CN112125975B - PD-L1 and CD47 bispecific fusion protein and medical application thereof - Google Patents

PD-L1 and CD47 bispecific fusion protein and medical application thereof Download PDF

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CN112125975B
CN112125975B CN202010581258.0A CN202010581258A CN112125975B CN 112125975 B CN112125975 B CN 112125975B CN 202010581258 A CN202010581258 A CN 202010581258A CN 112125975 B CN112125975 B CN 112125975B
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bispecific fusion
fusion protein
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CN112125975A (en
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花海清
包如迪
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Jiangsu Hansoh Pharmaceutical Group Co Ltd
Shanghai Hansoh Biomedical Co Ltd
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Abstract

The invention relates to a PD-L1 and CD47 bispecific fusion protein and medical application thereof. In particular to a bispecific fusion protein aiming at PD-L1 and CD47, a preparation method and application thereof. The bispecific fusion protein has the function of simultaneously combining PD-L1 and CD47, and can enhance immune response. The bispecific fusion proteins are useful for treating diseases characterized by immunosuppression.

Description

PD-L1 and CD47 bispecific fusion protein and medical application thereof
Technical Field
The invention relates to a bispecific fusion protein aiming at PD-L1 and CD47, a preparation method and application thereof, in particular to application thereof in tumor treatment.
Background
Immune surveillance is an important means of preventing tumor cell growth in humans. The human autoimmune system recognizes and kills tumor cells, which in order to grow, often evade immune surveillance by some means. Currently known approaches include three: 1) Immune surveillance of T lymphocytes was avoided. Tumor cells bind to receptor protein PD-1 on the surface of T cell membrane through high expression of programmed death ligand proteins PD-L1 and PD-L2 to induce T cell apoptosis. 2) Immune surveillance by Natural Killer (NK) cells was avoided. The NK cell has a protein NKG2D on the cell membrane, which can activate the NK cell to kill tumor cell after the NKG2D is combined with MICA/MICB protein on tumor cell membrane. However, tumor cells themselves have a mechanism that promotes the shedding of MICA/MICB from the cell membrane, thereby avoiding NK cell killing. 3) Immune surveillance by macrophages was avoided. Almost all tumor cells express CD47 on their cell membrane surface, and CD47 can bind to signal regulator protein (sirpa) on the macrophage membrane surface, thereby inducing an inhibitory signal, inhibiting phagocytic function of macrophages on tumor cells. Accordingly, the immune suppression means of tumor cells can be relieved, and the elimination effect of the human immune system on the tumor cells can be re-activated.
Antibody drugs against programmed death protein (PD-1) and programmed death ligand protein (PD-L1) can relieve the inhibition of T cells by tumors, and have been used clinically. In clinical application, only part of tumor patients respond to PD-1/PD-L1 antibody medicines, and after a period of treatment, the tumor patients have drug resistance, and the diseases continue to progress. In order to solve the problems, the invention designs a series of bispecific fusion proteins which can simultaneously aim at PD-L1 and CD47, and can further relieve the immunosuppression of macrophages on the basis of relieving the immunosuppression of T cells, and compared with the traditional PD-1/PD-L1 antibody medicines, the bispecific fusion proteins have better anti-tumor effect.
CD47 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily members. Proteins that bind CD47 in humans include the signal regulatory protein (SIRP) family and thrombin-sensitive protein (TSP-1). SIRP is a transmembrane glycoprotein comprising three family members, sirpa (CD 172 a), sirpa (CD 172 b), sirpa gamma (CD 172 g). These three members have similar membrane outer ends, each of which contains three immunoglobulin (Ig) -like regions, the first of which belongs to the Ig-V region and the second and third of which belong to the Ig-C region. SIRP proteins are expressed primarily in macrophages (mΦ), dendritic Cells (DCs), and neuronal cells.
CD47 has a variety of biological functions including cell migration, T cell, dendritic cell activation, axon development, and the like. In addition, CD47 inhibits phagocytosis of macrophages by interacting with sirpa. CD47 transmits a so-called "Don't eat me" signal in this way, which protects normal cells such as blood cells from phagocytosis by macrophages.
It was found that many tumor cells overexpress CD47 in addition to normal tissue cells, and that phagocytosis of tumor cells by macrophages is prevented by binding to sirpa on the surface of the macrophages, which is considered a mechanism by which tumors evade immune surveillance of the body. Studies have shown that antibodies specific for CD47 that have activity in blocking the binding of CD 47-SIRPalpha have anti-tumor effects. However, binding of antibodies to CD47 also causes injury to normal cells by macrophages, resulting in side effects. Among the most significant side effects observed clinically are hemolysis and anemia caused by blood cell death. And the natural protein binding to CD47 is utilized to block the binding of CD 47-SIRPalpha, so that the excessively strong side effect is hopeful to be avoided. Fusion proteins designed and prepared using sirpa, such as sirpa-Fc, have been previously reported (Lee WY et al, 2007), but have insufficient affinity for the target CD 47.
Disclosure of Invention
The invention relates to a bispecific fusion protein of PD-L1 and CD47, which can achieve the purpose of killing tumors through two mechanisms, namely blocking the inhibitory signals of T cells induced by tumor cells and blocking the inhibitory signals of macrophages induced by the tumor cells. The bispecific fusion protein comprises:
(a) An antibody or antigen-binding fragment thereof that binds to human programmed death ligand 1 (PD-L1);
(b) A CD 47-binding protein selected from the group consisting of proteins represented by the following sequences: SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 and SEQ ID NO:23, preferably said CD 47-binding protein is linked to the heavy chain of said antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1).
In one embodiment of the invention, the heavy chain variable region of the antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) comprises the following CDR sequences: SEQ ID NO:1, seq ID NO:2 and SEQ ID NO:3.
in one embodiment of the invention, the light chain variable region of the antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) comprises the following CDR sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
In one embodiment of the invention, the heavy chain amino acid sequence of the antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is SEQ ID NO:7, the light chain sequence is SEQ ID NO:8.
in one embodiment of the invention, the heavy chain amino acid sequence of the bispecific fusion protein of PD-L1 and CD47 is selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, seq ID NO:14, SEQ ID NO:16, SEQ ID NO:18, seq ID NO:20, SEQ ID NO:22 and SEQ ID NO:24.
in one embodiment of the invention, the light chain amino acid sequence of the bispecific fusion protein of PD-L1 and CD47 is selected from the group consisting of SEQ ID NO:8.
in a preferred embodiment of the invention, the bispecific fusion protein of PD-L1 and CD47 comprises a light chain and a heavy chain, wherein:
1) The heavy chain amino acid sequence is SEQ ID NO:10, the light chain sequence is SEQ ID NO:8, 8;
2) The heavy chain amino acid sequence is SEQ ID NO:12, the light chain sequence is SEQ ID NO:8, 8;
3) The heavy chain amino acid sequence is SEQ ID NO:14, the light chain sequence is SEQ ID NO:8, 8;
4) The heavy chain amino acid sequence is SEQ ID NO:16, the light chain sequence is SEQ ID NO:8, 8;
5) The heavy chain amino acid sequence is SEQ ID NO:18, the light chain sequence is SEQ ID NO:8, 8;
6) The heavy chain amino acid sequence is SEQ ID NO:20, the light chain sequence is SEQ ID NO:8, 8;
7) The heavy chain amino acid sequence is SEQ ID NO:22, the light chain sequence is SEQ ID NO:8, 8;
8) The heavy chain amino acid sequence is SEQ ID NO:24, the light chain sequence is SEQ ID NO:8.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain variable region of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) comprises the following CDR sequences: SEQ ID NO:1, seq ID NO:2 and SEQ ID NO:3, the light chain variable region comprises the following CDR sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; the CD47 binding protein has the sequence of SEQ ID NO:9.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain variable region of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) comprises the following CDR sequences: SEQ ID NO:1, seq ID NO:2 and SEQ ID NO:3, the light chain variable region comprises the following CDR sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; the CD47 binding protein has the sequence of SEQ ID NO:11.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain variable region of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) comprises the following CDR sequences: SEQ ID NO:1, seq ID NO:2 and SEQ ID NO:3, the light chain variable region comprises the following CDR sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; the CD47 binding protein has the sequence of SEQ ID NO:13.
In a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain variable region of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) comprises the following CDR sequences: SEQ ID NO:1, seq ID NO:2 and SEQ ID NO:3, the light chain variable region comprises the following CDR sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; the CD47 binding protein has the sequence of SEQ ID NO:15.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain variable region of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) comprises the following CDR sequences: SEQ ID NO:1, seq ID NO:2 and SEQ ID NO:3, the light chain variable region comprises the following CDR sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; the CD47 binding protein has the sequence of SEQ ID NO:17.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain variable region of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) comprises the following CDR sequences: SEQ ID NO:1, seq ID NO:2 and SEQ ID NO:3, the light chain variable region comprises the following CDR sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; the CD47 binding protein has the sequence of SEQ ID NO:19.
In a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain variable region of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) comprises the following CDR sequences: SEQ ID NO:1, seq ID NO:2 and SEQ ID NO:3, the light chain variable region comprises the following CDR sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; the CD47 binding protein has the sequence of SEQ ID NO:21.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain variable region of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) comprises the following CDR sequences: SEQ ID NO:1, seq ID NO:2 and SEQ ID NO:3, the light chain variable region comprises the following CDR sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; the CD47 binding protein has the sequence of SEQ ID NO:23.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain sequence of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is SEQ ID NO:7, the light chain sequence is SEQ ID NO:8, the sequence of the CD47 binding protein is SEQ ID NO:9.
In a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain sequence of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is SEQ ID NO:7, the light chain sequence is SEQ ID NO:8, the sequence of the CD47 binding protein is SEQ ID NO:11.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain sequence of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is SEQ ID NO:7, the light chain sequence is SEQ ID NO:8, the sequence of the CD47 binding protein is SEQ ID NO:13.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain sequence of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is SEQ ID NO:7, the light chain sequence is SEQ ID NO:8, the sequence of the CD47 binding protein is SEQ ID NO:15.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain sequence of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is SEQ ID NO:7, the light chain sequence is SEQ ID NO:8, the sequence of the CD47 binding protein is SEQ ID NO:17.
In a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain sequence of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is SEQ ID NO:7, the light chain sequence is SEQ ID NO:8, the sequence of the CD47 binding protein is SEQ ID NO:19.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain sequence of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is SEQ ID NO:7, the light chain sequence is SEQ ID NO:8, the sequence of the CD47 binding protein is SEQ ID NO:21.
in a preferred embodiment of the invention, in the bispecific fusion protein of the invention, the heavy chain sequence of an antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is SEQ ID NO:7, the light chain sequence is SEQ ID NO:8, the sequence of the CD47 binding protein is SEQ ID NO:23.
in one embodiment of the invention, in the bispecific fusion protein of the invention, the antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is linked to a protein that binds CD47 by a polypeptide linker.
In one embodiment of the invention, the bispecific fusion protein comprises a polypeptide linker of 5-30 amino acids.
In one embodiment of the invention, the bispecific fusion protein comprises a polypeptide linker (GGGGS) n Where n=1-6.
In one embodiment of the invention, the bispecific fusion protein comprises a polypeptide linker (GGGGS) n Where n=4.
The invention provides a nucleic acid for encoding the bispecific fusion protein.
The invention provides an expression vector containing the nucleic acid.
The present invention provides a host cell transformed with the above-described expression vector.
In one embodiment of the invention, the host cell is a bacterium, preferably E.coli.
In one embodiment of the invention, the host cell is a yeast, preferably pichia pastoris.
In one embodiment of the invention, the host cell is a mammalian cell, preferably a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney (HEK) 293 cell.
The invention also provides a pharmaceutical composition comprising the bispecific fusion protein and at least one pharmaceutically acceptable excipient, diluent or carrier. The compositions of the present invention may be used alone or in combination with other therapeutic agents to improve efficacy or reduce potential side effects.
The present invention provides a method of treating cancer using the pharmaceutical composition of the present invention, comprising administering to a patient or subject an effective amount of the bispecific fusion protein or pharmaceutical composition thereof. In one embodiment, the methods are used to treat immunosuppression-related tumors or cancers, including but not limited to lung cancer, breast cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gall bladder cancer, lymphoma, myeloma, and melanoma.
Drawings
Fig. 1: conformation of the bispecific fusion protein;
fig. 2: detecting the blood toxicity of the bispecific fusion protein;
fig. 3: in vivo efficacy of bispecific fusion proteins.
Detailed Description
Detailed Description
1. Terminology
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless otherwise defined explicitly in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The three-letter and one-letter codes for amino acids used in the present invention are described in J.biol. Chem,243, p3558 (1968).
The term "antibody" as used herein refers to an immunoglobulin that is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains joined by interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, i.e., igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α and ε chains, respectively. The Ig of the same class can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, for example, igG can be classified into IgG1, igG2, igG3 and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each of the five classes of Ig may have either a kappa chain or a lambda chain.
The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of the parent antibody (parental antibody). The antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen binding fragments include, but are not limited to: fab, fab ', F (ab') 2, fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson (2005) nat biotechnol.23: 1126-1136.
The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds through the hydrophobic effect of the CH3 domain.
The terms "specifically bind", "selectively bind" and "selectively bind" as used herein refer to the binding of an antibody or fusion protein to an epitope on a predetermined antigen.
The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking.
The term "inhibit growth" (e.g., involving a cell) is intended to include any measurable decrease in cell growth.
Methods for producing and purifying antibodies and antigen binding fragments are well known and can be found in the art, e.g., in the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15.
The engineered bispecific fusion proteins of the invention can be prepared and purified using conventional methods. The recombinant protein expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the FC region. Stable clones were obtained by expressing antibodies that specifically bound to human antigens. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. The soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.
"administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
"treatment" means administration of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the invention, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. While embodiments of the present invention (e.g., therapeutic methods or articles of manufacture) may be ineffective in alleviating the symptoms of each patient suffering from the disease of interest, they should alleviate the symptoms of the disease of interest in a statistically significant number of patients as determined by any statistical test methods known in the art, such as Student t test, chi-square test, U test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test.
The term "consisting essentially of … …" or variations thereof as used throughout the specification and claims is meant to include all such elements or groups of elements, and optionally other elements of similar or different nature to those described, which do not significantly alter the basic or novel nature of a given dosing regimen, method or composition. As a non-limiting example, a binding compound consisting essentially of the mentioned amino acid sequences may also include one or more amino acids that do not significantly affect the properties of the binding compound.
The term "naturally occurring" as applied to an object in the present invention refers to the fact that the object may be found in nature. For example, polypeptide sequences or polynucleotide sequences that are present in organisms (including viruses) that can be isolated from natural sources and that have not been intentionally modified by man in the laboratory are naturally occurring.
An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the route of administration and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.
"exogenous" refers to a substance that is to be produced by background outside an organism, cell or human. "endogenous" refers to substances produced in cells, organisms or humans according to background.
"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, a comparison is made when two sequences are aligned to give the greatest percent homology.
The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include their progeny. Thus, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or unintentional mutations. Including mutant progeny having the same function or biological activity as screened in the original transformed cell. Where different names are meant, they are clearly visible from the context.
"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may be, but need not be, present.
"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.
The invention will be further described with reference to the following examples, which are not intended to limit the scope of the invention. The experimental method without specific conditions being noted in the embodiment of the invention is generally according to conventional conditions, such as an antibody technical laboratory manual and a molecular cloning manual of cold spring harbor; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.
Example 1: PD-L1 antibody construction and preparation
Encoding SEQ ID NO:7 and SEQ ID NO: the 8-sequence DNA sequence was synthesized by CRO Genscript and cloned into pCDNA3.1 vector (Invitrogen) to become the heavy chain vector (P1 HC) and the light chain vector (P1 LC) of antibody P1, respectively. CHO cells were co-transfected with P1HC and P1LC, cell expression supernatant samples were high speed centrifuged to remove impurities, buffer replaced with PBS and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with PBS containing 5mM imidazole and washed 2-5 column volumes. The replaced supernatant samples were loaded onto a column. The column was washed with PBS containing 5mM imidazole to A 280 The reading drops to baseline. The column was then rinsed with PBS+10mM imidazole to remove non-specifically bound heteroproteins and the effluent was collected. The target protein was eluted with a PBS solution containing 300mM imidazole, and the elution peaks were collected. The collected eluate was further purified by ion exchange (SP column). Preparing solution A: 0.01M PB, pH8.0. Preparing a solution B: solution A+1M NaCl. Firstly, the target protein is eluted by the PBS solution of imidazole to be replaced by the A solution, the A solution is used for balancing an SP column, the sample is loaded, the concentration gradient of the B solution is 0-100 percent, the elution is performed by 10 times of column volume, and each elution peak is collected. And (3) carrying out electrophoresis on the obtained protein, identifying the protein as correct, and then sub-packaging the protein to obtain the PD-L1 antibody P1.
HCDR1
SYWMH
SEQ ID NO：1
HCDR2
RIGPNSGFTSYNEKFKN
SEQ ID NO：2
HCDR3
GGSSYDYFDY
SEQ ID NO：3
LCDR1
RASESVSIHGTHLMH
SEQ ID NO：4
LCDR2
AASNLES
SEQ ID NO：5
LCDR3
QQSFEDPLT
SEQ ID NO：6
P1HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO：7
P1LC
DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO：8
Example 2: construction and preparation of bispecific fusion protein P1P47A1
A DNA sequence encoding a polypeptide linker ggggsggggsggsggggs (SEQ ID NO: 25) and a DNA sequence encoding SEQ ID NO:9 is synthesized by CRO corporation Genscript, cloned into vector P1HC in sequence, and the DNA sequence encoding the polypeptide linker ggggsggggsggggsggggggs is cloned into the DNA sequence encoding SEQ ID NO:7, encoding the 3' end of the DNA sequence of SEQ ID NO:9 to the 3' end of the DNA sequence encoding the polypeptide linker GGGGSGGGGSGGGGSGGGGS, as a vector P1P47A1HC comprising the DNA sequence encoding the PD-L1 heavy chain-polypeptide linker-CD 47 binding protein. CHO cells were co-transfected with P1P47A1HC and P1LC, cell expression supernatant samples were high speed centrifuged to remove impurities, and buffer replaced with PBS and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with PBS containing 5mM imidazole and washed 2-5 column volumes. Will replace the upper partAnd (5) loading the clear sample on a column. The column was washed with PBS containing 5mM imidazole to A 280 The reading drops to baseline. The column was then rinsed with PBS+10mM imidazole to remove non-specifically bound heteroproteins and the effluent was collected. The target protein was eluted with a PBS solution containing 300mM imidazole, and the elution peaks were collected. The collected eluate was further purified by ion exchange (SP column). Preparing solution A: 0.01M PB, pH8.0. Preparing a solution B: solution A+1M NaCl. Firstly, the target protein is eluted by the PBS solution of imidazole to be replaced by the A solution, the A solution is used for balancing an SP column, the sample is loaded, the concentration gradient of the B solution is 0-100 percent, the elution is performed by 10 times of column volume, and each elution peak is collected. The obtained protein is split after being identified as correct by electrophoresis, and the PD-L1 and CD47 bispecific fusion protein A1 (P1P 47A 1) is obtained.
P47A1
GEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
SEQ ID NO：9
P1P47A1HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
SEQ ID NO：10
The light chain (P1P 47A1 LC) of the PD-L1 and CD47 bispecific fusion protein A1 is identical to P1 LC.
Example 3: construction and preparation of bispecific fusion proteins P1P47A1N
A DNA sequence encoding a polypeptide linker GGGGSGGGGSGGGGSGGGGS and a sequence encoding SEQ ID NO:11 is synthesized by CRO Genscript, and then cloned into vector P1HCAnd the DNA sequence encoding the polypeptide linker GGGGSGGGGSGGGGSGGGGS is cloned into a DNA sequence encoding SEQ ID NO:7, encoding the 3' end of the DNA sequence of SEQ ID NO:11 to a DNA sequence encoding a polypeptide linker GGGGSGGGGSGGGGSGGGGS, as a vector P1P47A1NHC comprising a DNA sequence encoding a PD-L1 heavy chain-polypeptide linker-CD 47 binding protein. CHO cells were co-transfected with P1P47A1NHC and P1LC, cell expression supernatant samples were high speed centrifuged to remove impurities, and buffer replaced with PBS and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with PBS containing 5mM imidazole and washed 2-5 column volumes. The replaced supernatant samples were loaded onto a column. The column was washed with PBS containing 5mM imidazole to A 280 The reading drops to baseline. The column was then rinsed with PBS+10mM imidazole to remove non-specifically bound heteroproteins and the effluent was collected. The target protein was eluted with a PBS solution containing 300mM imidazole, and the elution peaks were collected. The collected eluate was further purified by ion exchange (SP column). Preparing solution A: 0.01M PB, pH8.0. Preparing a solution B: solution A+1M NaCl. Firstly, the target protein is eluted by the PBS solution of imidazole to be replaced by the A solution, the A solution is used for balancing an SP column, the sample is loaded, the concentration gradient of the B solution is 0-100 percent, the elution is performed by 10 times of column volume, and each elution peak is collected. And (3) carrying out electrophoresis on the obtained protein, identifying the protein as correct, and then sub-packaging the protein to obtain the PD-L1 and CD47 bispecific fusion protein A1N (P1P 47A 1N).
P47A1N
GEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
SEQ ID NO：11
P1P47A1NHC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
SEQ ID NO：12
The light chain (P1P 47A1 NLC) of the PD-L1 and CD47 bispecific fusion proteins A1N is identical to P1 LC.
Example 4: construction and preparation of bispecific fusion protein P1P47A2
A DNA sequence encoding a polypeptide linker GGGGSGGGGSGGGGSGGGGS and a sequence encoding SEQ ID NO:13 is synthesized by CRO corporation Genscript, cloned into vector P1HC in sequence, and the DNA sequence encoding the polypeptide linker ggggsggggsggggsggggggs is cloned into the DNA sequence encoding SEQ ID NO:7, encoding the 3' end of the DNA sequence of SEQ ID NO:13 to a DNA sequence encoding a polypeptide linker ggggsggggsggsggggs, as a vector P1P47A2HC comprising a DNA sequence encoding a PD-L1 heavy chain-polypeptide linker-CD 47 binding protein. CHO cells were co-transfected with P1P47A2HC and P1LC, cell expression supernatant samples were high speed centrifuged to remove impurities, and buffer replaced with PBS and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with PBS containing 5mM imidazole and washed 2-5 column volumes. The replaced supernatant samples were loaded onto a column. The column was washed with PBS containing 5mM imidazole to A 280 The reading drops to baseline. The column was then rinsed with PBS+10mM imidazole to remove non-specifically bound heteroproteins and the effluent was collected. The target protein was eluted with a PBS solution containing 300mM imidazole, and the elution peaks were collected. The collected eluate was further purified by ion exchange (SP column). Preparing solution A: 0.01M PB, pH8.0. Preparing a solution B: solution A+1M NaCl. Firstly, the target protein is eluted by the PBS solution of imidazole to be replaced by the A solution, the A solution is used for balancing an SP column, the sample is loaded, the concentration gradient of the B solution is 0-100 percent, the elution is performed by 10 times of column volume, and each elution peak is collected. The obtained protein is split after being identified as correct by electrophoresis, and the PD-L1 and CD47 bispecific fusion protein A2 (P1P 47A 2) is obtained.
GEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS
SEQ ID NO：13
P1P47A2HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS
SEQ ID NO：14
The light chain (P1P 47A2 LC) of the PD-L1 and CD47 bispecific fusion protein A2 is consistent with P1LC
Example 5: construction and preparation of bispecific fusion protein P1P47A2T
A DNA sequence encoding a polypeptide linker GGGGSGGGGSGGGGSGGGGS and a sequence encoding SEQ ID NO:15 is synthesized by CRO corporation Genscript, cloned into vector P1HC in sequence, and the DNA sequence encoding the polypeptide linker ggggsggggsggggsggggggs is cloned into the DNA sequence encoding SEQ ID NO:7, encoding the 3' end of the DNA sequence of SEQ ID NO:15 to a DNA sequence encoding a polypeptide linker GGGGSGGGGSGGGGSGGGGS, as a vector P1P47A2THC comprising a DNA sequence encoding a PD-L1 heavy chain-polypeptide linker-CD 47 binding protein. CHO cells were co-transfected with P1P47A2THC and P1LC, cell expression supernatant samples were high speed centrifuged to remove impurities, buffer exchanged with PBS and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with PBS containing 5mM imidazole and washed 2-5 column volumes. The replaced supernatant samples were loaded onto a column. The column was washed with PBS containing 5mM imidazole to A 280 The reading drops to baseline. The column was then rinsed with PBS+10mM imidazole to remove non-specifically bound heteroproteins and the effluent was collected. The target protein was eluted with a PBS solution containing 300mM imidazole, and the elution peaks were collected. The collected eluate was further purified by ion exchange (SP column). Preparing solution A: 0.01M PB, pH8.0. Preparing a solution B: solution A+1M NaCl. Will first The target protein is eluted by the PBS solution of imidazole to be replaced by solution A, and the solution A is used for balancing an SP column, the sample is loaded, the concentration gradient of the solution B is 0-100 percent, the elution is performed by 10 times of column volume, and each elution peak is collected. And (3) carrying out electrophoresis on the obtained protein, identifying the protein as correct, and then sub-packaging the protein to obtain the PD-L1 and CD47 bispecific fusion protein A2T (P1P 47A 2T).
P47A2T
GEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSGGGGSGGGGSGGGGSGRFYVVMWK
SEQ ID NO：15
P1P47A2THC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSGGGGSGGGGSGGGGSGRFYVVMWK
SEQ ID NO：16
The light chain of the PD-L1 and CD47 bispecific fusion protein A2T (P1P 47A2 TLC) is identical to that of P1LC
Example 6: construction and preparation of bispecific fusion protein P1P47TA2
A DNA sequence encoding a polypeptide linker GGGGSGGGGSGGGGSGGGGS and a sequence encoding SEQ ID NO:17 is synthesized by CRO corporation Genscript, cloned into vector P1HC in sequence, and the DNA sequence encoding the polypeptide linker ggggsggggsggggsggggggs is cloned into the DNA sequence encoding SEQ ID NO:7, encoding the 3' end of the DNA sequence of SEQ ID NO:17 to a DNA sequence encoding a polypeptide linker ggggsggggsggsggggs, as a vector P1P47TA2HC comprising a DNA sequence encoding a PD-L1 heavy chain-polypeptide linker-CD 47 binding protein. Co-transfection of CHO with P1P47TA2HC and P1LCCells, cell expression supernatant samples were high speed centrifuged to remove impurities, buffer exchanged for PBS, and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with PBS containing 5mM imidazole and washed 2-5 column volumes. The replaced supernatant samples were loaded onto a column. The column was washed with PBS containing 5mM imidazole to A 280 The reading drops to baseline. The column was then rinsed with PBS+10mM imidazole to remove non-specifically bound heteroproteins and the effluent was collected. The target protein was eluted with a PBS solution containing 300mM imidazole, and the elution peaks were collected. The collected eluate was further purified by ion exchange (SP column). Preparing solution A: 0.01M PB, pH8.0. Preparing a solution B: solution A+1M NaCl. Firstly, the target protein is eluted by the PBS solution of imidazole to be replaced by the A solution, the A solution is used for balancing an SP column, the sample is loaded, the concentration gradient of the B solution is 0-100 percent, the elution is performed by 10 times of column volume, and each elution peak is collected. And (3) carrying out electrophoresis on the obtained protein, identifying the protein as correct, and then sub-packaging the protein to obtain the PD-L1 and CD47 bispecific fusion protein TA2 (P1P 47TA 2).
P47TA2
GRFYVVMWKGGGGSGGGGSGGGGSGEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS
SEQ ID NO：17
P1P47TA2HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGRFYVVMWKGGGGSGGGGSGGGGSGEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS
SEQ ID NO：18
The light chain of the PD-L1 and CD47 bispecific fusion protein TA2 (P1P 47TA2 LC) corresponds to P1LC example 7: construction and preparation of bispecific fusion protein P1P47B1
A DNA sequence encoding a polypeptide linker GGGGSGGGGSGGGGSGGGGS and a sequence encoding SEQ ID NO:19 is synthesized by CRO corporation Genscript, cloned into vector P1HC in sequence, and the DNA sequence encoding the polypeptide linker ggggsggggsggggsggggggs is cloned into the DNA sequence encoding SEQ ID NO:7, encoding the 3' end of the DNA sequence of SEQ ID NO:19 to a DNA sequence encoding a polypeptide linker GGGGSGGGGSGGGGSGGGGS, as a vector P1P47B1HC comprising a DNA sequence encoding a PD-L1 heavy chain-polypeptide linker-CD 47 binding protein. CHO cells were co-transfected with P1P47B1HC and P1LC, cell expression supernatant samples were high speed centrifuged to remove impurities, buffer replaced with PBS and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with PBS containing 5mM imidazole and washed 2-5 column volumes. The replaced supernatant samples were loaded onto a column. The column was washed with PBS containing 5mM imidazole to A 280 The reading drops to baseline. The column was then rinsed with PBS+10mM imidazole to remove non-specifically bound heteroproteins and the effluent was collected. The target protein was eluted with a PBS solution containing 300mM imidazole, and the elution peaks were collected. The collected eluate was further purified by ion exchange (SP column). Preparing solution A: 0.01M PB, pH8.0. Preparing a solution B: solution A+1M NaCl. Firstly, the target protein is eluted by the PBS solution of imidazole to be replaced by the A solution, the A solution is used for balancing an SP column, the sample is loaded, the concentration gradient of the B solution is 0-100 percent, the elution is performed by 10 times of column volume, and each elution peak is collected. The obtained protein is split after being identified as correct by electrophoresis, and the PD-L1 and CD47 bispecific fusion protein B1 (P1P 47B 1) is obtained.
P47B1
GEDELQVIQPEKSVSVAAGESATLRCtvTSLIPVGPIQWFRGAGAGRELIYNQKEGHFPRVTTVSEsTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
SEQ ID NO：19
P1P47B1HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGEDELQVIQPEKSVSVAAGESATLRCtvTSLIPVGPIqWFRGAGAGRELIYNQKEGHFPRVTTVSEsTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS
SEQ ID NO：20
The light chain of the PD-L1 and CD47 bispecific fusion protein B1 (P1P 47B1 LC) is identical to P1 LC.
Example 8: construction and preparation of bispecific fusion protein P1P47B2
A DNA sequence encoding a polypeptide linker GGGGSGGGGSGGGGSGGGGS and a sequence encoding SEQ ID NO:21 is synthesized by CRO corporation Genscript, cloned into vector P1HC in sequence, and the DNA sequence encoding polypeptide ggggsggggsggggsggggggs is cloned into the DNA sequence encoding SEQ ID NO:7, encoding the 3' end of the DNA sequence of SEQ ID NO:21 to a DNA sequence encoding a polypeptide linker ggggsggggsggsggggs, as a vector P1P47B2HC comprising a DNA sequence encoding a PD-L1 heavy chain-polypeptide linker-CD 47 binding protein. CHO cells were co-transfected with P1P47B2HC and P1LC, cell expression supernatant samples were high speed centrifuged to remove impurities, buffer replaced with PBS and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with PBS containing 5mM imidazole and washed 2-5 column volumes. The replaced supernatant samples were loaded onto a column. The column was washed with PBS containing 5mM imidazole to A 280 The reading drops to baseline. The column was then rinsed with PBS+10mM imidazole to remove non-specifically bound heteroproteins and the effluent was collected. The target protein was eluted with a PBS solution containing 300mM imidazole, and the elution peaks were collected. The collected eluate was further purified by ion exchange (SP column). Preparing solution A: 0.01M PB, pH8.0. Preparing a solution B: solution A+1M NaCl. Firstly, the target protein is eluted by the PBS solution of imidazole to be replaced by the A solution, the A solution is used for balancing an SP column, the sample is loaded, the concentration gradient of the B solution is 0-100 percent, the elution is performed by 10 times of column volume, and each elution peak is collected. And (3) carrying out electrophoresis on the obtained protein, identifying the protein as correct, and then sub-packaging the protein to obtain the PD-L1 and CD47 bispecific fusion protein B2 (P1P 47B 2).
P47B2
GEEELQVIQPDKSISVAAGESATLHCTVTSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDsTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDHVEFKSGAGTELSVRAKPS
SEQ ID NO：21
P1P47B2HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGEEELQVIQPDKSISVAAGESATLHCTVTSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDsTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDHVEFKSGAGTELSVRAKPS
SEQ ID NO：22
The light chain of the PD-L1 and CD47 bispecific fusion protein B2 (P1P 47B2 LC) corresponds to P1LC example 9: construction and preparation of bispecific fusion protein P1P47G
A DNA sequence encoding a polypeptide linker GGGGSGGGGSGGGGSGGGGS and a sequence encoding SEQ ID NO:23 is synthesized by CRO corporation Genscript, cloned into vector P1HC in sequence, and the DNA sequence encoding the polypeptide linker ggggsggggsggggsggggggs is cloned into the DNA sequence encoding SEQ ID NO:7, encoding the 3' end of the DNA sequence of SEQ ID NO:23 to the 3' end of the DNA sequence encoding the polypeptide GGGGSGGGGSGGGGSGGGGS, as a vector P1P47GHC comprising the DNA sequence encoding the PD-L1 heavy chain-polypeptide linker-CD 47 binding protein. CHO cells were co-transfected with P1P47GHC and P1LC, cell expression supernatant samples were high speed centrifuged to remove impurities, buffer exchanged for PBS, imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with PBS containing 5mM imidazole and washed 2-5 column volumes. The replaced supernatant samples were loaded onto a column. The column was washed with PBS containing 5mM imidazole to A 280 The reading drops to baseline. Washing the column with PBS+10mM imidazole to remove nonspecifically bound heteroproteins, and collecting the flowAnd (5) discharging liquid. The target protein was eluted with a PBS solution containing 300mM imidazole, and the elution peaks were collected. The collected eluate was further purified by ion exchange (SP column). Preparing solution A: 0.01M PB, pH8.0. Preparing a solution B: solution A+1M NaCl. Firstly, the target protein is eluted by the PBS solution of imidazole to be replaced by the A solution, the A solution is used for balancing an SP column, the sample is loaded, the concentration gradient of the B solution is 0-100 percent, the elution is performed by 10 times of column volume, and each elution peak is collected. And (3) carrying out electrophoresis on the obtained protein, identifying the protein as correct, and then sub-packaging the protein to obtain the PD-L1 and CD47 bispecific fusion protein G (P1P 47G).
P47G
GEELQMIQPEKLLLVTVGKTATLHCTVTSLLPVGPVQWFRGVGPGRELIYNQKEGHFPRVTTVSDsTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTELSVRAKPS
SEQ ID NO：23
P1P47GHC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGEELQMIQPEKLLLVTVGKTATLHCTVTSLLPVGPVqWFRGVGPGRELIYNQKEGHFPRVTTVSDsTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTELSVRAKPS
SEQ ID NO：24
The light chain (P1P 47 GLC) of the PD-L1 and CD47 bispecific fusion protein G is identical to P1 LC.
Example 10: binding of bispecific fusion proteins to PD-L1 proteins
PD-L1 protein (Sino Biological Inc., cat# 10084-HNAH) was diluted to a concentration of 1. Mu.g/ml with PBS pH7.4, added to a 96-well high affinity ELISA plate at a volume of 100. Mu.l/well and incubated overnight (16-20 hours) in a refrigerator at 4 ℃. After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 150. Mu.l/well of a blocking solution of 3% Bovine Serum Albumin (BSA) diluted with PBST was added, and incubated at room temperature for 1 hour for blocking. After blocking, the blocking solution was discarded and the plate was washed 4 times with PBST buffer. The protein to be tested was diluted with 3% BSA in PBST, 1. Mu.M starting, 10-fold gradient, 10 doses, 100. Mu.l/well into the ELISA plate and incubated at room temperature for 1 hour. After incubation, plates were washed 4 times with PBST, 100. Mu.l/well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab 97225) diluted with 3% BSA in PBST was added and incubated for 1 hour at room temperature. After washing the plate 4 times with PBST, 100. Mu.l/well TMB chromogenic substrate (Cell Signaling Technology, cat# 7004S) was added, incubated at room temperature for 1 minute in the absence of light, the reaction was stopped by adding 100. Mu.l/well Stop Solution (Cell Signaling Technology, cat# 7002S), the absorbance was read at 450nm using a microplate reader (BioTek, model number Synergy H1), and the data was analyzed. The concentration signal value curve analysis results are shown in the following table 1:
TABLE 1
Example 11: binding of bispecific fusion proteins to CD47 proteins
CD47-His protein (Sino Biological Inc., cat# 12283-H08H) was diluted to a concentration of 1. Mu.g/ml with PBS pH7.4, added to a 96-well high affinity ELISA plate at a volume of 100. Mu.l/well and incubated overnight (16-20 hours) in a refrigerator at 4 ℃. After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 150. Mu.l/well of a blocking solution of 3% Bovine Serum Albumin (BSA) diluted with PBST was added, and incubated at room temperature for 1 hour for blocking. After blocking, the blocking solution was discarded and the plate was washed 4 times with PBST buffer. The protein to be tested was diluted with 3% BSA in PBST, 1. Mu.M starting, 10-fold gradient, 10 doses, 100. Mu.l/well into the ELISA plate and incubated at room temperature for 1 hour. After incubation, plates were washed 4 times with PBST, 100. Mu.l/well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab 97225) diluted with 3% BSA in PBST was added and incubated for 1 hour at room temperature. After washing the plate 4 times with PBST, 100. Mu.l/well TMB chromogenic substrate (Cell Signaling Technology, cat# 7004S) was added, incubated at room temperature for 1 minute in the absence of light, the reaction was stopped by adding 100. Mu.l/well Stop Solution (Cell Signaling Technology, cat# 7002S), the absorbance was read at 450nm using a microplate reader (BioTek, model number Synergy H1), and the data was analyzed. The concentration signal value curve analysis results are shown in the following table 2:
TABLE 2
Sample of Binding to CD47 EC 50 (nM)
SIRPa-Fc 43.2
P1 >100
P1P47A1 0.799
P1P47A1N 0.636
P1P47A2T 0.859
P1P47TA2 0.493
P1P47B1 5.283
P1P47G 0.286
Example 12: detection of hematological toxicity of bispecific fusion proteins
Using Lymphoprep TM (Axis-Shield PoC AS, cat#AS 1114547) red blood cells were separated from mixed healthy human blood by density gradient centrifugation. Red blood cells were resuspended using PBS and diluted proportionally to the appropriate density. The red blood cell suspension was added to a 96-well cell culture plate. The diluted bispecific fusion protein and Hu5F9 (a CD47 antibody at the stage of clinical study) were added to the corresponding wells of the culture plate. Incubate at room temperature for about 1 hour. The agglutination of erythrocytes at various concentrations was observed and stored by photographing using a gel imaging system (Tanon 1600R). The experimental results are shown in FIG. 2. In contrast to the CD47 antibody Hu5F9, the bispecific fusion protein was not or only slightly hematotoxic.
Example 13: in vivo efficacy of bispecific fusion proteins
To study the anti-tumor effect of the bispecific fusion proteins, the anti-tumor effect of the bispecific fusion proteins of the present invention was evaluated after subcutaneous inoculation of MC38-hPD-L1/hCD47 (intestinal cancer cell line carrying human PD-L1 and CD47 genes) cells in transgenic mice carrying human PD-L1, SIRPA and CD47 genes to form subcutaneous tumors. Will be 5x10 5 The MC38-hPD-L1/hCD47 cells were injected subcutaneously into mice and, after 2 weeks, the bispecific fusion protein was injected twice a week at a dose of 6mg/kg. Human IgG1 protein was used as control at a dose of 6mg/kg. Control or dosing groups each had 6 mice. Tumor inhibition was calculated by measuring tumor volume. Tumor inhibition = 100% - (day 21 dosing group tumor volume-day 0 dosing group tumor volume)/(day 21 control group tumor volume-day 0 control group tumor volume). The experimental results are shown in fig. 3 and table 3 below. Bispecific fusion proteins showed stronger anti-tumor effects compared to PD-L1 antibodies.
TABLE 3 Table 3
Administration group Tumor inhibition rate
P1 6mg/kg 54.5％
P1P47A1 6mg/kg 86.5％

Claims (11)

1. A bispecific fusion protein of PD-L1 and CD47, comprising:
(a) An antibody or antigen-binding fragment thereof that binds to human programmed death ligand 1 (PD-L1); the heavy chain amino acid sequence of the antibody or antigen binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is SEQ ID NO:7, the light chain sequence is SEQ ID NO:8, 8;
(b) A CD 47-binding protein selected from the group consisting of proteins represented by the following sequences: SEQ ID NO:9, the CD 47-binding protein is linked to the heavy chain C-terminus of the antibody or antigen binding fragment thereof that binds human programmed death ligand 1 (PD-L1);
Wherein the antibody or antigen-binding fragment thereof that binds to human programmed death ligand 1 (PD-L1) is linked to a protein that binds CD47 by a polypeptide linker, said polypeptide linker being (GGGGS) n Where n=4.
2. A bispecific fusion protein of PD-L1 and CD47, wherein the bispecific fusion protein of PD-L1 and CD47 comprises a heavy chain and a light chain, wherein:
1) The heavy chain amino acid sequence is SEQ ID NO:10, the light chain sequence is SEQ ID NO:8.
3. nucleic acid encoding the bispecific fusion protein of PD-L1 and CD47 of any one of claims 1-2.
4. An expression vector comprising the nucleic acid of claim 3.
5. A host cell transformed with the expression vector of claim 4.
6. The host cell of claim 5, wherein the host cell is a bacterial, yeast, or mammalian cell.
7. The host cell of claim 6, wherein the host cell is an escherichia coli, pichia pastoris, chinese Hamster Ovary (CHO) cell, or Human Embryonic Kidney (HEK) 293 cell.
8. A pharmaceutical composition comprising the bispecific fusion protein of PD-L1 and CD47 of any one of claims 1-2, and a pharmaceutically acceptable carrier.
9. The pharmaceutical composition of claim 8, wherein the carrier is an excipient.
10. The pharmaceutical composition of claim 8, wherein the carrier is a diluent.
11. Use of the bispecific fusion protein of PD-L1 and CD47 of any one of claims 1-2 or the pharmaceutical composition of any one of claims 8-10 in the manufacture of a medicament for the treatment or prevention of a disease caused by immunosuppression, wherein said disease is lung cancer, breast cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gall bladder cancer, myeloma and melanoma.
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