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CN109306017B - Recombinant protein prepared based on SIRP-alpha D1 mutant and application thereof - Google Patents

Recombinant protein prepared based on SIRP-alpha D1 mutant and application thereof Download PDF

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CN109306017B
CN109306017B CN201811187563.0A CN201811187563A CN109306017B CN 109306017 B CN109306017 B CN 109306017B CN 201811187563 A CN201811187563 A CN 201811187563A CN 109306017 B CN109306017 B CN 109306017B
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王雷
黄苏萍
尹建河
张楠
董丽莎
赵永浩
汤春
彭继荣
张晓东
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Beierda Pharmacy Suzhou Co ltd
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Abstract

The invention discloses a recombinant protein prepared based on a SIRP alpha D1 mutant and application thereof, wherein a mutant I is obtained based on surface contact amino acid multi-point mutation of an IgV region (SIRP alpha D1) at the outer end of a SIRP alpha membrane, a mutant II is obtained by performing deglycosylation mutation on 80 th amino acid asparagine (N) on the basis of the mutant I, and a recombinant protein CD001/CD002 is obtained by performing fusion expression on a coupling human IgG1-Fc region. Compared with wild type or deglycosylated single-point mutants, the mutants have higher affinity with the recombinant protein CD47 and various cancer cell lines, and have better blocking effect on the combination of CD47 and SIRPa on the protein level and the cellular level; no coagulation to white blood cells (erythrocytes); functionally, the phagocytosis of tumor cells by macrophages can be activated more effectively. The CD001/CD002 has better target binding activity, better target blocking effect and stronger immune activation function, and has no side effect on erythrocytes.

Description

Recombinant protein prepared based on SIRP-alpha D1 mutant and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant protein prepared based on a SIRP-alpha D1 mutant and application thereof.
Background
CD47, also known as integrin-associated protein (IAP), is widely expressed on the cell surface and interacts with signal regulatory protein α (SIRP α), thrombospondin (TSP 1) and integrins (integrins) to mediate a series of responses such as apoptosis, proliferation, and immunity. CD47 is a 5-transmembrane protein with molecular weight of about 50kDa, belongs to immunoglobulin superfamily, and has IgV structure domain in the extracellular N-terminal. CD47 was first identified as a tumor antigen for human ovarian cancer in the 80's 19 th century, and CD47 was subsequently found to be expressed in various types of human tumors, including Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Acute Lymphocytic Leukemia (ALL), non-hodgkin's lymphoma (NHL), Multiple Myeloma (MM), Bladder Cancer (BC), and other solid tumors.
CD47 is a "self" marker, representing a "do not eat me" signal. The human body needs 20-30 trillion red blood cells to maintain efficient oxygen transport throughout the body. The life cycle of erythrocytes is short, only 120 days, with 100 billion erythrocytes produced per hour, and numerous aged erythrocytes phagocytosed and cleared by macrophages. However, macrophages are how to differentiate between young and senescent erythrocytes, and the mechanism of attacking only senescent erythrocytes is unknown. Until 2000, Oldenborg et al demonstrated that CD47 is an important "self" marker on the cell surface and an important signal for regulating macrophage phagocytosis. CD47 can bind to macrophage surface SIRP alpha, phosphorylate its ITIM, and subsequently recruit SHP-1 protein, resulting in a cascade of reactions that inhibit phagocytosis by macrophages. Young erythrocytes express higher CD47 releasing the "self-eat me" signal to macrophages, while senescent erythrocytes, CD47, are down-regulated and eventually cleared by macrophages.
Tumor cells have a series of protocols to evade the pursuit of the human immune system, including secretion of immunosuppressive factors, down-regulation of MHCI expression, and up-regulation of PD-L1 to inhibit CD8+ T cell activity. Clever tumor cells do not, of course, pass the perfect mask of CD47, and they evade phagocytosis by high expression of CD 47. Different studies have shown that almost all tumor cells and tissues highly express CD47, 3-fold higher than corresponding normal cells and tissues. By means of the "self" signal of CD47, tumor cells effectively evade phagocytosis by macrophages.
Various studies have shown that at the cellular level, the phagocytosis of cancer cells by macrophages can be effectively activated by blocking the correlation between sirpa-CD 47, and the growth of tumors in mice can be effectively inhibited by blocking the correlation between sirpa-CD 47 in vivo by CD47 antibodies. In addition, the structural and single-point mutation (fig. 1C) researches show that I33, H56 and S66 can affect the combination of sirpa D1 and CD47, and in a C' Dloop ring, K53 and E54 may have certain influence on the structural stability after combination with CD 47; in sirpa D1, there is potential N80 glycosylation that may have some effect on binding to CD 47.
The Fc receptor exists on the surface of various immune cells, such as B lymphocytes, natural killer cells (NK cells), macrophages, neutrophils and other cells with immune protection functions, and after the Fc receptor is combined, the Fc receptor can stimulate the macrophages to phagocytose pathogens or target cells and stimulate cytotoxic lymphocyte T cells, thereby mediating macrophage or antibody dependent cytotoxicity to kill the pathogens, cancer cells and the like.
There are many kinds of inhibitors for blocking SIRP alpha-CD 47 signal path, including anti-CD 47 monoclonal antibody Hu5F9-G4 of FortySevern company, TRILLIUM (TM) HERAPEUTICS, bifunctional fusion protein TTI-621 of INC company, and HY03M of Yongke biological medicine technology (Shanghai) Limited company, but these inhibitors have the disadvantages of side effect of binding with erythrocyte (hemolysis) or low targeting affinity with cancer cell.
The invention patent with patent application number 201510203619 discloses a gene recombinant fusion protein, which can achieve the purpose of eliminating tumor through two mechanisms, namely blocking the inhibitory signal of macrophage induced by tumor cells, and directly activating the phagocytosis of the macrophage. The protein is formed by connecting a first Ig-like region (SIRP alpha D1) at the outer end of a membrane of human Signal-regulatory protein (Signal-regulatory proteins) alpha (SIRP alpha) with an Fc fragment of human IgG1 (SIRP alpha D1-Fc). The invention provides a nucleotide molecule for coding recombinant bifunctional fusion protein, an expression vector for expressing the protein, a method for preparing the protein and a method for treating diseases related to over-expression of CD 47. The protein belongs to a homodimer and has a molecular weight of 90 kDa. However, this patent does not improve the targeting affinity to cancer cells, nor does it functionally improve the phagocytosis of cancer cells by macrophages.
Disclosure of Invention
The invention aims to provide a recombinant protein prepared based on a SIRPa D1 mutant, which changes the affinity of SIRPa D1 with cancer cells and erythrocytes by carrying out surface amino acid point mutation and combined deglycosylation point mutation on SIRPa D1, thereby improving tumor inhibition and reducing the side effect of erythrocyte hemolysis. The invention relates to 2 recombinant fusion proteins, which comprise SIRP alpha D1 contact surface amino acid mutation combination coupled human IgG1-Fc (CD001), and the recombinant protein CD001 is subjected to N80 deglycosylation to obtain CD 002. The CD001 and the CD002 can kill the tumor cells through two mechanisms, namely blocking SIRP alpha-CD 47 signals to activate phagocytosis of the tumor cells by macrophages, and combining Fc with Fc receptors of the macrophages to activate immunization of the macrophages. The invention discovers that CD001 and CD002 have better target binding activity, better target blocking effect and stronger immune activation function, have no side effect on erythrocytes, and are equivalent to a control (IgG1-Fc or PBS). The affinity of CD001, CD002 and target CD47 is improved by 120 times on the protein level, the binding capacity with a plurality of cancer cells is also obviously improved on the cell level, the target blocking effect is improved by 150-300 times, the phagocytosis (ADCP) of the cancer cells by activated macrophages is obviously improved, and the effect of no toxic or side effect is achieved on the hemolysis of white blood cells (red blood cells).
The invention discloses a recombinant protein, which comprises a mutant I obtained by carrying out multi-point mutation on amino acids on the outer end contact surface of a signal regulatory protein SIRP alpha membrane and an immunoglobulin Fc region connected with the mutant I, and/or a mutant II obtained by combining mutation of individual amino acids on the basis of the mutant I and an immunoglobulin Fc region connected with the mutant II, and then the recombinant protein CD001/CD002 is obtained by carrying out fusion expression by coupling the immunoglobulin Fc region.
Preferably, the mutant I is obtained by carrying out multi-point mutation on the contact surface amino acid of an IgV region (SIRP alpha D1) at the outer end of the SIRP alpha membrane. And carrying out multi-point mutation on the contact surface amino acid of the IgV region at the outer end of the SIRP alpha membrane of the signal regulatory protein.
In any of the above embodiments, preferably, mutant II is obtained by mutating the 80 th amino acid asparagine based on mutant I.
Preferably in any of the above embodiments, CD001 is deglycosylated with N80 to obtain CD 002.
Preferably, in any of the above embodiments, the signal-regulating protein SIRP alpha is a mature wild-type human signal-regulating protein.
Preferably, in any of the above embodiments, the immunoglobulin Fc region is human IgG 1-Fc.
The invention also provides an amino acid encoding the recombinant protein of any one of the above.
The invention also provides a polynucleotide encoding the amino acid.
The invention also provides an expression vector, which comprises the polynucleotide.
The invention also provides a host cell which comprises the expression vector.
The invention also provides an application of the recombinant protein in preparing a CD47 over-expression medicament for treating tumor diseases.
Preferably, the neoplastic disease includes at least one of a hematological tumor, a solid tumor disease, the hematological tumor being at least one of acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, mixed leukemia, non-hodgkin's lymphoma; the solid tumor is at least one of lung cancer, bladder cancer, breast cancer, renal cancer, colon cancer, prostate cancer, ovarian cancer, colon cancer, and pancreatic cancer.
The invention also provides application of the recombinant protein in preparing an immune medicament for blocking the interaction between CD47 and SIRPa and activating immune cells such as macrophages.
Advantageous effects
The invention discloses a recombinant protein prepared based on a SIRP alpha D1 mutant, which is characterized in that a mutant I is obtained based on surface contact amino acid multi-point mutation of an IgV region (SIRP alpha D1) at the outer end of a SIRP alpha membrane, a mutant II is obtained by performing deglycosylation mutation on 80 th amino acid asparagine (N) on the basis of the mutant I, and a human IgG1-Fc region is coupled for fusion expression to obtain recombinant proteins CD001 and CD 002. Compared with a wild type or deglycosylated single-point mutant, the mutant has higher affinity with the recombinant protein CD47 and various cancer cell lines, and has better blocking effect on the combination of CD47 and SIRPa on the protein level and the cellular level; no coagulation to white blood cells (erythrocytes); functionally, the phagocytosis of tumor cells by macrophages can be activated more effectively. The invention provides amino acid and nucleotide sequences of recombinant proteins CD001 and CD002, construction of mutant expression vectors and a preparation method of fusion protein. The invention discovers that CD001 and CD002 have better target binding activity, better target blocking effect and stronger immune activation function, have no side effect on erythrocytes, and are equivalent to a control (IgG1-Fc or PBS). The affinity of CD001 and CD002 with a target CD47 is improved by 120 times, the binding capacity with a plurality of cancer cells is also obviously improved, the target blocking effect is improved by 150-300 times, the phagocytosis (ADCP) of activated macrophages to the cancer cells is obviously improved, and the effect of no toxic or side effect is achieved in the aspect of hemolysis of white blood cells (red blood cells).
The invention relates to 2 recombinant proteins, which comprise a combination of SIRPa D1 contact surface amino acid point mutation, a human IgG1-Fc (CD001) and CD002 obtained by carrying out deglycosylation point mutation (N80A) on CD001, wherein the recombinant proteins can kill tumor cells through two mechanisms, namely blocking SIRPa-CD 47 signals to activate phagocytosis of the tumor cells by macrophages, and combining Fc with Fc receptors of the macrophages to activate immunization of the macrophages. The invention utilizes human embryonic kidney cells (293F) to express the recombinant protein and purify and prepare the recombinant protein, finds that the mutant has better target binding activity and macrophage ADCP activating function, and has lower hemolysis degree on erythrocytes.
The invention obtains the recombinant protein CD001(I31/K53/E54/H56/S66) by carrying out point mutation on partial surface amino acids of a binding region of SIRP alpha D1 and CD47, or carries out deglycosylation on the 80 th amino acid mutation of the mutant CD001 to obtain CD002 (I31/K53/E54/H56/S66/N80).
The invention researches the affinity of the mutant and SIRPa ligand CD47 on protein level, the affinity is improved by 120 times compared with wild type SIRPa D1-Fc (IgG1), and the affinity is improved by 30 times compared with deglycosylated single-point mutant SIRPa D1-Fc (N80A); meanwhile, the CD001 and CD002 have stronger blocking effect in the aspect of target blocking effect, and the blocking effect is improved by 150-300 times compared with SIRP alpha D1-Fc (IgG1) and SIRP alpha D1-Fc (N80A); the affinity with a stable cell line CHO-K1 expressing CD47 is researched on a cellular level, the affinity is remarkably improved compared with that of a wild type SIRP alpha D1-Fc (IgG1) and a deglycosylated single-point mutant SIRP alpha D1-Fc (N80A), and the affinity is also remarkably improved in the binding capacity with a plurality of cancer cells (Jurkat, HL-60, PC-3, CCRF-CEM and Raji); in addition, the invention researches the immune activation capability of the mutants on human macrophages, and can more effectively activate the phagocytosis (ADCP) of human macrophages on cancer cells; in vitro research on side effects of erythrocyte coagulation is carried out in the invention, CD001 and CD002 do not cause hemolysis on erythrocytes, while H5F9-G4 and SIRP alpha-Fc generate hemolysis when the concentration reaches 0.78 mu G/ml, and the invention still has no hemolysis side effect when the concentration reaches 200 mu G/ml.
Drawings
FIG. 1 is a schematic representation of the action of a SIRPa-CD 47 molecule and surface contact of important amino acids, wherein FIG. 1A is a schematic representation of CD47 and SIRPa molecules, FIG. 1B is a schematic representation of the immune escape of tumor cells by SIRPa-CD 47, and FIG. 1C is an important surface contact amino acid affecting SIRPa-CD 47 interaction;
FIG. 2 is a schematic diagram of the CD001/CD002 design of the present invention;
FIG. 3 is an SDS-PAGE pattern of seven fusion proteins;
FIG. 4 is target binding activity (ELISA);
FIG. 5 shows seven cell target binding activities (FACS);
FIG. 6 is target inhibition activity (ELISA);
FIG. 7 is target inhibitory activity (FACS);
FIG. 8 is FcrR binding activity (ELISA);
FIG. 9A is the results of the identification of human MDM after detachment activation; FIG. 9B is an ADCP activation activity assay of MDM by several fusion proteins;
FIG. 10 is an analysis of the coagulation effect of several fusion proteins on erythrocytes.
Detailed Description
The following examples are further illustrative of the present invention as to the technical content of the present invention, but the essence of the present invention is not limited to the following examples, and one of ordinary skill in the art can and should understand that any simple changes or substitutions based on the essence of the present invention should fall within the protection scope of the present invention.
As shown in figure 1 and figure 2, the invention obtains CD001(I31/K53/E54/H56/S66) by carrying out combined mutation on part of surface amino acids of a binding region of SIRPa D1 and CD47, or obtains CD002(I31/K53/E54/H56/S6/N80) by carrying out mutation and deglycosylation on the 80 th amino acid of the mutant CD 001. It is speculated that a short peptide sequence 'SCAWSGVAG' may be helpful for improving the activity of SIRPa D1, so the invention adds the short peptide sequence at the N-terminal of wild type SIRPa D1 to prepare CD003(SCAWSGVAG) as a reference study. In the aspect of amino acid mutation selection, the invention adopts the principle of similar amino acid mutation or amino acid structure similarity, such as K/R, E/Q, T/S, V/I, and the like, and the deglycosylation mutation is to mutate N into A. The recombinant protein is expressed by using human embryonic kidney cells (293F) and purified, the affinity of the mutants with a SIRP alpha ligand CD47 is studied on the protein level, the mutants have obvious improvement (up to 120 times) compared with wild type SIRP alpha D1-Fc (IgG1) and a deglycosylated single-point mutant SIRP alpha D1-Fc (N80A), the binding capacity with a CHO-K1 stable cell line expressing CD47 is obviously improved, the binding capacity with a plurality of cancer cells (Jurkat, HL-60, PC-3, CCRF-CEM and Raji) is obviously improved, and the phagocytosis of the cancer cells by human-derived macrophages can be more effectively activated; in the aspect of side effect, hemolysis on erythrocytes is negligible, while H5F9-G4 and SIRPa-Fc generate hemolysis when the concentration reaches 0.78 mu G/ml, and the invention still has no coagulation side effect when the concentration reaches 200 mu G/ml.
The invention provides the amino acid sequence and the nucleotide sequence of the mutant CD001, CD002 and CD003, an expression vector for expressing the mutant and a preparation mode of recombinant protein.
The technical scheme of the invention for further optimization is that based on the combination of SIRP alpha D1 contact surface amino acid point mutations and IgG1-Fc coupling to obtain recombinant protein CD001, deglycosylation is carried out on the mutant CD001 to obtain recombinant protein CD002, 293F cells are used for expressing the recombinant protein and purifying, compared with wild type SIRP alpha D1-Fc (IgG1), the mutants have higher affinity with target CD47, higher affinity with cancer cell lines and side effects on erythrocyte hemolysis.
The technical scheme of the invention for further optimization is that compared with wild type SIRP alpha D1-Fc (IgG1), the mutants of CD001 and CD002 have stronger blocking effect on target spots CD47 and SIRP alpha, and the blocking performance is improved by 150-300 times.
The technical scheme of the invention is that the recombinant proteins CD001(SEQ ID NO:16, SEQ ID NO:17) and CD002(SEQ ID NO:18 and SEQ ID NO:19) have stronger tumor killing activity, can effectively activate ADCP immune function of macrophage, and has two points of action mechanism, namely blocking SIRP alpha-CD 47 signal to activate phagocytosis of macrophage on tumor cells, and the other is that Fc combines with Fc receptor of macrophage to activate immune function of macrophage. The mutant can be applied to the treatment of tumors with over-expression of CD 47.
The technical scheme of the invention for further optimization is that the recombinant proteins CD001(SEQ ID NO:16, SEQ ID NO:17) and CD002(SEQ ID NO:18, SEQ ID NO:19) have lower side effect and do not cause hemolysis to erythrocytes. The mechanism of action may be that the signaling pathway leading to hemolysis of red blood cells cannot be activated due to reduced epitope affinity. The mutant has higher safety.
The technical scheme of the invention is that the recombinant fusion protein comprises a step of coupling IgG1-Fc to obtain CD001 and a step of obtaining CD002 by CD001 deglycosylation point mutation based on the combination of SIRP alpha extracellular region IgV sample region (SIRP alpha D1) contact surface amino acid point mutation, wherein the SIRP alpha D1 or a mutant thereof can be combined with CD47, and the IgG1-Fc can be combined with FcrR to activate the immunity of macrophages.
The technical scheme of the invention for further optimization is that the signal regulatory protein SIRP alpha in the recombinant protein is a mature wild type endogenous human signal regulatory protein, the IgV-like region of the SIRP alpha extracellular region is preferably human SIRP alpha D1, and the IgG1-Fc fragment is preferably human IgG1-Fc fragment.
The technical scheme of the invention for further optimization is that the recombinant protein CD001 has an amino acid sequence with at least 95% similarity to SEQ ID NO. 10; in one embodiment, the recombinant fusion protein CD002 of the present invention has an amino acid sequence with at least 95% similarity to SEQ ID NO. 10;
the technical scheme of the invention for further optimization is that the invention provides an amino acid sequence and a nucleotide sequence for coding the recombinant proteins (CD001 and CD002), an expression vector for expressing the recombinant proteins and a preparation mode of the recombinant proteins.
The technical scheme of the invention for further optimization is that the invention provides a host cell for expressing the recombinant protein.
The invention provides an application of recombinant protein in treating tumor diseases of CD47 overexpression.
The invention is applied to tumor diseases including blood tumor and solid tumor diseases, wherein the blood tumor is selected from acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, mixed leukemia and non-Hodgkin's lymphoma; the solid tumor is selected from lung cancer, bladder cancer, breast cancer, renal cancer, colon cancer, prostate cancer, ovarian cancer, colon cancer, and pancreatic cancer.
The specific embodiment is as follows:
example 1
The invention relates to 2 recombinant proteins, which comprise a combination of SIRPa D1 contact surface amino acid point mutation, a human IgG1-Fc (CD001) and CD002 obtained by carrying out deglycosylation point mutation (N80A) on CD001, wherein the recombinant proteins can kill tumor cells through two mechanisms, namely blocking SIRPa-CD 47 signals to activate phagocytosis of the tumor cells by macrophages, and combining Fc with Fc receptors of the macrophages to activate immunization of the macrophages. The invention utilizes human embryonic kidney cells (293F) to express the recombinant protein and purify and prepare the recombinant protein, finds that the mutant has better target binding activity and macrophage ADCP activating function, and has lower hemolysis degree on erythrocytes.
The invention obtains CD001(I31/K53/E54/H56/S66) by carrying out point mutation on partial surface amino acids of a binding region of SIRP alpha D1 and CD47, or obtains CD002(I31/K53/E54/H56/S6/N80) by carrying out mutation deglycosylation on the 80 th amino acid of the mutant CD 001. CD003 was prepared by adding the short peptide sequence "SCAWSGVAG" to the N-terminus of wild type SIRP α D1 for reference study.
The invention researches the affinity of the mutant and SIRPa ligand CD47 on protein level, the affinity is improved by 120 times compared with wild type SIRPa D1-Fc (IgG1), and the affinity is improved by 30 times compared with deglycosylated single-point mutant SIRPa D1-Fc (N80A); meanwhile, the CD001 and CD002 have stronger blocking effect in the aspect of target blocking effect, and the blocking effect is improved by 150-300 times compared with SIRP alpha D1-Fc (IgG1) and SIRP alpha D1-Fc (N80A); the affinity with a stable cell line CHO-K1 expressing CD47 is researched on a cellular level, the affinity is remarkably improved compared with that of a wild type SIRP alpha D1-Fc (IgG1) and a deglycosylated single-point mutant SIRP alpha D1-Fc (N80A), and the affinity is also remarkably improved in the binding capacity with a plurality of cancer cells (Jurkat, HL-60, PC-3, CCRF-CEM and Raji); in addition, the invention researches the immune activation capability of the mutants on human macrophages, and can more effectively activate the phagocytosis (ADCP) of human macrophages on cancer cells; in vitro research on side effects of erythrocyte coagulation is carried out in the invention, CD001 and CD002 do not cause hemolysis on erythrocytes, while H5F9-G4 and SIRP alpha-Fc generate hemolysis when the concentration reaches 0.78 mu G/ml, and the invention still has no hemolysis side effect when the concentration reaches 200 mu G/ml.
1. Test materials and methods
Constructing expression vectors of SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A), CD001, CD002 and CD 003; human SIRP alpha possesses two pairs of wild type V1 or V2, the amino acid sequence 27-504 constitutes mature V1 (NP-542970.1 NCBI), V2 and V1 have 13 amino acid difference, the amino acid sequence 30-504 constitutes mature V2(CAA71403.1GeneBank), the invention selects the amino acid sequence 31-148(SEQ ID NO:1) of wild type 2V 2 and SIRP alpha D1, the mutant is mutated according to the preset amino acid, CD001(I31/K53/E54/H56/S66), CD002 (I31/K53/E54/H56/S58003/N80) and CD SCAWSGVAG are respectively added at the N-terminal of SIRP alpha D1 (I1). For the selection of Fc, human IgG1(SEQ ID NO:2) and human IgG4 Fc (S228P, SEQ ID NO:3) were used. And adding an optimized signal peptide to the N end of the recombinant protein amino acid for secretory expression, performing codon optimization on the recombinant protein amino acid, adding a Kozak sequence GCCGCCACC to the 5' end of the nucleotide, adding EcoRI/HindIII enzyme cutting sites of pcDNA3.4 to the two ends of the nucleotide, and connecting the synthetic gene to the pcDNA3.4 after enzyme cutting.
2. Protein production
Expression vectors of SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A), CD001, CD002, CD003 and positive control antibody H5F9-G4(SEQ ID NO:4, SEQ ID NO:5) were amplified and extracted by E.coli and endotoxin was removed according to the plasmid: Expi-293F cells were transfected at 1 μ g/ml medium. The transfection reagent used was Expifactamine 293transfection kit (Theromfisher, Lot #: A14524), the cell density at transfection was 25 × 105cells/ml, the expression enhancers Enhancer1 and Enhancer2 were added 16-18h after transfection, and cell supernatants were collected 5 days after transfection.
Purifying with ProteinA, centrifuging at 4 deg.C at 10000rpm/min for 30min to remove cell debris, balancing 10 column volumes with balancing solution (0.02MPB, 0.15MNaCl, pH7.0), flowing the supernatant through the column at 2ml/min, washing 5 column volumes with the balancing solution, eluting with eluent (0.02MPB, 0.15MNaCl, pH3.0), and dripping the eluent into a collecting tube containing neutralizing solution (1MTris, pH 9.0). The protein eluate was collected, concentrated by ultrafiltration using an ultrafiltration tube (MilliporerUFC 903096)4000G and buffer was replaced with PBS (HyCloneSH30256.01), and stored at-20 ℃ after SDS-PAGE detection. Removing endotoxin, filtering and sterilizing, and detecting the purity of SDS-PAGE.
3. Target binding Activity (ELISA)
The binding activity of SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4 to its target CD47 was tested by ELISA.
The specific implementation is as follows:
hCD47-his (Cat # CD7-H5227, Lot # C56P1-737F1-FA) was coated at 1. mu.g/ml, the coating buffer selected from PBS (HyCloneLot: AC13298279), 100. mu.l/well, coating at room temperature (25 ℃) for 16-18H, washing the plate with TBST for 2 times, blocking with PBS + 3% BSA, blocking at 200. mu.l/well, blocking at room temperature (25 ℃) for 16-18H, washing the plate with TBST for 1 time, draining, and drying at 37 ℃ for 2 hours.
Dilution of CD47 series of inhibitors:
SIRP α -Fc, SIRP α D1-Fc (IgG1), SIRP α D1-Fc (IgG4), SIRP α D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4 were dispensed in 330 μ l100 μ G/ml, with 100 μ G/ml being the first gradient and a 4-fold dilution gradient was carried out, for example the second gradient was 80 μ l of the first gradient added to 240 μ l PBS and so on for a total of 11 gradient concentrations. Incubate at 37 ℃ for 1 hour. After washing the plate 3 times with the automatic plate washer PBST, 100. mu.l of 1: 20000 dilutions of goat anti-human HRP secondary antibody (abcam Lot #: ab98624) were incubated for 45 min at 37 ℃. After incubation, the plate is washed for 3 times by an automatic plate washing machine, and after the last washing, the residual liquid is completely removed from the ELISA plate to the absorbent paper. 100. mu.l of TMB developing solution was added to each well. The reaction was kept in the dark for 3-5 minutes, 50. mu.l of 1% H was added to each well2SO4The reaction was terminated. Setting the light absorption values of reading by an MD (I3X) microplate reader to be 450nm and 630nm, and storing data after automatically reading the values.
4. Target binding Activity (FACS)
The binding activity of SIRP α -Fc, SIRP α D1-Fc (IgG1), SIRP α D1-Fc (IgG4), SIRP α D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4 to cancer cell lines such as CHO-K1 stable cell line expressing human or cynomolgus CD47, Jurkat, HL-60, PC-3, CCRF-CEM, Raji was measured by flow cytometry (BDFACSCCelestaCellAnalyzer).
The specific implementation is as follows:
in this example, 2 stable cell lines (CHO-K1-Cyno-CD47/CHO-K1-Human-CD47) expressing CD47 and 5 cancer cell lines (CHO-K1-Cyno-CD47/CHO-K1-Human-CD47 from Nanjing Kingsler Biotech, Inc. ((Jurkat) ((R))
Figure BDA0001826643590000101
TIB-152TM) Belonging to the group of T-lymphocytic leukemia cells, HL-60: (
Figure BDA0001826643590000102
CCL-240TM) Belongs to human promyelocytic leukemia cells, PC-3 (Shanghai national academy of sciences cell bank) belongs to human prostate cancer cells, CCRF-CEM (Shanghai national academy of sciences cell bank) belongs to human acute lymphoblastic leukemia T lymphocytes, and Raji (Shanghai national academy of sciences cell bank) belongs to human lymphoma cells.
After the cells listed were digested separately (suspension cells were not digested), centrifuged at room temperature at 1000rpm for 5mins, supernatant was discarded, washed with PBS, and PBS was resuspended in a flow tube with cell concentration adjusted to 1 × 106cells/ml. The volume of PBS in each tube is 250 mu L, the reaction concentration of the recombinant protein or antibody is 3 gradients, 1 mu g/ml, 0.1 mu g/ml and 0.01 mu g/ml, and after the concentration of the protein or antibody is adjusted, 250 mu L of the corresponding protein is added in each tube; after 1h incubation at room temperature, PBS was washed 3 times (1500rpm, 5 mins); mu.L of a secondary antibody, Goatanti-humanIgG/Alexa647(BiossLot: AE041526) was added to each tube, and after incubation at room temperature for 30mins in the absence of light, washed 3 times with 1mLPBS (1500rpm, 5 mins); 500. mu.L of LPBS was added to each tube to resuspend the cells and test them on the machine.
5. Target inhibition Activity (ELISA)
Biotin-labeled SIRP alpha-Fc (Cat #4546-SA-050, R)&DSystem), CD47(Acro, CD7-H5227-1mg, Histag) were coated at 1. mu.g/ml in 100. mu.l/well in PBS buffer, mixed with different concentrations of the test sample, added to a 96-well plate precoated with CD47 antigen, and incubated at 37 ℃ for 45 minutes. After incubation, the plate is washed for 5 times by an automatic plate washer, and after the last washing, the enzyme label plate is used for absorbing waterAnd (4) covering the paper with clean residual liquid. Mu.l of Streptavidin-HRP was added to each well and incubated at 37 ℃ for 45 minutes. After incubation, the plate is washed for 5 times by an automatic plate washing machine, and the residual liquid is completely removed from the water absorption paper by an enzyme label plate after the last washing. 100. mu.l of TMB developing solution was added to each well. Reaction is carried out for 3-5 minutes in the dark, and 50 mul of 1% H is added into each well2SO4The reaction was terminated. Setting the light absorption values of reading by an MD (I3X) microplate reader to be 450nm and 630nm, and storing data after automatically reading the values.
Wherein the sample to be detected comprises SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4, B6H12(SEQ ID NO.:6 and SEQ ID NO.:7) and Human-IgG1, the initial concentration is 10 mu G/ml, the dilution is carried out by 3 times of gradient, and 12 concentration gradients are totally included.
6. Target inhibition Activity (FACS)
Jurkat cells were centrifuged at room temperature at 1000rpm for 5mins, the supernatant was discarded, washed with PBS, and the PBS was resuspended in a cell flow tube at a cell concentration of 1 × 107cells/ml. Biotin-labeled SIRP alpha-Fc (Cat #4546-SA-050, R)&DSystem) mixed with samples to be tested at different concentrations respectively and added to Jurkat cells (2.5 × 10)5cells/action). Each reaction system is 250 μ L, the reaction concentration of the recombinant protein or antibody is 10 gradients, the initial concentration is 300nM, and 3-fold gradient dilution is performed.
After incubation for 1h at 4 ℃, PBS was washed 3 times (1500rpm, 5 mins); 100 μ L of Anti-SAiFlour647 (gentript, 3 μ g/mL) was added to each tube, incubated at 4 ℃ in the dark for 30mins, and washed 3 times with 1mLPBS (1500rpm, 5 mins); 500. mu.L of LPBS was added to each tube to resuspend the cells and test them on the machine.
The sample to be detected comprises SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4 and Human-IgG 1.
Fc γ R binding Activity
Coating FcrRI, FcrRIIa, FcrRIIb and FcrRIIIa (1 mu g/ml and 100 mu l/hole) with PBS respectively, labeling a sample to be detected with Streptavidin-HRP, and detecting the binding activity of each sample to the FcrRI, FcrRIIa, FcrRIIb and FcrRIIIa by indirect ELISA;
the samples to be tested were added to 96-well plates precoated with FcrRI, FcrRIIa, FcrRIIb, FcrRIIIa, respectively, and incubated at 37 ℃ for 45 minutes. After incubation, the plate is washed for 5 times by an automatic plate washing machine, and the residual liquid is completely removed from the water absorption paper by an enzyme label plate after the last washing. 100. mu.l of TMB developing solution was added to each well. Reaction is carried out for 3-5 minutes in the dark, and 50 mul of 1% H is added into each well2SO4The reaction was terminated. Setting the light absorption values of reading by an MD (I3X) microplate reader to be 450nm and 630nm, and storing data after automatically reading the values.
The sample to be tested comprises SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4, B6H12 and Human-IgG1, the initial concentration is 10 mu G/ml, 3 times of gradient dilution is carried out, and 12 concentration gradients are totally formed.
8. In vitro function detection (activated macrophage phagocytosis experiment ADCP)
MDM isolation induction of effector cells: extracting 20 persons of venous blood to separate PBMC, separating monocyte, adding M-CSF to induce differentiation MDM, and identifying differentiated macrophage MDM by CD11b, CD14, CD45, CD163 and CD206 biomarkers after 2 weeks;
ADCP: removing target cell HL-60 from the incubator, collecting the cells into a 15ml centrifuge tube, centrifuging, discarding the supernatant, resuspending the cells in PBS, counting, staining the target cells with PKH26(SIGMA-ALDRICH), standing at 37 deg.C&5％CO2Culturing overnight; taking out the target cells from the culture medium the next day, centrifugally collecting the cells, discarding the supernatant, using the complete culture medium for resuspension, and counting; taking target cells, adding a complete culture medium, adding the cells and a sample to be detected into a corresponding 96-well plate, wherein the cells and the sample to be detected are 10000 cells/well; incubating for 0.5h at room temperature; taking out effector cells (MDM) from a culture medium, collecting the supernatant, adding PBS for washing, adding Accutase for digesting the MDM, removing the MDM from the wall, adding complete culture medium with the same volume for terminating digestion, transferring the cell suspension into a centrifuge tube, and centrifuging the cell supernatant and the digested cells for 10min according to 300 g; MDM cells were added to a 96-well plate corresponding to the target cells by adding a corresponding volume of MDM cells to the complete medium. Effector cells: target cells were incubated at 37 ℃ for 1h at 5: 1;
adding Accutase, observing whether the adherent cells are digested under a microscope (about 15min), taking out the cells, transferring the cells to another plate, and adding the removed cell suspension into the corresponding hole again after the cells are completely digested; centrifuging, adding detection antibody CD11b, and incubating at 4 deg.C for 15 min; adding PBS to each hole, centrifuging, removing supernatant, and adding PBS for heavy suspension; detecting by a flow cytometer;
and (3) data analysis: % Phagocytosis byMDMs { (PKH26+ & CD11b + cells)/AllPKH26+ cells }. times.100%.
The sample to be tested comprises SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4, B6H12, Human-IgG1, Human-IgG4 and anti-SIRPaAb, the initial concentration is 10 mu G/ml, 5-fold gradient dilution is carried out, and 7 concentration gradients are totally adopted. Samples at each concentration were replicated in duplicate wells.
9. Agglutination test of erythrocytes (hemagglutination activity)
Hemagglutination activity of SIRP α -Fc, SIRP α D1-Fc (IgG1), SIRP α D1-Fc (IgG4), SIRP α D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4 on blood cells was determined using erythrocytes from healthy humans.
The specific implementation is as follows:
collecting whole blood with anticoagulant such as sodium citrate; putting the whole blood into a 15ml centrifuge tube, supplementing PBS to 15ml, centrifuging at room temperature, centrifuging at 200 Xg for 10mins, and discarding the supernatant; make up RBCs to 15mL with PBS, mix well, centrifuge at room temperature, 1500rpm, 5 mins. Washing for 3 times; after the last washing, the concentration of RBCs is adjusted to 2% by PBS (for example, 49ml of PBS is added into 1ml of RBCs, the recombinant protein and the positive antibody are diluted by 2-fold gradient according to 200 mu g/ml, 15 concentration gradients are counted, a 96-hole round bottom plate is used, 50 mu l of recombinant protein or monoclonal antibody with corresponding concentration and 50 mu l of RBCs are added into each hole, and the reaction result is observed and recorded after the incubation for 2h at room temperature.
The experimental results are as follows:
1. construction of expression vectors
And constructing the optimized nucleotide sequence on a pcDNA3.4 vector after EcoRI/HindIII enzyme digestion according to the recombinant protein consisting of the selected and designed SIRPa, SIRP alpha D1, SIRP alpha D1 mutant and Fc, and performing target gene sequencing on the constructed expression vector.
The amino acid and nucleotide composition of each fusion protein is as follows:
SIRP alpha-Fc has 1722 nucleotides (SEQ ID NO.9) and encodes 574 amino acids (SEQ ID NO: 8); SIRP α D1-Fc (IgG1) has a total of 1035 nucleotides (SEQ ID NO:11) and encodes 345 amino acids (SEQ ID NO: 10); SIRP α D1-Fc (IgG4) has 1041 nucleotides (SEQ ID NO:13) in total and encodes 347 amino acids (SEQ ID NO: 12); SIRP α D1-Fc (N80A) has a total of 1122 nucleotides (SEQ ID NO:15) and encodes 374 amino acids (SEQ ID NO: 14); CD001 has 1035 total nucleotides (SEQ ID NO:17) and encodes 345 amino acids (SEQ ID NO: 16); CD002 has 1035 total nucleotides (SEQ ID NO:19) and encodes 345 amino acids (SEQ ID NO: 18); CD003 has a total of 1062 nucleotides (SEQ ID NO:21) encoding 354 amino acids (SEQ ID NO: 20);
2. protein production
The theoretical molecular weight of the expressed recombinant protein is as follows:
SIRP alpha-Fc-127.2 Kd, SIRP alpha D1-Fc (IgG4) -76.7 Kd, SIRP alpha D1-Fc (IgG4) -80.4 Kd, SIRP alpha D1-Fc (N80A) -82.2 Kd, CD 001-76.7 Kd, CD 002-76.7 Kd, and CD 003-78.3 Kd. The protein is expressed and purified by 293F cells and identified by SDS-PAGE, as shown in figure 3, the actual molecular weight is close to the theoretical molecular weight, and simultaneously, the invention is compared with SIRPa-Fc prepared by R & D and ACRO expression, and the molecular weight detected by SDS is consistent. Meanwhile, the mutant recombinant protein related to the invention has no influence on the expression in host cells due to the change of partial amino acids.
3. Target binding Activity (ELISA)
The mutant recombinant proteins were analyzed for their binding activity to their target CD47 by ELISA, and the results are shown in figure 4. We found that the affinity of SIRPaD1-Fc was superior to SIRPa-Fc, while that of SIRPaD1 deglycosylated mutant SIRPaD1-Fc (N80A) was superior to wild-type SIRPaD1-Fc, i.e., target binding activity SIRPaD1-Fc (N80A) > SIRPaD1-Fc > SIRPa-Fc, consistent with that described in patent CN 201510203619.7. And the binding activity of the part contacted with the surface amino acid mutant CD001 or the surface amino acid combined deglycosylated mutant CD002 can be obviously improved with the target CD 47. Compared with the wild type, the affinity of CD001/CD002 is improved by 120 times, the affinity of CD001/CD002 is improved by 10-30 times compared with SIRP alpha D1-Fc (N80A), and the affinity of CD001/CD002 is improved by 5 times compared with the H5F9-G4 target spot. In addition, the addition of SCAWSGVAG (CD003) to the N-terminal of the wild type can also improve the target affinity to a certain extent, and compared with the wild type, the target affinity is improved by about 15 times.
Overall target binding activity of CD 47:
(CD001/CD002)>CD003>SIRPαD1-Fc(N80A)>SIRPaD1-Fc>SIRPa-Fc
4. target binding Activity (FACS)
We determined the binding activity of SIRP α -Fc, SIRP α D1-Fc (IgG1), SIRP α D1-Fc (IgG4), SIRP α D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4 to cancer cell lines such as CHO-K1 stable cell line, Jurkat, HL-60, PC-3, CCRF-CEM, Raji expressing human or cynomolgus monkey CD47 by flow cytometry. The results are shown in FIG. 5.
The results show that the affinity of CD001/CD002 to the tested cell strains is significantly higher than that of SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A) to the tested cell strains, and the affinity is improved or leveled compared with the CD47 antibody H5F 9-G4. The affinity of CD003 for the cell lines tested was not significantly altered compared with SIRP α -Fc, SIRP α D1-Fc (IgG1), SIRP α D1-Fc (IgG4), SIRP α D1-Fc (N80A).
The results show that the SIRPaD1 contact surface amino acid mutant CD001 and the deglycosylation mutant CD002 can obviously improve the binding activity of the SIRPaD1 and the cancer cells. While the pure wild-type deglycosylation mutant SIRP alpha D1-Fc (N80A) can not effectively improve the binding activity with target cells; mutant CD003 obtained by adding short peptide sequence "SCAWSGVAG" to the N-terminus of wild type did not improve the binding activity to target cells.
5. Target inhibition activity
The invention utilizes Biotin to mark SIRPa-Fc (Cat #4546-SA-050, R & DSystem), and detects the blocking effect of 11 samples to be detected, such as SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4, B6H12 and the like, on the combination of SIRPa and CD 47. The results are shown in FIG. 6.
The result shows that the siRNA has stronger target blocking effect on CD001 and CD002 compared with SIRP alpha D1-Fc (IgG1) and SIRP alpha D1-Fc (N80A), and the blocking effect is improved by 150-300 times compared with SIRP alpha D1-Fc (IgG 1). The target binding inhibition activity of the deglycosylated single-point mutant SIRP alpha D1-Fc (N80A) is slightly improved compared with that of the SIRP alpha D1-Fc (IgG1), and the target binding inhibition activity of CD003 is obviously reduced.
It was shown that deglycosylated mutant SIRP alpha D1-Fc (N80A) or the N-terminal addition of the short peptide sequence CD003 did not improve the target inhibitory activity at the protein level. And the contact surface amino acid mutant CD001 can effectively enhance the target binding inhibition activity, and the N80A mutant CD002 can further enhance the target binding inhibition activity on the basis.
6. Target inhibition Activity (FACS)
The invention utilizes Biotin to mark SIRPa-Fc (Cat #4546-SA-050, R & DSystem), and detects the blocking effect of 10 samples to be detected, such as SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A), CD001, CD002, CD003, H5F9-G4 and the like, on the combination of SIRPa and Jurkat cells. The results are shown in FIG. 7. The result shows that compared with SIRP alpha D1-Fc (IgG1), CD001 and CD002 have stronger blocking effect, and the blocking effect is improved by 40-80 times. The cell binding inhibition activity of the deglycosylated single-point mutant SIRP alpha D1-Fc (N80A) is slightly reduced, and the cell binding inhibition activity of CD003 is not changed.
It was shown that deglycosylated mutant SIRP alpha D1-Fc (N80A) or N-terminal plus short peptide sequence CD003 did not improve target inhibition activity at cellular level. And the contact surface amino acid mutant CD001 (recombinant protein) can effectively enhance the cell binding inhibition activity, and the N80A mutant CD002 (recombinant protein) can further enhance the cell binding inhibition activity on the basis.
Fc γ R binding Activity
The binding activity of the selected Fc (IgG1) to Fc receptors FcrRI, FcrRIIa, FcrRIIb, and FcrRIIIa was verified, and the results are shown in fig. 8. It was shown that there was no significant difference from sirpa D1-Fc (IgG1) -derived Fc, presumably no significant difference in the effect of ADCP and ADCC or CDC on activation by Fc receptors.
8. In vitro functional assays
To analyze the ADCP activity of CD001/CD002 on activated macrophages, MDM was isolated and induced to amplify from PBMCs of 20 healthy persons, and the isolated induced MDM was confirmed by multiple biomarkers.
Further, the present invention performed experimental analysis of ADCP activating activity of mutant fusion proteins, in this experiment, the positive control of the present invention used various CD47 inhibitors, including antibodies B6H12, H5F9-G4 of CD47, a fusion protein SIRPa related to SIRPa, SIRP α -Fc, SIRP α D1-Fc (IgG1), SIRP α D1-Fc (IgG4), SIRP α D1-Fc (N80A), and a monoclonal antibody of SIRPa, and each sample tested used 7 gradient duplicate replicates.
The MDM biomarker detection results indicate that MDM detection markers activated by isolation from human blood are positive, meeting experimental requirements, as shown in fig. 9A and 9B.
In ADCP activation (as shown in figure 10), CD001, CD002 were significantly stronger than SIRPa-Fc, SIRPa D1-Fc (IgG1), SIRPa D1-Fc (IgG4) and deglycosylated single-point mutant SIRPa D1-Fc (N80A), with CD002 being most significant, at least 20% greater ADCP activity against MDM, whether compared to control antibody or control fusion protein. In addition, the ADCP activating activity of the deglycosylated single-point mutant SIRP alpha D1-Fc (N80A) and CD003 was not improved compared with that of SIRP alpha D1-Fc (IgG1), indicating that the ADCP activating activity of the deglycosylated single-point mutant SIRP alpha D1-Fc (N80A) or the N-terminal addition of the short peptide sequence CD003 was not improved. And the contact surface amino acid mutation CD001 can effectively enhance the ADCP activity, and the N80A mutation CD002 performed on the basis can further enhance the ADCP activity. Meanwhile, the ADCP activity of the SIRP alpha D1-Fc (IgG4) is lower than that of other fusion proteins, and the fact that the receptor activities of the SIRP alpha D1 and the Fc act together can maximize the activity of activating macrophage ADCP.
9. Agglutination test of erythrocytes (hemagglutination activity)
The invention detects the agglutination of red blood cells of SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4), SIRP alpha D1-Fc (N80A), CD001, CD002, CD003 and H5F9-G4 with the blood of a healthy person.
The results show that SIRP alpha-Fc, SIRP alpha D1-Fc (IgG4) and H5F9-G4 all produce different degrees of erythrocyte hemolysis (different hemolysis degrees according to protein concentration), while CD001 and CD002 have the same result as the negative control when the concentration reaches 200 mu G/ml, and have no hemolysis, and in addition, the results are consistent with the results of three fresh blood replicates of healthy people. The safety of CD001 and CD002 is better than that of SIRP alpha-Fc, SIRP alpha D1-Fc (IgG4) and H5F9-G4, and the safety is equivalent to that of SIRP alpha D1-Fc (IgG 1).
Conclusion analysis:
1. and (4) conclusion:
the invention obtains CD001 by mutating the contact surface amino acid of SIRP alpha D1 and obtains CD002 by mutating CD001 with N80A, so as to improve the target binding activity and reduce the side effect caused by erythrocyte coagulation. Meanwhile, the antibodies H5F9-G4 and B6H12, the recombinant fusion protein SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4) and SIRP alpha D1-Fc (N80A) are taken as research references.
The research finds that the binding activity of CD001/CD002 is obviously higher than that of SIRP alpha-Fc, SIRP alpha D1-Fc (IgG1), SIRP alpha D1-Fc (IgG4) and SIRP alpha D1-Fc (N80A) with CD47 recombinant protein or a plurality of cancer cell lines no matter the CD47 recombinant protein or the various cell lines (2 strains express CD47 stable cell lines and 5 cancer cell lines), the affinity of CD003 with CD47 recombinant protein is improved to a certain extent, but the binding with target cells is not improved to a great extent;
in the aspect of binding inhibition of CD47 and a ligand SIRPa thereof (IC50), CD001/CD002 has a better effect, and is improved by 150-300 times compared with SIRP alpha D1-Fc (IgG1), and a deglycosylated single-point mutant SIRP alpha D1-Fc (N80A) has no obvious improvement in target binding inhibition compared with SIRP alpha D1-Fc (IgG 1);
in the aspect of immune function activation, CD001/CD002 can more remarkably activate phagocytosis of human macrophages (MDM) on cancer cells; of these, CD002 was most significant, and at least 20% increased ADCP activity against MDM, whether compared to control antibodies or control fusion proteins. In addition, the ADCP activating activity of the deglycosylated single-point mutant SIRP alpha D1-Fc (N80A) and CD003 was not improved compared with that of SIRP alpha D1-Fc (IgG1), indicating that the ADCP activating activity of the deglycosylated single-point mutant SIRP alpha D1-Fc (N80A) or the N-terminal addition of the short peptide sequence CD003 was not improved. And the contact surface amino acid mutation CD001 can effectively enhance the ADCP activity, and the N80A mutation CD002 performed on the basis can further enhance the ADCP activity. Meanwhile, the ADCP activity of the SIRP alpha D1-Fc (IgG4) is lower than that of other fusion proteins, and the fact that the receptor activities of the SIRP alpha D1 and the Fc act together can maximize the activity of activating macrophage ADCP.
In terms of safety, neither CD001/CD002/CD003 nor SIRPa D1-Fc (IgG1) caused erythrolysis at a concentration of 200. mu.g/ml, while H5F9-G4 and SIRPa-Fc produced hemolysis at a concentration of 0.78. mu.g/ml; primate studies (LiuJ, WangL, ZhaoF, TsengS, NarayananC, shuraal, et. (2015)) found that effective concentrations of CD47 inhibitor in blood needed to be 50-150 μ g/ml, and thus CD001/CD002/CD003 and sirpa D1-Fc (IgG1) were more safe as CD47 inhibitors.
The present invention can be further illustrated based on the examples, but is not limited to the examples, and the examples do not limit the scope of the present invention. Various modifications, substitutions and equivalents may occur to those skilled in the art based on the disclosure within the scope of the appended claims.
Sequence listing
<110> times Dada pharmaceutical industries (Suzhou) Co., Ltd
<120> recombinant protein prepared based on SIRP-alpha D1 mutant and application
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Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225
<210> 4
<211> 117
<212> PRT
<213> Homo sapiens
<400> 4
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Thr Ile Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Tyr Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 5
<211> 112
<212> PRT
<213> Homo sapiens
<400> 5
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 6
<211> 118
<212> PRT
<213> Homo sapiens
<400> 6
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr
20 25 30
Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Ile Asp Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Phe Cys
85 90 95
Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 7
<211> 107
<212> PRT
<213> Homo sapiens
<400> 7
Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly
1 5 10 15
Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr
20 25 30
Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
35 40 45
Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro
65 70 75 80
Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Gly Phe Pro Arg
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 8
<211> 574
<212> PRT
<213> Homo sapiens
<400> 8
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg
115 120 125
Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe
130 135 140
Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu
145 150 155 160
Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr
165 170 175
Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His
180 185 190
Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro
195 200 205
Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr
210 215 220
Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val
225 230 235 240
Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp
245 250 255
Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr
260 265 270
Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn
275 280 285
Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His
290 295 300
Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala
305 310 315 320
His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser
325 330 335
Asn Glu Arg Asn Glu Phe Glu Pro Lys Ser Cys Asp Lys Thr His Thr
340 345 350
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
355 360 365
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
370 375 380
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
385 390 395 400
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
405 410 415
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
420 425 430
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
435 440 445
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
450 455 460
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
465 470 475 480
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
485 490 495
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
500 505 510
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
515 520 525
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
530 535 540
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
545 550 555 560
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 9
<211> 1722
<212> DNA
<213> Homo sapiens
<400> 9
gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60
gctatcctgc attgcaccgt gaccagcctg attcccgtgg gaccaatcca gtggttcaga 120
ggagccggac cagccagaga gctgatctac aaccagaagg agggccactt ccccagagtg 180
acaacagtgt ccgagagcac caagcgggag aacatggact tcagcatcag catcagcaac 240
atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300
accgagttca agagcggagc cggaacagag ctgagcgtga gagccaaacc tagcgctcca 360
gtggtgtctg gaccagcagc tagagccaca cctcagcaca ccgtgtcctt cacttgcgag 420
agccacggat tcagccccag agacatcacc ctgaagtggt tcaagaacgg caacgagctg 480
agcgacttcc agaccaacgt ggacccagtg ggagagagcg tgtcctacag catccactcc 540
accgccaagg tggtgctgac aagagaggac gtgcacagcc aggtcatttg cgaggtggca 600
cacgtgaccc tgcagggaga tcctctgaga ggaaccgcca acctgagcga gaccatcaga 660
gtgcctccta ccctggaagt gacacagcag ccagtgaggg ccgagaacca ggtcaacgtg 720
acttgccagg tccggaagtt ctacccccag agactgcagc tgacttggct ggagaacggc 780
aacgtgtcta gaaccgagac cgcctctacc gtgaccgaga acaaggacgg cacctacaat 840
tggatgtctt ggctgctggt gaacgtgtcc gcccataggg acgacgtgaa gctgacttgc 900
caggtggaac acgacggaca gcctgctgtg tctaagagcc acgacctgaa ggtgtccgcc 960
catcctaagg agcagggatc taataccgcc gccgagaaca ccggaagcaa cgagcggaac 1020
gagttcgagc ccaagtcttg cgacaagacc cacacttgcc ctccttgtcc agccccagaa 1080
ctgctgggag gccctagcgt gtttctgttc cctcccaagc ccaaggacac cctgatgatc 1140
agcaggaccc cagaagtgac ttgcgtggtg gtggacgtgt ctcacgagga tcccgaggtc 1200
aagttcaatt ggtacgtgga cggcgtggag gtgcacaacg ctaagaccaa gcccagggag 1260
gagcagtaca acagcaccta ccgggtggtg tccgtgctga cagtgctgca ccaggattgg 1320
ctgaacggca aggagtacaa gtgcaaggtg tccaacaagg ccctgccagc ccctatcgag 1380
aagaccatca gcaaggccaa gggccagcct agagagcctc aggtgtacac actgcctcct 1440
agcagggacg agctgaccaa gaaccaggtg tccctgactt gcctcgtgaa gggcttctac 1500
cccagcgata tcgccgtgga gtgggagtct aatggccagc ccgagaacaa ctacaagacc 1560
acccctccag tgctggatag cgacggctct ttcttcctgt acagcaagct gaccgtggac 1620
aagagcaggt ggcagcaggg caacgtgttc tcttgcagcg tgatgcacga ggccctgcat 1680
aaccactaca cccagaagag cctgagcctg agcccaggca ag 1722
<210> 10
<211> 345
<212> PRT
<213> Homo sapiens
<400> 10
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
115 120 125
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 11
<211> 1035
<212> DNA
<213> Homo sapiens
<400> 11
gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60
gctatcctgc attgcaccgt gaccagcctg attcccgtgg gaccaatcca gtggttcaga 120
ggagccggac cagccagaga gctgatctac aaccagaagg agggccactt ccccagagtg 180
acaacagtgt ccgagagcac caagcgggag aacatggact tcagcatcag catcagcaac 240
atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300
accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagcgacaag 360
acccacacct gtcctccttg tccagcccca gaactgctgg gaggaccaag cgtgttcctg 420
ttccctccca agcccaagga caccctgatg atcagcagga ccccagaagt gacttgcgtg 480
gtggtggacg tgtctcacga ggatcccgaa gtgaagttca attggtacgt ggacggcgtg 540
gaggtgcaca acgctaagac caagcccagg gaggagcagt acaacagcac ctaccgggtg 600
gtgtccgtgc tgacagtgct gcaccaggat tggctgaacg gcaaggagta caagtgcaag 660
gtgtccaata aggccctgcc agcccctatc gagaagacca tcagcaaggc caagggccag 720
cctagagagc ctcaggtgta caccctgcct cctagcaggg acgagctgac caagaaccag 780
gtgtccctga cttgcctcgt gaagggcttc taccccagcg atattgccgt cgagtgggag 840
tctaacggcc agcccgagaa caactacaag accacacctc cagtgctgga tagcgacggc 900
agcttcttcc tgtacagcaa gctgaccgtg gacaaaagcc gctggcagca gggcaacgtg 960
ttttcttgca gcgtgatgca cgaagccctg cacaaccact acacccagaa gagcctgagc 1020
ctgtctccag gcaag 1035
<210> 12
<211> 347
<212> PRT
<213> Homo sapiens
<400> 12
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
115 120 125
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
130 135 140
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
145 150 155 160
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
165 170 175
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
180 185 190
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
195 200 205
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
210 215 220
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
225 230 235 240
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
245 250 255
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
260 265 270
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
275 280 285
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
290 295 300
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
305 310 315 320
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
325 330 335
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 13
<211> 1041
<212> DNA
<213> Homo sapiens
<400> 13
gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60
gctatcctgc attgcaccgt gaccagcctg attcccgtgg gaccaatcca gtggttcaga 120
ggagccggac cagccagaga gctgatctac aaccagaagg agggccactt ccccagagtg 180
acaacagtgt ccgagagcac caagcgggag aacatggact tcagcatcag catcagcaac 240
atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300
accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagcgagagc 360
aagtacggcc ctccttgtcc tccttgtcca gctccagagt ttctgggcgg ccctagcgtg 420
tttctgtttc ctcccaagcc caaggacacc ctgatgatca gccggacccc agaagtgact 480
tgcgtggtgg tggacgtgtc tcaggaggat ccagaggtgc agttcaattg gtacgtggac 540
ggcgtggagg tgcacaacgc taagaccaag cccagggagg agcagttcaa cagcacctac 600
cgggtggtgt cagtgctgac agtgctgcac caggattggc tgaacggcaa ggagtacaag 660
tgcaaggtgt ccaacaaggg cctgcctagc agcatcgaga agaccatcag caaggccaag 720
ggccagccta gagagcctca ggtgtacacc ctgcctccta gccaggagga gatgaccaag 780
aaccaggtgt ccctgacttg cctcgtgaag ggattctacc ccagcgacat cgcagtcgag 840
tgggaaagca acggccagcc cgagaacaac tacaagacca cccctccagt gctggatagc 900
gacggcagct tcttcctgta cagccggctg accgtggaca aaagtcgctg gcaggagggc 960
aacgtgttca gttgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 1020
ctgagcctga gcctgggaaa g 1041
<210> 14
<211> 374
<212> PRT
<213> Homo sapiens
<400> 14
Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu Glu Leu Gln Val Ile
1 5 10 15
Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu
20 25 30
His Cys Thr Val Thr Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe
35 40 45
Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly
50 55 60
His Phe Pro Arg Val Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn
65 70 75 80
Met Asp Phe Ser Ile Ser Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly
85 90 95
Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe
100 105 110
Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser Ala
115 120 125
Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln His Glu Pro
130 135 140
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
145 150 155 160
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
165 170 175
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
180 185 190
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
195 200 205
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
210 215 220
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
225 230 235 240
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
245 250 255
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
260 265 270
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
275 280 285
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
290 295 300
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
305 310 315 320
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
325 330 335
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
340 345 350
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
355 360 365
Ser Leu Ser Pro Gly Lys
370
<210> 15
<211> 1122
<212> DNA
<213> Homo sapiens
<400> 15
tcttgcgctt ggagcggagt ggcaggagaa gaagagctgc aggtcatcca gcccgacaaa 60
agcgtgagcg tggcagccgg agaaagcgct attctgcatt gcaccgtgac cagcctgatc 120
ccagtgggac caatccagtg gttcagagga gccggaccag ccagggagct gatctacaac 180
cagaaggagg gccacttccc cagagtgacc acagtgtccg agagcaccaa gcgggagaac 240
atggacttca gcatcagcat cagcgccatc accccagcag acgccggcac atactattgc 300
gtgaagttcc ggaagggcag cccagatacc gagtttaaga gcggcgccgg aacagagctg 360
agcgtgagag ctaagcctag cgctccagtg gtgtcaggac cagccgctag agctacacct 420
cagcacgagc ccaagtcttg cgacaagacc cacacttgcc ctccttgtcc agccccagaa 480
ctgctgggag gccctagcgt gtttctgttc cctcccaagc ccaaggacac cctgatgatc 540
agcaggaccc cagaagtgac ttgcgtggtg gtggacgtgt ctcacgagga tcccgaggtc 600
aagttcaatt ggtacgtgga cggcgtggag gtgcacaacg ctaagaccaa gcccagggag 660
gagcagtaca acagcaccta ccgggtggtg tccgtgctga cagtgctgca ccaggattgg 720
ctgaacggca aggagtacaa gtgcaaggtg tccaacaagg ccctgccagc ccctatcgag 780
aagaccatca gcaaggccaa gggccagcct agagagcctc aggtgtacac actgcctcct 840
agcagggacg agctgaccaa gaaccaggtg tccctgactt gcctcgtgaa gggcttctac 900
cccagcgata tcgccgtgga gtgggagtct aatggccagc ccgagaacaa ctacaagacc 960
acccctccag tgctggatag cgacggctct ttcttcctgt acagcaagct gaccgtggac 1020
aagagcaggt ggcagcaggg caacgtgttc tcttgcagcg tgatgcacga ggccctgcat 1080
aaccactaca cccagaagag cctgagcctg agcccaggca ag 1122
<210> 16
<211> 345
<212> PRT
<213> Homo sapiens
<400> 16
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
115 120 125
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 17
<211> 1035
<212> DNA
<213> Homo sapiens
<400> 17
gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60
gctatcctgc attgcaccgt gaccagcctg tttcccgtgg gaccaatcca gtggttcaga 120
ggagccggac cagccagaga gctgatctac aaccagaggc agggcccttt ccctagagtg 180
acaaccgtgt ccgagaccac caagagggag aacatggact tcagcatcag catcagcaac 240
atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300
accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagcgacaag 360
acccacacct gtcctccttg tccagcccca gaactgctgg gaggaccaag cgtgttcctg 420
ttccctccca agcccaagga caccctgatg atcagcagga ccccagaagt gacttgcgtg 480
gtggtggacg tgtctcacga ggatcccgaa gtgaagttca attggtacgt ggacggcgtg 540
gaggtgcaca acgctaagac caagcccagg gaggagcagt acaacagcac ctaccgggtg 600
gtgtccgtgc tgacagtgct gcaccaggat tggctgaacg gcaaggagta caagtgcaag 660
gtgtccaata aggccctgcc agcccctatc gagaagacca tcagcaaggc caagggccag 720
cctagagagc ctcaggtgta caccctgcct cctagcaggg acgagctgac caagaaccag 780
gtgtccctga cttgcctcgt gaagggcttc taccccagcg atattgccgt cgagtgggag 840
tctaacggcc agcccgagaa caactacaag accacacctc cagtgctgga tagcgacggc 900
agcttcttcc tgtacagcaa gctgaccgtg gacaaaagcc gctggcagca gggcaacgtg 960
ttttcttgca gcgtgatgca cgaagccctg cacaaccact acacccagaa gagcctgagc 1020
ctgtctccag gcaag 1035
<210> 18
<211> 345
<212> PRT
<213> Homo sapiens
<400> 18
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
115 120 125
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 19
<211> 1035
<212> DNA
<213> Homo sapiens
<400> 19
gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60
gctatcctgc attgcaccgt gaccagcctg tttcccgtgg gaccaatcca gtggttcaga 120
ggagccggac cagccagaga gctgatctac aaccagaggc agggcccttt ccctagagtg 180
acaaccgtgt ccgagaccac caagagggag aacatggact tcagcatcag catcagcgcc 240
atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300
accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagcgacaag 360
acccacacct gtcctccttg tccagcccca gaactgctgg gaggaccaag cgtgttcctg 420
ttccctccca agcccaagga caccctgatg atcagcagga ccccagaagt gacttgcgtg 480
gtggtggacg tgtctcacga ggatcccgaa gtgaagttca attggtacgt ggacggcgtg 540
gaggtgcaca acgctaagac caagcccagg gaggagcagt acaacagcac ctaccgggtg 600
gtgtccgtgc tgacagtgct gcaccaggat tggctgaacg gcaaggagta caagtgcaag 660
gtgtccaata aggccctgcc agcccctatc gagaagacca tcagcaaggc caagggccag 720
cctagagagc ctcaggtgta caccctgcct cctagcaggg acgagctgac caagaaccag 780
gtgtccctga cttgcctcgt gaagggcttc taccccagcg atattgccgt cgagtgggag 840
tctaacggcc agcccgagaa caactacaag accacacctc cagtgctgga tagcgacggc 900
agcttcttcc tgtacagcaa gctgaccgtg gacaaaagcc gctggcagca gggcaacgtg 960
ttttcttgca gcgtgatgca cgaagccctg cacaaccact acacccagaa gagcctgagc 1020
ctgtctccag gcaag 1035
<210> 20
<211> 354
<212> PRT
<213> Homo sapiens
<400> 20
Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu Glu Leu Gln Val Ile
1 5 10 15
Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu
20 25 30
His Cys Thr Val Thr Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe
35 40 45
Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly
50 55 60
His Phe Pro Arg Val Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn
65 70 75 80
Met Asp Phe Ser Ile Ser Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly
85 90 95
Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe
100 105 110
Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser Asp
115 120 125
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
130 135 140
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
145 150 155 160
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
165 170 175
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
180 185 190
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
195 200 205
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
210 215 220
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
225 230 235 240
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
245 250 255
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
260 265 270
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
275 280 285
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
290 295 300
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
305 310 315 320
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
325 330 335
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
340 345 350
Gly Lys
<210> 21
<211> 1062
<212> DNA
<213> Homo sapiens
<400> 21
tcttgcgctt ggagcggagt ggcaggagaa gaagagctgc aggtcatcca gcccgacaaa 60
agcgtgagcg tggcagccgg agaaagcgct attctgcatt gcaccgtgac cagcctgatc 120
ccagtgggac caatccagtg gttcagagga gccggaccag ccagggagct gatctacaac 180
cagaaggagg gccacttccc cagagtgacc acagtgtccg agagcaccaa gcgggagaac 240
atggacttca gcatcagcat cagcaacatc accccagcag acgccggcac atactattgc 300
gtgaagttcc ggaagggcag cccagatacc gagtttaaga gcggcgccgg aaccgaactg 360
agcgtgagag ctaagcccag cgacaagacc cacacttgcc ctccttgtcc agccccagaa 420
ctgctgggag gccctagcgt gtttctgttc cctcccaagc ccaaggacac cctgatgatc 480
agcaggaccc cagaagtgac ttgcgtggtg gtggacgtgt ctcacgagga tcccgaggtc 540
aagttcaatt ggtacgtgga cggcgtggag gtgcacaacg ctaagaccaa gcccagggag 600
gagcagtaca acagcaccta ccgggtggtg tccgtgctga cagtgctgca ccaggattgg 660
ctgaacggca aggagtacaa gtgcaaggtg tccaacaagg ccctgccagc ccctatcgag 720
aagaccatca gcaaggccaa gggccagcct agagagcctc aggtgtacac actgcctcct 780
agcagggacg agctgaccaa gaaccaggtg tccctgactt gcctcgtgaa gggcttctac 840
cccagcgata tcgccgtgga gtgggagtct aatggccagc ccgagaacaa ctacaagacc 900
acccctccag tgctggatag cgacggctct ttcttcctgt acagcaagct gaccgtggac 960
aagagcaggt ggcagcaggg caacgtgttc tcttgcagcg tgatgcacga ggccctgcat 1020
aaccactaca cccagaagag cctgagcctg agcccaggca ag 1062

Claims (2)

1. A recombinant protein comprises a mutant I obtained by carrying out multi-point mutation on contact surface amino acids of an IgV region at the outer end of a mature wild type human signal regulatory protein SIRP alpha membrane and an immunoglobulin Fc region connected with the mutant I, and then coupling human IgG1-Fc for fusion expression to obtain a recombinant protein CD 001; or mutant II obtained by mutating the asparagine at the 80 th amino acid on the basis of the mutant I and an immunoglobulin Fc region connected with the mutant II are coupled with human IgG1-Fc for fusion expression to obtain recombinant protein CD 002; the nucleotide sequence of the gene for coding CD001 is shown as SEQ ID NO. 17 in the sequence table, and the amino acid sequence thereof is shown as SEQ ID NO. 16 in the sequence table; the nucleotide sequence of the gene coding CD002 is shown as SEQ ID NO. 19 in the sequence table, and the amino acid sequence thereof is shown as SEQ ID NO. 18 in the sequence table.
2. Use of the recombinant protein according to claim 1 for the preparation of a medicament for the treatment of a neoplastic disorder in which CD47 is overexpressed, said neoplastic disorder being selected from at least one of a hematologic neoplasm, which is at least one of acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, mixed leukemia, non-hodgkin's lymphoma; the solid tumor is at least one of lung cancer, bladder cancer, breast cancer, renal cancer, colon cancer, prostate cancer, ovarian cancer, colon cancer, and pancreatic cancer.
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CN106146670B (en) * 2015-04-24 2019-01-15 宜明昂科生物医药技术（上海）有限公司 A kind of new recombination double functions fusion protein and its preparation and application
CN111087473B (en) * 2019-12-11 2022-06-14 上海百英生物科技有限公司 SIRPa-Fc-IL21 fusion protein and application thereof
KR102557016B1 (en) * 2020-09-22 2023-07-20 비피진 주식회사 CD47 binder and liposome complex for treatment cancer
CN116178561B (en) * 2021-11-26 2025-07-18 杭州尚健生物技术有限公司 Fusion proteins comprising SIRPalpha mutants
CN118302437A (en) * 2021-11-19 2024-07-05 杭州尚健生物技术有限公司 SIRP alpha variants and uses thereof

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HUE050875T2 (en) * 2012-01-17 2021-01-28 Univ Leland Stanford Junior High affinity SIRP-alpha reagents
MX395275B (en) * 2015-08-07 2025-03-25 Alx Oncology Inc CONSTRUCTIONS WITH A SIRP-ALPHA DOMAIN OR ITS VARIANTS

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