CN112010979B - Anti-human SIRP alpha monoclonal antibody and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, relates to an anti-human SIRP alpha monoclonal antibody and application thereof, and also provides a coding nucleic acid molecule, an expression vector, a host cell and a method for expressing the antibody. Antibody immunoconjugate, bispecific molecule, chimeric antigen receptor, pharmaceutical composition, and diagnostic and therapeutic methods comprising the antibodies of the invention are also provided. The monoclonal antibody of the invention can specifically recognize human SIRP alpha, the binding affinity of the antibody and the human SIRP alpha is stronger than that of the existing anti-human SIRP alpha monoclonal antibody, and the sequence is novel.

Description

Anti-human SIRP alpha monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a high-affinity and functional anti-human SIRP alpha monoclonal antibody or antibody fragment. The invention also provides a coding nucleic acid molecule of the antibody, an expression vector, a host cell and a method for expressing the antibody. Antibody immunoconjugate conjugates, bispecific molecules, chimeric and antigen receptors, pharmaceutical compositions, and diagnostic and therapeutic methods comprising the antibodies of the invention are also provided.

Background

Signal regulatory protein alpha (Signal regulatory protein alpha), also known as SHPS-1 or SIRP alpha, is a typical inhibitory immunoreceptor in the SIRP family, and is mainly expressed on the surface of myeloid cells such as macrophages and dendritic cells, as well as neuronal cell membranes, while other cells in the body express less (Kharitonenkov, A.et al. (1997) 'A family of proteins at a high signaling protein receptors'. As a transmembrane protein, there are a total of three immunoglobulin-like domains in the extracellular region, whose amino-terminal domain can interact with CD47 to mediate signal transduction; the cytoplasmic tail of SIRP alpha is closely related to kinases and adaptor proteins; and the intracellular domain has typical immune receptor tyrosine inhibitory motif, can be phosphorylated, thereby generating accumulation reaction, and is activated by cytolytic protein tyrosine phosphatase SHP-1 and SHP-2 connected with the intracellular domain. SHP-1 and SHP-2 can modulate the signaling pathways in the cell and the efficacy of their downstream effectors by dephosphorylation of the proximal substrate (Mi, Z.P.et al (2000). "Expression of a kinase-associated membrane Protein, P84/SHPS-1, and its ligand, IAP/CD47, in motor derivatives". J.Comp.Neurol.416, 335-344; Ohnishi, H.et al. (1999) "transcription and association of BIT with SHP-2induced by proteins". J.Neurocm.72, 1402-1408; van den Berg, T.K.et al ". Nonetworked J.7788. 7788).

CD47, also known as integrin-associated proteins (IAPs), membrane proteins belonging to the immunoglobulin superfamily, are highly expressed on the cell membranes of various tumors, with a single immunoglobulin-like extracellular domain and five transmembrane regions. CD47 is a natural ligand for sirpa, and the CD 47-sirpa signaling pathway plays a role in a variety of physiological mechanisms. Among them, the most characteristic is the action exerted in the phagocytic process of macrophages in vivo. When the SIRP alpha on the surface of the macrophage is combined with a CD47 molecule on the surface of a tumor cell, the SIRP alpha mediates a signal of ' Don't eat me ', so that the tumor cell is prevented from being phagocytized by the macrophage, immune escape occurs, and further the tumor progresses. In one study, the experimenter designed high affinity variants of sirpa that antagonized CD47 on cancer cells and caused increased phagocytosis of cancer cells (Weiskopf k. et al (2013). "Engineered sirpa variants as immunological addenda to anticancer antibodies". science.341(6141): 88-91). Another study (in mice) found that Anti-SIRP α antibodies alone or in synergy with other cancer treatments could help macrophage function and reduce cancer growth and metastasis (functional new cancer metastasis cancers-defining cells. Feb 2017; Yanagita T. (2017). "Anti-SIRP α antibodies as a Potential new cell for cancer immunology". JCI insight.2(1): e 89140).

There has been little research on anticancer monotherapy or combination therapy using anti-sirpa antibodies. Most work on anti-sirpa antibodies is on the mechanistic study of the CD 47-sirpa interaction, and has been performed using murine anti-sirpa antibodies; for example, mice 12C4 and 1.23A increased neutrophil-mediated ADCC of trastuzumab (trastuzumab) -conditioned SKBR3 cells (zhao. et al (2011)' CD47-signal regulatory protein- α (SIRP α) interaction for antibody-mediated regulator cell determination ". pnas.108(45), 18342-18347). WO2015/138600 discloses a murine anti-human SIRP alpha antibody KWAR23 and its use to enhance in vitro phagocytosis of cetuximab (cetuximab)A chimeric Fab fragment. A humanized KWAR23 with a human IgG1 Fc portion comprising a N297A mutation is disclosed in WO 2018/026600. WO2013/056352 discloses IgG₄29AM4-5 and other IgG₄A human anti-SIRP α antibody. IgG₄29AM4-5 was administered three times a week at 8mg/kg for four weeks, reducing leukemia transplantation (engraftment) of primary human AML cells injected into the right femur of NOD Severe combined immunodeficiency disease (scid) gamma (NSG) mice. Anti-sirpa antibodies known in the art are not suitable for monotherapy or combination therapy against sirpa because they are not specific for human sirpa, or they are too specific. The existing antibodies KWAR23, SE5a5, 29AM4-5 and 12C4 are not specific as they also bind human SIRP γ. Binding to SIRP γ expressed on T cells may negatively affect T cell proliferation and recruitment. Other anti-sirpa antibodies also have limited specificity.

Although antibody drugs targeting sirpa have been developed and approved, development of mabs with higher affinity and other pharmacological properties against sirpa targets is still highly desirable and of significant medical value.

Disclosure of Invention

In order to overcome the defects, the invention aims to provide an anti-human SIRPa monoclonal antibody and an application thereof, wherein the antibody has high affinity and functionality which are superior to the existing anti-human SIRPa monoclonal antibody.

In order to achieve the above object, the present invention provides an anti-human sirpa monoclonal antibody, comprising: heavy and light chains;

the heavy chain and the light chain both comprise a variable region comprising a complementarity determining region;

the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain are represented by CDR-H1, CDR-H2 and CDR-H3, respectively;

the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain are represented by CDR-L1, CDR-L2 and CDR-L3, respectively;

the amino acid sequence of the CDR-H1 is SEQ ID NO: 3 is shown in the specification;

the amino acid sequence of the CDR-H2 is SEQ ID NO: 4 is shown in the specification;

the amino acid sequence of the CDR-H3 is SEQ ID NO: 5 is shown in the specification;

the amino acid sequence of CDR-L1 is SEQ ID NO: 6 is shown in the specification;

the amino acid sequence of CDR-L2 is SEQ ID NO: 7 is shown in the specification;

the amino acid sequence of CDR-L3 is SEQ ID NO: shown in fig. 8.

The heavy chain variable region amino acid sequence is SEQ ID NO: 1 is shown in the specification; the amino acid sequence of the light chain variable region is SEQ ID NO: 2, respectively.

A nucleotide molecule encoding said anti-human sirpa monoclonal antibody;

the sequence of the nucleotide molecule is selected from SEQ ID NO: 9 and SEQ ID NO: 10;

sequence SEQ ID NO: 9 encodes the heavy chain variable region of said antibody;

sequence SEQ ID NO: 10 encodes the light chain variable region of said antibody.

An expression vector containing the nucleotide molecule.

A host cell comprising said expression vector.

A preparation method of an anti-human SIRP alpha monoclonal antibody comprises the following steps:

step 1: preparing an expression vector containing a nucleotide molecule for expressing the anti-SIRPa monoclonal antibody;

step 2: transfecting a eukaryotic host cell with the expression vector of step 1;

and step 3: culturing the eukaryotic host cell transfected in step 2;

and 4, step 4: separating and purifying to obtain the antibody.

The invention also relates to antibody immunoconjugate conjugates, bispecific molecules, chimeric antigen receptors or pharmaceutical compositions comprising the aforementioned anti-human sirpa monoclonal antibodies.

Both the heavy and light chains also include a constant region that is a constant region of murine IgG, preferably IgG 1.

The heavy chain and the light chain both comprise constant regions, and the constant regions of the heavy chain are shown as SEQ ID NO: 11 is shown in the figure; the light chain constant region is shown as SEQ ID NO: shown at 12.

Compared with the prior art, the invention has the following advantages: the monoclonal antibody of the invention can specifically recognize human SIRP alpha, the binding affinity of the antibody and the human SIRP alpha is stronger than that of the existing anti-human SIRP alpha monoclonal antibody, and the sequence is novel.

Drawings

FIG. 1 is a capture ELISA assay for the binding ability of antibodies to human SIRPa protein;

FIG. 2 is a graph of an indirect ELISA assay for the binding ability of antibodies to human SIRPa protein;

FIG. 3 is a graph of an indirect ELISA assay for the cross-reaction of antibodies to human SIRP beta protein;

FIG. 4 is a reference antibody blocking ELISA;

FIG. 5 is a flow cytometry evaluation of antibodies blocking the binding of human SIRPa protein to cell membrane surface human CD47 protein;

FIG. 6 shows BIACORE surface plasmon resonance method for determining the affinity of mouse anti-SIRP alpha monoclonal antibody.

Detailed Description

The technical solution of the present invention is further described below with reference to examples.

Firstly, obtaining an antibody:

example 1 obtaining a mouse monoclonal antibody specific against SIRP alpha by the fusion hybridoma technique

1.1 animal immunization

Mice were immunized according to methods commonly used in the literature (E Harlow, D.Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998). The immunogen is recombinant human SIRP alpha (amino acid Met 1-Arg 370) protein with a C-terminal containing human IgG1 Fc tag (self-produced by a company, the used plasmid is CD 172-ECD-Fc; the carrier is pCP; and the inserted amino acid sequence is MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNER). The His-tagged protein of recombinant human SIRP alpha (produced by the company, the plasmid used is SRPA-ECD-His; the vector is pCMV-C-His; the amino acid sequence inserted between EcoR I and Xba I is

MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNER) as a test antigen for determination of serum titers and hybridoma screening. Briefly, the appropriate amount of Freund's adjuvant was removed into a 1.5ml EP tube and mixed well with a shaker. And (3) preparing a human SIRP alpha-Fc antigen protein solution by using PBS. Mixing adjuvant and protein antigen solution according to required amount, emulsifying antigen by injector to form stable water-in-oil solution, and injecting into animal. According to the result of serum titer measurement, 2 to 3 times of boosting immunization is usually required after the first immunization to achieve good immune effect. And (3) selecting an immune mouse with high serum titer for intraperitoneal injection, and performing cell fusion after final immunization.

1.2 hybridoma fusion and screening

Prior to cell fusion, mouse myeloma cells (SP2/0-Ag14, ATCC # CRL-1581) were cultured in logarithmic growth phase. Immunized mice were sacrificed and spleens were harvested in sterile environment and splenocytes B cells and SP2/0 myeloma cells were chemically fused using PEG according to literature procedures (E Harlow, D.Lane, Antibody: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Kohler G, and Milstein C, "Continuous cultures of fused cell secreted antibodies of predefined specificity," Nature,256: 495-. After the fusion, the cells are plated in 96-well cell culture plates, and the growth of the viable hybridoma cells can be observed under a microscope after 7 to 10 days. Two weeks after cell plating, culture supernatants from each well were collected and hybridoma screened using an ELISA method using human SIRP α -Fc protein antigen. Briefly, 60. mu.l of goat anti-mouse containing 1. mu.g/ml was usedMouse IgG F (ab')₂Specific fragments (Jackson Immunoresearch Laboratories, Inc., Cat # 115-. After washing the plate 1 time with 0.05% Tween 20 in PBS (i.e., 1xPBST), 200. mu.l/well of 5% skim milk powder in PBST was added and blocked at 37 ℃ for 2 hours. The plate was washed again, 60. mu.l/well of hybridoma supernatant was added, incubated at 37 ℃ for 40 minutes, and then the plate was washed 4 times. Add 100. mu.l/well of biotin-labeled human SIRP α Fc protein solution (1: 5000 dilution in PBST containing 2.5% nonfat dry milk), incubate at 37 ℃ for 40 minutes, wash the plate 4 times, and pat dry. Then add 1: horse radish peroxidase-labeled streptavidin (Jackson ImmunoResearch Laboratories, Inc., Cat # 016. sup. 030. sup. 084) diluted 5000 in PBST solution containing 5% skim milk powder was washed 4 times after incubation at 37 ℃ for 40 minutes and blotted dry. The absorbance of each well at 450 nm was measured by adding 100. mu.l/well of TMB (Innoreagents, Cat # TMB-S-002) chromogenic substrate, developing at room temperature for 5 to 15 minutes, followed by stopping with 1M sulfuric acid solution. The ELISA binding positive hybridoma well cells were picked out, transferred to a 24-well plate, and cultured. And performing a second round of re-screening by an ELISA method, screening out hybridomas which specifically recognize the SIRP alpha antigen and can block the combination of CD47/SIRP alpha (the result is shown in Table 1, wherein A2D9D1B1 is the hybridomas obtained by the invention, and the hybridomas express the anti-human SIRP alpha monoclonal antibodies prepared by the invention), and subcloning by a limiting dilution method to obtain the target monoclonal cell strain. Then expressing the produced mouse monoclonal antibody in small quantity, and carrying out the next functional evaluation analysis on the purified antibody.

TABLE 1 fused hybridoma supernatant detection ELISA data

II, an in vitro analysis method:

example 2in vitro assay for determining the functional Activity of SIRP α monoclonal antibodies

2.1 determination of the binding Capture ELISA-based antibody binding Capacity

Formulation of goat anti-mouse IgG F (ab') with 1xPBS₂A specific secondary antibody (Jackson ImmunoResearch Laboratories, Inc., Cat # 115-. After coating, the plates were washed 1 time with 1xPBST, 200. mu.l/well of PBST solution containing 5% skim milk powder was added and blocked at 37 ℃ for 2 hours. The plate was washed again with 100. mu.l/well of diluted antibody solution and Benchmark (i.e., KWAR23 antibody, available from Inc. with the heavy chain amino acid as

EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLEWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCARWGAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；

The light chain amino acid is

QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPRTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC), incubated at 37 degrees celsius for 40 minutes, and then the plates were washed 4 times. Biotin-labeled human SIRP α Fc protein solution (1: 10000 diluted in PBST containing 2.5% skim milk powder) was added to 100 μ l/well, incubated at 37 ℃ for 40 min, and the plate was washed 4 times. Add 1: 10000 dilutions of horseradish peroxidase-labeled streptavidin (Jackson ImmunoResearch Laboratories, Inc., Cat # 016-. 100. mu.l/well of an ELISA chromogenic substrate TMB (Innoreagents, Cat # TMB-S-002) was added, development was carried out at room temperature for 5 to 15 minutes, and then the development was stopped with a 1M sulfuric acid solution, and the absorbance of each well at 450 nm was measured. Data processing was performed using GraphPad Prism software, the results are shown in fig. 1. As can be seen from the figure, the A1E7F8G7 monoclonal antibody of the invention can identify human SIRP alpha antigen, and the binding activity is equivalent to that of Benchmark (abbreviated as BM).

2.2 Indirect ELISA to determine the binding Capacity of antibodies

Human SIRP alpha-Fc antigen was prepared with 1 Xcarbonate/bicarbonate buffer to a final concentration of 2. mu.g/ml, added at 100. mu.l/well to a 96-well microplate, and coated at 37 ℃ for 2 hours. After coating was completed, the plate was washed 4 times with 1xPBST, and 200. mu.l/well of PBST solution containing 5% skim milk powder was added and blocked at 37 ℃ for 2 hours. The plate was washed again, and after adding a 100. mu.l/well gradient diluted antibody solution, the plate was incubated at 37 ℃ for 40 minutes and then washed 4 times. Add 1: a5000 dilution of horseradish peroxidase-labeled goat anti-mouse Fc γ specific fragment (Jackson ImmunoResearch Laboratories, Inc., Cat # 115-. After 100. mu.l/well of an ELISA color developing substrate TMB was added and developed at room temperature for 3 to 10 minutes, the color development was terminated with a 1M sulfuric acid solution, and the absorbance of each well at 450 nm was measured. Data processing was performed using GraphPad Prism software, and the results are shown in fig. 2. As can be seen from the figure, the A1E7F8G7 monoclonal antibody of the invention can identify human SIRP alpha protein.

2.3 Indirect ELISA for determining the Cross-reactivity of antibodies with human SIRP beta

1x carbonate/bicarbonate buffer is used for preparing a human SIRP beta-ECD-His antigen (the plasmid is SIRP beta-ECD-His; the vector is pCMV-C-His; the inserted amino acid sequence between EcoR I and Xba I is

MDMRVPAQLLGLLLLWFPGSRCEDELQVIQPEKSVSVAAGESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTTVSELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAVRATPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPAGDSVSYSIHSTARVVLTRGDVHSQVICEIAHITLQGDPLRGTANLSEAIRVPPTLEVTQQPMRAENQANVTCQVSNFYPRGLQLTWLENGNVSRTETASTLIENKDGTYNWMSWLLVNTCAHRDDVVLTCQVEHDGQQAVSKSYALEISAHQKEHGSDITHEAALAPTAPLHHHHHHHHHH), the final concentration was adjusted to 2. mu.g/ml, and 100. mu.l/well was applied to a 96-well plate and coated at 37 ℃ for 2 hours. After coating was completed, the plate was washed 4 times with 1xPBST, and 200. mu.l/well of PBST solution containing 5% skim milk powder was added and blocked at 37 ℃ for 2 hours. The plate was washed again, and after adding 100. mu.l/well of the gradient diluted antibody solution and BM, the plate was incubated at 37 ℃ for 40 minutes and then washed 4 times. Add 1: the goat anti-mouse Fc γ specific fragment labeled with horseradish peroxidase at 5000 dilution was incubated at 37 ℃ for 40 minutes, followed by plate washing 4 times and patting dry. After 100. mu.l/well of an ELISA color developing substrate TMB was added and developed at room temperature for 3 to 10 minutes, the color development was terminated with a 1M sulfuric acid solution, and the absorbance of each well at 450 nm was measured. Data processing was performed using GraphPad Prism software, and the results are shown in fig. 3. As can be seen from the figure, the A1E7F8G7 monoclonal antibody of the invention can cross-react with human SIRP beta antigen, and the binding activity is close to that of Benchmark.

2.4 detection of SIRP alpha antibody blocking Activity

2.4.1 reference antibody blocking ELISA

The ability of the sirpa antibody to block the reference antibody/sirpa antigen binding was assessed by a competition ELISA method. Briefly, reference antibody (BM) was prepared at a final concentration of 2. mu.g/ml using 1xPBS, applied to a 96-well microplate at 100. mu.l/well, and coated overnight at 4 ℃. The next day, after washing the plate 1 time with PBST, 200. mu.l/well of PBST solution containing 5% skim milk powder was added, blocked at 37 ℃ for 2 hours, and the plate was washed 4 times again. The SIRP alpha antibody or the control antibody is diluted in a gradient in a human SIRP alpha Fc protein solution containing a biotin label, and is pre-incubated for 40 minutes at room temperature after being prepared. The incubated antibody and SIRP α Fc biotin solution was then applied to the reference antibody coated plate at 100 μ l/well and after incubation at 37 ℃ for 40 minutes, the plate was washed 4 times again. Then add 1: 10000 horseradish peroxidase-labeled streptavidin diluted in 5% skimmed milk powder-containing PBST solution, incubated at 37 ℃ for 40 minutes, and then the plate was washed 4 times and patted dry. TMB was added to the reaction solution to develop color and 1M sulfuric acid was added to terminate the reaction, and the absorbance at 450 nm was measured. Data processing using GraphPad Prism software to derive IC for antibody blocking reference antibody/SIRPa binding₅₀The concentration values are shown in FIG. 4. As can be seen from the figure, the A1E7F8G7 monoclonal antibody of the invention can specifically block the SIRPa protein from binding to the reference antibody thereofThe Benchmark has the blocking capacity equivalent to that of the Benchmark, and the A1E7F8G7 monoclonal antibody is similar to the Benchmark in antigen binding epitope.

2.4.2 flow cytometry evaluation of antibodies to block the binding of SIRPa protein to cell membrane surface CD47 protein

The SIRP alpha antibody or the contrast antibody is diluted in a gradient manner in a human SIRP alpha Fc protein solution containing a biotin label, and is pre-incubated for 40 minutes at room temperature after being prepared. 293F cells overexpressing human CD47 protein on the membrane surface were harvested in logarithmic growth phase (internal construction of the company, transfection of human CD47 full-length protein into 293F cells with lipofectamine 3000 transfection reagent, pCMV-T-P as vector, inserted sequence at EcoR I and Xba I

MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRNN, the cells were washed 2 times with PBS and then resuspended in FACS buffer (PBS containing 2% fetal bovine serum). Adjust density to 1x10⁵The cells/well were plated in a 96-well U-bottom plate, centrifuged at 300g for 5 minutes, the supernatant was decanted, and a solution of incubated antibody and human SIRPa Fc biotin was added at 100. mu.l/well to a 293F cell plate whose membrane surface overexpressed human CD 47. After incubation at 4 ℃ for 40 minutes, 300g were centrifuged for 5 minutes and the supernatant was decanted. The cells were washed 2 times with FACS buffer, 100. mu.L/well SA-PE (Jackson Immunoresearch, Cat # 016. sup. 110-one 084, 1: 500 dilution) was added, after incubation at 4 ℃ for 40 minutes, the cells were centrifuged at 300g for 5 minutes and the supernatant was decanted. The cells were washed 2 more times with FACS buffer. Then 100. mu.L of FACS buffer was added to each well for resuspension, and cells were blown down and tested on the machine. The mean fluorescence intensity MFI value per well of cells was determined using a BD Canto II model flow cytometer. Data processing using GraphPad Prism software gave antibody-bound cell ECs₅₀The concentration values (i.e., the corresponding antibody concentration values at which 50% of the maximum fluorescence binding signal of the antibody was reached) were shown in FIG. 5. As can be seen from the figure, the A1E7F8G7 monoclonal antibody of the present inventionThe human CD47 ligand capable of specifically blocking the binding of human SIRP alpha protein to the cell membrane surface has slightly better blocking capability than that of Benchmark, and the suggestion is that the A1E7F8G7 of the invention is a functional antibody with slightly better SIRP alpha/CD 47 blocking activity than a reference antibody.

2.5BIACORE surface plasmon resonance method for determining mouse anti SIRP alpha monoclonal antibody affinity experimental flow as follows:

1) balancing: the 10 Xbuffer (HBS-EP) was diluted to 1X with ultrapure water, the left hand side sample Tube A was inserted into the run 1 Xbuffer bottle, and the right hand side sample Tube was inserted into fresh ultrapure water. Opening a Biacore T200Control Software2.0, loading a CM5 chip coupled with anti-mouse IgG anti body protein, running a 'Prime' program twice, automatically switching the instrument into a Standby mode after running, and balancing the chip and a buffer solution overnight;

2) optimizing the capture amount: the optimized concentration was used to flow on-chip the murine anti-A1E 7F8G7 of the present invention.

3) And (3) affinity determination: solutions of human SIRP α -his antigen were prepared at 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, respectively. Editing an affinity determination program, setting capture time and a capture channel at a capture flow rate of 10 mul/min, setting a sample injection flow rate of 30 mul/min, a binding time of 120s and a dissociation time of 600s, selecting a proper sample tank, placing an analyte in the sample tank according to a position set in the program, and operating the program;

4) the binding rate constant K of murine anti-A1E 7F8G7 to human SIRP alpha-his antigen was calculated by Biacore T200 Evaluation software2.0_a(1/Ms), dissociation Rate constant K_d(1/s), affinity constant K_D(M)。

The study evaluated the affinity of the A1E7F8G7 monoclonal antibody of the invention for human sirpa-his antigen, and the specific data are shown in the following table and figure 6. Biacore affinity assessment data indicate: murine anti-A1E 7F8G7 affinity constant K for human SIRP alpha-his antigen_D8.19E-10 and Benchmark 2.94E-09, indicating that A1E7F8G7 has an approximately 3.6-fold stronger affinity than Benchmark.

TABLE 2 affinity Biacore evaluation of anti-SIRP alpha mouse monoclonal antibodies

Example 3 binding Activity of anti-SIRP α mouse monoclonal antibodies

Following the assay described in example 2, the binding activity of sirpa antibodies was assessed and summarized in table 3 below.

Where sirpa Benchmark was used as a control. As can be seen from Table 3, the anti-human SIRP alpha monoclonal antibody of the present invention has slightly better binding activity with human SIRP alpha antigen than that of Benchmark, and can cross-react with SIRP beta antigen.

TABLE 3 binding Activity of SIRP alpha antibodies

Example 4 functional blocking assay (see assay 2.4) data for antibody functional assay evaluation were obtained

TABLE 4 functional evaluation results of anti-SIRP alpha mouse monoclonal antibodies

Table 4 shows that the competitive ELISA experiment of the antibody on the Benchmark/human SIRP alpha-Fc shows that the A1E7F8G7 antibody can specifically block the SIRP alpha protein from binding to the Benchmark, and the blocking capability of the antibody is equivalent to that of the Benchmark; meanwhile, cell blocking FACS (FACS) experiments of the human CD47 cell/human SIRP alpha-Fc antibody show that the A1E7F8G7 antibody can specifically block the binding of human SIRP alpha protein to human CD47 protein, the blocking capability of the antibody is slightly better than that of Benchmark, and the A1E7F8G7 is a functional antibody which has similar binding epitope to that of the Benchmark and can block the binding of SIRP alpha/CD 47.

Example 5DNA cloning and sequencing, variable region sequencing of anti-SIRPa mouse monoclonal antibodies

Total R was extracted from the cultured mouse monoclonal Cell strain using Fastpure Cell/Tissue Total RNA Isolation Kit (Vazyme, catalog # RC101)And (4) NA. The procedure is briefly described below, with centrifugation collecting 1.5X10⁶The cells were transferred to a 1.5ml centrifuge tube and the supernatant was blotted. To the cell pellet was added 500. mu.l Buffer RL1 and vortexed. The treated cells were transferred to gDNA-Filter Columns (which had been placed in the collection tube), centrifuged at 13,000g for 2min at room temperature, and the supernatant in the collection tube was retained. Add 1.6 volumes of Buffer RL2 and mix gently. The mixture was transferred to RNAPure Columns, centrifuged at 13,000g for 1min at room temperature, and the waste liquid was discarded. To RNAcure Columns was added 500. mu.l Buffer RW1, and 13,000g was centrifuged at room temperature for 1min, and the waste liquid was discarded. Add 700. mu.l Buffer RW2, centrifuge at 13,000g for 1min at room temperature, discard the waste. Then 700. mu.l of Buffer RW2 was added, and the mixture was centrifuged at 13,000g at room temperature for 1min, and the waste liquid was discarded. Centrifugation at 13,000g for 2min was performed to completely remove Buffer RW2 remaining in RNAcure Columns. The column was transferred to a new RNase-free Collection Tubes 1.5ml centrifuge tube, and 50. mu.l of RNase-free ddH2O was suspended and dropped into the center of the column. The mixture was left at room temperature for 2min, and centrifuged at 13,000g at room temperature for 1min to elute RNA.

Next, the cDNA kit for reverse transcription from Takara (catalog #6210A) was used to convert the total RNA into CDNA. The experimental system was prepared as follows, 5. mu.l total RNA + 0.5. mu.l Oligo (dT) Primer + 0.5. mu.l Random 6mers + 1.0. mu.l dNTP mix + 3.0. mu.l RNase-free ddH₂O (10. mu.l total). The mixture was pre-denatured at 65 ℃ for 5min and then placed on ice for 2 min. Further add 4. mu.l 5 XPrimeScript II Buffer + 0.5. mu.l RNase Inhibitor + 1.0. mu.l PrimeScript II RTase + 4.5. mu.l RNase-free ddH₂O (20 mul system in total), mixing uniformly, operating a PCR instrument at 40 ℃ for 50min, and then operating at 70 ℃ for 10min to complete the cDNA synthesis.

The cDNA was further added with Poly G at the 3' end, and the reaction system was prepared as follows: 5 μ l cDNA +33.5 μ l ddH₂O + 5. mu.l of 10x TdT Buffer + 5. mu.l of CoCl +1. mu.l of dGTP + 0.5. mu.l of Terminal TransPerase (total 50. mu.l system), mixed well, run at 37 ℃ for 30min by using a PCR instrument, then shift to 70 ℃ for 10min, and the Poly G tailing addition is completed.

Further, gene amplification of the antibody variable region was carried out using the tailed cDNA as a template. For the amplification of the heavy chain variable region sequence of the antibody, a Novozam kit (catalog # P525-03) is selected to prepare a PCR reaction system: 25 μ l 2x Phanta Max Master Mix (Dye Plus) +2.0 μ l BSJ-Pri3

AntisenseP-mIgG1 (nucleotide sequence GCATCCCAGGGTCACCATGGAGTTAGTT) + 2.0. mu.l BSJ-Pri1 Universal C1 (nucleotide sequence GCATCCCAGGGTCACCATGGAGTTAGTT)

AAGCAGTGGTATCAACGCAGAGTCATCCCCCCCCCCCCCCCC) + 1.25. mu.l cDNA plus Poly G tail + 19.75. mu.l ddH₂O (total 50. mu.l system). For the amplification of the light chain variable region sequence of the antibody, a PCR reaction system was first prepared using a kit of Takara (catalog # R010A): 10 μ L of 5 × PrimeSTAR Buffer +0.5 μ L of PrimeSTAR +1.25 μ L of BSJ-Pri1 Universal C1 (nucleotide sequence AAGCAGTGGTATCAACGCAGAGTCATCCCCCCCCCCCCCCCC) +1.25 μ L of BSJ-Pri19 (nucleotide sequence GCACACGACTGAGGCACCTCCAGATGTT) +4 μ L of abrrant-L-R2 (nucleotide sequence GCACACGACTGAGGCACCTCCAGATGTT)

TCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGC) +4. mu.l dNTP + 1.25. mu.l cDNA plus Poly G tail + 27.75. mu.l ddH₂O (total 50. mu.l system).

The temperature cycles for PCR amplification of the variable regions of the antibody heavy chains were as follows:

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℃*∞

the temperature cycles for PCR amplification of antibody light chain variable regions are as follows:

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℃*∞

the PCR products were analyzed by 1% agarose gel electrophoresis, bands of DNA segments of corresponding sizes (about 500bp for VH, about 500bp for Vkappa light chain) were excised, and DNA was extracted using the gel DNA recovery kit from TIANGEN (catalog # DP 209-03). Briefly described as follows: the gel was weighed, an equal volume of solution PN was added, followed by incubation at 50 ℃ for 10min until the gel was completely dissolved. The resulting solution was transferred to a CA2 adsorption column (adsorption column placed in collection tube), left at room temperature for at least 2min, centrifuged at 12,000rpm at room temperature for 30sec, and the waste liquid was discarded. 600. mu.l of the rinsing solution PW was added to the column, and then centrifuged at 12,000rpm at room temperature for 30sec, to discard the waste liquid. Then, 600. mu.l of the rinsing solution PW was added to the column again, and the column was left to stand for 5min, centrifuged at 12,000rpm for 30sec at room temperature, and the waste liquid was discarded. And centrifuged again at 12,000rpm at room temperature for 2min to remove the liquid residue from the column, and left at room temperature for at least 2min to completely dry the residual liquid. The column was placed in a clean centrifuge tube, 35. mu.l of elution buffer EB was added and left at room temperature for at least 2 min. The prepared DNA sample was obtained by centrifugation at 12,000rpm for 2 min. The sequences of the variable regions of the antibodies obtained by sequencing the purified PCR products are shown in Table 5, the full-length amino acid sequence of the SIRPa mouse monoclonal antibody is shown in the following, and the nucleotide sequence of the SIRPa mouse monoclonal antibody is shown in the following.

TABLE 5 variable region amino acid sequences and CDR regions of SIRP alpha mouse monoclonal antibodies

The full-length amino acid sequence of the SIRP alpha mouse monoclonal antibody is as follows:

amino acid sequence of A1E7F8G7 mouse SIRP alpha monoclonal antibody

VH (variable heavy chain)

QAQLQQSGPELVKPGTSVKMSCKASGYTFTDYVITWVKQKTGQGLEWIGEIYPGSVSTYYNEKFKGKATLTADKSSNTVYMQLSGLTSEDSAVYFCARFDYRYDRYYVMDYWGQGTSVTVSS(SEQ ID No.:1)

CH (heavy chain constant region)

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID No.:11)

VL (light chain variable region)

DIVMTQSQKFMSTSVGDRVTVTCKASQNVDTNVVWYQQKPGQSPKTLIYSTSYRYSGVPDRITGSGSGTYFTLTISNVQSEDLAEYFCQQFSSYPWTFGGGTKLEIK(SEQ ID No.:2)

CL (light chain constant region)

RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC(SEQ ID No.:12)

The coding DNA sequence of the SIRP alpha mouse monoclonal antibody is as follows:

DNA sequence of heavy chain variable region

5’-CAGGCTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGACTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTGACTATGTTATAACTTGGGTGAAGCAGAAAACTGGACAGGGCCTTGAATGGATTGGAGAGATTTATCCTGGAAGTGTTAGCACTTACTACAATGAGAAGTTTAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGTCTACATGCAGCTCAGTGGTCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATTCGACTATAGGTACGACCGGTACTATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA-3’(SEQ ID No.:9)

DNA sequence of heavy chain constant region

5’-GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA-3’(SEQ ID No.:13)

DNA sequence of light chain variable region

5’-GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCACCGTCACCTGCAAGGCCAGTCAGAATGTGGATACTAATGTAGTCTGGTATCAACAGAAACCAGGCCAATCTCCTAAAACACTGATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGAATCACAGGTAGTGGATCTGGGACATATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAATATTTCTGTCAACAATTTAGCAGCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA-3’(SEQ ID No.:10)

DNA sequence of light chain constant region

5’-CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACTAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAGAGTGT-3’(SEQ ID No.:14)

In conclusion, the monoclonal antibody A1E7F8G7 capable of specifically recognizing human SIRP alpha has stronger binding affinity with human SIRP alpha and novel sequence, can specifically block the binding of human SIRP alpha and human CD47, and is a novel monoclonal functional antibody against human SIRP alpha.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

<110> Boaoxin Biotechnology (Nanjing) Ltd

<120> anti-human SIRP alpha monoclonal antibody and application thereof

<160> 14

<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gln Ala Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Val Ile Thr Trp Val Lys Gln Lys Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Ser Val Ser Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Val Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Phe Asp Tyr Arg Tyr Asp Arg Tyr Tyr Val Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 2

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Ser Thr Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Ile Thr Gly

50 55 60

Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Phe Ser Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 3

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Asp Tyr Val Ile Thr

1 5

<210> 4

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Glu Ile Tyr Pro Gly Ser Val Ser Thr Tyr Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 5

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Phe Asp Tyr Arg Tyr Asp Arg Tyr Tyr Val Met Asp Tyr

1 5 10

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Lys Ala Ser Gln Asn Val Asp Thr Asn Val Val

1 5 10

<210> 7

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ser Thr Ser Tyr Arg Tyr Ser

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gln Gln Phe Ser Ser Tyr Pro Trp Thr

1 5

<210> 9

<211> 366

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caggctcagc tgcagcagtc tggacctgag ctggtgaagc ctgggacttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact gactatgtta taacttgggt gaagcagaaa 120

actggacagg gccttgaatg gattggagag atttatcctg gaagtgttag cacttactac 180

aatgagaagt ttaagggcaa ggccacactg actgcagaca aatcctccaa cacagtctac 240

atgcagctca gtggtctgac atctgaggac tctgcggtct atttctgtgc aagattcgac 300

tataggtacg accggtacta tgttatggac tactggggtc aaggaacctc agtcaccgtc 360

tcctca 366

<210> 10

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcacc 60

gtcacctgca aggccagtca gaatgtggat actaatgtag tctggtatca acagaaacca 120

ggccaatctc ctaaaacact gatttactcg acatcctacc ggtacagtgg agtccctgat 180

cgaatcacag gtagtggatc tgggacatat ttcactctca ccatcagcaa tgtgcagtct 240

gaagacttgg cagaatattt ctgtcaacaa tttagcagct atccgtggac gttcggtgga 300

ggcaccaagc tggaaatcaa a 321

<210> 11

<211> 324

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 12

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 13

<211> 975

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60

tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 120

tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180

ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 240

acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 300

gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360

cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420

gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480

gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540

agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600

aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660

aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720

agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780

aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840

tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 900

acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 960

tctcctggta aatga 975

<210> 14

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60

ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120

tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180

agcaaagaca gcacctacag catgagcagc accctcacgt tgactaagga cgagtatgaa 240

cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300

agcttcaaca ggggagagtg t 321

Claims (9)

1. An anti-human sirpa monoclonal antibody, wherein said antibody comprises a heavy chain and a light chain;

the heavy and light chains each comprise a variable region comprising a complementarity determining region;

the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain are represented by CDR-H1, CDR-H2 and CDR-H3, respectively;

the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain are represented by CDR-L1, CDR-L2 and CDR-L3, respectively;

the amino acid sequence of the CDR-H1 is shown as SEQ ID NO: 3 is shown in the specification;

the amino acid sequence of the CDR-H2 is shown as SEQ ID NO: 4 is shown in the specification;

the amino acid sequence of the CDR-H3 is shown as SEQ ID NO: 5 is shown in the specification;

the amino acid sequence of the CDR-L1 is shown in SEQ ID NO: 6 is shown in the specification;

the amino acid sequence of the CDR-L2 is shown in SEQ ID NO: 7 is shown in the specification;

the amino acid sequence of the CDR-L3 is shown in SEQ ID NO: shown in fig. 8.

2. The anti-human sirpa monoclonal antibody of claim 1, wherein the heavy chain variable region amino acid sequence is set forth in SEQ ID NO: 1 is shown in the specification; the variable region amino acid sequence of the light chain is shown as SEQ ID NO: 2, respectively.

3. The anti-human sirpa monoclonal antibody of claim 1, wherein both the heavy and light chains comprise a constant region, and the heavy chain constant region is as set forth in SEQ ID NO: 11 is shown in the figure; the light chain constant region is shown as SEQ ID NO: shown at 12.

4. A nucleotide molecule encoding the anti-human SIRPa monoclonal antibody of any one of claims 1-3.

5. The nucleotide molecule of claim 4, wherein the sequence of the nucleotide molecule comprises SEQ ID NO: 9 and SEQ ID NO: 10;

sequence SEQ ID NO: 9 encodes the heavy chain variable region of said antibody;

sequence SEQ ID NO: 10 encodes the light chain variable region of said antibody.

6. An expression vector comprising the nucleotide molecule of claim 4 or 5.

7. A host cell comprising the expression vector of claim 6.

8. A preparation method of an anti-human SIRP alpha monoclonal antibody is characterized by comprising the following steps:

step 1: preparing an expression vector containing a nucleotide molecule that expresses the anti-human sirpa monoclonal antibody of claim 1;

step 2: transfecting a eukaryotic host cell with the expression vector of step 1;

and step 3: culturing the eukaryotic host cell transfected in the step 2;

and 4, step 4: separating and purifying to obtain the antibody.

9. An antibody immunoconjugate, bispecific molecule, chimeric antigen receptor, or pharmaceutical composition comprising the anti-human sirpa monoclonal antibody of claim 1.

Images