Patent Application
20230312709
The present application claims priority to Chinese patent application CN 202011544262.6, filed to the China National Intellectual Property Administration on Dec. 23, 2020 and entitled “Antibody Targeting CD47 and Application thereof”, the application of which is hereby incorporated by reference in its entirety.
The present application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy is named PN182539_Sequence_listing.txt and is 14,554 bytes in size, which is identical to the sequence listing filed in the corresponding international application No. PCT/CN2021/140404 filed on Dec. 21, 2021, except that the description of the artificial sequences has been added, and no new matter is added.
The present application belongs to the technical field of biological medicines. Specifically, it relates to an antibody targeting CD47 and an application thereof, and more specifically, relates to an anti-CD47 antibody or an antigen-binding fragment thereof, a nucleic acid, a vector, a cell, a composition, an application and a kit.
Cancer immunotherapy is a major event in the field of biological sciences in recent years. Immune checkpoint inhibitor therapies of T-cell-based CTLA4 antibody, PD-1 antibody, PD-L1 antibody and the like and cell therapies of CAR-T, TCR-T and the like are all hot immunotherapies in recent years. These revolve around how to restore T cell functions without exception, and in other words, how to improve the acquired immune system capacity. However, a road of using an immune checkpoint as a target point, and activating the T cell function, to improve the capacity of an acquired immune system so as to conquer a cancer is still full of twists and turns. However, the action of an innate immune system in tumor immunotherapy is not exerted for a long time. In fact, in the entire tumor-infiltrating area, macrophages account for about 50% of tumor tissues. More importantly, the number of the macrophages is inversely related to the prognosis of tumors, which further indicates that the macrophages have very important effect in the tumors. The phagocytosis exerted by the macrophages requires two signals to act simultaneously: one is the activation of an “eat me” signal on the surface of a target cell, and the other is the inactivation of a “don't eat me” signal on the surface of the same target. The absence of any one signal is not sufficient to trigger the phagocytosis. It is indicated from more and more evidences that CD47 is a type of the “don't eat me” signal, and it inhibits the phagocytosis of the macrophages by mutually combining with a signal regulatory protein α (SIRPα) on the surface of the macrophages. Tumor cells may also escape the macrophage phagocytosis through the expression of CD47 (for example, referring to EP2242512 and related documents cited therein).
CD47, also known as an integrin-associated protein (IAP), is a 50 kDa membrane protein with an amino-terminal immunoglobulin domain and a carboxy-terminal multiple transmembrane domain. It interacts with a plurality of ligands, including but not limited to SIRPα, SIRPγ, an integrin and thrombospondin-1 (TSP-1). SIRPα is mainly expressed on myeloid cells, including a macrophage, a myeloid dendritic cell (DC), a granulocyte, a mast cell and a precursor thereof, and including a hematopoietic stem cell. CD47/SIRPα interaction transmits the “don't eat me” signal, and prevents autophagy. The analysis of patient tumors and matched adjacent normal (non-tumor) tissues shows that the CD47 protein is overexpressed on cancer cells, and this effectively helps them escape innate immune surveillance and elimination. Blocking the interaction of CD47-SIRPα with an anti-CD47 antibody has already shown to effectively induce the phagocytosis of the tumor cells in vitro and inhibit the growth of various hematological and solid tumors in vivo. Therefore, CD47 is an effective target for cancer therapy, and an appropriate antagonist thereof is required to prepare human therapeutic agents.
A CD47 monoclonal antibody has the higher targeting binding property to red blood cells, and it is easy to cause the red blood cell agglutination, so that the therapeutic effect of the corresponding antibody is greatly reduced, and drug side effects are caused. The present application aims to provide an antibody targeting CD47, and the antibody may block the binding of CD47 to SIRPα, and promote the phagocytosis of macrophages. What is more commendable is that it may prevent red blood cell blood from agglutinating to a certain extent, and the safety is good.
A purpose of the present application is to provide an anti-CD47 antibody or an antigen-binding fragment thereof, and the antibody contains the following complementarity determining regions (CDRs):
Another purpose of the present application is to provide a nucleic acid, a vector, a cell or a pharmaceutical composition related to the anti-CD47 antibody or the antigen-binding fragment thereof.
The present application further relates to an application of the anti-CD47 antibody or the antigen-binding fragment thereof, and the related nucleic acid, vector, cell or pharmaceutical composition in preparation of a drug for treating and/or preventing a CD47-positive tumor.
The present application further relates to an application of the anti-CD47 antibody or the antigen-binding fragment thereof, and the related nucleic acid, vector or cell in preparation of a kit for detecting CD47 or diagnosing a CD47-related disease.
The present application further provides a kit for detecting CD47, and the kit contains the anti-CD47 antibody or the antigen-binding fragment thereof.
The present application further provides a kit for diagnosing a CD47-related disease, and the kit contains the anti-CD47 antibody or the antigen-binding fragment thereof.
The present application is further described below in combination with the specific embodiments, but the embodiments do not limit the present application in any form. Unless otherwise specified, reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.
Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.
The present application relates to an anti-CD47 antibody or an antigen-binding fragment thereof, and the antibody contains the following CDRs:
An important advantage of the antibody or the antigen-binding fragment thereof is its high affinity to CD47.
An important advantage of the antibody or the antigen-binding fragment thereof is that it has the activity of blocking the binding of CD47 to SIR Pa.
An important advantage of the antibody or the antigen-binding fragment thereof is its ability to promote a macrophage to phagocytize a tumor cell.
An important advantage of the antibody or the antigen-binding fragment thereof is that it has the effect of significantly reducing agglomeration of red blood cell blood.
An important advantage of the antibody or the antigen-binding fragment thereof is that it shows a very weak level of low or no binding to a red blood cell.
Because of the above properties, the antibody or the antigen-binding fragment thereof may preferably be used as an antibody drug.
In the present application, a technical term “antibody or antigen-binding fragment thereof” is a protein that binds to a specific antigen, and it generally refers to all proteins and protein fragments containing CDRs. “Antibody” specifically refers to a full-length antibody. A term “full-length antibody” includes a polyclonal antibody and a monoclonal antibody.
A term “antigen-binding fragment” is a substance containing a part or all of CDRs of an antibody, and it lacks at least some of amino acids present in a full-length chain but is still capable of specifically binding to an antigen. This type of the fragment has the biological activity, because it binds to a target antigen, and may compete with other antigen-binding molecules (including a complete antibody) for binding to a given epitope. In some embodiments, the antigen-binding fragment has a function of specifically recognizing and binding to CD47. In some embodiments, the antigen-binding fragment is a fragment which has a function of blocking the binding of CD47 to its ligand, and activating an immune cell, and in one aspect, this type of the fragment is selected from Fab (consisting of a complete light chain and Fd), Fv (consisting of VH and VL), ScFv (a single-chain antibody, VH and VL are linked by a linker peptide) or a single-domain antibody (consisting of VH only). The fragment may be generated by a recombinant nucleic acid technology, or may be generated by enzymatic lysis or chemical cleavage of the antigen-binding molecules (including the complete antibody).
A term “complementarity determining region” or “CDR” refers to a hypervariable region of a heavy chain and a light chain of an immunoglobulin. There are three heavy chain CDRs and three light chain CDRs. Here, depending on the situation, terms “CDR” and “CDRs” are used to refer to a region containing one or more or even all of major amino acid residues that contribute to the binding affinity of the antibody or the antigen-binding fragment thereof to an antigen or an epitope recognized by it. In another specific embodiment, a CDR region or CDR refers to the hypervariable region of the heavy chain and the light chain of the immunoglobulin defined by the international immunogenetics information system (IMGT).
In the present application, the complementarity determining region of the heavy chain is represented by HCDR, and the complementarity determining region of the light chain is represented by LCDR. Common CDR marking methods used in the field include: a Kabat numbering scheme, a Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of an immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for an immunoglobulin variable region. In their compilation of “Sequences of Proteins of Immunological Interest”, the amino acid sequences of light chain (λ, κ) variable regions and antibody heavy chains, as well as the variable regions of T cell receptors (α, β, γ, and δ) are aligned and numbered. In the past few decades, the accumulation of sequences has led to the creation of a KABATMAN database, and the Kabat numbering scheme is generally considered as a widely adopted standard for numbering the antibody residues. The present application adopts a Kabat annotation standard to mark the CDR regions, but the CDR regions marked by other methods also belong to the protection scope of the present application.
Terms “specific recognizing”, “selective binding”, “selectively binding” and “specifically binding” or the like refer to the binding of the antibody or the antigen-binding fragment thereof to a predetermined epitope on an antigen. Typically, the antibody or the antigen-binding fragment thereof binds with an affinity (KD) of less than about 10−6 M, for example, less than about 10−7 M, 10−8 M, 10−9 M, or 10−10 M or less.
In the present application, an object specifically recognized by the provided antibody or antigen-binding fragment thereof may be CD47 derived from various genera, such as human, mouse, and monkey (such as, cynomolgus monkey).
In some embodiments, the antibody contains at least one of a heavy chain variable region sequence and a light chain variable region sequence, at least a part of at least one of the heavy chain variable region sequence and the light chain variable region sequence is from at least one of a mouse-derived antibody, a humanized antibody, a primate-derived antibody or a mutant thereof.
In some embodiments, the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO:5˜9 or a sequence having at least 95% of the identity with any one of SEQ ID NO:5˜9.
In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO:14˜18 or a sequence having at least 95% of the identity with any one of SEQ ID NO:14˜18.
In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:5, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:14; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:14; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:15; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:16; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:17; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:18; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:14; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:14; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:16; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:16; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:16; or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:18.
In some embodiments, the antibody has a constant region, and the heavy chain constant region is selected from any one of IgGI, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is a κ or λ chain.
In some embodiments, the species source of the constant region is selected from mouse, rabbit, sheep, monkey, or human.
In some embodiments, the antibody is any one or more of a CDR-transplanted antibody, a multimeric antibody or a bispecific antibody.
In some embodiments, the antigen-binding fragment is any one or more of F (ab′)2, Fab, scFv, Fv, and a single-domain antibody.
In some embodiments, the CD47 is human CD47, mouse CD47 or monkey CD47.
The present application further relates to a nucleic acid, and the nucleic acid encodes the anti-CD47 antibody or the antigen-binding fragment thereof.
The nucleic acid is usually a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA), and a nucleic acid molecule may be single-stranded or double-stranded, but is preferably a double-stranded DNA. The nucleic acid is “operably linked” while it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or an enhancer affects the transcription of a coding sequence, the promoter or the enhancer is operably linked to the coding sequence. The DNA nucleic acid is preferably used while it is linked into a vector.
Furthermore, since the antibody is a membrane protein, the nucleic acid generally carries a signal peptide sequence.
The present application further relates to a vector, and the vector carries the above nucleic acid.
A term “vector” refers to a nucleic acid delivery tool into which a polynucleotide may be inserted. While the vector may express the protein encoded by the inserted polynucleotide, the vector is called as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection, so that a genetic material element carried by it may be expressed in the host cell. The vector is well-known to those skilled in the art and includes, but is not limited to: a plasmid; a phagemid; a cosmid; an artificial chromosome, such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1-derived artificial chromosome (PAC); and a phage such as a λ phage or an M13 phage and an animal virus. The animal virus that may be used as the vector includes, but is not limited to, a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpesvirus (such as, a herpes simplex virus), a poxvirus, a baculovirus, a papillomavirus, and a papovavirus (such as SV40). In some embodiments, the vector described in the present application contains a regulatory element commonly used in genetic engineering, such as the enhancer, the promoter, an internal ribosome entry site (IRES) and other expression control elements (for example, a transcription termination signal, or a polyadenylation signal and a polymeric U sequence).
The present application further relates to a cell, and the cell carries the above nucleic acid or contains the above vector. The nucleic acid encoding the anti-CD47 antibody or the antigen-binding fragment thereof is used to link to the vector, then expressed in the cell and the corresponding antibody may be obtained. The vector may be introduced into a eukaryotic cell, especially a mammalian cell, to construct and obtain the cells capable of expressing the anti-CD47 antibody or the antigen-binding fragment thereof described in the present application.
As used herein, expressions of “cell”, “cell line” and “cell culture” are used interchangeably, and all such names include a progeny. Thus, words “transformant” and “transformed cell” include a primary test cell and a culture derived therefrom, regardless of the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progenies may not be exactly the same in terms of DNA content. A mutant progeny that has the same function or biological activity as screened in the original transformed cell are included. In the case that a different name is meant, it is clear from the context.
The present application further relates to a pharmaceutical composition, and the pharmaceutical composition contains the anti-CD47 antibody or the antigen-binding fragment thereof, the nucleic acid, the vector or the cell.
In some embodiments, the composition further contains a pharmaceutically acceptable carrier. The “pharmaceutically acceptable carrier” may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents and the like that are physiologically compatible, to prolong the shelf life or efficacy of the antibody.
An application of the anti-CD47 antibody or the antigen-binding fragment thereof, the nucleic acid, the vector, the cell or the composition in preparation of a drug for treating and/or preventing a CD47-positive tumor should also be within the protection scope of the present application.
The drug may reduce the red blood cell blood agglutination and reduce its binding activity with the red blood cells.
An application of the anti-CD47 antibody or the antigen-binding fragment thereof, the nucleic acid, the vector or the cell in preparation of a kit for detecting CD47 or diagnosing a CD47-related disease should also be within the protection scope of the present application.
The present application further provides a kit for detecting CD47, and the kit contains the anti-CD47 antibody or the antigen-binding fragment thereof.
The present application further provides a kit for diagnosing a CD47-related disease, and the kit contains the anti-CD47 antibody or the antigen-binding fragment thereof.
The present application has the following beneficial effects.
The present application provides the anti-CD47 antibody or the antigen-binding fragment thereof. The antibody does not agglutinate the red blood cell in vitro, and more commendably, it shows the very weak level of low or no binding to the red blood cell; it may effectively block the binding of CD47 to SIRPα, and activate and mediate the phagocytic activity of the macrophages on the tumor cells, it shows the significant targeting specificity with the CD47-positive tumor cell, is high in affinity, and strong in specificity, without causing the red blood cell agglutination, and shows the extremely weak binding to human red blood cells and platelets, the safety is good; and therefore, the anti-CD47 antibody or the antigen-binding fragment thereof may be used as a very promising target site in a tumor immunotherapy system, play a strong role in human tumor treatment, and have a broad application prospect in preparation of the drug for treating the CD47-positive tumors.
Embodiments of the present application are described in detail below in combination with the embodiments.
1. Construction of hCD47-ECD-HIS Recombinant Expression Plasmid
The DNA sequence encoding human CD47 extracellular region (hCD47-ECD) following by 6×HIS tag sequence was synthesized based on a template (CEJ 94640.1) from Gen Bank, and then was cloned into an expression vector pCDNA3.4 (Thermo Company), to establish a recombinant eukaryotic expression plasmid of a CD47 extracellular full-length protein, named hCD47-ECD-HIS recombinant plasmid DNA.
2. Expression and Purification of hCD47-ECD-HIS Recombinant Protein
The expression and purification of the hCD47-ECD-HIS recombinant protein includes the following steps.
On the day of transfection, the Expi293 cells were counted with a cell counter (Countstar; IC1000), and the cell density was diluted and adjusted to 2.9*106 with fresh Dynamis culture solution; it was ready for the transfection; PEI:DNA=3:1; mixed uniformly for 5 min, the two were gently mixed for 20 times and standing for 15˜30 min; a DNA-PEI mixture was added to the Expi293 cells, mixed uniformly, and placed in the cell culture shaker (Adolf Kuhner; ISF4-XC) and cultured at 37° C., 8% CO2, and 120 rpm; and after 4 h of the transfection, a double-antibody (gibco; 15140122) and an anticoagulant (gibco; 0010057) were supplemented.
3. Screening of Anti-CD47 Mouse Monoclonal Antibody
The hCD47-ECD-HIS recombinant protein (hereinafter referred to as an hCD47 antigen) purified in the step 2 was used for immunization of BALB/C mice (purchased from the Guangdong Experimental Animal Center). A specific method is as follows.
Amino acid sequences of mouse antibody light chain CDRs are shown in SEQ ID NOs:1-3:
Amino acid sequences of mouse antibody heavy chain CDRs are shown in SEQ ID NOs:10˜12:
An amino acid sequence of a mouse antibody light chain variable region (>7A11 D3D3-VL) was shown in SEQ ID NO:4.
An amino acid sequence of a mouse antibody heavy chain variable region (>7A11 D3D3-VH) was shown in SEQ ID NO:13.
The humanized design was performed on the mouse antibody, 5 humanized sequences, 7A11-L01˜05 respectively, were designed for a 7A11 D3D3 mouse monoclonal antibody light chain, and amino acid sequences of humanized light chain variable regions (7A11-L01˜05) were shown in SEQ ID NOs:5˜9; and 5 humanized sequences, 7A11-H01˜05 respectively, were designed for a heavy chain, and amino acid sequences of humanized heavy chain variable regions (7A11-H01˜05) were shown in SEQ ID NOs:14˜18.
The above amino acid sequences were combined, and the obtained antibodies and the sequences corresponding to the heavy chain variable regions (VH) and light chain variable regions (VL) thereof were shown in Table 1.
A humanized variable domain was combined with a secretion signal and human κ and human FcIgG4S228P constant domains, cloned into a mammalian expression system, and transfected into a 293 cell to generate humanized mAb. A humanized variant was expressed as a full-length IgG molecule, secreted into a culture medium and purified with the protein A.
Humanized antibody and positive control antibody Hu5F9-G4 (a Hu5F9-G4 sequence has already disclosed in the US patent US2015/0183874A1) were transiently expressed in Expi293 cells and purified.
For the transient expression of the antibody in the Expi293 cells, a vector pCDNA3.4 was used, and a heavy chain and a light chain of the antibody were firstly cloned into the separate pCDNA3.4 vector, a PEI (purchased from Polysciences) chemical transfection reagent was used, the pCDNA3.4 vectors with the heavy chain and the light chain of the antibody molecule were transferred into the Expi293 cells according to a chemical transfection method, and the transferred and cultured Expi293 cells were transiently transfected according to a scheme provided by a manufacturer.
On the day before transient transfection, the Expi293 (ThermoFisher Scientific; A14635) cells were passaged, inoculated with a Dynamis medium (gibco; A2617502) at a density of 2E6 in a 1 L shake flask (conning; 431147), and placed and cultured in a cell culture shaker (Adolf Kuhner; ISF4-XC) at 37° C., 8% CO2 and 120 rpm.
On the day of transfection, the Expi293 cells were counted with a cell counter (Countstar; IC1000), and the cell density was diluted and adjusted to 2.9E6 with fresh Dynamis culture solution; it was ready for the transfection; PEI:DNA=3:1 mixed uniformly for 5 min, the two were gently mixed for 20 times and standing for 15˜30 min; the DNA-PEI mixture was added to the Expi293 cells, mixed uniformly, and placed in the cell culture shaker (Adolf Kuhner; ISF4-XC) and cultured at 37° C., 8% CO2, and 120 rpm; and after 4 h of the transfection, a double-antibody (gibco; 15140122) and an anticoagulant (gibco; 0010057) were supplemented.
Purification of harvested supernatant: the transfection was continuously cultured for 7 days, and then the sample was collected. First it was centrifuged at a low speed of 1000 rpm, 10 min and 4° C. (Xiangyi H2050R), and then centrifuged at a high speed of 12000 rpm, 30 min and 4° C.; and a cell culture supernatant was collected, and filtered at 0.22 urn. The culture supernatant was used in a Protein A Sepharose column (GE Healthcare). The column was washed with PBS, and eluted with an elution buffer (0.1 M sodium citrate buffer, pH 3.0) to remove the protein. A collected component was neutralized with 1 M Tris pH 9.0. Finally, the purified sample was dialyzed with PBS.
1. Experimental Method
An equilibrium dissociation constant (KD) of the antibody of the present application binding to human CD47 (hCD47) was determined by a biofilm interferometry (ForteBio). ForteBio affinity determination was performed according to a method in the prior art (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs; 2013.5 (2): p. 270-8), and it was specifically as follows: a sensor was used to equilibrate offline for 30 minutes in an analyzing buffer and detected online for 60 seconds to establish a baseline. The purified antibody obtained in Example 3 was loaded online to an AHC sensor (Forte Bio) for ForteBio affinity determination, and then the sensor with the loaded antibody was exposed in 100 nM of the CD47 antigen and acted for 5 minutes, then the sensor was transferred to the analyzing buffer for dissociation for 5 minutes and used for dissociation rate measurement. Kinetic analysis was performed with a 1:1 binding model.
2. Experimental Result
CD47 antibody affinity determination results were shown in Table 2, and the results showed that the CD47 antibody has high affinity.
1. Experimental Method
Binding of the CD47 antibody of the present application to human CD47 was measured in a measurement method based on a flow cytometry. The specific steps were as follows.
A cell line CCRF-CEM (Cell Bank of Chinese Academy of Sciences, Shanghai) expressing human CD47 was used, which belongs to human acute lymphoblastic leukemia T lymphocytes. CCRF-CEM cells (0.1×106 cells) were mixed with experimental antibodies (the CD47 antibody of the present application and a Hu5F9-G4 antibody) at different concentrations (the highest concentration is 30 μg/mL, it was three-fold dilution, and there were 10 concentrations in total) in PBS containing 3% bovine serum albumin (BSA) and incubated on ice for 30 minutes. Then, the cells were washed at least twice, a PE Goat anti human IgG Fc (1:500× dilution) fluorescent secondary antibody was prepared with a FCM buffer (1×PBS+3% BSA), added to a corresponding 96-well plate according to 100 μL/well, and incubated in a refrigerator at 4° C. for 30 min. The 96-well plate was taken out, and centrifuged at 250 g for 5 min, after a supernatant was carefully removed, the FCM buffer was added at 200 μL/well, and it was centrifuged again at 250 g for 5 min, the supernatant was carefully removed. The cells were washed at least twice and resuspended using 100 μL/well 1×PBS, and analyzed by the flow cytometry, and a concentration-dependent curve was fitted with GraphPad according to the mean fluorescence intensity (MFI) thereof. The Hu5F9-G4 antibody was used as a positive control antibody.
2. Experimental Result
EC50 results of the CD47 antibody binding to human CD47 were shown in Table 3, the average fluorescence intensity of the CD47 antibody (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to human CD47 was shown in
1. Experimental Method
By transfecting a pCDNA3.4 vector carrying the full-length monkey CD47, a stable CHO cell line (CHO-cynoCD47 cell) overexpressing the monkey CD47 was generated, and the CHO-cynoCD47 cells (0.1×106 cells) were mixed with experimental antibodies (the CD47 antibody of the present application and a Hu5F9-G4 antibody) at different concentrations (the highest concentration is 10 μg/mL, it was three-fold dilution, and there were 10 concentrations in total) in PBS containing 3% BSA, and incubated on ice for 30 minutes. Then, the cells were washed at least twice, a PE Goat anti human IgG Fc (1:500× dilution) fluorescent secondary antibody was prepared with a FCM buffer (1×PBS+3% BSA), added to a corresponding 96-well plate according to 100 μL/well, and incubated in a refrigerator at 4° C. for 30 min. The 96-well plate was taken out, and centrifuged at 250 g for 5 min, after a supernatant was carefully removed, the FCM buffer was added at 200 μL/well, and it was centrifuged again at 250 g for 5 min, the supernatant was carefully removed. The cells were washed at least twice and resuspended with 1×PBS at 100 μL/well, and analyzed by the flow cytometry, and a concentration-dependent curve was fitted with GraphPad according to MFI thereof. The Hu5F9-G4 antibody was used as a positive control antibody.
2. Experimental Result
EC50 results of the CD47 antibody binding to monkey CD47 were shown in Table 4, the average fluorescence intensity of the CD47 antibody (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, 7A11H52, positive control antibody Hu5F9-G4 and hIgG4-isotype control) binding to monkey CD47 was shown in
1. Experimental Method
The ability of the CD47 antibody of the present application to block the binding of human CD47 to SIRPα was determined by a flow cytometry. The specific steps were as follows:
Antibody dilution: an FCM buffer (1×PBS+3% BSA) was used to dilute the CD47 antibody of the present application and the control antibody Hu5F9-G4 to 90 μg/mL, then it was diluted to 10 concentrations in a 3-fold gradient, and a subtype control hIgG4 is diluted to 30 μg/mL, 1.1 μg/mL, and 0.04 μg/mL, and a ligand hSIRP α-mFC (AcroBiosystems) is diluted to 10 μg/mL.
CCRF-CEM (Cell Bank of Chinese Academy of Sciences, Shanghai) cells were added to a 96-well V-type plate at 0.1×106 cells/well, and hSIRPα-mFC binding was monitored under a condition of increasing the amount of the CD47 antibody. Bound SIRPα was determined with a PE Goat anti mouse IgG Fc secondary antibody (Biolegend). The Hu5F9-G4 antibody was used as a positive control antibody.
2. Experimental Result
Results of CD47 antibody inhibition of CD47/SIRP α binding to human CD47 (7A11H11, 7A11H12, 7A11H22, 7A11H32, 7A11H42, Hu5F9-G4, and hIgG4-isotype control) were shown in
1. Experimental Method
The ability of the CD47 antibody of the present application to promote macrophage phagocytosis of tumor cells was measured in a measurement method based on a flow cytometry. The specific steps were as follows:
Fresh blood of a donor was taken and separated to obtain peripheral blood mononuclear cells (PBMC), and CD14-positive mononuclear cells were separated from PBMC by an hCD14 magnetic bead (Miltenyi/130-050-201). A complete medium containing rhGM-CSF (R&D; 7954-GM-010) was prepared, the final concentration of rhGM-CSF was 50 ng/mL, and the concentration of the CD14 positive mononuclear cells was 5E5/mL, it was added to a cell culture dish at 20 mL/dish; it was transferred to a cell culture incubator at 5% CO2 and 37° C., and the medium (containing 50 ng/mL GM-CSF) was replaced in half every 3 days; and it was continued to culture for 4 days. On the 8th day, the macrophage supernatant was aspirated into a 15 mL centrifuge tube, and pre-cooled DPBS was added at the same time, the cells were directly collected with a cell scraper.
The tumor cell line Jurkat (Cell Bank of Chinese Academy of Sciences, Shanghai) with high expression of human CD47 was selected as a target cell type, and the target tumor cells were fluorescently labeled according to instructions of a CellTrace™ CFSE kit. The labeled tumor cells were co-cultured with the above differentiated macrophages at a ratio of 1:1, and at the same time, the antibodies at final concentrations of 10 μg/mL, 1 μg/mL, and 0.1 μg/mL were added to incubate at 37° C. for 2 hours. Then, the cells were washed at least twice, and carefully blown down, and allophycocyanin (APC)-labeled CD14 antibody (purchased from Biolegend; B259538) was added and incubated in PBS containing 0.1% BSA on ice (protected from light) for 30 minutes. The cells were washed at least twice and analyzed by a flow cytometry. The phagocytosed cell population was a population of cells that are CD14-positive in living cells and also positive for fluorescent dye CFSE (carboxyfluorescein diacetate, and succinimidyl ester). The Hu5F9-G4 antibody was used as a positive control antibody.
2. Experimental Result
Measurement results of the ability of CD47 antibody to promote macrophage phagocytosis of tumor cells were shown in
1. Experimental Method
Hemagglutination analysis of RBC was performed to characterize the RBC agglutination ability of the CD47 antibody. The CD47 antibodies were screened for RBC agglutination by observing the ability of the antibodies to avoid sedimentation of human RBCs. A specific method was as follows.
RBCs were diluted to 2% in PBS, and the CD47 antibody (the concentration is 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 1.5625 μg/mL, 0.78125 μg/mL, 0.390625 μg/mL, 0.195313 μg/mL, and 0.097656 μg/mL successively) was dropwise added and incubated in a round-bottom 96-well plate at 37° C. for 2 hours. The presence of unprecipitated RBCs was an evidence of RBC hemagglutination, and the unprecipitated RBCs were haze-like compared to the unagglomerated RBCs that form clear red dots. The Hu5F9-G4 antibody was used as a positive control antibody.
2. Experimental Result
Measurement results of the RBC agglutination ability of the CD47 antibody were shown in
1. Experimental Method
The CD47 monoclonal antibody has the property of binding to human RBC. For a CD47 antibody inhibitor, there is a potential risk of drug efficacy being interfered by RBCs and tumor off-target. If an antibody with low binding activity to RBC may be screened, the risk of off-target may be reduced, and its safety may be improved. Specific steps were as follows.
2. Experimental Result
Measurement results of the binding ability of the CD47 antibody to human RBC were shown in
1. Experimental Method
Like the CD47 monoclonal antibody that binds to the human red blood cells, the CD47 monoclonal antibody has the active characteristics of binding to the platelets, and there are many side effects caused by reduction of the platelets. The antibody with low platelet binding may reduce the risk of off-target and improve its safety. A specific method was as follows.
Antibody dilution: the antibody was diluted to 20 μg/ml with a FACS buffer, and the volume was 240 μL.
Cell counting and plating: whole blood cells were diluted by 20 times, and equally distributed to a 96-well V-type plate according to 100 μL/tube.
The above diluted antibody was added to the cells at 100 μL/well, and incubated at 2° C.-8° C. for 0.5 h.
The 96-well plate was taken out, and centrifuged at 250 g for 5 min, after the supernatant was carefully removed, the FACS buffer was added at 200 μL/well, and it was centrifuged again at 250 g for 5 min, the supernatant was carefully removed.
A PE fluorescent secondary antibody (1:500 dilution) (PE goat anti-human IgG Fc; biolegend; 409304) was prepared with the FACS buffer, and added to a corresponding 96-well plate at 100 μL/well, and 1 μL of APC anti human CD61 (biolegend; 336411) was added to each well at the same time, the cells were resuspended, and incubated at 2° C.-8° C. for 30 min.
The 96-well plate was taken out, and centrifuged at 250 g for 5 min, after a supernatant was carefully removed, the FACS buffer was added at 200 μL/well, and it was centrifuged again at 250 g for 5 min, the supernatant was carefully removed.
It was resuspended with 1×PBS at 200 μL/well, and an FACS detection was performed. The Hu5F9-G4 antibody was used as a positive control antibody.
2. Experimental Result
Binding analysis results of the CD47 antibody and platelets were shown in
1. Experimental Method
A detection method is the same as that of Example 8, the red blood cells were replaced with tumor cells as a target cell, and it was detected whether the CD47 antibody of the present application has an activating effect on the phagocytosis of the red blood cells by the macrophages. The Hu5F9-G4 antibody was used as a positive control antibody.
2. Experimental Result
Results of the analysis of the effect of the CD47 antibody on the phagocytosis of the red blood cells by the activated macrophages were shown in
1. Experimental Method
70 NOD SCID female mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were selected to construct a human B lymphocyte subcutaneous xenograft model (Raji) and a human malignant melanoma model (A375), and the antitumor activity of the CD47 antibody of the present application is evaluated. In those studies, the Hu5F9-G4 antibody analog and TJC-4 antibody analog were used as positive control antibodies, and hIgG4 was used as an isotype control antibody.
2. Experimental Result
Anti-tumor results of the CD47 antibody on the human B lymphocyte subcutaneous xenograft model were shown in
Anti-tumor results of the CD47 antibody on the human malignant melanoma model were shown in
The above embodiments are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above embodiments, and any other changes, modifications, replacements, combinations, simplifications made without departing from the spirit essence and principles of the present application should be equivalent substitution modes and are all included in the protection scope of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011544262.6 | Dec 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/140404 | 12/22/2021 | WO |
