CN109897111A

CN109897111A - The bispecific antibody of anti-PD-1/CD47 and its application

Info

Publication number: CN109897111A
Application number: CN201711298703.7A
Authority: CN
Inventors: 黄莺; 张发明; 席甘
Original assignee: Hangzhou Hice Biological Medicine Co Ltd
Current assignee: Hangzhou Hice Biological Medicine Co Ltd
Priority date: 2017-12-08
Filing date: 2017-12-08
Publication date: 2019-06-18
Anticipated expiration: 2037-12-08
Also published as: CN109897111B

Abstract

The invention proposes a kind of recombinant antibodies.The recombinant antibodies include: the antibody of anti-PD-1；And people's SIRPA extracellular fragment, the N-terminal of the people SIRPA extracellular fragment are connected with the C-terminal of the heavy chain of the antibody of the anti-PD-1.The recombinant antibodies target PD-1 and CD47 simultaneously, can dramatically increase the ability of the stimulation immune system of the two, show stronger tumor suppression ability than single target antibody.

Description

The bispecific antibody of anti-PD-1/CD47 and its application

Technical field

The present invention relates to immunologys and antibody engineering technical field, and in particular to the bispecific antibody of anti-PD-1 and CD47 And its application, more particularly it relates to recombinant antibodies, nucleic acid, construct, preparation and reorganization antibody method and treatment cancer The therapeutic combination of disease.

Background technique

Cancer becomes one of the main reason for whole world leads to death in recent years.According to World Health Organization, 2015 The whole world has accounted for nearly 1/6th of current year total death toll because number of cancer deaths reaches nearly 8,800,000.2015 China there are about 429.2 ten thousand cancer new cases, 281.4 ten thousand cancer mortality cases, and also cancer morbidity is still in rising trend in China.

At present for the treatment means of cancer still to be performed the operation, based on radiation treatment and chemotherapy, traditional is controlled Treatment means have the shortcomings that tumour-specific difference and toxic side effect are big.Therefore, people are for more safe and efficient and high specificity Means carry out treating cancer and have strong demand.In recent years, leading to as mechanism understanding of the people for cancer is constantly deepened Specific antibody is crossed to inhibit the molecular targeted therapy of tumour gradually to show up prominently.Antibody drug on this basis is with cell The drug of antibody engineering technology preparation based on engineering technology and technique for gene engineering has specificity height, significant in efficacy, poison The advantages that few side effects, there is boundless prospect to oncotherapy.In recent years, some immunologic test points (immunogicalcheckpoint) constantly discover, is developed resistance for the specific antibody of these immunologic test points Immunotherapy is pushed to new wave height tide by the process of disconnected immunosurveillance escape.

In immune system, the activation process of T cell be it is complicated, the of MHC-TCR complex offer is provided One stimulus signal, it is also necessary to the second signal (namely " costimulatory signal ") that some molecules of Antigen Presenting Cell surface provide. Lacking this kind of costimulatory signal will lead to the unresponsive of T cell, tolerance and apoptosis^[1].Programmed death receptor -1 (programmed death-1, PD-1, also known as CD279) and its ligand programmed death receptors ligand 1 and 2 (PD-L1 and PD- L2) be so a kind of inhibition costimulatory molecules.

The PD-1 of people belongs to the I type transmembrane glycoprotein of CD28 family member, and molecular weight is about 55kDa.The born of the same parents of people PD-1 There are two tyrosine residues (Y223 and Y248) for inner region, and an immunity receptor tyrosine for participating in constituting N-terminal respectively inhibits Motif (immunoreceptor tyrosine-based inhibitory motif, ITIM, motif VDYGEL) and C-terminal One immunity receptor tyrosine rely on conversion motif (immunoreceptor tyrosin-based switch motif, ITSM, motif TEYATI).ITIM motif can recruit the albumen in SHP-2 intracellular and phosphorylation downstream, reduce BCR or TCR by Body signal, the generation and the stagnation of inducing cell division cycle of the final proliferation and cell factor for inhibiting lymphocyte^[2].Extracellular region There is an IgV spline structure domain, glycosylated containing multiple glycosylation sites and by severe, be primarily involved in and ligand binding, to send out Wave the function of inhibiting T cell activation.With dimeric forms, there are different, the extracellular region shortages half of PD-1 from other costimulatory molecules Cystine residue, therefore PD-1 can exist in the form of monomer.PD-1 is expressed in the T and B cell of activation, also thin in monokaryon Table in born of the same parents, Dendritic Cells (dendritic cells, DCs) and regulatory T cells (T regulatory cells, Tregs) It reaches.

Two kinds of ligands of PD-1, PD-L1 (B7-H1, also known as CD274) and PD-L2 (B7-DC), are I type glycoprotein, belong to B7 family protein member^[3].The major ligand PD-L1 of PD-1 contains IgV sample area, IgC sample area, transmembrane region and cytoplasmic region, wherein born of the same parents Matter area participates in intracellular signal transduction, and IgV sample area and IgC sample area then participate in intercellular signal transduction.It is expressed by some inflammation The positive regulation of inflammation factor, such as interleukin-4 (IL-4), tumor necrosis factor α (TNF-α) and interferon gamma (IFN-γ) Deng^[4].In addition to expressing other than the T cell of activation, B cell, macrophage, Dendritic Cells and kinds of tumor cells, PD-L1 is also Wide expression is in organs such as non-lymphoid organs such as heart, blood vessel, placenta, skeletal muscle, lung, liver, spleen and thymus gland.PD-L2 is main only It is expressed on the macrophage of activation, DCs and a small number of tumour cells^[5]。

The immunosuppressive action of PD-1/PD-L1 signal path has the generation of panimmunity dysfunctional disease with development Important function.Such as autoimmune disease.The mouse of missing PD-1 will appear retardance, organ specific autoimmune.PD- 1 deletion condition equal body under the LPR model for the lupus erythematosus for being related to autoimmune disease and the NOD mouse model of diabetes Reveal the tissue specificity autoimmunity characterization of acceleration^[6].Furthermore in chronic infection's body of some virus HIV and HBV etc. Inside have been found that cytotoxic T (cytotoxic lymphocyte, CTL) cell of the functional defect of overexpression PD-1, and Blocking PD-1 signal then can be reversed " afunction " state of CTL and removes virus^[7]。

Tumour cell can implement immunologic escape using the immunosuppressive action of PD-1/PD-L1 signal path.Kinds of tumors Cell meeting up-regulated expression PD-L1, such as non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC), melanin Tumor, lymthoma, breast cancer, leukaemia and various urological cancers, tumor in digestive tract, system genitale tumour etc.^[8].Overexpression PD- L1 is phosphorylated the tyrosine in the ITSM structural domain of PD-1, in turn to the PD-1 acceptor interaction with T cell surface Cause the dephosphorylation of downstream phosphatidyl-inositol 3-kinase (PI3K) and tyrosine kinase (Syk), inhibits downstream AKT, ERK etc. logical The activation on road, the transcription and translation of gene needed for finally inhibiting T cell activation and cell factor^[5].On the other hand, research shows that PD-L1 causes PD-1 in the accumulation of T cell, leads to cell cycle arrest, makes largely to accumulate in the cell of G0/G1 phase^[9].In vitro Experiment and mouse model also found that activation PD-1/PD-L1 signal path causes specific CTL tune to be died, and kill the cell of CTL Hurt the decline of effect sensibility, promotes tumour cell that immunologic escape occurs^[10].In conclusion tumour cell by expression PD-L1 and The PD-1 on T cell surface is acted on, and is able to suppress activation, proliferation and the killing to tumour of T cell.

Therefore, PD-1/PD-L1 signal path becomes recruit's target of immunotherapy of tumors, PD-1/PD-L1 signal Access play the role of in tumour immunity it is key, block PD-1/PD-L1 signal path can block inhibiting tumour cells cell Immune main force's T cell.The hot spot that the exploitation of anti-PD-1 and anti-PD-L1 antibody has become in immunotherapy of tumors research is ground Study carefully direction.U.S. FDA, which has been approved by the monoclonal antibody medicine for PD-1 target spot used, at present Merck & Co., Inc. (Merck) The Opdivo of Keytruda (pembrolizumab) and Bristol-Myers Squibb Co. (Bristol-Myers Squibb) (Nivolumab).There are Tecentriq (Atezolizumab), the brightness of Roche (Roche) for the monoclonal antibody medicine of PD-L1 target spot What the Bavencio (avelumab) and AstraZeneca (AstraZeneca) of auspicious (Pfizer) and Merck (Merck) production were produced Imfinzi(Durvalumab).These monoclonal antibody medicines since most clinically for treating after melanoma and chemotherapy still The kinds of tumors such as the metastatic squamous non-small cell lung cancer of progress are used for Hodgkin lymphoma, kidney, gastric cancer, anus till now The tumor types such as cancer, liver cancer, colorectal cancer all have been achieved for preferable clinical test results.In addition there are also multiple anti-PD-1 and The antibody of anti-PDL1 enters treatment of the clinical test for kinds of tumors.

But the maximum of immunotherapy is limited in efficient too low at present, and PD-1's is efficient from 15%-50% etc..Have Research evidence shows that can successful immunotherapy for cancer be widely immunized accordingly depending on trigger system, rather than only triggers The local influence of tumour itself, the response that stimulation immune system generates more system are expected to dramatically increase the validity of immunotherapy.

Summary of the invention

The present invention is directed to solve at least some of the technical problems in related technologies.

For this purpose, the invention proposes a kind of recombinant antibodies in the first aspect of the present invention.According to an embodiment of the invention, The recombinant antibodies include: the antibody of anti-PD-1；And people's SIRPA extracellular fragment, the N-terminal of the people SIRPA extracellular fragment with it is described The C-terminal of the heavy chain of the antibody of anti-PD-1 is connected.Recombinant antibodies according to an embodiment of the present invention target PD-1 and CD47 simultaneously, can be with The ability for dramatically increasing the stimulation immune system of the two, shows stronger tumor suppression ability than single target antibody.

According to an embodiment of the invention, above-mentioned recombinant antibodies can further include following additional technical feature at least it One:

According to an embodiment of the invention, the antibody of the anti-PD-1 is the IgG class antibody of anti-PD-1.Preferably, IgG class is anti- Body is IgG4 hypotype.IgG4 incomplete antibody easy to form (exchange that Fab-arm occurs), has carried out the IgG4 hypotype of S228P transformation Antibody can effectively reduce the exchange of Fab-arm, effectively avoid the effect of ADCC and CDC.

According to an embodiment of the invention, the antibody of the anti-PD-1 is H8.H8 can be found in patent 201610207741.6 with And the content that PCT/CN2016/103814 is recorded.

According to an embodiment of the invention, further comprise link peptide, the antibody of the N-terminal of the link peptide and the anti-PD-1 Heavy chain C-terminal be connected, the C-terminal of the link peptide is connected with the N-terminal of the people SIRPA extracellular fragment.And then it can avoid albumen Influencing each other for steric hindrance, further promotes protein folding.

According to an embodiment of the invention, the link peptide has amino acid sequence shown in SEQ ID NO:1.

GGGGSGGGGSERGETGP(SEQ ID NO:1)。

In the second aspect of the present invention, the invention proposes a kind of recombinant antibodies.According to an embodiment of the invention, described heavy The light chain of group antibody has amino acid sequence shown in SEQ ID NO:2, and the heavy chain of the recombinant antibodies has SEQ ID NO:3 Shown in amino acid sequence.

DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTL NINPMEEEDTAMYFCQQSKEVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 2)。

EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKN NLYLQMNSLRAEDTALYYCARQKGEAWFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSERGETGP EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSIS ISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS(SEQ ID NO:3)。

Recombinant antibodies according to an embodiment of the present invention target PD-1 and CD47 simultaneously, and the stimulation that can dramatically increase the two is exempted from The ability of epidemic disease system shows stronger tumor suppression ability than single target antibody.

In the third aspect of the present invention, the invention proposes a kind of nucleic acid.According to an embodiment of the invention, the nucleic acid is compiled The mentioned-above recombinant antibodies of code.The recombinant antibodies of nucleic acid encode according to an embodiment of the present invention target PD-1 and CD47 simultaneously, The ability that the stimulation immune system of the two can be dramatically increased, shows stronger tumor suppression ability than single target antibody.

According to an embodiment of the invention, above-mentioned nucleic acid can further include at least one following additional technical feature:

According to an embodiment of the invention, the nucleic acid has nucleotide sequence shown in SEQ ID NO:4 and 5.

gacatcgtgctgacccagtcccctgcttccctggctgtgtcccctggacagagggccaccatcacatgccgggcctc cgagtccgtggacaactacggcatctccttcatgaactggttccagcagaagcccggccagcctcccaagctgctga tctacgccgcctccaacaagggcacaggcgtgcctgccaggttttccggttctggctccggcaccgacttcaccctg aacatcaaccctatggaagaggaagacaccgccatgtacttctgccagcagtccaaggaggtgccttggacattcgg cggcggcaccaagctggagatcaagcggaccgtggccgctccaagcgtcttcatttttcccccttccgacgaacagc tgaagagtgggacagcctcagtggtctgtctgctgaacaatttctaccctagagaggctaaggtgcagtggaaagtc gataacgcactgcagtctggcaatagtcaggagtcagtgacagaacaggacagcaaggattccacttattctctgtc tagtacactgactctgtctaaagccgactacgaaaagcacaaagtgtatgcttgtgaagtgacccaccaggggctgt Ccagtcccgtgaccaaatctttcaataggggcgagtgt (SEQ ID NO:4).

gaggtgcagctggtccagagcggaggcggactggtccagcctggcggcagcctgaagctcagctgtgccgccagcgg attcaccttctcctcctacggaatgtcctgggtccggcaggctcctggcaaaggactggactgggtggctaccatct ccggcggaggaagggacacctactaccccgactccgtcaagggcaggttcaccatctcccgggacaatagcaagaac aacctgtatctccagatgaacagcctgcgggctgaggacaccgccctgtactactgcgctcggcagaagggcgaagc ctggttcgcctattggggacagggcacactggtgaccgtgagcgccgccagcacaaaaggccccagcgtgttccccc tggctccctgttccaggagcaccagcgagtccaccgctgctctgggctgcctggtgaaggactatttccctgagccc gtcaccgtcagctggaatagcggcgccctgaccagcggagtccacacattccccgccgtgctgcaaagcagcggcct gtactccttatcttctgtcgtgaccgtgccctccagcagcctgggaaccaagacctatacctgcaacgtggaccaca agcccagcaacaccaaggtggataagcgggtcgaatccaagtacggccccccttgtcctccttgtcccgctcctgag ttcctgggaggacccagcgtgtttctgttccctcctaagcccaaggacaccctgatgatcagccggacccccgaggt cacctgtgtggtggtggacgtgtcccaggaggaccccgaggtgcagtttaactggtacgtggacggcgtggaagtgc acaatgccaagaccaagcccagggaggagcagttcaacagcacctaccgggtggtgtccgtgctgaccgtgctgcac caggactggctgaacggcaaggagtacaagtgcaaagtgtccaacaaaggcctgcccagctccatcgagaagaccat ctccaaggccaagggccaacctcgggagccccaagtgtatacactgcccccttcccaggaagagatgaccaagaacc aggtcagcctcacctgtctggtgaagggcttctatcccagcgacatcgccgtcgaatgggaatccaacggccagccc gagaacaattacaagaccaccccccccgtgctggattccgacggctccttctttctgtatagccggctcaccgtgga caagagcaggtggcaggagggcaacgtgttctcctgtagcgtcatgcacgaggccctgcacaaccactacacccaga aatccctgtccctgtccctgggaaagggcggcggcggctccggcggaggaggcagcgaaaggggcgaaaccggccct gaggaggagttacaagtgatccagcccgacaagtccgtgtccgtggctgctggcgagtccgctatcctgcactgcac cgtgacctccctgatccccgtgggccctatccagtggttcaggggagctggccccgctagggagctgatctacaacc agaaggagggccacttccccagggtgaccaccgtgtccgagagcaccaagagggagaacatggacttctccatcagc atctccaacatcacccccgctgacgccggcacctactactgcgtgaagttcaggaagggcagccccgacaccgagtt Caagtccggcgctggcaccgagctgtccgtgagggccaaaccctcc (SEQ ID NO:5).

The recombinant antibodies of encoded by nucleic acid according to an embodiment of the present invention target PD-1 and CD47 simultaneously, can dramatically increase The ability of the stimulation immune system of the two shows stronger tumor suppression ability than single target antibody.

In the fourth aspect of the present invention, the invention proposes a kind of constructs.According to an embodiment of the invention, the building Body includes: the first nucleic acid molecules, the antibody of the anti-PD-1 of the first nucleic acid molecule encoding；Second nucleic acid molecules, second core Acid molecule encoding human SIRPA extracellular fragment.After construct according to an embodiment of the present invention imports recipient cell, the recombinant antibodies of expression PD-1 and CD47 are targeted simultaneously, the ability of the stimulation immune system of the two can be dramatically increased, shown than single target antibody stronger Tumor suppression ability.

According to an embodiment of the invention, above-mentioned construct can further include following additional technical feature at least it One:

According to an embodiment of the invention, further comprising: the first promoter, first promoter and first nucleic acid Molecule is operably connected.First promoter can efficiently start the expression of the first nucleic acid molecules and the second nucleic acid molecules, Jin Ershi The expression of existing recombinant antibodies noted earlier.

According to an embodiment of the invention, first promoter is selected from U6, H1, CMV, EF-1, LTR or RSV promoter.Hair Bright people's discovery, U6, H1, CMV, EF-1, LTR or RSV promoter, which can efficiently start, expresses the first, second nucleic acid molecules, first, The expression efficiency of second nucleic acid molecules significantly improves.

According to an embodiment of the invention, the construct further comprises: third nucleic acid molecules, the third nucleic acid molecules It is arranged between first nucleic acid molecules and second nucleic acid molecules, and the third nucleic acid molecule encoding link peptide. And then the albumen that the albumen of the first nucleic acid molecules expression can be expressed with the second nucleic acid molecules is linked together by link peptide, is become Fusion protein plays a role.

According to an embodiment of the invention, the carrier of the construct is non-pathogenic virus carrier.In the embodiment of the present invention Construct carrier pathogenic sites by modification or mutation, lost the pathogenic of virus, and then implemented according to the present invention The safety for the treatment of under the non-pathogenic virus of example is carrier mediated is higher.

According to an embodiment of the invention, the viral vectors includes being selected from retrovirus vector, slow virus carrier or gland At least one of viral related viral vectors.Above-mentioned carrier can realize entrained nucleic acid in the high efficient expression of recipient cell, treatment It is high-efficient.

In the fifth aspect of the invention, the invention proposes a kind of methods for preparing mentioned-above recombinant antibodies.According to The embodiment of the present invention, which comprises money is bought into the construct described in you and is introduced into mammalian cell；By the food in one's mouth Newborn zooblast is cultivated under conditions of being suitable for protein expression and secretion, to obtain the recombinant antibodies.Utilize basis The method of the embodiment of the present invention can simply, efficiently obtain mentioned-above recombinant antibodies, as previously mentioned, the recombinant antibodies are same When target PD-1 and CD47, the ability for the stimulation immune system that both can be dramatically increased is shown stronger than single target antibody Tumor suppression ability.

According to an embodiment of the invention, the above method can further include at least one following additional technical feature:

According to an embodiment of the invention, the mammalian cell includes being selected from CHOK1, CHOS, 293F, 293T at least One of.

In the sixth aspect of the present invention, the invention proposes a kind of therapeutic combinations for treating cancer.According to this hair Bright embodiment, the therapeutic combination include: mentioned-above construct, mentioned-above recombinant antibodies or noted earlier Nucleic acid.Therapeutic combination according to an embodiment of the present invention further significantly improves the therapeutic effect of cancer.

According to an embodiment of the invention, being supplied to the therapeutic combination of the embodiment of the present invention of patient, preferably it is applied to Bio-compatible solution or acceptable pharmacy delivery vehicle.Various therapeutic combinations as preparation are suspended or are dissolved in medicine Upper or physiologically acceptable carrier, such as physiological saline；Isotonic salting liquid or other people's being proficient in the knowledge of is obvious In formula.Carrier appropriate depends greatly on administration route.Other have water and anhydrous isotonic sterile injection liquid and There are water and anhydrous sterile suspensions, is pharmaceutically acceptable carrier.

To sum up, the purpose of the present invention is to provide a kind of new difunctional specificity that can target PD1 and CD47 simultaneously is anti- Body.

CD47 is also referred to as integrin associated protein (integrin associated protein, IAP), be it is a kind of across Membrane glycoprotein, molecular weight are immunoglobulin (Ig) superfamilies in 50kDa or so, include an extracellular N-terminal IgV structural domain, 5 The transmembrane segment of a very hydrophobic and carboxy-terminal cytosolic area.CD47 can be with signal adjusting protein alpha (Signal regulatory Protein α, SIRP α), thrombospondin (thrombospondin-1, TSP1) and integrin (integrins) phase Interaction participates in the adjusting of the immunocytes cross-film such as T cell and monocyte migration and phagocytosis.

The ligand SIRP α albumen of CD47 is also referred to as CD172a or SHPS-1 (SH2domain-containing Protein tyrosine phosphatase substrate-1), it is a kind of transmembrane glycoprotein, belongs to immunoglobulin and surpass house Race, the wherein combination of N-terminal and CD47.In DC, macrophage, mast cell, in granulocyte, nerve cell and candidate stem cell Equal wide expression is then expressed less in other cells.SIRP α is after in conjunction with CD47, the immunity receptor junket of C-terminal cytoplasmic region Propylhomoserin inhibits motif (ITIM) that phosphorylation occurs, and raises and causes tyrosine phosphatase SHP-1 and SHP-2 phosphorylation, and swashs Downstream signaling pathway living, to play the role of inhibiting macrophage phagocytosis.

CD47 wide expression discharges the signal of a kind of " not eating me " on various types of cells surface.CD47 is cell surface one Important " self " label, is a signal of interest for adjusting macrophage phagocytosis.CD47 can be with Macrophage Surface SIRP α combine, its ITIM of phosphorylation then recruits SHP-1 albumen, generates a series of cascade reaction α albumen and inhibits macrophage The phagocytosis of cell.CD47 high expression in young red blood cell, and the low expression in the red blood cell of aging, so that macrophage is thin Born of the same parents targetedly attack and can remove the red blood cell of aging.Tumour cell is in order to escape the immune system attack of body then Utilize the label for playing CD47 this " one of own side ".It is different studies have shown that CD47 is identified for the first time from the eighties in 19th century as people The tumour antigen of class oophoroma, then CD47 is found in a variety of human tumor types and expresses, including acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), Fei Huojin lymthoma (NHL), multiple Myeloma (MM) and other solid tumors are much higher than common normal cell.Therefore the signal path of CD47 and its ligand SIRP α albumen Become a kind of novel targets that can inhibit immunosurveillance escape.The drug development based on this signal path was continuous in recent years It attracts attention.

There are a variety of machines for process by the combination of drugs block CD47 and SIRP α to reach inhibition tumor immune escape System.First, the combination of physical blocking CD47 and SIRP α can release inhibiting effect of the SIRP α releasing to macrophage, this work With the cellulotoxic effect for not depending on antibody Fc mediation, belongs to extrinsic and scope is immunized.It is found that the anti-CD47 of missing Fc segment Antibody still is able to the removing for promoting macrophage to tumour.Second, it is straight that macrophage can not depended on for the blocking of CD47 Connect the apoptosis of induction tumour cell.Third, antibody are realized antigen presentation by activation T cell and DC cell and started antitumor Adaptive immunity.DC cell and T cell hypotype can express SIRP α.By blocking CD47 and SIRP alpha signal access that can release Inhibit the maturation of DC and generates cell factor.DC swallows tumour cell by CD47 antibody and parent's phagocytosis molecule synergistic effect, and Offer tumor associated antigen and give CD8+T cell, and then plays the specific killing action of CD8+T cells against tumor.

So CD47 antibody or its ligand SIRP α are after PD1/PD-L1 antibody, next-generation tumour immunity inhibitor is produced Product weight pound drug.The two has similarity, and CD47 and PD-L1 are by the regulation of transcription factor myc；And both extensively each It is expressed in class tumour cell.For some aspects, CD47 antibody may more have prospect than PD1/PD-L1 antibody.It is first CD47 ratio PD-L1 has more extensive expression, and the nearly all high expression in all tumour cells, and represent it has more For the effect of wide spectrum.Second, CD47 antibody has the inhibition tumour mechanism of diversification more than PD-1 antibody.PD-1,CTLA-4 Etc. tumour immunities inhibitor why only play a role to sub-fraction, perhaps an important reason be exactly PD-1 or The traditional immunizations inhibitor antibody such as CTLA-4, can not form the killer T cell of tumour-specific, and CD47 antibody can not only start Macrophage-mediated non-habitual immunologic process, and can be by macrophage, the antigen transmitting starting of DC etc. is thin to tumour The specific killing of born of the same parents.However, CD47 is expressed since it is extensive, the non-immunity Signal Regulation of different tissues is also related to, is hindered Disconnected CD47 signal may cause that macrophage widely attacks the risk of itself normal tissue or certain non-immunities are adjusted Disorder.Macrophage plays phagocytosis, needs " not eating me " signal and calreticulin that CD47 is this kind of " eating me " signal such as (calreticulin, CRT) synergistic effect.Under normal conditions tumour cell height expression CRT and normal cell " eating me " signal such as CRT is not expressed, so while CD47 is also extensively in the health tissues such as the cerebral cortex of people and cerebellum, The blocking effect of CD47 antibody is than relatively limited in the normal tissue.However, observation I phase clinic clinical data, receive radiotherapy with Patient after chemotherapy can also raise " eating me " signal.After CD47 antibody treatment, CD47+ red blood cell is exhausted, causes the poor of transience Blood is its main adverse reaction.

CD47 antibody treatment is to play Tumor cytotoxicity by DC cell and CD8+T.DC cell passes through CD47 antibody With parent's phagocytosis molecule synergistic effect, tumour cell is swallowed, and offer tumor associated antigen to CD8+T, and then play CD8+T to swollen The specific killing action of tumor, meanwhile, tumour cell raises the expression of CD47, cheats macrophage with this.So pass through CD47 The signal of antibody block this " not eating me " makes macrophage play phagocytosis.PD-1 and CD47, Ke Yixian are targeted simultaneously The ability for increasing the stimulation immune system of the two is write, significant effect enhancing is the immune sentry post inhibitor of a new generation.

Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description Obviously, or practice through the invention is recognized.

Detailed description of the invention

Fig. 1 is the SDS-PAGE qualification result figure of HX009-5 according to an embodiment of the present invention；

Fig. 2 is the SEC-HPLC qualification result figure of HX009-5 according to an embodiment of the present invention；

Fig. 3 is the experimental result picture of H8 and HX009-5 according to an embodiment of the present invention to the affinity of PD-1；

Fig. 4 is that HX009-5 and H8 according to an embodiment of the present invention inhibit Pd-1 and the active result figure of PdL1；

Fig. 5 is that HX009-5 and H8 according to an embodiment of the present invention inhibit Pd-1 and the active result figure of PdL2；

Fig. 6 is experimental result picture of the HX009-5 according to an embodiment of the present invention in conjunction with people CD47；

Fig. 7 is the experimental result picture for the combination that HX009-5 according to an embodiment of the present invention inhibits CD47 and SIRPA；

Fig. 8 is the experimental result picture that HX009-5 according to an embodiment of the present invention and CD16a are not bound with；

Fig. 9 is the experimental result picture that HX009-5 according to an embodiment of the present invention and CD32a are not bound with；

Figure 10 is the experimental result picture that HX009-5 according to an embodiment of the present invention and CD32b are not bound with；

Figure 11 is the experimental result picture that HX009-5 according to an embodiment of the present invention and CD64 are not bound with；

Figure 12 is the experiment knot that antibody H8 and HX009-5 stimulation T cell according to an embodiment of the present invention secretes IL-2 secretion Fruit figure；

Figure 13 is the experimental result that Hx009-5 sample according to an embodiment of the present invention will not generate red cell agglutination Figure；

Figure 14 is the result figure that gross tumor volume according to an embodiment of the present invention changes over time；And

Figure 15 is the result figure that gross tumor volume according to an embodiment of the present invention changes over time.

Specific embodiment

The solution of the present invention is explained below in conjunction with embodiment.

It will be understood to those of skill in the art that the following examples are merely to illustrate the present invention, and it should not be regarded as limiting this The range of invention.Particular technique or condition are not specified in embodiment, according to the literature in the art described technology or item Part (such as being write with reference to J. Pehanorm Brooker etc., " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated, the third edition, Science Press) Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition Conventional products.

Used cell line and basic experiment technology are as described below in the examples below:

The protein expression of the bispecific antibody of embodiment 1PD-1/CD47

On the basis of humanized antibody H8 (the IgG class antibody of anti-PD-1), pass through special Linker (GGGGSGGGGSERGETGP) the film outer portion (32aa-137aa) of people SIRPA (NP_542970.1) is connected to H8 heavy chain C-terminal obtains bispecific antibody HX009-5, makes it while targeting target protein PD-1 and CD47.

In practical operation, the nucleic acid sequence of the light chain of full genome composite coding humanized antibody H8 is first coupled to expression and carries Expression vector 1 is obtained in body, while the nucleic acid sequence of the heavy chain of full genome synthesis H8 and Linker-SIRPA (108mer) respectively, Directly the sequence of heavy chain of H8 is connected to and obtains the expression vector 2 for expressing the monoclonal antibody H8 of anti-PD1 in expression vector 1.It is logical After crossing Over-lap PCR fusion, it is connected to the expression vector 3 that expression bispecific antibody HX009-5 is obtained in expression vector 1. The DNA for extracting expression vector 2 and 3, transfects respectively to 293 cell of mammalian cell.After cell transfecting, antibody is in mammal Cell inner expression, and be secreted into extracellular.Then, by antibody A affinity column, purify the antibody of expression, i.e., acquisition H8 and HX009-5 albumen.HX009-5 is carried out after Quality Identification for subsequent with SDS-PAGE and SEC-HPLC standard analytical techniques Pharmacodynamic study.

Wherein, SDS-PAGE the and SEC-HPLC qualification result of HX009-5 is shown in Fig. 1 and Fig. 2 respectively.

The SDS-PAGE qualification result of HX009-5 is shown in Fig. 1.As shown in Figure 1, swimming lane 1:HX009-5 is restored；Swimming lane 2:H8 is also It is former；Swimming lane M: protein standard substance (18.4KDa 25KDa 35KDa 45KDa 66.2KDa)；Swimming lane 3:BSA.As shown in Figure 1, it waits Select antibody HX009-5 population of samples purity higher.

The SEC-HPLC qualification result of HX009-5 is shown in Fig. 2.As shown in Fig. 2, integral quantitatively confirms that the total purity of the antibody is 98.2%.

HX009-5 heavy chain amino acid sequence:

evqlvqsggglvqpggslklscaasgftfssygmswvrqapgkgldwvatisgggrdtyypdsvkgrftisrdnskn nlylqmnslraedtalyycarqkgeawfaywgqgtlvtvsaastkgpsvfplapcsrstsestaalgclvkdyfpep vtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygppcppcpape flggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlh qdwlngkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqp ennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslslgkggggsggggsergetgp eeelqviqpdksvsvaagesailhctvtslipvgpiqwfrgagpareliynqkeghfprvttvsestkrenmdfsis Isnitpadagtyycvkfrkgspdtefksgagtelsvrakps (SEQ ID NO:3), wherein the part of underscore mark For antibody variable region.

Encode the nucleic acid sequence of HX009-5 heavy chain:

HX009-5 light-chain amino acid sequence:

divltqspaslavspgqratitcrasesvdnygisfmnwfqqkpgqppklliyaasnkgtgvparfsgsgsgtdftl ninpmeeedtamyfcqqskevpwtfgggtkleikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkv Dnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvt ksfnrgec (SEQ ID NO: 2), wherein the part of underscore mark is antibody variable region.

Encode the nucleic acid sequence of HX009-5 light chain:

Embodiment 2HX009-5 bispecific antibody ELISA Binding experiment

1, H8, HX009-5ELISA PD-1 Binding experiment

For the H8 antibody that embodiment 1 prepares, compared with being carried out head to head with HX009-5, including PD1 Binding experiment and PDL1 competitive assay, specific as follows:

Specific step is as follows:

1) envelope antigen: 0.25 μ g/ml of hPD-1-his antigen, 100 holes μ l/, 4 DEG C of coatings are overnight；

2) it closes 2 hours for 37 DEG C of 1%BSA (PBS dilution), 1 × PBST (Tween-20,1%) is washed 3 times, is gently patted dry；

3) primary antibody: 2 μ g/ml, 1:5 7 gradient concentrations of gradient dilution, blank control group PBS, 37 DEG C are incubated for 1 hour；

4) secondary antibody: PBST is washed 3 times, is gently patted dry, and the diluted HRP enzyme mark goat-anti people of 1:10000 is added in every 100 μ l of hole IgG (H+L) secondary antibody, 37 DEG C are incubated for 1 hour；

5) develop the color: PBST is washed 3 times, is gently patted dry；TMB color developing agent is added in every 100 μ l of hole, reacts at room temperature 5~15min；

6) colour developing terminates: 2M H is added in 50 holes μ l/₂SO₄The reaction of solution color development stopping；

7) it reads: in microplate reader, the light absorption value in each hole is detected with absorbance 450nm.

It the results are shown in Table 1 and Fig. 3, H8 and HX009-5 can be calculated to the EC of PD-1₅₀Value is respectively 0.05nM and 0.05nM. From the figure 3, it may be seen that merging upper Linker-SIRPA (108mer) in C-terminal does not influence antibody HX009-5 and PD-1 affinity.

Table 1:

2, HX009-5, H8 and PDL1 competitive ELISA are tested

Specific step is as follows:

1) 0.5 μ g/ml of hPD-1-hIgGFc antigen, 50 holes μ l/, 4 DEG C of coatings envelope antigen: are coated in 96 hole elisa Plates Overnight；

2) PBST board-washing 3 times, gently pat dry, and 37 DEG C of 1%BSA (PBS dilution) closing 2 hours, 1 × PBST is added (Tween-20,1%) it washs 3 times；

3) primary antibody: 6 μ g/ml, 1:3 7 gradient concentrations of gradient dilution, blank control group PBS, 50 holes μ l/ are added to packet By on good ELISA Plate, it is incubated at room temperature 10min；

4) ligand: 0.6 μ g/ml of PDL1-mIgG2aFc solution is added, 50 holes μ l/, 37 DEG C are incubated for 1 hour；

5) secondary antibody: PBST is washed 3 times, is gently patted dry；The diluted HRP enzyme mark sheep anti-mouse igg of 1:5000 is added in every 50 μ l of hole (H+L) secondary antibody, 37 DEG C are incubated for 1 hour；

5) develop the color: PBST is washed 3 times, is gently patted dry；TMB color developing agent is added in every 50 μ l of hole, reacts at room temperature 5~15min；

6) terminate: 2M H is added in 50 holes μ l/₂SO₄Color development stopping reaction；

It the results are shown in Table 2 and Fig. 4, and HX009-5 and H8 inhibits the IC of Pd-1 and PdL1₅₀Value be respectively 1.5nM and 1.0nM.It follows that merging the knot that upper Linker-SIRPA (108mer) inhibits Pd-1 and PdL1 to antibody HX009-5 in C-terminal Conjunction has no significant effect.

Table 2:

3, HX009-5, H8 and PDL2 competitive ELISA are tested

Specific step is as follows:

1) hPD-1-hIgGFc antigen 1 .0 μ g/ml, 100 holes μ l/, 4 DEG C of packets envelope antigen: are coated in 96 hole elisa Plates It is stayed overnight；

2) PBST board-washing 3 times, gently pat dry, and 37 DEG C of 1%BSA (PBS dilution) closing 2 hours, 1 × PBST is added (Tween-20,1%) it washs 4 times；

3) primary antibody: 20 μ g/ml, 1:3 7 gradient concentrations of gradient dilution, blank control group PBS, 50 holes μ l/ are added to packet By on good ELISA Plate, it is incubated at room temperature 10min；

4) ligand: 0.6 μ g/ml of PDL2-his tag solution is added, 50 holes μ l/, 37 DEG C are incubated for 1 hour；

5) secondary antibody: PBST is washed 5 times, is gently patted dry；It is small that the anti-his tag of the diluted HRP enzyme mark of 1:750 is added in every 50 μ l of hole Mouse monoclonal antibody secondary antibody, 37 DEG C are incubated for 1 hour；

6) develop the color: PBST is washed 6 times, is gently patted dry；TMB color developing agent is added in every 100 μ l of hole, reacts at room temperature 30min；

7) terminate: 2M H is added in 50 holes μ l/₂SO₄Color development stopping reaction；

8) it reads: in microplate reader, the light absorption value in each hole is detected with absorbance 450nm.

It the results are shown in Table 3 and Fig. 5, and HX009-5 and H8 inhibits the IC of Pd-1 and PdL2₅₀Value be respectively 1.5nM and 2.7nM. is it follows that merge the knot that upper Linker-SIRPA (108mer) inhibits Pd-1 and PdL2 to antibody HX009-5 in C-terminal Conjunction has no significant effect.

Table 3:

4, HX009-5 ELISA in conjunction with CD47 is tested

For the HX009-5 antibody that embodiment 1 prepares, carries out the ELISA in conjunction with CD47 and test, specific as follows:

Specific step is as follows:

1) envelope antigen: 0.25 μ g/ml of CD47 antigen, 100 holes μ l/, 4 DEG C of coatings are overnight；

3) primary antibody: 10 μ g/ml, 1:5 7 gradient concentrations of gradient dilution, blank control group PBS, 37 DEG C are incubated for 1 hour；

It the results are shown in Table 4 and Fig. 6, HX009-5 is in conjunction with people CD47, EC₅₀For 0.6nM.

Table 4:

5, HX009-5 and SIRPA competitive ELISA is tested

Specific step is as follows:

1) envelope antigen: 0.25 μ g/ml of CD47 antigen is coated in 96 hole elisa Plates, 100 holes μ l/, 4 DEG C of coatings are overnight；

3) primary antibody: 30 μ g/ml, 1:3 7 gradient concentrations of gradient dilution, blank control group PBS, 50 holes μ l/ are added to packet By on good ELISA Plate, it is incubated at room temperature 10min；

4) ligand: 0.6 μ g/ml of SIRPA-his tag solution is added, 50 holes μ l/, 37 DEG C are incubated for 1 hour；

It the results are shown in Table 5 and Fig. 7, HX009-5 can inhibit the combination of CD47 and SIRPA, IC₅₀For 21nM.

Table 5:

The research of embodiment 3HX009-5Fc end effect

To HX009-5 (embodiment 1 prepares) carry out the research of Fc end effect, with Fc receptor CD16, CD32a, CD32b with And the affinity constant plan row of CD64, it is specific as follows to judge the binding ability of HX009-5 Yu Fc receptor:

1, the affinity constant measurement of HX009-5 and CD16a

Fc receptor CD16a (also known as Fc γ RIIIa) can participate in antibody-dependant cell and mediate in conjunction with the end Fc of IgG antibody Cytotoxicity (ADCC).Capacity of the therapeutic monoclonal antibodies in conjunction with Fc receptor to the antibody safety and have Effect property.This experiment detects the affinity constant of HX009-5 and CD16a using ELISA method, to evaluate HX009-5 and Fc receptor The binding ability of CD16a.

The HX009-5 antibody and HX006 antibody (for IgG1 hypotype) prepared for embodiment 1 carries out and CD16a It is tested in conjunction with ELISA, specific as follows:

Specific step is as follows:

1) envelope antigen: 0.5 μ g/ml of CD16a antigen, 100 holes μ l/, 4 DEG C of coatings are overnight；

4) secondary antibody: PBST is washed 3 times, is gently patted dry, and the diluted HRP enzyme mark goat anti-human igg of 1:8000 is added in every 100 μ l of hole (H+L) secondary antibody, 37 DEG C are incubated for 1 hour；

5) develop the color: PBST is washed 3 times, is gently patted dry；TMB color developing agent is added in every 100 μ l of hole, reacts at room temperature 30min；

It the results are shown in Table 6 and Fig. 8, HX009-5 is not bound with CD16a.

Table 6:

2, the affinity constant measurement of HX009-5 and CD32a

The HX009-5 antibody and HX006 antibody (for IgG1 hypotype) prepared for embodiment 1 carries out and CD32a It is tested in conjunction with ELISA, specific as follows:

Specific step is as follows:

1) envelope antigen: 0.5 μ g/ml of CD32a antigen, 100 holes μ l/, 4 DEG C of coatings are overnight；

It the results are shown in Table 7 and Fig. 9, HX009-5 is not bound with CD32a.

Table 7:

3, the affinity constant measurement of HX009-5 and CD32b

The HX009-5 antibody and HX006 antibody (for IgG1 hypotype) prepared for embodiment 1 carries out and CD32b It is tested in conjunction with ELISA, specific as follows:

Specific step is as follows:

1) envelope antigen: 0.5 μ g/ml of CD32b antigen, 100 holes μ l/, 4 DEG C of coatings are overnight；

It the results are shown in Table 8 and Figure 10, HX009-5 is not bound with CD32b.

Table 8:

4, the affinity constant measurement of HX009-5 and CD64

The HX009-5 antibody and HX006 antibody (for IgG1 hypotype) prepared for embodiment 1 carries out and CD64 It is tested in conjunction with ELISA, specific as follows:

Specific step is as follows:

1) envelope antigen: 0.5 μ g/ml of CD64 antigen, 100 holes μ l/, 4 DEG C of coatings are overnight；

It the results are shown in Table 9 and Figure 11, HX009-5 is not bound with CD64.

Table 9:

Embodiment 4 mixes the anti-PD1 biological activity of lymph reaction detection bispecific antibody

It is tested using mixed lymphocyte reaction (MLP) (MLR) and detects and compare H8 (embodiment 1 prepares) and HX009-5 thorn Swash T lymphocyte and secretes IL-2 and IFNgamma secretion capacity, specific as follows:

MLR experiment is mixed using the T cell (TC) and Dendritic Cells (DC) in different people source, anti-using DC cell Body offers ability stimulation T cell and secretes IL-2 and IFNgamma.First with single in cell factor GM-CSF and IL-4 inducing blood Monocyte differentiation is then immature DC cell maturation with TNFa stimulation at Dendritic Cells.DC after maturation with it is of the same race different After the TC cell in source carries out mixing 5 days, the secretion level of the IL-2 and IFNgamma in cell conditioned medium are detected.It is mixed in 96 orifice plates TC and DC is closed, TC 1 × 10 is added by every hole⁵With DC 1 × 10⁴, antibody concentration totally 8 ladders from 10M to 0.09765625nM are set Degree, after hybrid reaction 5 days, with IL-2 detection kit quantitative detection supernatant IL-2 content.

It is as shown in figure 12 that antibody H8 and HX009-5 stimulate T cell to secrete IL-2 secretion level.As seen from Figure 12, antibody H8 T cell can be effectively stimulated to secrete IL-2 with HX009-5, it follows that it is right to merge upper Linker-SIRPA (108mer) in C-terminal The ability that antibody HX009-5 stimulation T cell secretes IL-2 does not influence.

Embodiment 5HX009-5 hemagglutination reaction research

Specific step is as follows:

1, the peripheral blood 5ml for extracting volunteer, is picked up with 5ml anticoagulant heparin pipe, so that it is come into full contact with liver after gently shaking up It after element, is added in 15ml centrifuge tube, adds the PBS of 9ml, soft to mix, 2100rpm is centrifuged 10min；

2, the plasma supernatant containing leucocyte of red blood cell layer or more is discarded, 12ml PBS is added and is resuspended, 1500rpm, centrifugation 5min；

3, the above supernatant of red blood cell layer is discarded, 12ml PBS is added and is resuspended, 1500rpm is centrifuged 5min；

4, step 3 is repeated twice；

5, supernatant is abandoned, the red cell suspension for drawing 1ml is added in 15ml centrifuge tube, and the PBS for adding 9ml is mixed, preparation It is spare at 10% red blood cell.

6, take prepare 10% red blood cell 1ml that 15ml centrifuge tube is added, the PBS for adding 9ml is resuspended, and is prepared into 1% red blood cell is spare；

7, prepared by each sample solution:

1) HX009-5 is diluted to 0.9mg/ml from 9.1mg/ml, then is successively diluted by 3X, totally 12 concentration gradients；

2) H8 is diluted to 0.9mg/ml from 10mg/ml, then is successively diluted by 3X, totally 12 concentration gradients；

3) potato agglutinin extracting solution is successively diluted by 3X, totally 8 concentration gradients；

4) PBS is as blank control；

One 96 hole U floor cells culture plates are taken out, first 1% red blood cell of 50 μ l is added in each hole to B1 to G12, then presses as follows The sample of 50ml is added in figure sequence scheme, after mixing, puts 37 DEG C, 5% carbon dioxide incubator is incubated overnight.

After for 24 hours, the observation of 96 orifice plates is taken out, and take pictures under gel image analyser, as shown in the following table 10 and Figure 13, potato Agglutinin extracting solution has apparent red cell agglutination as 4 concentration gradients before positive control；H8 and HX009-5 and blank Each gradient is compareed to occur without agglutinating reaction；So as to obtain, HX009-5 sample will not generate red cell agglutination.

Table 10:

Embodiment 6HX009-5 is in KARPAS-299 people's primary cutaneous type subcutaneous transplantation MiXeno model Antitumor action

Human tumour transplantation model is established using NSG mouse, research H8 and HX009-5 (embodiment 1 prepares) exists Antitumor action in KARPAS-299 people's primary cutaneous type subcutaneous transplantation MiXeno model, specific as follows:

NSG mouse have NOD, Prkdcscid, IL2rgnull missing/variation features, be current immune deficiency degree most Tool mouse that is high, being most suitable for human archeocyte transplanting, to human archeocyte and tissue almost without rejection.Therefore, inventor Selection, which is adopted, transfers graft-versus-host reaction (GVHD) mould constructed by human peripheral blood mononuclear cell (PBMC) to NSG mouse Type, and thus measure the internal pharmacodynamics of HX009-5.Inventor establishes human tumour transplantation model (Mixeno with NSG mouse Model), research HX009-5 is antitumor in KARPAS-299 people's primary cutaneous type subcutaneous transplantation MiXeno model Effect.

KARPAS-299 cell is inoculated in 30 NCG mouse in right side dorsal sc within 0th day (Day 0), inoculated tumour Reach 60mm when mean tumor volume within 6 days (Day 6) after cell³, uniformly it is divided into 5 groups, every group of 6 mouse.It is transplanted from tail vein PBMC due to the source PBMC difference, is divided into donorA and donorB in 30 NCG mouse (1-5 group mouse), and every group 6 Mouse is divided into a and b each 3, and cell is resuspended in PBS (0.1ml is inoculated with volume).Test is divided into test medicine HX009-5 0.1mg/ Kg, 1mg/kg and 10mg/kg, positive control H8 (namely HX008 referred to below) 10mg/kg group and Isotype antibody Human IgG4 5mg/kg control group.Tail vein injection administration is administered, gives altogether for the 6th, 9,13,16,19,22 day after inoculated tumour cell Medicine six times (being shown in Table 11).According to Relative tumor inhibiting rate (TGI_RTV) therapeutic evaluation is carried out, according to the weight of animals variation and dead feelings Condition carries out safety evaluatio.

Table 11: antitumor action experimental design of the test medicine HX009-5 in KARPAS-299Mixeno tumor model

Note: administered volume is 10 μ l/g；N: animal number of elements；On the day of Day 0 is tumor cell inoculation；I.v.: tail vein is given Medicine.

The gross tumor volume of each group changes over time shown in following Figure 14 and 15, and the homotype that relative comparison group is vaccinated with PBMC is anti- Body (Human lgG4), test medicine HX009-5 and H8 show apparent tumor-inhibiting action.Wherein, HX009-5 has significantly Dosage correlation, dosage is bigger, and tumor control rate is higher.Under comparable sodium, HX009-5 ratio H8 shows stronger tumour Restraint illustrates the bis- target spot antibody of PD1/CD47 better than the mono- target spot antibody of PD1.

In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples It closes and combines.

Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, modifies, replacement and variant.

SEQUENCE LISTING

<110>Hangzhou Han Si biological medicine Co., Ltd

<120>bispecific antibody of anti-PD-1/CD47 and its application

<130> PIDC3175932

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 17

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of link peptide

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Arg Gly Glu Thr Gly

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Pro

<210> 2

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<213> Artificial

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<223>amino acid sequence of the light chain of recombinant antibodies

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Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

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Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile Asn

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Pro Met Glu Glu Glu Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys

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Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

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

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Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

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Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

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Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

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His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

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Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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<223>amino acid sequence of the heavy chain of recombinant antibodies

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

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Asn Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Gln Lys Gly Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

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Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

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

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

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Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

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Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

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

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Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

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Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

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Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

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Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

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Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

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His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly

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Gly Ser Gly Gly Gly Gly Ser Glu Arg Gly Glu Thr Gly Pro Glu Glu

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Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly

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Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val Gly

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Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr

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Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Ser

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Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser

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<210> 4

<211> 654

<212> DNA

<213> Artificial

<220>

<223>nucleic acid sequence of recombinant antibodies light chain is encoded

<400> 4

gacatcgtgc tgacccagtc ccctgcttcc ctggctgtgt cccctggaca gagggccacc 60

atcacatgcc gggcctccga gtccgtggac aactacggca tctccttcat gaactggttc 120

cagcagaagc ccggccagcc tcccaagctg ctgatctacg ccgcctccaa caagggcaca 180

ggcgtgcctg ccaggttttc cggttctggc tccggcaccg acttcaccct gaacatcaac 240

cctatggaag aggaagacac cgccatgtac ttctgccagc agtccaagga ggtgccttgg 300

acattcggcg gcggcaccaa gctggagatc aagcggaccg tggccgctcc aagcgtcttc 360

atttttcccc cttccgacga acagctgaag agtgggacag cctcagtggt ctgtctgctg 420

aacaatttct accctagaga ggctaaggtg cagtggaaag tcgataacgc actgcagtct 480

ggcaatagtc aggagtcagt gacagaacag gacagcaagg attccactta ttctctgtct 540

agtacactga ctctgtctaa agccgactac gaaaagcaca aagtgtatgc ttgtgaagtg 600

acccaccagg ggctgtccag tcccgtgacc aaatctttca ataggggcga gtgt 654

<210> 5

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<223>sequence of the nucleic acid of recombinant antibodies heavy chain is encoded

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agctgtgccg ccagcggatt caccttctcc tcctacggaa tgtcctgggt ccggcaggct 120

cctggcaaag gactggactg ggtggctacc atctccggcg gaggaaggga cacctactac 180

cccgactccg tcaagggcag gttcaccatc tcccgggaca atagcaagaa caacctgtat 240

ctccagatga acagcctgcg ggctgaggac accgccctgt actactgcgc tcggcagaag 300

ggcgaagcct ggttcgccta ttggggacag ggcacactgg tgaccgtgag cgccgccagc 360

acaaaaggcc ccagcgtgtt ccccctggct ccctgttcca ggagcaccag cgagtccacc 420

gctgctctgg gctgcctggt gaaggactat ttccctgagc ccgtcaccgt cagctggaat 480

agcggcgccc tgaccagcgg agtccacaca ttccccgccg tgctgcaaag cagcggcctg 540

tactccttat cttctgtcgt gaccgtgccc tccagcagcc tgggaaccaa gacctatacc 600

tgcaacgtgg accacaagcc cagcaacacc aaggtggata agcgggtcga atccaagtac 660

ggcccccctt gtcctccttg tcccgctcct gagttcctgg gaggacccag cgtgtttctg 720

ttccctccta agcccaagga caccctgatg atcagccgga cccccgaggt cacctgtgtg 780

gtggtggacg tgtcccagga ggaccccgag gtgcagttta actggtacgt ggacggcgtg 840

gaagtgcaca atgccaagac caagcccagg gaggagcagt tcaacagcac ctaccgggtg 900

gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaggagta caagtgcaaa 960

gtgtccaaca aaggcctgcc cagctccatc gagaagacca tctccaaggc caagggccaa 1020

cctcgggagc cccaagtgta tacactgccc ccttcccagg aagagatgac caagaaccag 1080

gtcagcctca cctgtctggt gaagggcttc tatcccagcg acatcgccgt cgaatgggaa 1140

tccaacggcc agcccgagaa caattacaag accacccccc ccgtgctgga ttccgacggc 1200

tccttctttc tgtatagccg gctcaccgtg gacaagagca ggtggcagga gggcaacgtg 1260

ttctcctgta gcgtcatgca cgaggccctg cacaaccact acacccagaa atccctgtcc 1320

ctgtccctgg gaaagggcgg cggcggctcc ggcggaggag gcagcgaaag gggcgaaacc 1380

ggccctgagg aggagttaca agtgatccag cccgacaagt ccgtgtccgt ggctgctggc 1440

gagtccgcta tcctgcactg caccgtgacc tccctgatcc ccgtgggccc tatccagtgg 1500

ttcaggggag ctggccccgc tagggagctg atctacaacc agaaggaggg ccacttcccc 1560

agggtgacca ccgtgtccga gagcaccaag agggagaaca tggacttctc catcagcatc 1620

tccaacatca cccccgctga cgccggcacc tactactgcg tgaagttcag gaagggcagc 1680

cccgacaccg agttcaagtc cggcgctggc accgagctgt ccgtgagggc caaaccctcc 1740

Claims

1. a kind of recombinant antibodies characterized by comprising

The antibody of anti-PD-1；And

People's SIRPA extracellular fragment,

The N-terminal of the people SIRPA extracellular fragment is connected with the C-terminal of the heavy chain of the antibody of the anti-PD-1.

2. recombinant antibodies according to claim 1, which is characterized in that the antibody of the anti-PD-1 is the IgG class of anti-PD-1 Antibody,

Optionally, the antibody of the anti-PD-1 is H8,

It optionally, further comprise link peptide, the N-terminal of the link peptide is connected with the C-terminal of the heavy chain of the antibody of the anti-PD-1, The C-terminal of the link peptide is connected with the N-terminal of the people SIRPA extracellular fragment,

Optionally, the link peptide has amino acid sequence shown in SEQ ID NO:1.

3. a kind of recombinant antibodies, which is characterized in that the light chain of the recombinant antibodies has amino acid sequence shown in SEQ ID NO:2 The heavy chain of column, the recombinant antibodies has amino acid sequence shown in SEQ ID NO:3.

4. a kind of nucleic acid, which is characterized in that the described in any item recombinant antibodies of nucleic acid encode claims 1 to 3.

5. nucleic acid according to claim 4, which is characterized in that the nucleic acid has nucleosides shown in SEQ ID NO:4 and 5 Acid sequence.

6. a kind of construct, which is characterized in that the construct includes:

First nucleic acid molecules, the antibody of the anti-PD-1 of the first nucleic acid molecule encoding；

Second nucleic acid molecules, the second nucleic acid molecule encoding people SIRPA extracellular fragment.

7. construct according to claim 6, which is characterized in that further comprise:

First promoter, first promoter are operably connected with first nucleic acid molecules,

Optionally, first promoter be selected from U6, H1, CMV, EF-1, LTR or RSV promoter,

Optionally, the construct further comprises:

Third nucleic acid molecules, third nucleic acid molecules setting first nucleic acid molecules and second nucleic acid molecules it Between, and the third nucleic acid molecule encoding link peptide,

Optionally, the link peptide has amino acid sequence shown in SEQ ID NO:1,

Optionally, the carrier of the construct is non-pathogenic virus carrier,

Optionally, the viral vectors includes being selected from retrovirus vector, slow virus carrier or adeno-associated virus (AAV) carrier At least one of.

8. a kind of method for preparing the described in any item recombinant antibodies of claims 1 to 3 characterized by comprising

The described in any item constructs of claim 6~7 are introduced into mammalian cell；

The mammalian cell is cultivated under conditions of being suitable for protein expression and secretion, it is anti-to obtain the recombination Body.

9. according to the method described in claim 8, it is characterized in that, the mammalian cell include selected from CHOK1, CHOS, At least one of 293F, 293T.

10. a kind of therapeutic combination for treating cancer characterized by comprising

The described in any item constructs of claim 6~7, the described in any item recombinant antibodies of claims 1 to 3 or right are wanted Seek 4~5 described in any item nucleic acid.