CN109897111B - Bispecific antibody against PD-1/CD47 and application thereof - Google Patents

Abstract

The invention provides a recombinant antibody. The recombinant antibody comprises: an anti-PD-1 antibody; and an extracellular domain of human SIRPA, the N-terminus of the extracellular domain of human SIRPA being linked to the C-terminus of the heavy chain of the anti-PD-1 antibody. The recombinant antibody simultaneously targets PD-1 and CD47, can remarkably increase the capacity of stimulating the immune system of the two antibodies, and shows stronger tumor inhibition capacity than that of a single-target antibody.

Description

Bispecific antibody against PD-1/CD47 and application thereof

Technical Field

The present invention relates to the fields of immunology and antibody engineering, in particular to bispecific antibodies against PD-1 and CD47 and uses thereof, and more particularly to recombinant antibodies, nucleic acids, constructs, methods of making recombinant antibodies, and therapeutic compositions for treating cancer.

Background

Cancer has become one of the leading causes of death worldwide in recent years. According to the report of the world health organization, the death number of cancer in 2015 worldwide reaches nearly 880 ten thousand, and accounts for nearly one sixth of the total death number in the same year. In 2015, about 429.2 ten new cancer cases and 281.4 ten cancer death cases exist in China, and the incidence rate of cancer still tends to rise in China.

At present, the treatment means for cancer still mainly comprises operation, radiotherapy and chemotherapy, and the traditional treatment means has the defects of poor tumor specificity, large toxic and side effects and the like. Therefore, there is a strong need for safer, more effective and specific means for treating cancer. In recent years, with the mechanism of cancer becoming increasingly well understood, molecular targeted therapy for inhibiting tumors by specific antibodies is emerging. The antibody medicine based on the method is prepared by the antibody engineering technology taking the cell engineering technology and the genetic engineering technology as main bodies, has the advantages of high specificity, obvious curative effect, less toxic and side effects and the like, and has very wide prospect for treating tumors. In recent years, the discovery of several immune checkpoints (immunogicalcheckpoint) has led to the development of antibodies specific for these immune checkpoints to block the process of immune escape of tumor cells, pushing immunotherapy towards a new wave of surge.

In the immune system, the process of T cell activation is complex, depending not only on the first stimulatory signal provided by the MHC-TCR complex, but also on the second signal (i.e., the "co-stimulatory signal") provided by some molecule on the surface of the antigen presenting cell. Lack of such co-stimulatory signals results in T cell unresponsiveness, tolerance and apoptosis^[1]. Programmed death receptor-1 (PD-1, also known as CD279) and its ligands programmed death receptor ligands 1 and 2(PD-L1 and PD-L2) are such inhibitory co-stimulatory molecules.

Human PD-1 belongs to the type I transmembrane glycoprotein of a member of the CD28 family, and has a molecular weight of approximately 55 kDa. Two tyrosine residues (Y223 and Y248) exist in the intracellular domain of human PD-1, and are respectively involved in an immunoreceptor tyrosine-based inhibition motif (ITIM, motif VDYGEL) constituting the N-terminal and an immunoreceptor tyrosine-based switch motif (ITSM, motif TEYATI) constituting the C-terminal. The ITIM motif recruits intracellular SHP-2 and phosphorylates downstream proteins, decreases BCR or TCR receptor signaling, ultimately inhibits lymphocyte proliferation and cytokine production and induces cell division cycle arrest^[2]. The extracellular region has an IgV-like domain which contains a plurality of glycosylation sites and is heavily glycosylated, and is mainly involved in binding with a ligand, thereby playing a role in inhibiting T cell activation. Unlike other costimulatory molecules, which exist as dimers, the extracellular domain of PD-1 lacks cysteine residues, and thus PD-1 can exist as monomers. PD-1 is expressed in activated T and B cells, as well as monocytes, Dendritic Cells (DCs) and regulatory T cells (Tregs).

Two ligands of PD-1, PD-L1(B7-H1, also known as CD274) and PD-L2(B7-DC), are type I glycoproteins that are members of the B7 family of proteins^[3]. PD-L1, the main ligand of PD-1, contains an IgV-like region, an IgC-like region, a transmembrane region and a cytoplasmic region, wherein the cytoplasmic region is involved in intracellular signal transduction and the IgV-like region and the IgC-like region are involved in intercellular signal transduction. Its expression is positively regulated by several inflammatory factors, such as interleukin 4(IL-4), tumorTumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and the like^[4]. In addition to expression in activated T cells, B cells, macrophages, dendritic cells and various tumor cells, PD-L1 is also widely expressed in non-lymphoid organs such as heart, blood vessels, placenta, skeletal muscle, lung, liver, spleen and thymus. PD-L2 is mainly expressed only on activated macrophages, DCs and a few tumor cells^[5]。

The immunosuppression effect of the PD-1/PD-L1 signal channel plays an important role in the occurrence and development of various immune dysregulated diseases. Such as autoimmune diseases. Mice lacking PD-1 develop delayed, organ-specific autoimmunity. Deletion of PD-1 exhibits accelerated tissue-specific autoimmune characterization in both LPR and NOD mouse models of lupus erythematosus and diabetes involving autoimmune diseases^[6]. Furthermore, Cytotoxic T (CTL) cells that overexpress a functional defect in PD-1 are found in chronically infected individuals with some viruses such as HIV and HBV, and blocking PD-1 signaling can reverse the "dysfunctional" state of CTL and clear the virus^[7]。

Tumor cells will use the immunosuppressive effects of the PD-1/PD-L1 signaling pathway to effect immune escape. A plurality of tumor cells can up-regulate and express PD-L1, such as Non-Small Cell Lung Cancer (NSCLC), melanoma, lymphoma, breast Cancer, leukemia, various tumors of urinary system, digestive tract and reproductive system^[8]. The overexpression of PD-L1 interacts with a PD-1 receptor on the surface of a T cell, so that tyrosine in an ITSM domain of PD-1 is phosphorylated, further dephosphorylation of downstream phosphatidylinositol 3-kinase (PI3K) and tyrosine kinase (Syk) is caused, activation of downstream AKT, ERK and other pathways is inhibited, and transcription and translation of genes and cytokines required by T cell activation are finally inhibited^[5]. On the other hand, it was shown that PD-L1 causes accumulation of PD-1 in T cells, resulting in cell cycle arrest, and cells in G0/G1 are accumulated in large amounts^[9]. In vitro experiments and mouse models also find that activation of PD-1/PD-L1 signal channel leads to specific CTL apoptosis, reduces the cytotoxic and injurious effect sensitivity of CTL, and promotes tumor cells to generateImmune escape^[10]. In conclusion, the tumor cells can inhibit the activation and proliferation of T cells and the killing of tumors by expressing PD-L1 and the action of PD-1 on the surfaces of the T cells.

Therefore, the PD-1/PD-L1 signal channel becomes a new molecular target for tumor immunotherapy, the PD-1/PD-L1 signal channel plays a key role in tumor immunity, and the blocking of the PD-1/PD-L1 signal channel can block the tumor cells from inhibiting the dominant force of cell immunity against T cells. The development of anti-PD-1 and anti-PD-L1 antibodies has been a hot research direction in the study of tumor immunotherapy. Currently, the monoclonal antibodies against PD-1 targets that have been approved for use by the FDA in the United states are Keytruda (pembrolizumab) from Merck and Opdivo (Nivolumab) from Bristol-Myers Squibb. Monoclonal antibodies directed against the PD-L1 target are Tecntriq (Atezolizumab) by Roche, Bavencio (avelumab) by Pfizer and Merck (Fechizer) and Imfinzi (Durvalumab) by AstraZeneca. The monoclonal antibody medicaments are clinically used for treating various tumors such as metastatic squamous non-small cell lung cancer which still progresses after melanoma and chemotherapy from the beginning, and have better clinical test results when being used for Hodgkin lymphoma, kidney cancer, stomach cancer, anal cancer, liver cancer, colorectal cancer and other tumor types at present. In addition, a number of anti-PD-1 and anti-PDL 1 antibodies were introduced into clinical trials for the treatment of a variety of tumors.

But the biggest limitation of current immunotherapy is that the effective rate is too low, and the effective rate of PD-1 is not equal from 15% to 50%. There is evidence to suggest that successful cancer immunotherapy depends on being able to trigger a systemic broad immune response, rather than merely triggering local effects of the tumor itself, stimulating the immune system to produce a more systemic response is expected to significantly increase the effectiveness of immunotherapy.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.

To this end, in a first aspect of the invention, the invention proposes a recombinant antibody. According to an embodiment of the invention, the recombinant antibody comprises: an anti-PD-1 antibody; and an extracellular domain of human SIRPA, the N-terminus of the extracellular domain of human SIRPA being linked to the C-terminus of the heavy chain of the anti-PD-1 antibody. The recombinant antibody provided by the embodiment of the invention targets PD-1 and CD47 simultaneously, can remarkably increase the capacity of stimulating the immune system of the two antibodies, and shows stronger tumor inhibition capacity than that of a single-target antibody.

According to an embodiment of the present invention, the recombinant antibody may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the anti-PD-1 antibody is an anti-PD-1 IgG class antibody. Preferably, the IgG class antibody is of the IgG4 subtype. IgG4 is easy to form half antibody (Fab-arm exchange occurs), and the IgG4 subtype antibody modified by S228P can effectively reduce the Fab-arm exchange and effectively avoid the effects of ADCC and CDC.

According to an embodiment of the invention, the anti-PD-1 antibody is H8. H8 is described in patent 201610207741.6 and PCT/CN 2016/103814.

According to an embodiment of the invention, the antibody further comprises a linker peptide, wherein the N-terminal of the linker peptide is connected with the C-terminal of the heavy chain of the anti-PD-1 antibody, and the C-terminal of the linker peptide is connected with the N-terminal of the human SIRPA extracellular segment. Further, the mutual influence of the steric hindrance of the proteins can be avoided, and the protein folding is further promoted.

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

GGGGSGGGGSERGETGP(SEQ ID NO:1)。

In a second aspect of the invention, the invention features a recombinant antibody. According to an embodiment of the present invention, the light chain of the recombinant antibody has the amino acid sequence shown in SEQ ID NO. 2, and the heavy chain of the recombinant antibody has the amino acid sequence shown in SEQ ID NO. 3.

DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLNINPMEEEDTAMYFCQQSKEVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:2)。

EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCARQKGEAWFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSERGETGPEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS(SEQ ID NO:3)。

The recombinant antibody provided by the embodiment of the invention targets PD-1 and CD47 simultaneously, can remarkably increase the capacity of stimulating the immune system of the two antibodies, and shows stronger tumor inhibition capacity than that of a single-target antibody.

In a third aspect of the invention, the invention features a nucleic acid. According to an embodiment of the invention, the nucleic acid encodes a recombinant antibody as described above. The recombinant antibody coded by the nucleic acid according to the embodiment of the invention targets PD-1 and CD47 simultaneously, can remarkably increase the capacity of the two to stimulate the immune system, and shows stronger tumor inhibition capacity than a single-target antibody.

According to an embodiment of the present invention, the above-mentioned nucleic acid may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the nucleic acid has the sequence of SEQ ID NO: 4 and 5.

gacatcgtgctgacccagtcccctgcttccctggctgtgtcccctggacagagggccaccatcacatgccgggcctccgagtccgtggacaactacggcatctccttcatgaactggttccagcagaagcccggccagcctcccaagctgctgatctacgccgcctccaacaagggcacaggcgtgcctgccaggttttccggttctggctccggcaccgacttcaccctgaacatcaaccctatggaagaggaagacaccgccatgtacttctgccagcagtccaaggaggtgccttggacattcggcggcggcaccaagctggagatcaagcggaccgtggccgctccaagcgtcttcatttttcccccttccgacgaacagctgaagagtgggacagcctcagtggtctgtctgctgaacaatttctaccctagagaggctaaggtgcagtggaaagtcgataacgcactgcagtctggcaatagtcaggagtcagtgacagaacaggacagcaaggattccacttattctctgtctagtacactgactctgtctaaagccgactacgaaaagcacaaagtgtatgcttgtgaagtgacccaccaggggctgtccagtcccgtgaccaaatctttcaataggggcgagtgt(SEQ ID NO：4)。

gaggtgcagctggtccagagcggaggcggactggtccagcctggcggcagcctgaagctcagctgtgccgccagcggattcaccttctcctcctacggaatgtcctgggtccggcaggctcctggcaaaggactggactgggtggctaccatctccggcggaggaagggacacctactaccccgactccgtcaagggcaggttcaccatctcccgggacaatagcaagaacaacctgtatctccagatgaacagcctgcgggctgaggacaccgccctgtactactgcgctcggcagaagggcgaagcctggttcgcctattggggacagggcacactggtgaccgtgagcgccgccagcacaaaaggccccagcgtgttccccctggctccctgttccaggagcaccagcgagtccaccgctgctctgggctgcctggtgaaggactatttccctgagcccgtcaccgtcagctggaatagcggcgccctgaccagcggagtccacacattccccgccgtgctgcaaagcagcggcctgtactccttatcttctgtcgtgaccgtgccctccagcagcctgggaaccaagacctatacctgcaacgtggaccacaagcccagcaacaccaaggtggataagcgggtcgaatccaagtacggccccccttgtcctccttgtcccgctcctgagttcctgggaggacccagcgtgtttctgttccctcctaagcccaaggacaccctgatgatcagccggacccccgaggtcacctgtgtggtggtggacgtgtcccaggaggaccccgaggtgcagtttaactggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagggaggagcagttcaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggagtacaagtgcaaagtgtccaacaaaggcctgcccagctccatcgagaagaccatctccaaggccaagggccaacctcgggagccccaagtgtatacactgcccccttcccaggaagagatgaccaagaaccaggtcagcctcacctgtctggtgaagggcttctatcccagcgacatcgccgtcgaatgggaatccaacggccagcccgagaacaattacaagaccaccccccccgtgctggattccgacggctccttctttctgtatagccggctcaccgtggacaagagcaggtggcaggagggcaacgtgttctcctgtagcgtcatgcacgaggccctgcacaaccactacacccagaaatccctgtccctgtccctgggaaagggcggcggcggctccggcggaggaggcagcgaaaggggcgaaaccggccctgaggaggagttacaagtgatccagcccgacaagtccgtgtccgtggctgctggcgagtccgctatcctgcactgcaccgtgacctccctgatccccgtgggccctatccagtggttcaggggagctggccccgctagggagctgatctacaaccagaaggagggccacttccccagggtgaccaccgtgtccgagagcaccaagagggagaacatggacttctccatcagcatctccaacatcacccccgctgacgccggcacctactactgcgtgaagttcaggaagggcagccccgacaccgagttcaagtccggcgctggcaccgagctgtccgtgagggccaaaccctcc(SEQ ID NO：5)。

The recombinant antibody coded by the nucleic acid of the embodiment of the invention targets PD-1 and CD47 simultaneously, can remarkably increase the capacity of the two to stimulate the immune system, and shows stronger tumor inhibition capacity than a single-target antibody.

In a fourth aspect of the invention, the invention features a construct. According to an embodiment of the invention, the construct comprises: a first nucleic acid molecule encoding an anti-PD-1 antibody; a second nucleic acid molecule encoding an extracellular segment of human SIRPA. After the construct is introduced into a receptor cell, the expressed recombinant antibody targets PD-1 and CD47 at the same time, so that the capacity of stimulating the immune system of the two antibodies can be remarkably increased, and the recombinant antibody shows stronger tumor inhibition capacity than a single-target antibody.

According to an embodiment of the present invention, the above-mentioned construct may further comprise at least one of the following additional technical features:

according to an embodiment of the present invention, further comprising: a first promoter operably linked to the first nucleic acid molecule. The first promoter can efficiently promote the expression of the first nucleic acid molecule and the second nucleic acid molecule, thereby realizing the expression of the recombinant antibody.

According to an embodiment of the present invention, the above-mentioned construct may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the first promoter is selected from the group consisting of U6, H1, CMV, EF-1, LTR or RSV promoters. The inventor finds that the U6, H1, CMV, EF-1, LTR or RSV promoter can efficiently promote the expression of the first nucleic acid molecule and the second nucleic acid molecule, and the expression efficiency of the first nucleic acid molecule and the second nucleic acid molecule is obviously improved.

According to an embodiment of the invention, the construct further comprises: a third nucleic acid molecule disposed between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a linker peptide. Furthermore, the protein expressed by the first nucleic acid molecule and the protein expressed by the second nucleic acid molecule can be connected together through a connecting peptide to play a role as a fusion protein.

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

According to an embodiment of the invention, the vector of the construct is a non-pathogenic viral vector. The pathogenic site of the construct vector in the embodiments of the present invention has been modified or mutated to lose the pathogenicity of the virus, thereby providing greater safety in non-pathogenic viral vector mediated therapy according to embodiments of the present invention.

According to an embodiment of the invention, the viral vector comprises at least one selected from a retroviral vector, a lentiviral vector or an adeno-associated viral vector. The vector can realize the high-efficiency expression of the carried nucleic acid in receptor cells, and has high treatment efficiency.

In a fifth aspect of the invention, the invention provides a method of producing a recombinant antibody as described above. According to an embodiment of the invention, the method comprises: introducing the construct into a mammalian cell; culturing said mammalian cells under conditions suitable for protein expression and secretion so as to obtain said recombinant antibody. The recombinant antibody can be obtained simply and efficiently by using the method provided by the embodiment of the invention, and as mentioned above, the recombinant antibody simultaneously targets PD-1 and CD47, so that the capacity of stimulating the immune system of the two antibodies can be obviously increased, and the recombinant antibody shows stronger tumor inhibition capacity than a single-target antibody.

According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:

according to an embodiment of the invention, the mammalian cell comprises at least one selected from the group consisting of CHOK1, CHOS, 293F, 293T.

In a sixth aspect of the invention, a therapeutic composition for treating cancer is presented. According to an embodiment of the invention, the therapeutic composition comprises: the aforementioned construct, the aforementioned recombinant antibody or the aforementioned nucleic acid. The therapeutic effect of the therapeutic composition according to the embodiment of the present invention on cancer is further significantly improved.

According to embodiments of the invention, therapeutic compositions of embodiments of the invention are provided to a patient for application to a biocompatible solution or to an acceptable pharmaceutical carrier. Various therapeutic compositions are prepared to be suspended or dissolved in a pharmaceutically or physiologically acceptable carrier, such as physiological saline; isotonic saline solution or other more obvious formulations specific to the person in the field. The appropriate carrier will depend to a large extent on the route of administration. Other isotonic sterile injection solutions, both aqueous and anhydrous, and sterile suspensions, both aqueous and anhydrous, are pharmaceutically acceptable carriers.

In summary, the present invention aims to provide a novel bifunctional specific antibody capable of targeting both PD1 and CD 47.

CD47, also known as Integrin Associated Protein (IAP), is a transmembrane glycoprotein with a molecular weight around 50kDa, is an immunoglobulin (Ig) superfamily comprising an extracellular N-terminal IgV domain, 5 highly hydrophobic transmembrane segments and a carboxy-terminal cytoplasmic region. CD47 interacts with Signal regulatory protein alpha (sirpa), thrombospondin (TSP 1) and integrins (integrins) and is involved in the regulation of transmembrane migration and phagocytosis of immune cells such as T cells and monocytes.

The SIRP alpha protein, the ligand of CD47, also known as CD172a or SHPS-1(SH2 domain-associating protein type phospholipid mutant-1), is a transmembrane glycoprotein belonging to the immunoglobulin superfamily, in which the N-terminus binds to CD 47. It is widely expressed in DCs, macrophages, mast cells, granulocytes, nerve cells and hematopoietic stem cells, and less expressed in other cells. SIRP alpha after binding with CD47, the Immunoreceptor Tyrosine Inhibition Motif (ITIM) of the C-terminal cytoplasmic region is phosphorylated, thereby recruiting and causing phosphorylation of tyrosine phosphatases SHP-1 and SHP-2, and activating downstream signaling pathway, thereby inhibiting phagocytosis of macrophages.

CD47 is widely expressed on the surface of various types of cells, releasing a "eat me" signal. CD47 is an important "self" marker on the cell surface and an important signal for regulating macrophage phagocytosis. CD47 can bind to SIRP alpha on the surface of macrophages, phosphorylate its ITIM, and subsequently recruit SHP-1 protein, producing a series of cascade-reaction alpha proteins that inhibit phagocytosis of macrophages. CD47 is highly expressed in young erythrocytes and less expressed in senescent erythrocytes, allowing macrophages to specifically attack and eliminate only senescent erythrocytes. Tumor cells use the "self-human" marker CD47 in order to evade the immune system attack of the body. Various studies have shown that CD47 was first identified as a tumor antigen for human ovarian cancer since the 80's 19 th century, and CD47 was subsequently found to be expressed in a variety of human tumor types, including Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Acute Lymphocytic Leukemia (ALL), non-hodgkin's lymphoma (NHL), Multiple Myeloma (MM), and other solid tumors, much higher than normal cells. Therefore, the signal path of the CD47 and the ligand SIRP alpha protein thereof becomes a new target point which can inhibit the immune escape of tumor cells. Drug development based on this signaling pathway has received increased attention in recent years.

There are various mechanisms for achieving the goal of inhibiting tumor immune escape by blocking the binding of CD47 to sirpa through drugs. First, physical blockade of binding of CD47 to sirpa abolishes inhibition of macrophages by sirpa, an effect that is independent of antibody Fc-mediated cytotoxic effects, and falls into the category of non-innate immunity. It was found that anti-CD 47 antibodies lacking the Fc fragment still promote tumor clearance by macrophages. Second, blockade of CD47 can directly induce apoptosis of tumor cells independent of macrophages. Third, antibodies achieve antigen presentation and initiate anti-tumor adaptive immunity by activating T cells and DC cells. Both DC cells and T cell subsets express SIRP alpha. By blocking the CD47 and sirpa signaling pathways, DC maturation inhibition and cytokine production can be relieved. The DC phagocytizes tumor cells through the synergistic effect of the CD47 antibody and the phagocytophilic molecules, presents tumor-related antigens to CD8+ T cells, and further plays a role in specifically killing the tumors by the CD8+ T cells.

Therefore, the CD47 antibody or the ligand SIRP alpha thereof is a heavy drug of the next generation of tumor immunosuppressant products after the PD1/PD-L1 antibody. The two are similar, and the CD47 and the PD-L1 are both regulated by a transcription factor myc; and both are widely expressed in various types of tumor cells. In some aspects, the CD47 antibody may be more promising than the PD1/PD-L1 antibody. First, CD47 was more widely expressed than PD-L1 and was highly expressed in almost all tumor cells, indicating a broader spectrum of effects. Second, the CD47 antibody has a much more diverse tumor-inhibiting mechanism than the PD-1 antibody. The reason why tumor immunosuppressive agents such as PD-1, CTLA-4 and the like only act on a small part is probably that traditional immunosuppressive agent antibodies such as PD-1, CTLA-4 and the like cannot form tumor specific killer T cells, while CD47 antibody can not only start macrophage-mediated non-adaptive immune process, but also start specific killing on tumor cells through antigen transmission of macrophages, DCs and the like. However, CD47 is also involved in the modulation of non-immune signals in different tissues due to its widespread expression, and blocking CD47 signals may cause the risk of macrophages attacking normal tissues extensively, or certain non-immune regulatory disorders. Macrophages are responsible for their phagocytosis, requiring the synergy of "eat me" signals such as CD47 with Calreticulin (CRT) and other "eat me" signals. In general, since tumor cells highly express CRT and normal cells do not express "eat me" signals such as CRT, although CD47 is widely used in healthy tissues such as cerebral cortex and cerebellum of human, the blocking effect of CD47 antibody in normal tissues is limited. However, observing phase i clinical data, patients receiving radiation and chemotherapy also up-regulate the "eat me" signal. After the treatment with the CD47 antibody, CD47+ red blood cells are exhausted, and the main adverse reaction is transient anemia.

CD47 antibody treatment exerts a tumor killing effect by DC cells and CD8+ T. The DC cells phagocytize tumor cells through the synergistic effect of the CD47 antibody and the phagocytophilic molecules, present tumor-related antigens to CD8+ T, further exert the specific killing effect of the CD8+ T on tumors, and simultaneously, the tumor cells up-regulate the expression of CD47 so as to deceive macrophages. Then the "eat me" signal is blocked by the CD47 antibody, causing the macrophages to exert phagocytosis. The PD-1 and the CD47 are targeted at the same time, the ability of stimulating the immune system of the PD-1 and the CD47 can be obviously increased, the effect is obviously enhanced, and the sentinel card inhibitor is a new generation of immune sentinel card inhibitors.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a diagram showing the SDS-PAGE identification result of HX009-5 according to an embodiment of the present invention;

FIG. 2 is a graph of SEC-HPLC identification of HX009-5 according to an embodiment of the present invention;

FIG. 3 is a graph of experimental results of the affinity of H8 and HX009-5 for PD-1 according to an embodiment of the present invention;

FIG. 4 is a graph showing the results of inhibition of Pd-1 and PdL1 activity by HX009-5 and H8 according to an embodiment of the present invention;

FIG. 5 is a graph showing the results of inhibition of Pd-1 and PdL2 activity by HX009-5 and H8 according to an embodiment of the present invention;

FIG. 6 is a graph of experimental results of HX009-5 binding to human CD47 according to an embodiment of the present invention;

FIG. 7 is a graph of experimental results of HX009-5 inhibiting the binding of CD47 to SIRPA in accordance with an embodiment of the present invention;

FIG. 8 is a graph of experimental results showing no combination of HX009-5 with CD16a according to an embodiment of the present invention;

FIG. 9 is a graph of experimental results of no binding of HX009-5 with CD32a according to an embodiment of the present invention;

FIG. 10 is a graph of experimental results of no binding of HX009-5 with CD32b according to an embodiment of the present invention;

FIG. 11 is a graph of experimental results of no binding of HX009-5 with CD64 according to an embodiment of the present invention;

FIG. 12 is a graph showing the results of experiments in which antibodies H8 and HX009-5 stimulate secretion of IL-2 by T cells, according to an embodiment of the present invention;

FIG. 13 is a graph showing the results of experiments in which Hx009-5 samples according to an embodiment of the present invention did not produce hemagglutination;

FIG. 14 is a graph of the results of tumor volume versus time according to an embodiment of the present invention; and

FIG. 15 is a graph of the results of tumor volume over time according to an embodiment of the present invention.

Detailed Description

The scheme of the invention will be explained with reference to the examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The cell lines and basic experimental techniques used in the following examples are as follows:

example 1 protein expression of bispecific antibody for PD-1/CD47

On the basis of humanized antibody H8 (IgG class antibody against PD-1), the external membrane part (32aa-137aa) of human SIRPA (NP-542970.1) is connected to the C-terminal of H8 heavy chain by a special linker (GGGGSGGGGSERGETGP) to obtain bispecific antibody HX009-5, so that the bispecific antibody can be targeted to target proteins PD-1 and CD47 simultaneously.

In actual operation, a nucleic acid sequence of a light chain of a coded humanized antibody H8 is synthesized in a whole gene manner, firstly connected to an expression vector to obtain an expression vector 1, and simultaneously, a heavy chain of H8 and a nucleic acid sequence of Linker-SIRPA (108mer) are synthesized in a whole gene manner respectively, and the heavy chain sequence of H8 is directly connected to the expression vector 1 to obtain an expression vector 2 for expressing a monoclonal antibody H8 of anti-PD 1. After fusion by Over-lap PCR, the expression vector 3 expressing bispecific antibody HX009-5 was obtained by ligation into expression vector 1. The DNA of expression vectors 2 and 3 were extracted and transfected into mammalian cell 293 cells, respectively. After cell transfection, the antibody is expressed in mammalian cells and secreted out of the cells. Then, the expressed antibody is purified by an antibody A affinity chromatography column, namely H8 and HX009-5 protein are obtained. HX009-5 was mass-characterized by SDS-PAGE and SEC-HPLC standard analytical techniques and used for subsequent pharmacodynamic studies.

Among them, the SDS-PAGE and SEC-HPLC identification of HX009-5 are shown in FIG. 1 and FIG. 2, respectively.

The result of SDS-PAGE identification of HX009-5 is shown in FIG. 1. As shown in fig. 1, lane 1: HX009-5 reduction; lane 2: h8 reduction; lane M: protein standards (18.4KDa 25KDa 35KDa 45KDa 66.2 KDa); lane 3: BSA. As can be seen from FIG. 1, the sample of candidate antibody HX009-5 had a high overall purity.

The SEC-HPLC identification of HX009-5 is shown in FIG. 2. As shown in fig. 2, the total purity of the antibody was confirmed to be 98.2% by integral quantification.

HX009-5 heavy chain amino acid sequence:

evqlvqsggglvqpggslklscaasgftfssygmswvrqapgkgldwvatisgggrdtyypdsvkgrf tisrdnsknnlylqmnslraedtalyycarqkgeawfaywgqgtlvtvsaastkgpsvfplpcsttsestaalglcvptfpptyvpctvsvgalatsvsvgslvsvvvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvsvtvstpvvpctvpppcpfpipflvpflvpflfpvpkpfpldvsvtvvsvtvsvtvvsvvsvtvvplmvsvtvvtvvtvvtvvtvvtvvtvvtvvtvgpvkvsngpykvplayckvstpvkvsnqkvstqkvstqkvstqkvstfgvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvtvvsvtvvsvvsvtvkswestvmegvmksvmkswestvplmkswestvstgisvvstgisgpygpygpygpygpygpglgcvvstgprvsvsvsvsvplsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvmksvmksvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvmglvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvsvs.

Nucleic acid sequence encoding HX009-5 heavy chain:

gaggtgcagctggtccagagcggaggcggactggtccagcctggcggcagcctgaagctcagctgtgccgccagcggattcaccttctcctcctacggaatgtcctgggtccggcaggctcctggcaaaggactggactgggtggctaccatctccggcggaggaagggacacctactaccccgactccgtcaagggcaggttcaccatctcccgggacaatagcaagaacaacctgtatctccagatgaacagcctgcgggctgaggacaccgccctgtactactgcgctcggcagaagggcgaagcctggttcgcctattggggacagggcacactggtgaccgtgagcgccgccagcacaaaaggccccagcgtgttccccctggctccctgttccaggagcaccagcgagtccaccgctgctctgggctgcctggtgaaggactatttccctgagcccgtcaccgtcagctggaatagcggcgccctgaccagcggagtccacacattccccgccgtgctgcaaagcagcggcctgtactccttatcttctgtcgtgaccgtgccctccagcagcctgggaaccaagacctatacctgcaacgtggaccacaagcccagcaacaccaaggtggataagcgggtcgaatccaagtacggccccccttgtcctccttgtcccgctcctgagttcctgggaggacccagcgtgtttctgttccctcctaagcccaaggacaccctgatgatcagccggacccccgaggtcacctgtgtggtggtggacgtgtcccaggaggaccccgaggtgcagtttaactggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagggaggagcagttcaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggagtacaagtgcaaagtgtccaacaaaggcctgcccagctccatcgagaagaccatctccaaggccaagggccaacctcgggagccccaagtgtatacactgcccccttcccaggaagagatgaccaagaaccaggtcagcctcacctgtctggtgaagggcttctatcccagcgacatcgccgtcgaatgggaatccaacggccagcccgagaacaattacaagaccaccccccccgtgctggattccgacggctccttctttctgtatagccggctcaccgtggacaagagcaggtggcaggagggcaacgtgttctcctgtagcgtcatgcacgaggccctgcacaaccactacacccagaaatccctgtccctgtccctgggaaagggcggcggcggctccggcggaggaggcagcgaaaggggcgaaaccggccctgaggaggagttacaagtgatccagcccgacaagtccgtgtccgtggctgctggcgagtccgctatcctgcactgcaccgtgacctccctgatccccgtgggccctatccagtggttcaggggagctggccccgctagggagctgatctacaaccagaaggagggccacttccccagggtgaccaccgtgtccgagagcaccaagagggagaacatggacttctccatcagcatctccaacatcacccccgctgacgccggcacctactactgcgtgaagttcaggaagggcagccccgacaccgagttcaagtccggcgctggcaccgagctgtccgtgagggccaaaccctcc(SEQ ID NO：5)。

HX009-5 light chain amino acid sequence:

divltqspaslavspgqratitcrasesvdnygisfmnwfqqkpgqppklliyaasnkgtgvparfsg sgsgtdftlninpmeeedtamyfcqqskevpwtfgggtkleikrtvaapvftfifpsdeqlksgasvlclnnnfypreaakvqwkvnalqsggnsqesvtteqdskdtsslsltlskadyekhkvyacetvqglsspvtkksftrgec (SEQ ID NO: 2), wherein the underlined parts are the antibody variable regions.

Nucleic acid sequence encoding HX009-5 light chain:

gacatcgtgctgacccagtcccctgcttccctggctgtgtcccctggacagagggccaccatcacatgccgggcctccgagtccgtggacaactacggcatctccttcatgaactggttccagcagaagcccggccagcctcccaagctgctgatctacgccgcctccaacaagggcacaggcgtgcctgccaggttttccggttctggctccggcaccgacttcaccctgaacatcaaccctatggaagaggaagacaccgccatgtacttctgccagcagtccaaggaggtgccttggacattcggcggcggcaccaagctggagatcaagcggaccgtggccgctccaagcgtcttcatttttcccccttccgacgaacagctgaagagtgggacagcctcagtggtctgtctgctgaacaatttctaccctagagaggctaaggtgcagtggaaagtcgataacgcactgcagtctggcaatagtcaggagtcagtgacagaacaggacagcaaggattccacttattctctgtctagtacactgactctgtctaaagccgactacgaaaagcacaaagtgtatgcttgtgaagtgacccaccaggggctgtccagtcccgtgaccaaatctttcaataggggcgagtgt(SEQ ID NO：4)。

example 2HX009-5 bispecific antibody ELISA binding assay

1. H8, HX009-5ELISA PD-1 binding experiments

The H8 antibody prepared in example 1 was compared head-to-head with HX009-5, including PD1 binding experiments and PDL1 competition experiments, as follows:

the method comprises the following specific steps:

1) coating antigen: hPD-1-his antigen 0.25 μ g/ml, 100 μ l/well, coated overnight at 4 deg.C;

2) 1% BSA (PBS dilution) at 37 ℃ blocking for 2 hours, 1 xPBST (Tween-20, 1%) washing 3 times, light pat dry;

3) a first antibody: diluting with 1:5 gradient of 7 gradient concentrations at 2 μ g/ml, and incubating at 37 deg.C for 1 hr with PBS as blank control group;

4) secondary antibody: PBST is washed for 3 times, is lightly tapped to be dry, 100 microliter of each hole is added with HRP enzyme-labeled goat anti-human IgG (H + L) secondary antibody diluted by 1:10000, and is incubated for 1 hour at 37 ℃;

5) color development: PBST was washed 3 times, patted dry gently; adding TMB color developing agent into 100 mul of each hole, and reacting for 5-15 min at room temperature;

6) and (3) color development termination: 50 μ l/well 2M H was added₂SO₄Terminating the color development reaction by the solution;

7) reading: the absorbance of each well was measured on a microplate reader using absorbance 450 nm.

The results are shown in Table 1 and FIG. 3, and the EC for PD-1 for H8 and HX009-5 can be calculated₅₀Values were 0.05nM and 0.05nM, respectively. As can be seen from FIG. 3, Linker-SIRPA (108mer) fused to the C-terminus had no effect on the affinity of antibody HX009-5 to PD-1.

Table 1:

2. competitive ELISA assay for HX009-5, H8 and PDL1

The method comprises the following specific steps:

1) coating antigen: coating hPD-1-hIgGFc antigen 0.5 μ g/ml and 50 μ l/well on 96-well enzyme label plate, and coating overnight at 4 deg.C;

2) PBST washing plate 3 times, gently clap dry, add 1% BSA (PBS dilution) 37 degrees C blocking 2 hours, 1 x PBST (Tween-20, 1%) washing 3 times;

3) a first antibody: diluting with a 1:3 gradient of 6 μ g/ml and 7 gradient concentrations, adding 50 μ l/well of PBS as a blank control group to the coated ELISA plate, and incubating at room temperature for 10 min;

4) ligand: PDL1-mIgG2aFc solution 0.6. mu.g/ml, 50. mu.l/well was added and incubated at 37 ℃ for 1 hour;

5) secondary antibody: PBST was washed 3 times, patted dry gently; adding HRP enzyme-labeled goat anti-mouse IgG (H + L) secondary antibody diluted by 1:5000 into 50 mu L of each hole, and incubating for 1 hour at 37 ℃;

5) color development: PBST was washed 3 times, patted dry gently; adding 50 mu l of TMB color developing agent into each hole, and reacting for 5-15 min at room temperature;

6) and (4) terminating: 50 μ l/well 2M H was added₂SO₄Terminating the color development reaction;

7) reading: the absorbance of each well was measured on a microplate reader using absorbance 450 nm.

The results are shown in Table 2 and FIG. 4, and IC's for Pd-1 and PdL1 were inhibited by HX009-5 and H8₅₀Values were 1.5nM and 1.0nM, respectively. It can be seen that Linker-SIRPA (108mer) fused to the C-terminus had no significant effect on the inhibition of Pd-1 binding to PdL1 by antibody HX 009-5.

Table 2:

3. competitive ELISA assay for HX009-5, H8 and PDL2

The method comprises the following specific steps:

1) coating antigen: coating hPD-1-hIgGFc antigen 1.0 μ g/ml and 100 μ l/well on 96-well enzyme label plate, and coating overnight at 4 deg.C;

2) PBST washing plate 3 times, gently clap dry, add 1% BSA (PBS dilution) 37 degrees C blocking 2 hours, 1 x PBST (Tween-20, 1%) washing 4 times;

3) a first antibody: diluting with a 1:3 gradient of 20 μ g/ml and 7 gradient concentrations, adding 50 μ l/well of PBS as a blank control group to the coated ELISA plate, and incubating at room temperature for 10 min;

4) ligand: adding 0.6 mu g/ml PDL2-his tag solution and 50 mu l/well, and incubating at 37 ℃ for 1 hour;

5) secondary antibody: PBST washing 5 times, gently patting dry; adding HRP enzyme-labeled anti-his tag mouse monoclonal antibody secondary antibody diluted by 1:750 into 50 mu l of each hole, and incubating for 1 hour at 37 ℃;

6) color development: PBST was washed 6 times, patted dry gently; adding TMB color developing agent into 100 mul of each hole, and reacting for 30min at room temperature;

7) and (4) terminating: 50 μ l/well 2M H was added₂SO₄Terminating the color development reaction;

8) reading: the absorbance of each well was measured on a microplate reader using absorbance 450 nm.

The results are shown in Table 3 and FIG. 5, and IC's for Pd-1 and PdL2 were inhibited by HX009-5 and H8₅₀The values were 1.5nM and 2.7nM., respectively, and it can be seen that Linker-SIRPA (108mer) fused to the C-terminus had no significant effect on the inhibition of Pd-1 binding to PdL2 by antibody HX 009-5.

Table 3:

4. ELISA assay for binding of HX009-5 to CD47

ELISA binding experiments with CD47 were performed on HX009-5 antibody prepared in example 1 as follows:

the method comprises the following specific steps:

1) coating antigen: CD47 antigen 0.25 μ g/ml, 100 μ l/well, 4 ℃ coating overnight;

2) 1% BSA (PBS dilution) at 37 ℃ blocking for 2 hours, 1 xPBST (Tween-20, 1%) washing 3 times, light pat dry;

3) a first antibody: diluting with a 1:5 gradient of 7 gradient concentrations at 10 μ g/ml, and incubating at 37 deg.C for 1 hr with a blank control group of PBS;

4) secondary antibody: PBST is washed for 3 times, is lightly tapped to be dry, 100 microliter of each hole is added with HRP enzyme-labeled goat anti-human IgG (H + L) secondary antibody diluted by 1:10000, and is incubated for 1 hour at 37 ℃;

5) color development: PBST was washed 3 times, patted dry gently; adding TMB color developing agent into 100 mul of each hole, and reacting for 5-15 min at room temperature;

6) and (3) color development termination: 50 μ l/well 2M H was added₂SO₄Terminating the color development reaction by the solution;

7) reading: the absorbance of each well was measured on a microplate reader using absorbance 450 nm.

The results are shown in Table 4 and FIG. 6, where HX009-5 binds to human CD47, EC₅₀It was 0.6 nM.

Table 4:

5. competitive ELISA assay for HX009-5 and SIRPA

The method comprises the following specific steps:

1) coating antigen: coating 0.25 mu g/ml of CD47 antigen on a 96-well enzyme label plate, coating 100 mu l/well and coating overnight at 4 ℃;

2) PBST washing plate 3 times, gently clap dry, add 1% BSA (PBS dilution) 37 degrees C blocking 2 hours, 1 x PBST (Tween-20, 1%) washing 4 times;

3) a first antibody: diluting with 1:3 gradient of 30 μ g/ml for 7 gradient concentrations, adding 50 μ l/well of PBS as blank control group onto the coated enzyme label plate, and incubating at room temperature for 10 min;

4) ligand: adding SIRPA-his tag solution 0.6. mu.g/ml and 50. mu.l/well, and incubating at 37 ℃ for 1 hour;

5) secondary antibody: PBST washing 5 times, gently patting dry; adding HRP enzyme-labeled anti-his tag mouse monoclonal antibody secondary antibody diluted by 1:750 into 50 mu l of each hole, and incubating for 1 hour at 37 ℃;

6) color development: PBST was washed 6 times, patted dry gently; adding TMB color developing agent into 100 mul of each hole, and reacting for 30min at room temperature;

7) and (4) terminating: 50 μ l/well 2M H was added₂SO₄Terminating the color development reaction;

8) reading: the absorbance of each well was measured on a microplate reader using absorbance 450 nm.

The results are shown in Table 5 and FIG. 7, where HX009-5 inhibits the binding of CD47 to SIRPA, IC₅₀Was 21 nM.

Table 5:

example 3HX009-5Fc end Effect study

The affinity constants of HX009-5 (prepared in example 1) and Fc end effects of Fc receptors CD16, CD32a, CD32b and CD64 were studied to determine the binding ability of HX009-5 to Fc receptors, as follows:

1. determination of affinity constant of HX009-5 with CD16a

The Fc receptor CD16a (also known as Fc γ RIIIa) binds to the Fc-terminus of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of a therapeutic monoclonal antibody to bind to an Fc receptor affects the safety and efficacy of the antibody. The experiment used ELISA to test the affinity constant of HX009-5 to CD16a to assess the ability of HX009-5 to bind to the Fc receptor CD16 a.

The binding ELISA assay with CD16a was performed on HX009-5 antibody prepared in example 1 and HX006 antibody (IgG 1 subtype) as follows:

the method comprises the following specific steps:

1) coating antigen: CD16a antigen 0.5. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃;

2) 1% BSA (PBS dilution) at 37 ℃ blocking for 2 hours, 1 xPBST (Tween-20, 1%) washing 3 times, light pat dry;

3) a first antibody: diluting with a 1:5 gradient of 7 gradient concentrations at 10 μ g/ml, and incubating at 37 deg.C for 1 hr with a blank control group of PBS;

4) secondary antibody: PBST is washed for 3 times, is lightly tapped and dried, 100 microliter of each hole is added with HRP enzyme-labeled goat anti-human IgG (H + L) secondary antibody diluted by 1:8000, and is incubated for 1 hour at 37 ℃;

5) color development: PBST was washed 3 times, patted dry gently; adding TMB color developing agent into 100 mul of each hole, and reacting for 30min at room temperature;

6) and (3) color development termination: 50 μ l/well 2M H was added₂SO₄Terminating the color development reaction by the solution;

7) reading: the absorbance of each well was measured on a microplate reader using absorbance 450 nm.

The results are shown in Table 6 and FIG. 8, where HX009-5 was not combined with CD16 a.

Table 6:

2. determination of affinity constant of HX009-5 with CD32a

The binding ELISA assay with CD32a was performed on HX009-5 antibody prepared in example 1 and HX006 antibody (IgG 1 subtype), as follows:

the method comprises the following specific steps:

1) coating antigen: CD32a antigen 0.5. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃;

2) 1% BSA (PBS dilution) at 37 ℃ blocking for 2 hours, 1 xPBST (Tween-20, 1%) washing 3 times, light pat dry;

3) a first antibody: diluting with a 1:5 gradient of 7 gradient concentrations at 10 μ g/ml, and incubating at 37 deg.C for 1 hr with a blank control group of PBS;

4) secondary antibody: PBST is washed for 3 times, is lightly tapped and dried, 100 microliter of each hole is added with HRP enzyme-labeled goat anti-human IgG (H + L) secondary antibody diluted by 1:8000, and is incubated for 1 hour at 37 ℃;

5) color development: PBST was washed 3 times, patted dry gently; adding TMB color developing agent into 100 mul of each hole, and reacting for 30min at room temperature;

6) and (3) color development termination: 50 μ l/well 2M H was added₂SO₄Solution stop displayCarrying out color reaction;

7) reading: the absorbance of each well was measured on a microplate reader using absorbance 450 nm.

The results are shown in Table 7 and FIG. 9, where HX009-5 did not bind to CD32 a.

Table 7:

3. determination of affinity constant of HX009-5 with CD32b

The binding ELISA assay with CD32b was performed on HX009-5 antibody prepared in example 1 and HX006 antibody (IgG 1 subtype), as follows:

the method comprises the following specific steps:

1) coating antigen: CD32b antigen 0.5. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃;

2) 1% BSA (PBS dilution) at 37 ℃ blocking for 2 hours, 1 xPBST (Tween-20, 1%) washing 3 times, light pat dry;

3) a first antibody: diluting with a 1:5 gradient of 7 gradient concentrations at 10 μ g/ml, and incubating at 37 deg.C for 1 hr with a blank control group of PBS;

4) secondary antibody: PBST is washed for 3 times, is lightly tapped and dried, 100 microliter of each hole is added with HRP enzyme-labeled goat anti-human IgG (H + L) secondary antibody diluted by 1:8000, and is incubated for 1 hour at 37 ℃;

5) color development: PBST was washed 3 times, patted dry gently; adding TMB color developing agent into 100 mul of each hole, and reacting for 30min at room temperature;

6) and (3) color development termination: 50 μ l/well 2M H was added₂SO₄Terminating the color development reaction by the solution;

7) reading: the absorbance of each well was measured on a microplate reader using absorbance 450 nm.

The results are shown in Table 8 and FIG. 10, where HX009-5 was not combined with CD32 b.

Table 8:

4. determination of affinity constant of HX009-5 with CD64

The binding ELISA assay with CD64 was performed on HX009-5 antibody prepared in example 1 and HX006 antibody (IgG 1 subtype), as follows:

the method comprises the following specific steps:

1) coating antigen: CD64 antigen 0.5. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃;

2) 1% BSA (PBS dilution) at 37 ℃ blocking for 2 hours, 1 xPBST (Tween-20, 1%) washing 3 times, light pat dry;

3) a first antibody: diluting with a 1:5 gradient of 7 gradient concentrations at 10 μ g/ml, and incubating at 37 deg.C for 1 hr with a blank control group of PBS;

4) secondary antibody: PBST is washed for 3 times, is lightly tapped and dried, 100 microliter of each hole is added with HRP enzyme-labeled goat anti-human IgG (H + L) secondary antibody diluted by 1:8000, and is incubated for 1 hour at 37 ℃;

5) color development: PBST was washed 3 times, patted dry gently; adding TMB color developing agent into 100 mul of each hole, and reacting for 30min at room temperature;

6) and (3) color development termination: 50 μ l/well 2M H was added₂SO₄Terminating the color development reaction by the solution;

7) reading: the absorbance of each well was measured on a microplate reader using absorbance 450 nm.

The results are shown in Table 9 and FIG. 11, where HX009-5 did not bind to CD 64.

Table 9:

example 4 detection of bispecific antibody anti-PD 1 biological Activity by Mixed lymph reaction

The ability of H8 (prepared in example 1) and HX009-5 to stimulate T lymphocytes to secrete IL-2 and IFNgamma was tested and compared using a Mixed Lymphocyte Reaction (MLR) assay as follows:

MLR experiments were performed using a mixture of T Cells (TC) of different human origin and Dendritic Cells (DC) to stimulate IL-2 and IFNgamma secretion from T cells using the antibody-presenting ability of DC cells. The differentiation of monocytes into dendritic cells in blood is first induced with the cytokines GM-CSF and IL-4, and then stimulated with TNFaImmature DC cells mature. After the mature DCs were mixed with allogeneic TC cells for 5 days, the secretion levels of IL-2 and IFNgamma in the cell supernatant were measured. Mixing TC and DC in a 96-well plate, adding TC 1X 10 per well⁵And DC 1 × 10⁴ Setting 8 gradients of antibody concentration from 10M to 0.09765625nM, mixing and reacting for 5 days, and quantitatively detecting IL-2 content in supernatant with IL-2 detection kit.

The level of secretion of IL-2 by T cells stimulated by antibodies H8 and HX009-5 is shown in FIG. 12. As can be seen in FIG. 12, the antibodies H8 and HX009-5 were effective in stimulating IL-2 secretion from T cells, and it was thus clear that the Linker-SIRPA (108mer) fused to the C-terminus had no effect on the ability of the antibody HX009-5 to stimulate IL-2 secretion from T cells.

Example 5HX009-5 hemagglutination reaction study

The method comprises the following specific steps:

1. extracting 5ml of peripheral blood of a volunteer, inoculating the peripheral blood with a 5ml heparin anticoagulation tube, shaking up lightly to make the peripheral blood fully contact with heparin, adding the heparin into a 15ml centrifuge tube, adding 9ml of PBS, mixing lightly and uniformly, and centrifuging at 2100rpm for 10 min;

2. discarding the supernatant containing the leukocyte plasma above the erythrocyte layer, adding 12ml PBS for resuspension, 1500rpm, and centrifuging for 5 min;

3. discarding supernatant above erythrocyte layer, adding 12ml PBS for resuspension, 1500rpm, centrifuging for 5 min;

4. repeating the step 3 for two times;

5. discarding the supernatant, sucking 1ml of erythrocyte suspension, adding into a 15ml centrifuge tube, adding 9ml of PBS, and mixing uniformly to prepare 10% of erythrocytes for later use.

6. Taking 1ml of prepared 10% red blood cells, adding the red blood cells into a 15ml centrifuge tube, adding 9ml of PBS for resuspension, and preparing 1% red blood cells for later use;

7. preparation of each sample solution:

1) diluting HX009-5 from 9.1mg/ml to 0.9mg/ml, and sequentially diluting according to 3X to obtain 12 concentration gradients;

2) diluting H8 from 10mg/ml to 0.9mg/ml, and sequentially diluting according to 3X to obtain 12 concentration gradients;

3) sequentially diluting the potato agglutinin extract by 3X to obtain 8 concentration gradients;

4) PBS as blank control;

a96-well U-bottomed cell culture plate was removed, 50. mu.l of 1% red blood cells were added to each well of B1 to G12, 50ml of the sample was added in the following scheme, mixed well, and incubated overnight in a 5% carbon dioxide incubator at 37 ℃.

After 24h, taking out the 96-well plate for observation, and taking a picture under a gel imaging analyzer, wherein the 4 concentration gradients of the potato agglutinin extract as a positive control have obvious erythrocyte agglutination reaction as shown in the following table 10 and figure 13; no agglutination occurred in H8 and HX009-5 with the blank for each gradient; thus, it was found that no agglutination of erythrocytes was observed in the HX009-5 sample.

Table 10:

example 6 anti-tumor Effect of HX009-5 in the model of MiXeno subcutaneous transplantation of KARPAS-299 Interhuman degenerative Large cell lymphoma

A human tumor transplantation model is established by using NSG mice, and the anti-tumor effect of H8 and HX009-5 (obtained by preparation in example 1) in a MiXeno model for subcutaneous transplantation of KARPAS-299 human-induced large cell lymphoma is researched, which is concretely as follows:

the NSG mouse has NOD, Prkdcscid and IL2rgnull deletion/variation characteristics, is the tool mouse with the highest immunodeficiency degree and the best suitability for human cell transplantation at present, and has almost no rejection reaction on human cells and tissues. Thus, the inventors selected a model of graft versus host response (GVHD) constructed by adoptively transfusing human Peripheral Blood Mononuclear Cells (PBMC) into NSG mice and thereby measured the in vivo pharmacodynamics of HX 009-5. The inventor establishes a human tumor transplantation model (Mixeno model) by using NSG mice and researches the anti-tumor effect of HX009-5 in a MiXeno model implanted under KARPAS-299 human metaplastic large cell lymphoma.

Day 0 (Day 0) KARPAS-299 cells were subcutaneously inoculated in the right dorsal part in 30 NCG mice 6 days (Day 6) after tumor cell inoculationThe tumor volume reaches 60mm³Evenly divided into 5 groups of 6 mice each. PBMC were transplanted from tail vein into 30 NCG mice (groups 1-5) and divided into donorA and donorB due to different PBMC origins, and 6 mice in each group were divided into 3 mice each for a and b, and cells were resuspended in PBS (0.1ml inoculation volume). The test is divided into test drugs HX 009-50.1 mg/kg, 1mg/kg and 10mg/kg, a positive control H8 (also referred to as HX008 below) 10mg/kg group and an isotype antibody Human IgG 45 mg/kg control group. The injection was administered into the tail vein, and was administered six times on days 6, 9, 13, 16, 19, and 22 after tumor cell inoculation (see table 11). According to relative tumor inhibition ratio (TGI)_RTV) And evaluating the curative effect, and evaluating the safety according to the weight change and death condition of the animals.

Table 11: experimental design of antitumor effect of test drug HX009-5 in KARPAS-299Mixeno tumor model

Note: the administration volume was 10. mu.l/g; n: the number of animals; day 0; i.v.: administration is carried out in the tail vein.

Tumor volumes of each group varied over time as shown in FIGS. 14 and 15 below, and both test drugs HX009-5 and H8 showed significant tumor suppression compared to control group inoculated with isotype antibody to PBMC (Human lgG 4). Among them, HX009-5 has obvious dose correlation, and the larger the dose of administration is, the higher the tumor inhibition rate is. At the same concentration, HX009-5 showed stronger tumor suppressive power than H8, indicating that the PD1/CD47 double-target antibody is superior to the PD1 single-target antibody.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Hangzhou Hansi biomedical Co., Ltd

<120> bispecific antibody against PD-1/CD47 and application thereof

<130> PIDC3175932

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 17

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of linker peptide

<400> 1

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

1 5 10 15

Pro

<210> 2

<211> 218

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of light chain of recombinant antibody

<400> 2

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

1 5 10 15

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 3

<211> 580

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of heavy chain of recombinant antibody

<400> 3

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

130 135 140

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

275 280 285

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile Thr

530 535 540

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

545 550 555 560

Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg

565 570 575

Ala Lys Pro Ser

580

<210> 4

<211> 654

<212> DNA

<213> Artificial

<220>

<223> nucleic acid sequence encoding recombinant antibody light chain

<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

<211> 1740

<212> DNA

<213> Artificial

<220>

<223> sequence of nucleic acid encoding heavy chain of recombinant antibody

<400> 5

gaggtgcagc tggtccagag cggaggcgga ctggtccagc ctggcggcag cctgaagctc 60

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 (6)

1. A recombinant antibody, wherein the recombinant antibody comprises: an anti-PD-1 antibody; and a human SIRPA extracellular section, wherein the amino acid sequence of the light chain of the recombinant antibody is shown as SEQ ID NO. 2, and the amino acid sequence of the heavy chain of the recombinant antibody is shown as SEQ ID NO. 3.

2. A nucleic acid encoding the recombinant antibody of claim 1.

3. The nucleic acid of claim 2, wherein the nucleic acid sequence corresponding to the light chain of the recombinant antibody is as set forth in SEQ ID NO: 4, the nucleotide sequence corresponding to the heavy chain of the recombinant antibody is shown as SEQ ID NO: 5, respectively.

4. A method of producing the recombinant antibody of claim 1, comprising:

introducing the nucleic acid of claim 2 or 3 into a mammalian cell;

culturing said mammalian cells under conditions suitable for protein expression and secretion so as to obtain said recombinant antibody.

5. The method of claim 4, wherein the mammalian cell comprises one selected from the group consisting of CHOK1, CHOS, 293F, 293T.

6. A therapeutic composition for treating cancer, comprising:

a recombinant antibody according to claim 1 or a nucleic acid according to any one of claims 2 to 3.

Images