Monoclonal antibody with human SIRP alpha binding activity and application thereof

Abstract

The invention belongs to the technical field of biological medicine, relates to a monoclonal antibody with human SIRP alpha binding activity and application thereof, and provides a coding nucleic acid molecule, an expression vector and a host cell of the antibody and a method for expressing the antibody. Antibody immunoconjugates, bispecific molecules, chimeric antigen receptors, pharmaceutical compositions and diagnostic and therapeutic methods comprising the antibodies of the invention are also provided. The monoclonal antibody disclosed by the invention has relatively strong human SIRP alpha binding activity, can be in cross binding with cynomolgus monkey SIRP alpha, and is an anti-human SIRP alpha monoclonal antibody with a novel sequence.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a monoclonal antibody having human SIRPα binding activity and an application thereof in the preparation of anti-tumor drugs. Background Art

[0002] Significant progress has been made in the use of blockers against inhibitory immune checkpoints in cancer immunotherapy. For example, antibodies targeting CTLA-4, PD-1, or PD-L1 have significant therapeutic effects on a variety of cancers, and their clinical applications are gradually increasing. However, not all patients can achieve long-term clinical benefits, and significant side effects may occur, including immune dysfunction toxicity caused by immune system imbalance, also known as immune-related adverse events (IRAEs) (Michot J M. et al. (2016). "Immune-related adverse events with immune checkpoint blockade: a comprehensive review". Eur J Cancer. 54: 139-148).

[0003] The SIRPα-CD47 pathway belongs to the macrophage immune checkpoint pathway. Targeting this pathway can promote innate and adaptive immune responses to enhance anti-cancer immunity. Among them, SIRPα (signal regulatory proteinα), also known as protein tyrosine phosphatase 2 containing Src homology 2 domain (SHPS-1), is a plasma membrane protein mainly expressed by myeloid hematopoietic cells and neural cells. Its cytoplasmic domain contains immune receptor tyrosine inhibitory motifs (ITIMs). After phosphorylation, ITIMs recruit SHP-1 and SHP-2 to inhibit cell activation (Barclay AN. et al. (2006). "The SIRP family of receptors and immune regulation". Nat Rev Immunol. 6 (6): 457-464).

[0004] The natural ligand of SIRPα is CD47, also known as integrin-associated protein (IAP), which is widely expressed on the surface of various cells and has a single immunoglobulin-like extracellular domain and five transmembrane regions. CD47 binds to the extracellular domain of SIRPα expressed on immune cells (such as macrophages) through the IgSF (immunoglobulin superfamily) domain, transduces SIRPα-mediated signals, and regulates immune responses. However, when SIRPα on the surface of macrophages binds to CD47 molecules on the surface of tumor cells, it sends a "don't eat me" signal to the immune system to avoid being phagocytosed by macrophages and achieve immune escape.

[0005] In immunocompetent syngeneic mouse models, blocking SIRPα-CD47 interaction with anti-SIRPα antibodies significantly inhibited tumor formation in renal cell carcinoma or melanoma (Yanagita T. et al. (2017). "Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy". JCI Insight. 2(1): e89140). This shows that blocking the SIRPα-CD47 pathway is a promising cancer treatment method. However, due to the widespread expression of CD47, potential problems of anti-CD47 therapy include antigen silencing (antigen sink) and blood toxicity, such as anemia and thrombocytopenia. Considering that SIRPα expression is relatively limited, targeting SIRPα is another way to block the SIRPα-CD47 pathway, and the treatment dose required for this method will be lower, and the safety and efficacy may be higher.

[0006] A variety of anti-SIRPα monoclonal antibodies have been developed, among which the monoclonal antibody KWAR23 binds to human SIRPα with high affinity and destroys the binding of SIRPα-CD47. When used in combination with tumor-modulating antibodies (e.g., anti-CD20 antibodies, Rituximab), it can greatly enhance the anti-tumor activity of neutrophils and macrophages in vitro and in vivo (Ring NG. et al. (2017). "Anti-SIRPα antibody immunotherapy enhances neutrophiland macrophage antitumor activity". Proc Natl Acad Sci US A. 114 (49): E10578-E10585). The currently developed anti-human SIRPα monoclonal antibodies still have the problem of limited SIRPα specificity. Therefore, a new therapeutic candidate SIRPα antibody with higher affinity is urgently needed. Summary of the invention

[0007] In order to overcome the above-mentioned defects, the purpose of the present invention is to provide a monoclonal antibody with human SIRPα binding activity and its use, which has high human SIRPα binding affinity and functional activity, and can cross-bind with crab-eating monkey SIRPα, which has great advantages over existing anti-human SIRPα monoclonal antibodies.

[0008] The technical solution provided by the present invention is as follows:

[0009] A monoclonal antibody having human SIRPα binding activity, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region;

[0010] The heavy chain variable region comprises V H CDR1, V H CDR2 and V H CDR3, the light chain variable region comprises V L CDR1, V L CDR2 and V L CDR3;

[0011] The V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 2;

[0012] The V H The amino acid sequence of CDR2 is shown in SEQ ID NO: 4;

[0013] The V H The amino acid sequence of CDR3 is shown in SEQ ID NO: 6;

[0014] The V L The amino acid sequence of CDR1 is shown in SEQ ID NO: 12;

[0015] The V L The amino acid sequence of CDR2 is shown in SEQ ID NO: 14;

[0016] The V L The amino acid sequence of CDR3 is shown in SEQ ID NO:16.

[0017] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18.

[0018] Preferably, the heavy chain amino acid sequence is shown as SEQ ID NO: 10; and the light chain amino acid sequence is shown as SEQ ID NO: 20.

[0019] Preferably, both the heavy chain and the light chain further comprise a constant region, which is a constant region of mouse IgG, preferably a constant region of IgG1.

[0020] The present invention further provides a nucleotide molecule, which encodes the anti-human SIRPα monoclonal antibody;

[0021] Preferably, the sequence of the nucleotide molecule is selected from SEQ ID NO: 7 and SEQ ID NO: 17;

[0022] The sequence SEQ ID NO: 7 encodes the heavy chain variable region of the monoclonal antibody;

[0023] The sequence SEQ ID NO: 17 encodes the light chain variable region of the monoclonal antibody.

[0024] The present invention further provides an expression vector, which contains the nucleotide molecule.

[0025] The present invention further provides a host cell, wherein the host cell contains the expression vector.

[0026] Preferably, the host cell is a eukaryotic cell, preferably a mammalian cell.

[0027] The present invention further provides a method for preparing a monoclonal antibody having human SIRPα binding activity, the preparation method comprising the following steps:

[0028] Step 1: preparing an expression vector containing a nucleotide molecule for expressing the monoclonal antibody having human SIRPα binding activity;

[0029] Step 2: transfecting eukaryotic host cells with the expression vector obtained in step 1;

[0030] Step 3: culturing the eukaryotic host cells transfected in step 2;

[0031] Step 4: separation and purification to obtain the monoclonal antibody.

[0032] The present invention also relates to an antibody immunoconjugate, a bispecific molecule, a chimeric antigen receptor or a pharmaceutical composition comprising the aforementioned monoclonal antibody having human SIRPα binding activity.

[0033] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of the monoclonal antibody having human SIRPα binding activity, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0034] The present invention further provides the use of the monoclonal antibody having human SIRPα binding activity in the preparation of anti-tumor drugs.

[0035] Preferably, the tumor is lung cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer or ovarian cancer.

[0036] The present invention has the following advantages: the monoclonal antibody of the present invention can specifically recognize human SIRPα and cynomolgus monkey SIRPα, has higher human SIRPα binding affinity and functional activity, and has a novel sequence.

[0037] The high species specificity of biological products determines that pharmacologically relevant animal species need to be used for testing in preclinical drug research. Non-human primates are more closely related to humans, which makes them more commonly used in preclinical research of biological products, of which nearly 80% use cynomolgus monkeys. Therefore, monoclonal antibodies with cynomolgus monkey antigen binding ability are more conducive to subsequent evaluation experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The affinity of mouse anti-SIRPα monoclonal antibody was determined by BIACORE surface plasmon resonance method;

[0039] Figure 2 The ability of antibodies to bind to human SIRPα protein was determined by capture ELISA;

[0040] Figure 3 Indirect ELISA was used to determine the antibody's ability to bind to human SIRPα protein;

[0041] Figure 4 Indirect ELISA was used to measure the cross-reactivity of antibodies to human SIRPβ protein;

[0042] Figure 5 Blocking ELISA for reference antibody;

[0043] Figure 6 Flow cytometry was used to evaluate the antibody blocking the binding of human SIRPα protein to human CD47 protein on the cell membrane surface. DETAILED DESCRIPTION

[0044] the term

[0045] "Binding to SIRPα" or "binding to SIRPα" means being able to interact with human SIRPα. "Specific binding" means being able to bind to human SIRPα protein (and possibly SIRPα protein from one or more non-human species) but not substantially binding to non-SIRPα proteins.

[0046] “Antigen binding site” refers to one or more fragments of an antibody that have the ability to specifically bind to an antigen (eg, SIRPα protein).

[0047] "Monoclonal antibody" refers to a preparation of antibody molecules having a single amino acid composition, exhibiting a single binding specificity and affinity for a particular epitope. Monoclonal antibodies or antigen-binding fragments thereof can be prepared, for example, by hybridoma technology, recombinant DNA technology, phage display technology, synthetic technology such as CDR grafting technology, or a combination of such or other techniques known in the art.

[0048] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, herein, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between an antibody and an antigen. Affinity can be measured by common methods known in the art, including those described in the prior art and herein.

[0049] The term "competition" when used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) competing for the same epitope means competition between antigen binding proteins, which is determined by the following assay: in the assay, the antigen binding protein to be detected (e.g., an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., SIRPα or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another. Competitive inhibition is measured by measuring the amount of a marker bound to a solid surface or cell in the presence of the antigen binding protein being tested. Typically, the antigen binding protein being tested is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and antigen binding proteins that bind to adjacent epitopes that are sufficiently close to the binding epitope of the reference antigen binding protein, and the two epitopes sterically hinder each other from binding.

[0050] Methods for producing and purifying antibodies and antigen-binding fragments are well known and disclosed in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Technology. For example, mice can be immunized with human SIRPα or its fragments, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods.

[0051] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising a SIRPα antibody or an antigen-binding fragment thereof, to a patient who has one or more symptoms of a disease. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, either by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect on the patient. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by physicians or other health care professionals to evaluate the severity or progression of the symptoms.

[0052] An "effective amount" includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0053] "Pharmaceutical composition" refers to a mixture containing one or more SIRPα antibodies or antigen-binding fragments thereof described herein and other pharmaceutical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0054] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0055] Example 1 Obtaining mouse monoclonal antibodies specific to SIRPα by fusion hybridoma technology

[0056] 1.1 Animal immunization

[0057] Mice were immunized according to the common method in the literature (E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998). The immunogen was a recombinant human SIRPα (amino acid Met 1-Arg 370) protein containing a human IgG1Fc tag at the C-terminus, human SIRPα-Fc (produced by the company, the plasmid used was CD172-ECD-Fc; the vector was pCP; the inserted amino acid sequence was

[0058] MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQW

[0059] FRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFK

[0060] SGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVG

[0061] ESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVN

[0062] VTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNER, SEQ ID NO: 21). Recombinant human SIRPα protein with his tag, human SIRPα-his (produced by the company, the plasmid used is SRPA-ECD-His; the vector is pCMV-C-His; the amino acid sequence is inserted between EcoR I and XbaI

[0063] MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNER, SEQ ID NO: 21) was used as the detection antigen for determining serum titer and hybridoma screening. Briefly, take out an appropriate amount of Freund's adjuvant into a 1.5ml EP tube and mix it with an oscillator. Prepare a human SIRPα-Fc antigen protein solution with PBS. Add the calculated amount of antigen and adjuvant into a microcentrifuge tube, gently vortex and mix for 2 minutes, repeat the emulsification and mixing to form an oil-in-water solution, and obtain an adjuvant-antigen mixture. Then use a syringe to inhale an appropriate amount of the adjuvant-antigen mixture for subcutaneous injection immunization of each mouse. Perform 2 to 3 booster immunizations according to the serum titer. Before cell fusion, perform a final booster immunization on animals with better titers by intraperitoneal injection.

[0064] 1.2 Hybridoma fusion and screening

[0065] Before cell fusion, mouse myeloma cells (SP2 / 0-Ag14, ATCC#CRL-1581) were cultured in the logarithmic growth phase. The immune mice were sacrificed and the spleen was removed in a sterile environment. The spleen B cells and SP2 / 0 myeloma cells were fused using PEG chemistry according to the method in the literature (Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 256: 495-497 (1975)).

[0066] The fused cells were plated in a 96-well cell culture plate. Surviving hybridoma cells were observed growing under a microscope after 7 to 10 days. Two weeks after cell plating, the culture supernatant of each well was collected and hybridoma screening was performed using the capture ELISA method using human SIRPα-Fc protein antigen. The following is a brief description: 60 μl of affinity purified F(ab') containing 1 μg / ml 2 The ELISA plate was coated with a fragment-specific goat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc., Cat#115-005-072) in PBS solution at 4°C overnight. The plate was then washed once with PBST (PBS solution containing 0.05% v / v Tween-20), 200 μl / well blocking buffer (PBST containing 5% w / v skim milk) was added, and the plate was blocked at 37°C for 2 hours. The plate was washed again, 60 μl / well of hybridoma supernatant was added, incubated at 37°C for 40 minutes, and then washed 4 times. 100 μl / well of biotin-labeled human SIRPα-Fc protein solution (1:5000 diluted in PBST containing 2.5% w / v skim milk) was added, incubated at 37°C for 40 minutes, washed 4 times, and patted dry. Then add 100 μl / well of horseradish peroxidase-labeled streptavidin (Jackson ImmunoResearch Laboratories, Inc., Cat#016-030-084) diluted 1:5000 in blocking buffer milk, incubate at 37°C for 40 minutes, wash the plate 4 times, and pat dry. Add 100 μl / well of ELISA substrate TMB (InnoReagents, Cat#TMB-S-002) to the plate, color at room temperature for 5 to 15 minutes, then add 1M sulfuric acid solution to terminate the reaction, and measure the absorbance of each well at 450nm. Then select the positive hybridoma that secretes antibodies that bind to human SIRPα-Fc and transfer it to a 24-well plate.

[0067] The hybridoma clones producing antibodies that bind to human SIRPα with high specificity were subcloned by limiting dilution to ensure the monoclonal origin of the cell line and then purified. Briefly, the protein A agarose column was washed with 5-10 column volumes of PBS buffer. The cell supernatant of the hybridoma monoclonal antibody was passed through the column and then the column was washed with PBS buffer until the protein absorbance reached the baseline. The column was eluted with elution buffer (0.1M glycine-HCl, pH 2.7) and immediately collected in a 1.5ml tube containing a neutral buffer (1MTris-HCl, pH 9.0). The fractions containing immunoglobulins were mixed and dialyzed in PBS at 4°C overnight. Subsequently, the functional activity of the purified monoclonal antibodies was characterized in vitro as described below.

[0068] Example 2 Determination of the affinity of mouse anti-SIRPα monoclonal antibody using BIACORE surface plasmon resonance technology

[0069] The affinity kinetics of the anti-SIRPα mouse monoclonal antibody (mAb) produced by the hybridoma clones in Example 1 were characterized using the Biacore T200 system (GE healthcare, Pittsburgh, PA, USA).

[0070] Briefly, goat anti-mouse IgG was covalently linked to a CM5 chip (carboxymethyl dextran coated chip) via primary amine using a standard amine coupling kit provided by Biacore. Unreacted groups on the biosensor surface were blocked with ethanolamine. The purified mouse anti-SIRPα antibody or reference antibody (i.e., KWAR23 antibody, produced by the company, with a heavy chain amino acid sequence of EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLEWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCARWGAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK, SEQ ID NO: 22; light chain amino acid sequence is QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPRTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO: 23) at a concentration of 66.7 nM and a flow rate of 10 μL / min. Then, gradient dilution of recombinant human SIRPα-his protein or cynomolgus monkey SIRPα-his protein (produced by the company, the plasmid used is SRPA-ECD-His; the vector is pCMV-C-His; the amino acid sequence is inserted between EcoR I and Xba I

[0071] MEPAGPAPGRLGPLLCLLLTASCAWSGVLGEEELQVIQPEKSVSVAAGDSATLNCTVSSLIPVGPIQW

[0072] FRGAGPGRELIYNLKEGHFPRVTAVSDPTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDVELKSG

[0073] AGTELSVRAKPSAPVVSGPAVRATAEHTVSFTCESHGFSPRDITLKWFKNGNELSDVQTNVDPAGKS

[0074] VSYSIRSTARVLLTRRDVHSQVICEVAHVTLQGDPLRGTANLSEAIRVPPFLEVTQQSMRADNQVNV

[0075] TCQVTKFYPQRLQLTWLENGNVSRTEMASALPENKDGTYNWTSWLLVNVSAHRDDVKLTCQVEHDGQPAVNKSFSVKVSAHPKEQGSNTAAENTGTNER, SEQ ID NO: 24), with a starting concentration of 200 nM, in HBS-EP + The samples were diluted 2-fold in a buffer (provided by Biacore) and flowed through the chip at a flow rate of 30 μL / min. The antigen-antibody binding kinetics were observed for 2 minutes and the dissociation kinetics were observed for 10 minutes. The BIAcore evaluation software was used to fit the 1:1 Langmuir binding model. a , k d and K D The values ​​of are shown in Table 1.

[0076] Table 1. Kinetic parameters of mouse anti-SIRPα monoclonal antibody binding to human or cynomolgus monkey SIRPα determined by Biacore

[0077]

[0078]

[0079] like Figure 1 As shown in Table 1, the binding K of the monoclonal antibody A2C12G3H2 of the present invention to human SIRPα D The value is comparable to that of the reference antibody, indicating that it has a high affinity for human SIRPα. In addition, the binding K D The value is at the level of 1.0E-07, indicating that it can bind to cynomolgus monkey SIRPα.

[0080] Example 3 Study on Binding Activity of Mouse Anti-SIRPα Monoclonal Antibodies

[0081] The binding activity of the mouse anti-SIRPα monoclonal antibody produced by the hybridoma clones in Example 1 was further tested using the following method.

[0082] 3.1 Determination of antibody binding capacity based on capture ELSIA

[0083] Affinity purified F(ab') was used at a final concentration of 2 μg / ml 2 Fragment-specific goat anti-mouse IgG or affinity purified F(ab') 2 Fragment-specific goat anti-human IgG (Jackson ImmunoResearch Laboratories, Inc., Cat#115-005-072 or Cat#109-005-097) in PBS was added to a 96-well plate at 100 μl / well and coated at 37°C for 2 hours. After washing the plate once with PBST, 200 μl / well of PBST solution containing 5% w / v skim milk was added and blocked at 37°C for 2 hours. The plate was washed again, and 100 μl / well of the diluted antibody solution was added to the ELISA plate coated with goat anti-mouse IgG secondary antibody. The reference antibody or hIgG (initial concentration of 66.7 nM, 5-fold gradient dilution with PBST containing 2.5% w / v skim milk) was added to the ELISA plate coated with goat anti-human IgG secondary antibody, incubated at 37°C for 40 minutes, and then washed 4 times. Add biotin-labeled human SIRPα-Fc protein solution (83 ng / mL, in PBST containing 2.5% w / v skim milk, 100 μl / well), incubate at 37°C for 40 minutes, and then wash the plate 4 times. Add horseradish peroxidase-labeled streptavidin (1:10000 diluted in PBST buffer, Jackson ImmunoResearch Laboratories, Inc., Cat#016-030-084) at 100 μl / well, incubate at 37°C for 40 minutes, then wash the plate 4 times and pat dry. Add 100 μl / well of ELISA color substrate TMB (InnoReagents, Cat#TMB-S-002) to the plate, color develop at room temperature for 5 to 15 minutes, and then stop the color development with 1M sulfuric acid solution. The absorbance of each well was read in a microplate reader using dual wavelength mode, 450 nm for TMB and 630 nm as a reference wavelength. OD (450-630) values ​​and antibody concentrations were plotted. The data were analyzed using Graphpad Prism software to obtain EC values. 50 The result is Figure 2 .

[0084] As can be seen from the figure, the A2C12G3H2 monoclonal antibody of the present invention can specifically recognize human SIRPα antigen, and its binding activity is better than that of the reference antibody.

[0085] 3.2 Indirect ELISA to determine the binding ability of antibodies to human SIRPα

[0086] 100 μl / well of 2 μg / ml human SIRPα-Fc in carbonate / bicarbonate buffer (pH 9.6) was added to the 96-well plate and coated at 37°C for 2 hours. After coating, the plate was washed 4 times with PBST and then blocked with 200 μl / well of blocking buffer (PBST containing 5% w / v skim milk) for 2 hours at 37°C. The plate was washed again, and 100 μl / well of gradient diluted antibody (initial concentration was 66.7 nM, diluted 5 times with PBST containing 2.5% w / v skim milk) was added, incubated at 37°C for 40 minutes, and then washed 4 times. Incubate with 100 μl / well horseradish peroxidase-labeled affinity-purified Fcγ fragment-specific goat anti-mouse IgG (1:5000 diluted in PBST buffer, Jackson ImmunoResearch Laboratories, Inc., Cat#115-035-071) at 37°C for 40 minutes, then wash the plate 4 times and pat dry. Add 100 μl / well of ELISA colorimetric substrate TMB (InnoReagents) to the plate, color develop at room temperature for 3 to 10 minutes, then stop color development with 1M sulfuric acid solution, read the absorbance of each well in an ELISA reader, use dual wavelength mode, 450nm for TMB, and 630nm as a reference wavelength. Graph the OD (450-630) value and antibody concentration. Analyze the data using Graphpad Prism software to obtain the EC 50 value.

[0087] from Figure 3 It can be seen that the A2C12G3H2 monoclonal antibody of the present invention has a strong human SIRPα protein binding activity.

[0088] 3.3 Indirect ELISA to determine the cross-reactivity of antibodies with human SIRPβ

[0089] 100 μl / well of 2 μg / ml human SIRPβ-his protein in carbonate / bicarbonate buffer (pH 9.6) (produced by the company, the plasmid used is SIRPβ-ECD-His; the vector is pCMV-C-His; the amino acid sequence is inserted between EcoR I and Xba I) was added to the 96-well plate.

[0090] MDMRVPAQLLGLLLLWFPGSRCEDELQVIQPEKSVSVAAGESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTTVSELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAVRATPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTN VDPAGDSVSYSIHSTARVVLTRGDVHSQVICEIAHITLQGDPLRGTANLSEAIRVPPTLEVTQQPMRAENQANVTCQVSNFYPRGLQLTWLENGNVSRTETASTLIENKDGTYNWMSWLLVNTCAHRDDVVLTCQVEHDGQQAVSKSYALEISAHQKEHGSDITHEAALAPTAPLHHHHHHHHHH, SEQ ID NO: 25), coated at 37°C for 2 hours. After coating, wash the plate 4 times with PBST, and then block with 200 μl / well blocking buffer (PBST containing 5% w / v skim milk) at 37°C for 2 hours. Wash the plate again, add 100 μl / well gradient dilutions of antibody, reference antibody or hIgG (starting concentration is 66.7nM, 5-fold gradient dilution with PBST containing 2.5% w / v skim milk), incubate at 37°C for 40 minutes, and then wash the plate 4 times. Incubate with 100 μl / well horseradish peroxidase-labeled affinity-purified Fcγ fragment-specific goat anti-mouse IgG or horseradish peroxidase-labeled affinity-purified F(ab') 2 Fragment goat anti-human IgG, Fcγ fragment specific (1:5000 diluted in PBST buffer, Jackson ImmunoResearch Laboratories, Inc., Cat#115-035-071 or Cat#109-036-098), incubate at 37°C for 40 minutes, then wash the plate 4 times and pat dry. Add 100 μl / well of ELISA color substrate TMB (InnoReagents). After 3 to 10 minutes of color development at room temperature, stop the color development with 1M sulfuric acid solution, and read the absorbance of each well in an ELISA reader using dual wavelength mode, 450nm for TMB, and 630nm as a reference wavelength. Graph the OD (450-630) value and antibody concentration. Analyze the data using Graphpad Prism software to obtain EC 50 The result is Figure 4 And as shown in Table 2.

[0091] As can be seen from the figure, the A2C12G3H2 monoclonal antibody of the present invention can cross-react with the human SIRPβ antigen, and the binding activity is better than that of the reference antibody.

[0092] Table 2. Binding activity of mouse anti-SIRPα antibodies

[0093]

[0094] Example 4. Detection of blocking activity of mouse SIRPα antibody

[0095] 4.1 Reference Antibody Blocking ELISA

[0096] The ability of the anti-SIRPα antibody of the present invention to block the binding of the reference antibody to the SIRPα antigen was evaluated by the competitive ELISA method. Briefly described as follows, the reference antibody was prepared with PBS to a final concentration of 2 μg / ml, and 100 μl / well was added to a 96-well plate and coated overnight at 4°C. The next day, the plate was washed once with PBST, 200 μl / well of a PBST solution containing 5% w / v skim milk was added, blocked at 37°C for 2 hours, and the plate was washed again 4 times. SIRPα antibody or control biotin-labeled human SIRPα-Fc protein (8.3 ng / ml in PBST containing 2.5% w / v skim milk) was diluted to a starting concentration of 40 nM, 5-fold gradient dilution, and pre-incubated at room temperature for 40 minutes. Then the antibody / SIRPα-Fc mixture was added to the reference antibody-coated plate at 100 μl / well, incubated at 37°C for 40 minutes, and then washed again 4 times. Then add horseradish peroxidase-labeled streptavidin (100 μl / well, 1:10000 diluted in PBST containing 5% w / v skim milk), incubate at 37°C for 40 minutes, wash the plate 4 times with washing buffer, and pat dry. Add TMB for color development, terminate the reaction with 1M sulfuric acid, and read the absorbance in a microplate reader using dual wavelength mode, 450nm for TMB, and 630nm as a reference wavelength. Graph the OD (450-630) value and antibody concentration. Analyze the data using Graphpad Prism software to obtain the IC 50 The result is Figure 5 shown.

[0097] As can be seen from the figure, the monoclonal antibody A2C12G3H2 of the present invention can specifically block the binding of SIRPα protein to the reference antibody, and its blocking ability is better than that of the reference antibody, indicating that the epitope of the antigen bound by the antibody A2C12G3H2 is the same or similar to that of the reference antibody.

[0098] 4.2 Flow cytometry evaluation of antibody blocking the binding of SIRPα protein to CD47 protein on the cell membrane surface

[0099] 293F cells expressing human CD47 on the cell surface were used (constructed in-house by the company, and the full-length human CD47 protein was transfected into 293F cells using lipofectamine 3000 transfection reagent. The vector was pCMV-TP, and the sequence inserted in EcoR I and Xba I was

[0100] The ability of the SIRPα antibody of the present application to block the binding of human SIRPα-Fc protein to cell surface CD47 was evaluated by flow cytometry (FACS).

[0101] The SIRPα antibody, reference antibody or negative control hIgG (human immunoglobulin for intravenous injection (pH 4, Hualan Biotechnology Co., Ltd.) of the present application was diluted with biotin-labeled human SIRPα-Fc protein, with a starting concentration of 66.67 nM, 5-fold gradient dilution, and incubated at room temperature for 40 minutes. 293F cells in the logarithmic growth phase were collected from the cell culture flask, washed twice with PBS, and resuspended with FACS buffer (PBS solution containing 2% fetal bovine serum).

[0102] CD47-293F cells were cultured at 1×10 5 Cells / well were plated in a 96-well plate and incubated with 100 μl / well antibody / SIRPα-Fc mixture at 4°C for 40 minutes. The plate was washed twice with FACS buffer, and then 100 μl / well R-phycoerythrin streptavidin (Jackson Immunoresearch, Cat#016-110-084, diluted 1:500 in FACS) was added and incubated at 4°C in the dark for 40 minutes. The cells were washed twice and resuspended in FACS buffer. The mean fluorescence intensity (MFI) value of each well of cells was determined using a Becton Dickinson FACS Canto II-HTS device. Graphpad Prism software was used for data analysis to obtain IC50 The result is Figure 6 And as shown in Table 3.

[0103] It can be seen that the A2C12G3H2 antibody of the present invention can block the binding of human SIRPα protein to human CD47 ligand on the cell membrane surface with an activity superior to that of the reference antibody.

[0104] Table 3. Blocking activity test of mouse anti-SIRPα antibodies

[0105]

[0106] Example 5 DNA cloning and sequencing, variable region sequencing of anti-SIRPα mouse monoclonal antibody

[0107] Total RNA was extracted from cultured mouse monoclonal cell lines using FastPure Cell / Tissue Total RNA Isolation Kit (Vazyme, Cat#RC101). The process is briefly described as follows: 1.5x10 6 Transfer the cells to a 1.5ml centrifuge tube and remove the supernatant. Add 500μl Buffer RL1 to the cell pellet and mix by pipetting. Transfer the treated cells to gDNA-Filter Columns (gDNA-Filter Columns have been placed in the collection tube), centrifuge at 13,000g for 2 minutes at room temperature, and retain the supernatant in the collection tube. Add 1.6 times the volume of Buffer RL2 and mix gently. Transfer the mixture to RNAPure Columns, centrifuge at 13,000g for 1 minute at room temperature, and discard the waste liquid. Add 500μl Buffer RW1 to RNAPure Columns, centrifuge at 13,000g for 1 minute at room temperature, and discard the waste liquid. Add 700μl Buffer RW2, centrifuge at 13,000g for 1 minute at room temperature, and discard the waste liquid. Add another 700μl Buffer RW2, centrifuge at 13,000g for 1 minute at room temperature, and discard the waste liquid. Centrifuge at 13,000g for 2 minutes to completely remove the residual Buffer RW2 in the RNAPure Columns. Transfer the adsorption column to a new RNase-free Collection Tubes 1.5ml centrifuge tube and drop 50μl of RNase-free ddHO in the center of the adsorption column. 2 O. Leave at room temperature for 2 min, centrifuge at 13,000 g for 1 min at room temperature to elute RNA.

[0108] Next, Takara's reverse transcription cDNA kit (Cat#6210A) was used to convert total RNA into cDNA. The experimental system was prepared as follows: 5μl total RNA + 0.5μl Oligo(dT) Primer + 0.5μl Random 6mers + 1.0μl dNTPMixture + 3.0μl RNase-free ddH 2 O (total 10 μl). Pre-denature at 65°C for 5 min, then place on ice for 2 min. Further add 4 μl 5x PrimeScript II Buffer + 0.5 μl RNase Inhibitor + 1.0 μl PrimeScriptII RTase + 4.5 μl RNase-free ddH 2 O (a total of 20 μl system), mix well after preparation, run on a PCR instrument at 40°C for 50 min, then transfer to 70°C for 10 min to complete cDNA synthesis.

[0109] The cDNA was further treated with Poly G at the 3' end. The reaction system was prepared as follows: 5 μl cDNA + 33.5 μl ddH 2 O+5μl10x TdT Buffer+5μl CoCl+1μl dGTP+0.5μl Terminal TransPerase (a total of 50μl system), mix well after preparation, run at 37℃ for 30min using a PCR instrument, then transfer to 70℃ for 10min to complete Poly G tailing.

[0110] Further, the tailed cDNA was used as a template for gene amplification of the antibody variable region. For amplification of the antibody heavy chain variable region sequence, a kit from Novozymes (Cat#P525-03) was used to prepare the PCR reaction system: 25 μl 2x Phanta Max Master Mix (Dye Plus) + 2.0 μl BSJ-Pri3AntisenseP-mIgG1 (nucleotide sequence is GCATCCCAGGGTCACCATGGAGTTAGTT, SEQ ID NO: 27) + 2.0 μl BSJ-Pri1Universal C1 (nucleotide sequence is

[0111] AAGCAGTGGTATCAACGCAGAGTCATCCCCCCCCCCCCCCCCC, SEQ ID NO: 28)+1.25μl PolyG-tailed cDNA+19.75μl ddH 2O (a total of 50 μl system). For amplification of the antibody light chain variable region sequence, first use Takara's kit (Cat#R010A) to prepare the PCR reaction system: 10 μl 5x PrimeSTAR Buffer + 0.5 μl PrimeSTAR + 1.25 μl BSJ-Pri1Universal C1 (nucleotide sequence is AAGCAGTGGTATCAACGCAGAGTCATCCCCCCCCCCCCCCCC, SEQ ID NO: 28) + 1.25 μl BSJ-Pri19 (nucleotide sequence is GCAACCGACTGAGGCACCTCCAGATGTT, SEQ ID NO: 29) + 4 μl aberrant-L-R2 (nucleotide sequence is

[0112] TCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGC, SEQ ID NO: 30)+4μl dNTP+1.25μl cDNA with Poly G tail+27.75μl ddH 2 O (total 50 μl system).

[0113] The temperature cycle for PCR amplification of the antibody heavy chain variable region is as follows:

[0114] 98℃*5min+(98℃*10s+62℃*10s+72℃*1min)*2Cycle+(98℃*10s+60℃*10s+72℃*1min)*4Cycles+(98℃ *10s+58℃*10s+72℃*1min)*10Cycles+(98℃*10s+56℃*10s+72℃*1min)*20Cycles+72℃*10min+4℃*∞

[0115] The temperature cycle for PCR amplification of the antibody light chain variable region is as follows:

[0116] 98℃*5min+(98℃*10s+64℃*10s+72℃*1min)*2Cycle+(98℃*10s+62℃*10s+72℃*1min)*6Cycles+(98℃ *10s+60℃*10s+72℃*1min)*12Cycles+(98℃*10s+58℃*10s+72℃*1min)*16Cycles+72℃*10min+4℃*∞

[0117] The PCR products were analyzed by 1% agarose gel electrophoresis, and the DNA segments of corresponding sizes were cut out (about 500 bp for VH and about 500 bp for Vkappa light chain), and DNA was extracted using TIANGEN's gel DNA recovery kit (Cat# DP209-03). The brief description is as follows: the gel was weighed, an equal volume of solution PN was added, and then incubated at 50°C for 10 minutes until the gel was completely dissolved. The resulting solution was transferred to the CA2 adsorption column (the adsorption column was placed in the collection tube), left at room temperature for at least 2 minutes, centrifuged at 12,000 rpm at room temperature for 30 seconds, and the waste liquid was discarded. 600 μl of rinsing solution PW was added to the column, and then centrifuged at 12,000 rpm at room temperature for 30 seconds, and the waste liquid was discarded. 600 μl of rinsing solution PW was added to the column again, left to stand for 5 minutes, centrifuged at 12,000 rpm at room temperature for 30 seconds, and the waste liquid was discarded. Centrifuge again at 12,000 rpm for 2 minutes at room temperature to remove the residual liquid in the column, and leave it at room temperature for at least 2 minutes to completely dry the residual liquid. Place the adsorption column in a clean centrifuge tube, add 35 μl of elution buffer EB, and leave it at room temperature for at least 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to obtain the prepared DNA sample. The purified PCR product is sequenced to obtain the variable region sequence of the antibody.

[0118] The sequence information of the clones of the present invention is shown in Tables 4 to 6.

[0119] Table 4 Sequence information of anti-SIRPα antibodies

[0120]

[0121] NA: nucleotide; AA: amino acid.

[0122] Table 5. Amino acid sequences of anti-SIRPα antibodies

[0123]

[0124]

[0125] Table 6. Nucleotide sequences of anti-CD47 antibodies

[0126]

[0127]

[0128]

[0129] In summary, the monoclonal antibody A2C12G3H2 of the present invention that can specifically recognize human SIRPα has a strong human SIRPα binding affinity, can bind to crab-eating monkey SIRPα, and can specifically block the binding of human SIRPα protein to the human CD47 ligand on the cell membrane surface. It is a monoclonal antibody against human SIRPα with a novel sequence.

[0130] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A monoclonal antibody having human SIRPα binding activity, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises V H CDR1, V H CDR2 and V H CDR3, the light chain variable region comprises V L CDR1, V L CDR2 and V L CDR3; The V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 2; The V H The amino acid sequence of CDR2 is shown in SEQ ID NO: 4; The V H The amino acid sequence of CDR3 is shown in SEQ ID NO: 6; The V L The amino acid sequence of CDR1 is shown in SEQ ID NO: 12; The V L The amino acid sequence of CDR2 is shown in SEQ ID NO: 14; The V L The amino acid sequence of CDR3 is shown in SEQ ID NO:

16.

2. The monoclonal antibody having human SIRPα binding activity according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

18.

3. The monoclonal antibody having human SIRPα binding activity according to claim 1, characterized in that: The heavy chain amino acid sequence is shown in SEQ ID NO: 10; the light chain amino acid sequence is shown in SEQ ID NO:

20.

4. A nucleotide molecule, characterized in that The nucleotide molecule encodes the monoclonal antibody having human SIRPα binding activity according to any one of claims 1 to 3.

5. The nucleotide molecule according to claim 4, characterized in that The sequence of the nucleotide molecule is selected from SEQ ID NO: 7 and SEQ ID NO: 17; The sequence SEQ ID NO: 7 encodes the heavy chain variable region of the monoclonal antibody; The sequence SEQ ID NO: 17 encodes the light chain variable region of the monoclonal antibody.

6. An expression vector, characterized in that: The expression vector contains the nucleotide molecule according to claim 4 or 5.

7. A host cell, characterized in that The host cell contains the expression vector according to claim 6.

8. The method for preparing a monoclonal antibody having human SIRPα binding activity according to any one of claims 1 to 3, characterized in that: The following steps are included: preparing an expression vector containing a nucleotide molecule for expressing a monoclonal antibody having human SIRPα binding activity according to any one of claims 1 to 3; The obtained expression vector is transfected into eukaryotic host cells and cultured; The monoclonal antibody is obtained by separation and purification.

9. An antibody immunoconjugate, a bispecific molecule, a chimeric antigen receptor or a pharmaceutical composition comprising the monoclonal antibody having human SIRPα binding activity according to any one of claims 1 to 3.

10. Use of the monoclonal antibody having human SIRPα binding activity according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

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