation method of the monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody, which comprises the following steps:

(1) Obtaining an expression vector containing the gene fragment of the monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody through an artificial synthesis or molecular biological method;

(2) Transfecting the expression vector into cells for protein expression;

(3) The monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody is obtained through protein purification.

The corresponding antibody can be obtained by respectively constructing expression vectors containing heavy chain or light chain sequences of the antibody, transfecting proper cells for expression and purification.

As a preferred embodiment of the present invention, in the step (1), the expression vector for preparing the gene fragment containing the monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody by molecular biology is: the heavy or light chain nucleotide sequence of the antibody was inserted into pcdna3.4 expression vector.

As a preferred embodiment of the present invention, in the step (2), the cell for protein expression is an Expi-293F cell.

As a preferred embodiment of the present invention, the Protein purification method in the step (3) is purification by Protein G.

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

The invention not only prepares a monoclonal antibody capable of specifically recognizing human CD73 protein, but also provides a humanized recombinant antibody thereof. On the basis, the inventor further utilizes the CD73 specific recombinant antibody to prepare the multifunctional recombinant antibody which can not only recognize human CD73, but also has the IL15 function, and can effectively enhance the killing effect of the original antibody on tumor cells. Furthermore, the invention also obtains a modified specific CD73 monoclonal antibody-IL 15 difunctional molecule by introducing a mutated human IgG1 constant region, and the molecule can effectively shorten the metabolism period while maintaining the anti-tumor activity in vivo, thereby reducing the toxic and side effects in vivo and greatly improving the safety. The multifunctional recombinant antibody provided by the invention can identify CD73 in various tumor cells, and has a huge application prospect.

Drawings

FIG. 1 results of binding of antibody 3E10 to human CD 73.

FIG. 2 results of antibody 3E10 inhibiting the activity of NCI-H1975 cell CD73 enzyme.

FIG. 3 results of binding of antibody 3E10 to mouse, cynomolgus monkey CD73 antigen.

FIG. 4 results of binding of human antibody hu3E10 to human CD73

FIG. 5 results of inhibition of tumor cell membrane CD73 enzymatic activity by human antibody hu3E 10.

FIG. 6 results of binding of multifunctional recombinant antibodies SPGL003, SPGL004 to human CD 73.

FIG. 7 results for the simultaneous binding of CD73 and CD122/132 by each set of antibodies.

FIG. 8 multifunctional recombinant antibody SPGL003, SPGL004 inhibited tumor cell membrane CD73 enzyme activity.

FIG. 9 results of stimulation of CTLL2 cell proliferation by multifunctional recombinant antibodies SPGL003, SPGL 004; a is the detection result of SPGL003 and SPGL004, and B is the detection result of IL 2.

FIG. 10 shows the results of the change in volume of the mouse colorectal cancer cell MC38 engraftment tumor of each group over time.

FIG. 11 shows the results of the change in the volume of human lung cancer cell H1975 transplants over time.

FIG. 12 results of inhibition of human breast cancer cell JIMT-1 engraftment by the multifunctional recombinant antibody SPGL004 in combination with HER2 mab.

FIG. 13 in vivo metabolism of multifunctional recombinant antibodies SPGL003 and SPGL004 in huFcRn transgenic mice.

FIG. 14 results of flow-through detection of SPGL004 binding to various tumor cells CD 73.

FIG. 15 multifunctional recombinant antibody SPGL004 placed on 0 periprotein.

FIG. 16 multifunctional recombinant antibody SPGL004℃placed at 5 periprotein.

FIG. 17 multifunctional recombinant antibody SPGL004 placed at 37℃for 5 periprotein.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

EXAMPLE 1 preparation of monoclonal antibodies and hybridomas

1) Antigen immunized mice: human CD73-ECD protein (commercially available from Beijing Bai Pogostemon Co., acro BIOSYSTEMS, SEQ ID NO: 1) was used for recombinant expression.

On the first day, after fully mixing the soluble human CD73-ECD protein with Freund's complete adjuvant (CFA, sigma Co.) emulsion, balb/c mice were subcutaneously injected (50. Mu.g/mouse); after fully mixing the soluble human CD73-ECD protein with Freund's incomplete adjuvant emulsion (IFA, sigma Co.) on days 14 and 36, balb/c mice were subjected to subcutaneous booster immunization (50. Mu.g/mouse); on day 50, the mice spleen was taken for fusion experiments 3-4 days after intraperitoneal injection of human CD73-ECD protein 50 μg/mouse challenge.

2) Preparation and selection of hybridomas:

The spleen cells of the mice were fused with myeloma cells SP2/0 of the mice by PEG (PEG 1450, sigma company) method using conventional hybridoma technique 3-4 days after the last impact immunization of the mice. The fused cells were suspended uniformly in complete medium, i.e. RPMI1640 and DMEM F12 medium 1: 1% glutamine (Gibco Co.), 1% sodium pyruvate (Gibco Co.), 1% MEM-NEAA (minimum minimal medium-non-essential amino acid solution, gibco Co.), 1% penicillin-streptomycin (Gibco Co.), 50. Mu.M beta-mercaptoethanol (Gibco Co.) and 20% FBS (Gibco Co.). The cells were plated in 96-well plates at 10 ⁵ cells/100. Mu.L/well overnight. The next day, 100. Mu.L of complete medium containing 2 XHAT (Sigma Co.) was added to each well, and the volume of medium in the 96-well plate was 200. Mu.L/well (containing 1 XHAT). After 7-12 days, supernatants were harvested and hybridoma wells positive for human CD73-ECD protein binding activity were screened by indirect enzyme-linked immunosorbent assay (ELISA).

Wherein, the method for screening the hybridoma holes positive for the activity of the human CD73-ECD protein by using the indirect ELISA method comprises the following steps: recombinant human CD73-ECD protein was diluted to 1. Mu.g/mL with coating solution (50 mM carbonate coating buffer, pH 9.6), 100. Mu.L/well was added to the ELISA plate and coated overnight at 4 ℃. PBST plate washing 3 times, adding 200 u L/hole blocking solution (2%BSA-PBST), 37 ℃ after 1h PBST plate washing 1 time for use. The collected hybridoma supernatants were sequentially added to the blocked ELISA plates at 100. Mu.L/well and left at 37℃for 1 hour. PBST plates were washed 3 times, HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Abcam, cat. No. ab 6789) was added and left at 37℃for 30min; after PBST washing the plate for 5 times, the residual liquid drops are beaten as much as possible on the absorbent paper, 100 mu L TMB (KPL company) is added into each hole, and the plate is placed for 5 minutes at room temperature (20+/-5 ℃) in a dark place; and adding 50 mu L of 2M H ₂SO₄ stop solution into each well to stop the substrate reaction, reading an OD value at 450nm of an enzyme-labeled instrument, and analyzing the binding capacity of the antibody to be detected and the target antigen human CD73-ECD protein.

Amplifying and screening the obtained hybridoma cell strain in a serum-containing complete culture medium, centrifuging and changing the solution to a serum-free culture medium (SFM culture medium) to enable the cell density to be 1-2 multiplied by 10 ⁷/mL, culturing for 2 weeks at the temperature of 5% CO ₂ and 37 ℃, centrifuging to obtain a culture supernatant, purifying by Protein G affinity chromatography, and obtaining the mouse anti-human CD73-ECD Protein monoclonal antibody which is named as 3E10.

Example 2ELISA assay for binding Activity of 3E10 to human CD73

The experimental steps are as follows: human CD73 was pre-coated and diluted with coating solution (50 mM carbonate coating buffer, pH 9.6) to 5. Mu.g/mL plates overnight at 4 ℃; then the mixture is sealed by 5% skimmed milk powder at 37 ℃ for 2 hours. PBST plate was washed 3 times, and the antibody to be tested diluted with 1% BSA-PBST gradient was sequentially added to the blocked ELISA plate, 100. Mu.L/well, and left at 37℃for 1h. PBST plates were washed 3 times, HRP-labeled goat anti-mouse IgG secondary antibody (available from Millipore under the accession number AP 181P) was added, and left at 37℃for 30min; after PBST washing the plate for 3 times, the residual liquid drops are beaten as much as possible on the absorbent paper, 100 mu L TMB (KPL company) is added into each hole, and the plate is placed for 5min at room temperature (20+/-5 ℃) in a dark place; and adding 50 mu L of 2M H ₂SO₄ stop solution into each well to stop the substrate reaction, reading an OD value at 450nm of an enzyme-labeled instrument, and analyzing the binding capacity of the antibody to be detected and the target antigen human CD 73.

The experimental results are shown in FIG. 1. As can be seen from FIG. 1, the EC ₅₀ for antibody 3E10 binding CD73-ECD was 29.83ng/mL, i.e., 0.20nM; it was demonstrated that antibody 3E10 had good binding activity to CD 73-ECD.

EXAMPLE 3 murine antibody 3E10 inhibits NCI-H1975 cell CD73 enzymatic Activity

CD73 expressed by tumor cells has enzymatic activity and can decompose AMP into Ado. AMP can inhibit the chemiluminescent reaction caused by ATP, but Ado does not. Thus, the activity of CD73 can be detected by detecting the consumption of AMP, thereby detecting the inhibition of the activity of CD73 enzyme by monoclonal antibodies.

The experimental steps are as follows: human tumor cells NCI-H1975 were seeded at 30000 cells/well in 96-well cell culture plates (Corning, cat # 3599), incubated overnight in a CO ₂ cell incubator, the next day, the culture supernatant was aspirated off, the mab or control mab to be tested was added, 50. Mu.L/well, serial dilutions (10000 ng/mL starting, 10 gradients total at 3-fold dilution, lowest concentration to 0.5 ng/mL), incubated for 30min at 37℃and then added with 800. Mu.M AMP (Sigma, cat#A1752) solution in 50. Mu.L/well serum-free medium, incubated for 3H at 37 ℃. mu.L/well of the cell reaction solution was pipetted into a fluorescence detection plate (White Opaque 96-well Microplate, perkinelmer, cat# 6005290), then 25. Mu.L/well of 80. Mu.M ATP solution prepared without serum medium was added, 50. Mu.L/well CTG solution (CELLTITER GLO, promega, cat#G 7573) was added, and after incubation at room temperature for 5 minutes, the fluorescence value was detected by a chemiluminescent detector (BioTech).

The experimental results are shown in FIG. 2. The IC ₅₀ for antibody 3E10 to inhibit the CD73 enzymatic activity of tumor cell NCI-H1975 was 39.89ng/mL, i.e., 0.27nM.

Example 4 determination of Cross Activity of antibody 3E10 with mouse, cynomolgus monkey CD73 antigen

Experimental procedure similar to example 2, human CD73-ECD was replaced with mouse CD73-ECD (amino acid sequence shown as SEQ ID NO: 2) and cynomolgus monkey CD73-ECD (amino acid sequence shown as SEQ ID NO: 3), respectively, both purchased from Beijing Baiposis corporation. The binding capacity of antibody 3E10 to the mouse, cynomolgus monkey CD73 antigen was determined.

The experimental results are shown in FIG. 3. As can be seen from fig. 3, antibody 3E10 binds very well to cynomolgus monkey CD73 protein with EC ₅₀ of 33.63ng/mL, i.e. 0.22nM, comparable to human CD73 (EC ₅₀ of 0.20 nM); but mab 3E10 did not bind to mouse CD73.

EXAMPLE 5 preparation of humanized antibody hu3E10

The heavy chain variable region and the light chain variable region of hybridoma 3E10 were obtained by a molecular biology-related method, and a chimeric antibody was further constructed.

RNA from 3E10 hybridoma cells was extracted by Trizol and mRNA was reverse transcribed to obtain cDNA, which was then used as a template to carry out PCR with heavy and light chain degenerate primers of murine antibodies, respectively (Antibody Engineering, volume 1,Edited by Roland Kontermann and Stefan D ubel, combined primer sequences from page 323), the PCR products obtained were sequenced and analyzed by kabat database to determine the variable region sequence of murine antibodies. The relevant sequence information is as follows:

The gene sequence of the 3E10 heavy chain variable region has the total length of 366bp, codes 122 amino acid residues and has the nucleotide sequence shown in SEQ ID NO:4, the amino acid sequence is shown as SEQ ID NO:5 is shown in the figure; the gene sequence of the 3E10 light chain variable region has the total length of 333bp and codes 111 amino acid residues, and the nucleotide sequence is shown as SEQ ID NO:6, the amino acid sequence is shown as SEQ ID NO: shown at 7.

The amino acid sequences of the light chain variable region and the heavy chain variable region of the 3E10 antibody were analyzed to determine 3 Complementarity Determining Regions (CDRs) and 4 Framework Regions (FRs) of the murine antibody according to the Kabat rule. The amino acid sequence of the 3E10 heavy chain complementarity determining region is HCDR1: GYSFTSYW (SEQ ID NO: 10), HCDR2: IYPGNSDT (SEQ ID NO: 11), HCDR3: TREGSKGYDVWYFAV (SEQ ID NO: 12), the amino acid sequence of the light chain complementarity determining region being LCDR1: KSVSTSGYSY (SEQ ID NO: 13), LCDR2: LASNLES (SEQ ID NO: 14) and LCDR3: QHSRELPFT (SEQ ID NO: 15).

The humanized template that best matches the FR region of each murine antibody described above was selected from the Germline database. Then the CDR region of the murine antibody is transplanted onto the selected humanized template, the CDR region of the humanized template is replaced, the heavy chain variable region is recombined with the human IgG1 constant region (SEQ ID NO: 8), the light chain variable region is recombined with the human kappa chain constant region (SEQ ID NO: 9), and on the basis of the three-dimensional structure of the antibody, the embedded residues, the residues directly interacted with the CDR region and the residues having important influence on the conformation of the VL and VH of each antibody are subjected to back mutation, so that the humanized antibody hu3E10 is finally obtained.

The humanized antibody hu3E10 (the heavy chain variable region amino acid sequence is shown as SEQ ID NO:16, the heavy chain amino acid sequence is shown as SEQ ID NO:17, the light chain variable region amino acid sequence is shown as SEQ ID NO:18, and the light chain amino acid sequence is shown as SEQ ID NO: 19).

The heavy chain and light chain of humanized antibody hu3E10 are respectively constructed to pcDNA3.4 expression vectors, an Expi-293F cell is transfected, humanized antibody hu3E10 is obtained through Protein G purification, and the molecular weight of each antibody is determined to be correct and the purity is more than 95 percent through SDS-PAGE electrophoresis and SEC-HPLC.

EXAMPLE 6ELISA method for determining the binding Activity of humanized antibody hu3E10 to human CD73

Experimental method referring to example 2, the binding activity of humanized antibody hu3E10 to human CD73 was measured and the experimental results are shown in fig. 4.

As shown in FIG. 4, humanized antibody hu3E10 bound human CD73 with EC ₅₀ at 5.44ng/mL, i.e., 0.04nM, significantly lower than antibody 3E10 (with EC ₅₀ at 29.83ng/mL, i.e., 0.20nM, as shown in example 2). It was demonstrated that humanized antibody hu3E10 had good binding activity to human CD 73.

EXAMPLE 7 humanized antibody hu3E10 inhibition of tumor cell membrane CD73 enzymatic Activity assay

Experimental method referring to example 3, the effect of the humanized antibody hu3E10 on inhibiting the CD73 enzyme activity of tumor cell membrane was measured, and the experimental result is shown in fig. 5.

As can be seen from FIG. 5, the humanized antibody hu3E10 can inhibit the enzymatic activity of tumor cell CD73 with high efficiency, and the IC ₅₀ is 27.81ng/mL, namely 0.19nM, and the activity is higher than that of mouse monoclonal antibody 3E10 (IC ₅₀ 0.27 nM). The humanized antibody hu3E10 was shown to inhibit tumor cell membrane CD73 enzymatic activity.

Example 8 preparation of multifunctional recombinant antibodies

1. Functional region of human IL 15: human IL15Rsushi (amino acid sequence SEQ ID NO: 20) and human IL15 (amino acid sequence SEQ ID NO: 21) were joined by (GGGGS) ₆ to form single chain IL15, IL15sc, amino acid sequence SEQ ID NO: shown at 22.

2. Antibody functional region that recognizes human CD 73:

① Heavy chain of SPGL 003: splicing the hu3E10 heavy chain sequence (the amino acid sequence is shown as SEQ ID NO: 17) with the human IL15sc sequence through (GGGGS) ₃ to form a heavy chain of SPGL003 (the amino acid sequence is shown as SEQ ID NO: 23);

② Heavy chain of SPGL 004: the hu3E10 heavy chain VH sequence (amino acid sequence shown as SEQ ID NO: 16) was linked to a mutated human IgG1 constant region (amino acid sequence shown as SEQ ID NO: 24) to form a hu3E10mu heavy chain (amino acid sequence shown as SEQ ID NO: 25); the hu3E10mu heavy chain was spliced with the human IL15sc sequence by (GGGGS) ₃ to form the heavy chain of SPGL004 (amino acid sequence shown as SEQ ID NO: 26).

③ The light chain is identical to the light chain sequence of hu3E10 (the amino acid sequence is shown in SEQ ID NO: 19).

3. Preparation of multifunctional recombinant antibodies

The heavy chain and the light chain of SPGL003 (or the heavy chain and the light chain of SPGL 004) are transfected into an Expi-293F cell in a cotransfection way, and the multifunctional recombinant antibody SPGL003 (or the multifunctional recombinant antibody SPGL 004) can be obtained through Protein G purification. The molecular weight of each expressed antibody was determined to be correct by SDS-PAGE electrophoresis and SEC-HPLC, and the purity of the antibody was >95%. The prepared multifunctional antibody is quantitatively packaged and frozen at-80 ℃ for standby.

EXAMPLE 9 multifunctional recombinant antibody SPGL004 binds CD73-ECD

Experimental methods referring to example 2, the binding activity of the multifunctional recombinant antibodies SPGL003 and SPGL004, respectively, to human CD73-ECD was measured, and the experimental results are shown in FIG. 6.

As can be seen from FIG. 6, the EC ₅₀ of SPGL004 and SPGL003 binding to human CD73-ECD was 31.42ng/mL and 34.26ng/mL, respectively, i.e., 0.16nM and 0.18nM, consistent with hu3E10 (0.20 nM, as shown in example 6), demonstrating that hu3E10 did not affect binding to CD73 after forming a bifunctional molecule with IL15, and that mutations in IgG1 in the bifunctional molecule did not affect binding to CD 73.

EXAMPLE 10 multifunctional recombinant antibody SPGL004 simultaneously binds CD73-ECD and CD122/132

The experimental method comprises the following steps: pre-coating human CD73-ECD protein, diluting to 2 μg/mL coating plate with coating solution (50 mM carbonate coating buffer, pH 9.6), 4 ℃ overnight; sealing with 5% skimmed milk powder at 37deg.C for 2 hr; PBST plate was washed 3 times, and the antibody to be tested was diluted to 1. Mu.g/mL with a 1% BSA-PBST gradient, and the blocked ELISA plate was added at 100. Mu.L/well and left at 37℃for 1h. PBST plates were washed 3 times, gradient diluted biotin-labeled CD122/132 (from Beijing Baipessary Co.) was added, 37℃for 1 hour, and after 3 times PBST plates were washed, diluted HRP-labeled SA (Pierce Co.) was added, and left at 37℃for 30 minutes; after PBST washing the plate for 3 times, the residual liquid drops are beaten as much as possible on the absorbent paper, 100 mu L TMB (KPL company) is added into each hole, and the plate is placed for 5min at room temperature (20+/-5 ℃) in a dark place; the substrate reaction was stopped by adding 50. Mu.L of 2M H ₂SO₄ stop solution to each well, the OD was read at 450nm by a microplate reader, and the binding capacity of the antibody to be tested to human CD122/132 was analyzed.

The experimental results are shown in FIG. 7. As can be seen in fig. 7, under the present experimental conditions, EC ₅₀ of antibodies SPGL004 and SPGL003 binding to human CD122/132 protein was 15.85ng/mL and 16.84ng/mL, i.e. 0.08nM and 0.09nM, respectively, demonstrating good activity of both multifunctional recombinant antibodies SPGL004 and SPGL003 binding to CD73 and CD122/132, respectively; in contrast, the control hu3E10 antibody, while capable of binding CD73, was unable to bind CD122/132.

EXAMPLE 11 determination of the Activity of multifunctional recombinant antibodies SPGL003, SPGL004 against tumor cell membrane CD73 enzyme

Experimental method referring to example 3, the effect of the multifunctional recombinant antibodies SPGL004 and SPGL003 on inhibiting the activity of tumor cell membrane CD73 enzyme was determined. The experimental results are shown in FIG. 8.

As can be seen from FIG. 8, antibodies SPGL004 and SPGL003 inhibited the CD73 enzyme activity of tumor cell NCI-H1975 at 50.99ng/mL and 53.08ng/mL, i.e., 0.27nM and 0.28nM, respectively, with slightly weaker enzyme inhibition than hu3E10 (EC ₅₀ of 0.19 nM).

EXAMPLE 12 multifunctional recombinant antibodies SPGL003, SPGL004 stimulated CTLL2 cell proliferation

The experimental method comprises the following steps: CTLL2 cells were diluted to 5X 10 ⁴/mL in 1640 medium containing 10% FBS and 100. Mu.L/well added to the cell culture plate. IL2 is diluted to 30ng/mL by 1640 culture solution containing 10% FBS, and after the dilution of 3 times of the culture solution is divided into 8 gradients, the culture plates containing CTLL2 cells are respectively added; SPGL004 and SPGL003 were diluted to 5000ng/mL in 1640 medium containing 10% FBS, and after 3-fold specific dilution, 8 gradients were added to each of the plates containing CTLL2 cells; after 72 hours of incubation in a CO ₂ cell incubator, the relative cell numbers of each well were determined with CCK8 and EC ₅₀ was calculated to determine the activity of the samples. The experimental results are shown in FIG. 9.

As can be seen from FIG. 9A, the multifunctional recombinant antibodies SPGL004 and SPGL003 both stimulated the proliferation of CTLL2 cells, and EC ₅₀ was 104.8ng/mL and 99.3ng/mL, i.e., 0.55nM and 0.52nM, respectively, demonstrating that SPGL004 has biological activity consistent with that of SPGL 003. As a control, as can be seen from FIG. 9B, IL2 EC ₅₀ was 0.75ng/mL, i.e., 0.049nM.

EXAMPLE 13 the multifunctional recombinant antibody SPGL004 showed significantly reduced toxicity in mice compared to SPGL003

The experimental method comprises the following steps: the multifunctional recombinant antibodies SPGL004 and SPGL003 were injected into the abdominal cavity of C57BL/6 mice (Vetong Lihua Co.) 3 times on days 1, 3 and 5, the injection volume was 0.2 mL/time, SPGL004 and SPGL003 were administered at doses of 0.5mg/kg, 1mg/kg, 2mg/kg and 4mg/kg, the death of the experimental mice was observed every day, and the control group was administered with the same volume of PBS. The results are shown in Table 1.

Table 1 survival of experimental animals in each dose group

Antibodies to	0.5mg/kg	1.0mg/kg	2.0mg/kg	4.0mg/kg
					SPGL003	100％	50％	0％	0％
SPGL004	100％	100％	100％	10％

As shown in table 1, on day 7 of the experiment, all experimental animals in the SPGL003 samples in the 4.0mg/kg group and the 2.0mg/kg group died (survival rate 0%); the 1.0mg/kg group still had 50% of the experimental animals died, while the 0.5mg/kg group had no experimental animals died; SPGL004 sample 4.0mg/kg showed 90% of the experimental animal deaths, but none of the remaining dose groups had the experimental animal deaths. These results demonstrate that SPGL004 toxicity to experimental mice is significantly reduced following Fc mutation.

EXAMPLE 14 multifunctional recombinant antibody SPGL004 inhibits mouse colorectal cancer cell MC38 transplantation tumor growth

The experimental method comprises the following steps: in vitro cultured mouse colon cancer MC38 cells were collected and the cell suspension concentration was adjusted to 1X 10 ⁷ cells/mL. Under sterile conditions, 100. Mu.L of MC38 cell suspension was inoculated subcutaneously into the right flank of C57BL/6 mice. The diameter of the transplanted tumors is measured by a vernier caliper for subcutaneous transplantation of mice, and animals are randomly grouped into 6 animals/group after the average tumor volume grows to 100-200mm ³. SPGL004 was administered at 1.0mg/kg, 0.5mg/kg, SPGL003 was administered at 0.5mg/kg, 0.25mg/kg, and the same amount of PBS was administered in the control group, 3 times per week by intraperitoneal injection, and a volume of 0.2 mL/time for 2 weeks. Throughout the experiment, the diameter of the transplanted tumor was measured 3 times per week, and the mice were weighed. The calculation formula of Tumor Volume (TV) is (wherein a, b represent length, width respectively):

TV＝1/2×a×b2。

The relative tumor volume (relative tumor volume, RTV) was calculated from the results of the measurements, calculated as (where V ₀ is the tumor volume measured at d0 of the group administration; V _t is the tumor volume at each measurement):

RTV＝V_t/V₀。

the evaluation index of the anti-tumor activity is relative tumor proliferation rate T/C (%), and the calculation formula is as follows (wherein TRTV: treatment group RTV; CRTV: negative control group RTV):

T/C(％)＝(TRTV/CRTV)×100；

Tumor inhibition rate TGI (%) =100-T/C (%).

The measurement results are shown in FIG. 10. As can be seen from FIG. 10, SPGL004 and SPGL003 both show strong antitumor activity, the TGI of SPGL003 at doses of 0.5mg/kg and 0.25mg/kg was 60.6% and 22.9%, respectively, and the TGI of SPGL004 at doses of 1.0 and 0.5mg/kg was 72.2% and 46.1%, respectively.

Referring to example 13, SPGL003 showed lethal toxicity at 1.0mg/kg dose and SPGL004 showed lethal toxicity at 4.0mg/kg dose, it was demonstrated that SPGL004 containing Fc mutation had significantly improved safety while not affecting antitumor activity in vivo.

EXAMPLE 15 multifunctional recombinant antibody SPGL004 inhibits growth of human lung cancer cell NCI-H1975 graft tumor in nude mice

The experimental method comprises the following steps: human lung cancer NCI-H1975 cells cultured in vitro were collected and the cell suspension concentration was adjusted to 8X 10 ⁷ cells/mL. Under sterile conditions, 100 μl of the cell suspension was inoculated subcutaneously into the right flank of nude mice. After tumor cells formed solid tumors subcutaneously in mice, the diameters of the transplanted tumors were measured by vernier calipers, and animals were randomly grouped after the average tumor volume had grown to 100-200mm ³. The multifunctional recombinant antibody SPGL004 was administered 3 times per week and 6 times per week at doses of 1.0mg/kg, 0.3mg/kg and 0.1 mg/kg. Throughout the experiment, the diameter of the transplanted tumor was measured 2 times per week while weighing the mice. The procedure is as in example 14. The results are shown in FIG. 11.

As can be seen from fig. 11, SPGL004 exhibited strong antitumor activity in human lung cancer cells. SPGL004 had TGI of 59.5%, 58.3% and 44.3% at doses of 1.0mg/kg, 0.3mg/kg and 0.1mg/kg, respectively.

EXAMPLE 16 multifunctional recombinant antibody SPGL004 in combination with HER2 mab inhibit growth of human breast cancer cell JIMT-1 graft tumor in nude mice

The experimental method comprises the following steps: human breast cancer JIMT-1 cells cultured in vitro were collected, and the cell suspension concentration was adjusted to 8X 10 ⁷ cells/mL. Under sterile conditions, 100 μl of the cell suspension was inoculated subcutaneously into the right flank of nude mice. After tumor cells formed solid tumors subcutaneously in mice, the diameters of the transplanted tumors were measured by vernier calipers, and animals were randomly grouped after the average tumor volume had grown to 50-100mm ³. The single drug group HER2 mab (trastuzumab) was administered at a dose of 20mg/kg, and the combination group was given 6 times per week by fixed doses of 20mg/kg trastuzumab in combination with 1.0mg/kg and 0.3mg/kg SPGL004, respectively. Throughout the experiment, the diameter of the transplanted tumor was measured 2 times per week while weighing the mice. The procedure is as in example 14. The results are shown in FIG. 12.

As can be seen from fig. 12, the TGI of Qu Tuo bead single drug was 55.3%, the TGI of the trastuzumab with 1.0mg/kg and 0.3mg/kg dose of SPGL004 was 90.3% and 71.7%, respectively, significantly higher than Qu Tuo bead single drug, demonstrating that SPGL004 and Qu Tuo beads have good synergistic antitumor effect.

EXAMPLE 17 in vivo metabolism of multifunctional recombinant antibodies SPGL003 and SPGL004 in huFcRn transgenic mice

The experimental method comprises the following steps: the pharmacokinetics of SPGL003 and SPGL004 were determined using human FcRn transgenic mice. 8 mice were divided into two groups, and SPGL003 and SPGL004 were intraperitoneally injected at a dose of 1mg/kg, and blood was collected after 2 hours, 6 hours, 24 hours, 48 hours and 72 hours, to obtain serum. After the serum was properly diluted, the blood concentration was measured by ELISA, and the method was basically the same as in example 11. The results are shown in FIG. 13.

As can be seen from fig. 13, fc-mutated SPGL004 significantly accelerated metabolism in human FcRn transgenic mice compared to Fc-wild type SPGL003, with half-lives of 9 hours and 22 hours, respectively, differing significantly.

EXAMPLE 18SPGL004 detects the expression of CD73 by multiple tumor cells

The binding force of SPGL004 to various human tumor cells was determined by flow cytometry Fluorescence sorting (Fluorescence ACTIVATED CELL Sorting, FACS).

A plurality of tumor cells were cultured in vitro, 100. Mu.L of SPGL004 as primary antibody was incubated with 1X10 ⁵ tumor cells suspended in 100. Mu.L of RPMI-1640 serum-free medium (available from Gibco, cat. No. 22400089) at 4℃for 1h, the cells were washed with PBS twice to remove unbound SPGL004, and then with 100. Mu.L of 2. Mu.g/mL of Alexa Fluor 488-labeled anti-human IgG fluorescent secondary antibody (available from Thermo, invrotogen, cat. No. A11013) at 4℃for 30min, the cells were washed with PBS twice to remove unbound secondary antibody, finally the cells were resuspended in 100. Mu.L of PBS, the binding affinity of SPGL004 to the various cells was determined by flow cytometer, and the obtained MFI value was compared with the MFI value of the control group (not SPGL 004), and the ratio indicates the binding strength of SPGL 004.

As can be seen from fig. 14, SPGL004 can bind to various human lung cancer cells (H1975, calu-3, a549, H322, H292), to human breast cancer cells (JIMT-1), to human kidney cancer cells (a 498), to human skin cancer cells (a 431), indicating that the various tumors described above may become indications for SPGL 004.

Example 19SPGL004 stability detection

And detecting the change of the purity of the SPGL004 after the SPGL004 is placed for different times by adopting a molecular sieve high performance liquid chromatography (SEC-HPLC), and examining the stability of the SPGL 004.

The experimental method comprises the following steps: this was performed on HPLC Ultimate 3000 (Thermo company) chromatograph using a TSKgel G3000SWXL column (TSK company). The mobile phase was measured as PBS (pH 7.4), constant flow rate 0.8mL/min, and loading was 100. Mu.g/100. Mu.L. The purity was expressed by calculating the content (%) of the target protein in the total protein using the 280nM absorption peak integration method. The results are shown in fig. 15, 16 and 17.

As can be seen from fig. 15, 16 and 17, SPGL004 had a purity of 89.7% in PBS solution for 0 week, a purity of 87.8% after 5 weeks at 4 ℃ and a change rate <3%; after 5 weeks at 37 ℃, the purity is 87.6%, and the change rate is <3%, which shows that SPGL004 is stable in PBS buffer solution under the conditions of 4 ℃ and 37 ℃, and the SPGL004 has good stability.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A monoclonal antibody capable of recognizing human CD73-ECD protein; the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO:5, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: shown at 7.

2. A recombinant antibody, characterized in that the recombinant antibody is obtained by humanizing the monoclonal antibody of claim 1, and the amino acid sequence of the heavy chain variable region of the recombinant antibody is shown in SEQ ID NO:16, the amino acid sequence of the light chain variable region of the recombinant antibody is shown as SEQ ID NO: shown at 18.

3. A multifunctional recombinant antibody, characterized in that the heavy chain of the multifunctional recombinant antibody comprises an antibody functional region which recognizes human CD73, a human IgG1 constant region functional region, a functional region of human IL15, and a nonfunctional amino acid fragment for linking the functional regions; the amino acid sequence of the antibody functional region that recognizes human CD73 comprises the heavy chain variable region amino acid sequence of the recombinant antibody of claim 2;

The human IgG1 constant region functional region is a mutant human IgG1 constant region, and the amino acid sequence of the mutant human IgG1 constant region is shown in SEQ ID NO: shown at 24; the heavy chain amino acid sequence of the multifunctional recombinant antibody is shown as SEQ ID NO: 26; the amino acid sequence of the light chain of the multifunctional recombinant antibody is shown as SEQ ID NO: 19.

4. An expression vector comprising a nucleotide sequence encoding the monoclonal antibody of claim 1, or the recombinant antibody of claim 2, or the multifunctional recombinant antibody of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. Use of the multifunctional recombinant antibody according to claim 3 for the preparation of a biological agent for the treatment of colorectal cancer, lung cancer and breast cancer.

7. A biologic comprising the monoclonal antibody of claim 1, or the recombinant antibody of claim 2, or the multifunctional recombinant antibody of claim 3; or the expression vector of claim 4, or the host cell of claim 5.

8. The method for producing a monoclonal antibody according to claim 1 or a recombinant antibody according to claim 2 or a multifunctional recombinant antibody according to claim 3, comprising the steps of:

(1) Obtaining an expression vector containing a gene fragment for encoding the monoclonal antibody or the recombinant antibody or the multifunctional recombinant antibody through an artificial synthesis or molecular biological method;

(2) Transfecting the expression vector into cells for protein expression;