CN112480237B - Fusion protein and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the field of biopharmaceuticals, in particular to a truncated body of Siglec-10, fusion protein containing the truncated body, nucleic acid for encoding the truncated body or the fusion protein containing the truncated body, an expression vector containing the nucleic acid, a host cell containing the expression vector, and application of the truncated body or the fusion protein containing the truncated body.
Description
Technical Field
The invention relates to the field of biopharmaceuticals, in particular to a truncated body of Siglec-10, fusion protein containing the truncated body, nucleic acid for encoding the truncated body or the fusion protein containing the truncated body, an expression vector containing the nucleic acid, a host cell containing the expression vector, and application of the truncated body or the fusion protein containing the truncated body.
Background
Tumor immunotherapy is a therapeutic method for controlling and eliminating tumors by restarting and maintaining tumor-immune circulation and restoring the normal anti-tumor immune response of the body. However, in some cases, the variant cells can evade immune surveillance of the body through various mechanisms, proliferate rapidly in the body, and form tumors. In many cancers, tumor cells CD24 are in a highly expressed state and allow the tumor cells to evade phagocytosis by macrophages. Thus, CD24 plays an important role in tumor cell immune escape.
CD24 is a highly glycosylated glycosyl phosphatidylinositol anchored cell membrane protein that binds to sialic acid-binding immunoglobulin-like lectin 10 (Siglec-10) on the surface of tumor-associated macrophages, activating the SHP-1/SHP-2 mediated inhibitory signaling pathway. In 2017, zhang Pei et al mention that inhibition of Natural Killer (NK) cell function by Siglec-10 interacting with CD24 in hepatocellular carcinoma resulted in a reduction of patient survival, siglec-10 might be a new target for hepatocellular carcinoma treatment (Siglec-10 mediates tumor cell survival and inhibits natural killer cell function in hepatocellular carcinoma, chinese scientific paper on-line, 2016, 6-7). In addition, CD24 may be the therapeutic target of liver cancer patients (Siglec-10is associated with survival and natural killer cell dysfunction in hepatocellular carcinoma,The Journal of surgical research,2015.194 (1): 107-113). Recent studies have found that phagocytosis of macrophages in all CD24 expressing human tumors is significantly enhanced by gene ablation of CD24 or Siglec-10 or blocking CD24-Siglec-10 interactions with antibodies. Tumor-expressed CD24 promotes immune escape by interacting with the inhibitory receptor Siglec-10 expressed on tumor-associated macrophages, and the CD24-Siglec-10 signaling axis can serve as a novel "do not eat me" signal, and is a potential target for future immunotherapy (CD 24 signalling through macrophage Siglec-10is a target for cancer immunotherapy,Nature,2019.572 (7769): 392-39).
CD24 is highly expressed in various tumor cells (Cellular and molecular characteristics of basal cells in airway epithelium, exp Lung Res,2001.27:401-415;CD24 is an effector of HIF-1-driven primary tumor growth and canretastasis. Cancer Res,2012.72 (21): 5600-5612; CD24 (+) cells fuel rapid tumor growth and display high metastatic capacity. Breast Cancer Res, 2015.17:78), and therefore, drugs that block the CD24-Siglec-10 signaling axis are a research and development hotspot in the field of tumor immunotherapy. Considering the problems of potential side effects such as B cell exhaustion, red blood cell injury and the like of the traditional high-affinity monoclonal antibody, the fusion protein based on the humanized Siglec-10 for blocking the interaction between CD24 and Siglec-10 has certain advantages.
It has been reported in the literature that the truncated form of Siglec-10 comprising the complete extracellular sequence forms a Siglec10-Fc fusion protein with Fc that can effectively block the interaction of CD24 and Siglec-10 (CD 24 signalling through macrophage Siglec-10is a target for cancer immunotherapy,Nature,2019.572 (7769): 392-39), thus presumably binding of the fusion protein to CD24 would act to block the signal axis of CD24-Siglec-10, relieving the "do not eat me" signal.
Siglec-10is an inhibitory receptor in a member of the Siglec family, and the human Siglec-10 protein is 697 amino acid residues in full length (UniProt accession number Q96LC 7) and includes a signal peptide (1-16), an extracellular domain (5 immunoglobulin-like regions, 17-539), 1 transmembrane region (540-594), and 2 intracellular inhibitory signal domains (595-697). The ectodomain includes ectodomain 1 (D1 region, ig-like V-type,17-141, containing Linker), ectodomain 2 (D2 region, ig-like C2-type1,142-235, containing Linker) and ectodomain 3-5 (D3-D5 region, 236-539, containing Linker), wherein D1 region is a main functional fragment of Siglec-10 binding to sialic acid, and arginine at position 119 of D1 region is a key site shared by Siglec family binding to sialic acid, and at the same time, after mutation of this site, siglec-10 no longer binds to CD24 (CD 24 and Siglec-10 Selectively Repress Tissue Damage-Induced Immune Responses, science,2009 (323): 1722-1725) is reported in the literature. Thus, the D1 region is presumed to be the critical region for Siglec-10 binding to CD 24.
Because the amino acid sequence of the whole extracellular domain of the human Siglec-10 protein is longer (539 amino acid residues (containing signal peptide)), and the glycosylated amino acid sites are more, the clinical application value of the Siglec-10 protein containing the whole extracellular domain is very limited from the aspects of drug formation and economy, and the production cost is extremely high, the Siglec-10 truncations which not only keep the binding property of the Siglec-10 and CD24 but also meet the requirements of the pharmacy or the fusion proteins containing the Siglec-10 truncations are purposefully developed, and the potential clinical application value of the Siglec-10 truncations is further excavated, so that the method has important significance.
Disclosure of Invention
To solve the above problems, the present inventors developed, based on humanized Siglec-10, a Siglec-10 truncate or a mutant thereof that can block the CD24-Siglec-10 signal axis that plays an important role in tumor immune escape, and a fusion protein comprising the same. The inventors designed various Siglec-10 truncations and fused with Fc to form fusion proteins, but found that most of the Siglec-10 truncations-Fc fusion proteins have extremely low expression levels after recombination and even no expression is detected, so the inventors tried to mutate the Siglec-10 truncations in the Siglec-10 truncations-Fc fusion proteins, and screen the Siglec-10 truncations-Fc fusion proteins which not only retain the binding activity with CD24, but also have higher expression levels.
The invention solves the technical problems through the following technical proposal.
The invention provides a Siglec-10 truncated-Fc fusion protein which can block the interaction between CD24 and Siglec-10, wherein the Siglec-10 truncated can be a natural Siglec-10 truncated or a mutant thereof.
As used herein, a Siglec-10 truncate refers to a functional variant or mutant thereof obtained after selective truncation of a wild-type human Siglec-10 protein, for example: the functional variant is obtained by cutting off one amino acid or a plurality of amino acid residues in the extracellular domain of Siglec-10, and even a plurality of amino acid residues; or functional variants obtained after cleavage of several or more amino acid residues in the extracellular domain of Siglec-10; or a functional variant obtained by cleaving two or more amino acid residues in the extracellular domain of Siglec-10; or a combination of the above functional variants, i.e., functional variants formed by ligating these functional variants through a linker. So long as the Siglec-10 truncations have the function of blocking the interaction of CD24 with Siglec-10.
In a preferred embodiment of the invention, the Siglec-10 truncations comprise a Siglec-10 extracellular domain, preferably the Siglec-10 truncations comprise the D1 region of the Siglec-10 extracellular domain; or the D1 region and the D2 region of the Siglec-10 extracellular domain.
The different structural fragments of the extracellular domain of Siglec-10 contained in the truncations of Siglec-10 as described above (for example between the D1 region and the D2 region) are preferably linked by the original Linker (Linker) between the D1 region and the D2 region of the protein of Siglec-10, and also by a Linker (Linker) which may be conventional in the art, for example (G4S), or by mutants of this Linker 3 . The N end or the C end of the Siglec-10 truncated body is preferably provided with a signal peptide or a mutant of the signal peptide; the signal peptide may be a signal peptide conventional in the art, such as HSA. The mutant of the linker or signal peptide referred to herein means a mutant retaining the function of the linker or signal peptide before mutation.
The "mutation" in the Siglec-10 truncating mutants described in the present invention preferably occurs at the sialic acid binding site, the N-linked glycosylation site and/or the cysteine site. Such mutants may be obtained by conventional mutation means in the art, for example substitution, addition or deletion of one or more amino acids therein; the mutant of the present invention is preferably obtained by substituting individual or multiple amino acids in a truncated form. The substitutions preferably occur at one or more of amino acid residue positions 21F, 36C, 41C, 100N, 101C, 119R, 128Y, 164C and 173C of the sequence shown in accession number Q96LC 7.
The Fc region in the Siglec-10 truncate-Fc fusion protein as described above preferably comprises an Fc region native sequence or an Fc non-native sequence; more preferably, the Fc region is a human Fc region.
In another embodiment, the Fc region of the invention is conceptual, i.e., although it may not actually exist, antibody engineering may be performed according to the amino acid sequence of the desired variant Fc region to produce a polypeptide or fusion protein comprising such sequence or DNA encoding the amino acid sequence of the desired variant Fc region.
In another embodiment, the Fc region may be an Fc region variant. As used herein, "variant Fc region" refers to an Fc region obtained by modification of an Fc native amino acid sequence with one or more amino acid residues. Methods of modification are well known to those skilled in the art, and include, but are not limited to, site-directed mutagenesis of DNA sequences encoding the Fc region, e.g., using PCR mutagenesis and cassette mutagenesis to prepare Fc region variants. For example, one or more amino acid residues of the Fc region may be deleted in order to improve binding to FcR. For example, in one embodiment, amino acid insertion type Fc region variants may be prepared in order to alter the effector function of the Fc region.
In one embodiment, for example, at least one amino acid residue (e.g., 1-2 amino acid residues, typically no more than 10 amino acid residues) may be introduced near one or more Fc region sites identified as affecting FcR binding. "nearby" refers to within 1-2 amino acid residues from the identified Fc region site that affects FcR binding. Such Fc region variants may exhibit enhanced or reduced FcR binding and/or ADCC activity.
To prepare such insertional variants, the co-crystallized structure of a polypeptide comprising an FcR binding region (e.g., the extracellular domain of a target FcR) and the Fc region of an amino acid residue to be inserted may be assessed in order to relate to Fc region variants having, for example, enhanced FcR binding capacity. Such insertions are typically placed in loops of the Fc region.
In one embodiment, by introducing appropriate amino acid sequence modifications in the native Fc region, fc region variants can be made that mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and/or bind fcγ receptors (fcγr) with greater affinity in the presence of human effector cells than recombinant proteins containing the native Fc region. The Fc region variants of the invention typically comprise at least one amino acid modification in the Fc region. Preferably, multiple amino acid modifications are combined. For example, an Fc region variant may include substitutions of 2, 3, 4, 5 or more amino acid residues, as in the identified specific FcR binding site.
The native Fc region is preferably a human Fc region, such as the native sequence of the Fc region of human IgG1 (a or non-a isotype), igG2, igG3, or IgG 4.
The invention also provides a nucleic acid molecule encoding the Siglec-10 truncations as described above, and the Fc fusion protein.
The invention also provides an expression vector comprising a nucleic acid molecule as described above, preferably pcDNA3.4 (available from Thermo Fisher).
The invention also provides a host cell comprising an expression vector as described above, preferably an Expi293F (available from Thermo Fisher).
The invention also provides a preparation method of the Siglec-10 truncated body and the Siglec-10 truncated body-Fc fusion protein, and the preparation method comprises the expression by using the host cell.
The invention also provides a Siglec-10 truncated body as described above and application of the Siglec-10 truncated body-Fc fusion protein as described above in preparing medicines for treating diseases related to CD24 high expression.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the Siglec-10 truncated-Fc fusion protein not only maintains the binding activity with CD24, but also has higher expression quantity, and the fusion protein is easy to prepare medicines, low in preparation cost and potential in clinical application value.
Drawings
FIG. 1 is a schematic representation of the structure of a Siglec-10 truncate-Fc fusion protein of the present invention.
FIG. 2 is a SDS-PAGE result of Siglec-10 truncate-Fc fusion protein prepared according to the present invention.
FIG. 3 is a graph showing the binding assay of the Siglec-10 truncate-Fc fusion protein of the present invention to cells highly expressing human CD 24.
Examples
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
EXAMPLE 1 Siglec-10 truncate-Fc fusion protein and preparation thereof
For the structural features of the human Siglec-10 protein and the binding mode thereof with CD24, a truncated-Fc fusion protein of Siglec-10is designed, which consists of a structural fragment (such as a D1 region, a D2 region and the like) in the extracellular domain of the human Siglec-10, and Fc, such as a fusion protein consisting of the D1 region of Siglec-10 and Fc (hereinafter referred to as Siglec-10 (D1) -Fc), the sequence of which is shown as SEQ ID NO:1, a fusion protein consisting of the D1 region of Siglec-10 and the D2 region of which is shown as SEQ ID NO:2, and a fusion protein consisting of the D1 region of Siglec-10 and Fc, wherein the 36-position cysteine mutation in the D1 region of Siglec-10is shown as serine (hereinafter referred to as Siglec-10 (D1 m) -Fc), and the sequence of which is shown as SEQ ID NO: 3.
Fusion protein sequence and IgG subtype selection
Siglec-10 (D1) -Fc amino acid sequence (SEQ ID NO: 1)
Siglec-10 (D1+D2) -Fc amino acid sequence (SEQ ID NO: 2)
Siglec-10 (D1 m) -Fc amino acid sequence (SEQ ID NO: 3)
Human Siglec-10 extracellular domain amino acid sequence (SEQ ID NO: 4)
The underlined amino acids are the signal peptide sequences; the amino acid marked by the wavy line is the sequence of the human Siglec-10 D1 region; the double underlined amino acids are the sequence of the human Siglec-10 D2 region; the amino acid shown in bold italics is the amino acid sequence after mutation at position 36 in the extracellular domain of human Siglec-10; the amino acids indicated by the dashed lines are Fc sequences, and the amino acids indicated by the shaded lines are linkers.
The fusion protein coding fragments are connected with an expression vector and then transferred into a host cell line (such as an Expi 293F) for expression, the inventor unexpectedly discovers that the expression quantity of most Siglec-10 truncated-Fc fusion proteins is very low or even undetectable, and unexpectedly discovers that the expression quantity of Siglec-10 (D1 m) -Fc reaches 2-3mg/L while the binding activity with CD24 is reserved, and the detail is shown in Table 1.
TABLE 1 expression levels and purification efficiency of three fusion proteins in an Expi293F System
| Fusion proteins | Expression of | Yield (mg/L) | Purification (one-step purification) |
| Siglec-10(D1)-Fc | No expression | NA | NA |
| Siglec-10(D1+D2)-Fc | Low expression | <1 | 80％ |
| Siglec-10(D1m)-Fc | With expression of | 2-3 | 80％ |
The preparation method of the Siglec-10 truncated fusion protein comprises the following steps:
the nucleotide sequence of the expression Siglec-10 truncated-Fc fusion protein is cloned into an expression vector, which contains a Leader sequence, and the fusion protein is synthesized by PCR technology. Wherein, the N-terminal of the mammalian expression structure is designed with a related signal peptide sequence to ensure secretion related signal transduction and processing. The expression vector comprises: mammalian expression vector expressing CMV promoter. The present invention provides a host cell comprising an expression vector as described above; the host cell is a cell of mammalian origin.
Specific preparation method
As shown in FIG. 1, the structure of the Siglec-10 truncate-Fc fusion protein of the present invention is schematically shown. The fusion proteins were synthesized into DNA fragments by overlapping PCR using standard molecular biology techniques, and the DNA fragments were cloned into the expression vector pcDNA3.4. Expression was then performed in Expi293F cells. Finally, protein G affinity chromatography was used to purify the fusion protein, and the purified fusion protein was concentrated in ultrafiltration tubes and replaced into DPBS buffer at pH 7.4. The specific process is as follows:
(1) Expression of Siglec-10 truncate-Fc fusion proteins in mammalian cells
Plasmid transient transformation of the target protein was performed on an Expi293F cell line (available from ThermoFisher, cat No. a 14527). The fusion protein was purified using a GE protein purification column. Specifically, the Expi293F cells were cultured in Expi293 TM Expression Medium Medium (available from ThermoFisher, cat. No. A1435101) was supplemented with Expifectamine TM 293 transfection kit (purchased from Invitrogen, A14525) was transferred into plasmid DNA. After 1 week, the supernatant was harvested and purified. The method comprises the following steps:
A. the day before transfection, the Expi293F cells were passaged and fresh medium was added to a density of 2-3X 10 6 Cells/ml were incubated on a cell shaker at a speed of 120 rpm, 37℃and 8% carbon dioxide.
B. On the day of transfection, the cell density was diluted to 2.0X10 with fresh medium 6 Cells/ml were transfected at a ratio of 1 ml cells to 1. Mu.g of ExpiFectamine transfection reagent. After transfection, the cells were subjected to shaking culture at a rotation speed of 120 rpm, 37℃and 8% carbon dioxide
C. After 20 hours of transfection, the feed was added in proportion.
D. 5-7 days of transfection, detecting the cell activity rate, if the activity rate is less than 60%, placing the cells in a centrifuge tube for 10000 revolutions per minute, centrifuging, and collecting the supernatant.
(2) The specific steps of protein purification are as follows:
first, protein G affinity chromatography was used. The diluted supernatant was first loaded onto Protein G column (GE Healthcare) with binding buffer (PBS, pH 7.4) at a flow rate of 1-5 mL/min. The column was then washed with 5-10 column volumes of binding buffer (PBS, pH 7.4) and the proteins eluted with eluent (0.1M sodium citrate, pH 3.0) at a flow rate of 1-5 ml/min and the eluted fractions were collected.
Finally, the eluted fusion protein was concentrated using a Millipore Amicon Ultra (10 kD) ultrafiltration tube and the buffer was converted to PBS at pH 7.4. Purity of each protein was identified by SDS-PAGE and A280 was used to determine protein concentration.
Example 2 Siglec-10 (D1 m) -Fc fusion protein binding to CD24 at the cellular level
The affinity of Siglec-10 (D1 m) -Fc fusion protein and CD24 was determined by FACS technology.
The specific method comprises the following steps: fusion protein S10- (D1 m) -Fc was fused to 2X 10 at a concentration (40 ug/mL) 5 Individual human CD24 CHOK1 cells (purchased from bio-tech company, beginner Kang Yuanbo) were incubated in 100uL (2% bsa in PBS) and after 1 hour at 4 degrees, the supernatant was discarded by centrifugation and the cells were washed twice with buffer. Anti-human IgG fluorescent secondary antibody (purchased from Sigma) was then added, after 1 hour at 4 degrees, the supernatant was discarded by centrifugation and the cells were washed twice with buffer. Flow cytometry analysis (CantoII, BD bioscience) was performed on antibody-labeled cells to obtain data analysis as shown in FIG. 3. As shown in FIG. 3, the Siglec-10 (D1 m) -Fc fusion protein retained binding activity to CD24 at the cellular level.
Sequence listing
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Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
325 330 335
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
340 345 350
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
355 360 365
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380
<210> 4
<211> 546
<212> PRT
<213> unknown (Artificial sequence)
<400> 4
Met Leu Leu Pro Leu Leu Leu Ser Ser Leu Leu Gly Gly Ser Gln Ala
1 5 10 15
Met Asp Gly Arg Phe Trp Ile Arg Val Gln Glu Ser Val Met Val Pro
20 25 30
Glu Gly Leu Cys Ile Ser Val Pro Cys Ser Phe Ser Tyr Pro Arg Gln
35 40 45
Asp Trp Thr Gly Ser Thr Pro Ala Tyr Gly Tyr Trp Phe Lys Ala Val
50 55 60
Thr Glu Thr Thr Lys Gly Ala Pro Val Ala Thr Asn His Gln Ser Arg
65 70 75 80
Glu Val Glu Met Ser Thr Arg Gly Arg Phe Gln Leu Thr Gly Asp Pro
85 90 95
Ala Lys Gly Asn Cys Ser Leu Val Ile Arg Asp Ala Gln Met Gln Asp
100 105 110
Glu Ser Gln Tyr Phe Phe Arg Val Glu Arg Gly Ser Tyr Val Arg Tyr
115 120 125
Asn Phe Met Asn Asp Gly Phe Phe Leu Lys Val Thr Ala Leu Thr Gln
130 135 140
Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro Gly Gln Pro Val
145 150 155 160
Thr Val Ile Cys Val Phe Asn Trp Ala Phe Glu Glu Cys Pro Pro Pro
165 170 175
Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser Ser Gln Gly Thr Lys Pro
180 185 190
Thr Thr Ser His Phe Ser Val Leu Ser Phe Thr Pro Arg Pro Gln Asp
195 200 205
His Asn Thr Asp Leu Thr Cys His Val Asp Phe Ser Arg Lys Gly Val
210 215 220
Ser Ala Gln Arg Thr Val Arg Leu Arg Val Ala Tyr Ala Pro Arg Asp
225 230 235 240
Leu Val Ile Ser Ile Ser Arg Asp Asn Thr Pro Ala Leu Glu Pro Gln
245 250 255
Pro Gln Gly Asn Val Pro Tyr Leu Glu Ala Gln Lys Gly Gln Phe Leu
260 265 270
Arg Leu Leu Cys Ala Ala Asp Ser Gln Pro Pro Ala Thr Leu Ser Trp
275 280 285
Val Leu Gln Asn Arg Val Leu Ser Ser Ser His Pro Trp Gly Pro Arg
290 295 300
Pro Leu Gly Leu Glu Leu Pro Gly Val Lys Ala Gly Asp Ser Gly Arg
305 310 315 320
Tyr Thr Cys Arg Ala Glu Asn Arg Leu Gly Ser Gln Gln Arg Ala Leu
325 330 335
Asp Leu Ser Val Gln Tyr Pro Pro Glu Asn Leu Arg Val Met Val Ser
340 345 350
Gln Ala Asn Arg Thr Val Leu Glu Asn Leu Gly Asn Gly Thr Ser Leu
355 360 365
Pro Val Leu Glu Gly Gln Ser Leu Cys Leu Val Cys Val Thr His Ser
370 375 380
Ser Pro Pro Ala Arg Leu Ser Trp Thr Gln Arg Gly Gln Val Leu Ser
385 390 395 400
Pro Ser Gln Pro Ser Asp Pro Gly Val Leu Glu Leu Pro Arg Val Gln
405 410 415
Val Glu His Glu Gly Glu Phe Thr Cys His Ala Arg His Pro Leu Gly
420 425 430
Ser Gln His Val Ser Leu Ser Leu Ser Val His Tyr Ser Pro Lys Leu
435 440 445
Leu Gly Pro Ser Cys Ser Trp Glu Ala Glu Gly Leu His Cys Ser Cys
450 455 460
Ser Ser Gln Ala Ser Pro Ala Pro Ser Leu Arg Trp Trp Leu Gly Glu
465 470 475 480
Glu Leu Leu Glu Gly Asn Ser Ser Gln Asp Ser Phe Glu Val Thr Pro
485 490 495
Ser Ser Ala Gly Pro Trp Ala Asn Ser Ser Leu Ser Leu His Gly Gly
500 505 510
Leu Ser Ser Gly Leu Arg Leu Arg Cys Glu Ala Trp Asn Val His Gly
515 520 525
Ala Gln Ser Gly Ser Ile Leu Gln Leu Pro Asp Lys Lys Gly Leu Ile
530 535 540
Ser Thr
545
Claims (6)
1. A Siglec-10 truncated-Fc fusion protein can block the interaction between CD24 and Siglec-10, and has the amino acid sequence shown in SEQ ID NO. 3.
2. A nucleic acid molecule encoding the Siglec-10 truncate-Fc fusion protein of claim 1.
3. An expression vector comprising the nucleic acid molecule of claim 2.
4. A host cell comprising the expression vector of claim 3.
5. A method for preparing a Siglec-10 truncate-Fc fusion protein according to claim 1, comprising the step of expression using the host cell of claim 4.
6. Use of the Siglec-10 truncate-Fc fusion protein of claim 1 for the preparation of a medicament for the treatment of a disease associated with high expression of CD 24.
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| CN201910867066 | 2019-09-12 | ||
| CN2019108670663 | 2019-09-12 |
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| CN112480237A CN112480237A (en) | 2021-03-12 |
| CN112480237B true CN112480237B (en) | 2023-10-17 |
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| CN202010953136.XA Active CN112480237B (en) | 2019-09-12 | 2020-09-11 | Fusion protein and preparation method and application thereof |
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| Country | Link |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000053747A1 (en) * | 1999-03-11 | 2000-09-14 | Mount Sinai Hospital | Sialic acid-binding ig-like lectin (siglec) gene; ob-binding protein like (ob-bpl) |
| CN102869369A (en) * | 2010-04-28 | 2013-01-09 | 肿瘤免疫股份有限公司 | Methods of use of soluble CD24 for therapy of rheumatoid arthritis |
| CN108463229A (en) * | 2016-01-11 | 2018-08-28 | 斯坦福大学托管董事会 | Chimeric protein and immunotherapy method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8163281B2 (en) * | 2009-03-04 | 2012-04-24 | The Regents Of The University Of Michigan | Treatment of drug-related side effect and tissue damage by targeting the CD24-HMGB1-Siglec10 axis |
| AU2016331076B2 (en) * | 2015-10-01 | 2020-12-03 | Kopfkino Ip, Llc | Compositions and methods for adjoining type i and type ii extracellular domains as heterologous chimeric proteins |
| WO2017085166A1 (en) * | 2015-11-17 | 2017-05-26 | Innate Pharma | Siglec-10 antibodies |
-
2020
- 2020-09-11 CN CN202010953136.XA patent/CN112480237B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000053747A1 (en) * | 1999-03-11 | 2000-09-14 | Mount Sinai Hospital | Sialic acid-binding ig-like lectin (siglec) gene; ob-binding protein like (ob-bpl) |
| CN102869369A (en) * | 2010-04-28 | 2013-01-09 | 肿瘤免疫股份有限公司 | Methods of use of soluble CD24 for therapy of rheumatoid arthritis |
| CN108463229A (en) * | 2016-01-11 | 2018-08-28 | 斯坦福大学托管董事会 | Chimeric protein and immunotherapy method |
Non-Patent Citations (5)
| Title |
|---|
| CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy;Amira A Barkal等;Nature;第572卷;全文 * |
| CD24和Siglec-10选择性抑制组织损伤诱导的炎性反应;方宏亮;万涛;;中国肿瘤生物治疗杂志(第03期);全文 * |
| CD24在口腔医学中的研究进展;梁静;王凯;吴家媛;;国际口腔医学杂志(第05期);全文 * |
| Molecular characterization of porcine Siglec-10 and analysis of its expression in blood and tissues;Z. Escalona等;Developmental and Comparative Immunology;第48卷;全文 * |
| Siglec-10 is associated with survival and natural killer cell dysfunction in hepatocellular carcinoma;Pei Zhang等;Journal of Surgical Research;全文 * |
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| CN112480237A (en) | 2021-03-12 |
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