CN112480237A

CN112480237A - Fusion protein and preparation method and application thereof

Info

Publication number: CN112480237A
Application number: CN202010953136.XA
Authority: CN
Inventors: 刘倩; 廖诗骅; 王明晓; 宋利平; 范艺
Original assignee: Shanghai Jinmante Biological Technology Co ltd
Current assignee: Shanghai Jinmante Biological Technology Co ltd
Priority date: 2019-09-12
Filing date: 2020-09-11
Publication date: 2021-03-12
Anticipated expiration: 2040-09-11
Also published as: CN112480237B

Abstract

The invention relates to the field of biological pharmacy, in particular to a Siglec-10 truncation, a fusion protein containing the truncation, a nucleic acid encoding the truncation or the fusion protein containing the truncation, an expression vector containing the encoding nucleic acid, a host cell containing the expression vector, and application of the truncation or the fusion protein containing the truncation.

Description

Fusion protein and preparation method and application thereof

Technical Field

Background

Tumor immunotherapy is a therapeutic method for controlling and eliminating tumors by restarting and maintaining tumor-immune circulation and restoring normal anti-tumor immune response of the body. However, in some cases, the heterosis cells can escape immune surveillance of the body through multiple mechanisms, and rapidly proliferate in vivo to form tumors. In many cancers, the tumor cell CD24 is highly expressed and allows the tumor cell to evade phagocytosis by macrophages. Thus, CD24 plays an important role in tumor cell immune escape.

CD24 is a highly glycosylated glycosylphosphatidylinositol-anchored cell membrane protein that binds to immunoglobulin-like lectin 10(Siglec-10) bound to sialic acid on the surface of tumor-associated macrophages, activating SHP-1/SHP-2 mediated inhibitory signaling pathways. In 2017, Zhang et al mentioned that the interaction between Siglec-10 and CD24 inhibits the function of Natural Killer (NK) cells in hepatocellular carcinoma, thereby reducing the survival time of patients, and Siglec-10 may become a new target for hepatocellular carcinoma treatment (Siglec-10 mediates the survival of tumor cells and inhibits the function of NK cells in hepatocellular carcinoma, Chinese scientific and technological paper is on line, 2016, 6, 7). In addition, CD24 may be The therapeutic target of liver cancer patients (Signal-10 is associated with The liver function and natural killer cell dysfunction in hepatocellular cancer, The Journal of therapeutic research,2015.194(1): 107-113). Recent studies found that phagocytosis of macrophages was significantly enhanced in all human tumors expressing CD24 by gene ablation of CD24 or Siglec-10 or blocking CD24-Siglec-10 interactions using 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 new "eat me" signal, a potential target for future immunotherapy (CD24 signaling through macrophage Siglec-10is a target for cancer immunology, Nature,2019.572(7769): 392-39).

CD24 is highly expressed in various tumor cells (Cellular and molecular characterization of basal cells in air Cancer, Exp Lung Res,2001.27:401 415; CD24 is an effector of HIF-1-driven primary tumor growth and Cancer Res,2012.72(21): 5600-. Considering that the traditional high-affinity monoclonal antibody drugs have potential side effects such as B cell exhaustion and erythrocyte injury, the fusion protein for blocking the interaction between CD24 and Siglec-10 based on the human Siglec-10 has certain advantages.

It has been reported in the literature that the formation of a Siglec10-Fc fusion protein by a Siglec-10 truncation containing a complete extracellular sequence and Fc can effectively block the interaction between CD24 and Siglec-10 (CD24 signalling through mac Siglec-10is a target for cancer immunology, Nature,2019.572(7769):392-39), and therefore, it is speculated that the binding of the fusion protein to CD24 can play a role in blocking the CD24-Siglec-10 signal axis and releasing the 'eat me' signal.

Siglec-10is an inhibitory receptor in Siglec family members, and the human Siglec-10 protein has a total length of 697 amino acid residues (UniProt accession number Q96LC7), including a signal peptide (1-16), an extracellular domain (5 immunoglobulin-like regions, 17-539), 1 transmembrane region (540-594) and 2 intracellular inhibitory signal regions (595-697). Wherein the extracellular domain comprises extracellular domain 1(D1 region, Ig-like V-type, 17-141, containing Linker), extracellular domain 2(D2 region, Ig-like C2-type1,142-235, containing Linker), and extracellular domain 3-5(D3-D5 region, 236-539, containing Linker), wherein D1 region is the main functional fragment of Siglec-10 combined with sialic acid, and arginine 119 of D1 region is the key site shared by Siglec family to combine with sialic acid, meanwhile, lec-10is not combined with CD24 after the site Siglec mutation according to literature report (CD24 and Siglec-10 SetivelReques Tissue Damage-Induced Immune Responses, Science,2009, 323: 1722-. Therefore, the D1 region is presumed to be a key region for binding Siglec-10 to CD 24.

Because the complete extracellular domain of the human Siglec-10 protein has a long amino acid sequence (539 amino acid residues (including signal peptide)) and more glycosylated amino acid sites, the Siglec-10 protein containing the complete extracellular domain has very limited clinical application value and extremely high production cost from the aspects of druggability and economy, the Siglec-10 truncation which retains the binding property of Siglec-10 and CD24 and meets the druggability requirement or the fusion protein containing the Siglec-10 truncation is purposefully developed, and the potential clinical application value of the Siglec-10 truncation is further excavated, so that the important significance is achieved.

Disclosure of Invention

In order to solve the above problems, the present inventors developed a Siglec-10 truncation capable of blocking the CD24-Siglec-10 signal axis, which plays an important role in tumor immune escape, or a mutant thereof, and a fusion protein comprising the same, based on human Siglec-10. The inventors designed various Siglec-10 truncations and fused the Siglec-10 truncations with Fc to form fusion proteins, but found that most of the Siglec-10 truncation-Fc fusion proteins have extremely low expression level after recombination and even can not be expressed by detection, so the inventors tried to mutate and transform the Siglec-10 truncations in the Siglec-10 truncation-Fc fusion proteins and screen the Siglec-10 truncation-Fc fusion proteins which not only retain the binding activity with CD24, but also have higher expression level.

The present invention solves the above technical problems by the following technical solutions.

The invention provides a Siglec-10 truncation-Fc fusion protein which can block the interaction between CD24 and Siglec-10, wherein the Siglec-10 truncation can be a natural Siglec-10 truncation or a mutant thereof.

As used herein, a Siglec-10 truncation refers to a functional variant or mutant thereof obtained after selective truncation of a wild-type human Siglec-10 protein, such as: 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, even a plurality of amino acid residues; or a functional variant obtained by truncating several or more amino acid residues from the extracellular domain of Siglec-10; or a functional variant obtained by intercepting several or more amino acid residues in two or more extracellular domains of Siglec-10; or a combination of the above functional variants, i.e., functional variants formed by linking these functional variants via a linker. So long as the Siglec-10 truncation has the function of blocking the interaction between CD24 and Siglec-10.

In a preferred embodiment of the present invention, the Siglec-10 truncation comprises a Siglec-10 extracellular domain, and preferably, the Siglec-10 truncation comprises a D1 region of the Siglec-10 extracellular domain; or the D1 and D2 regions of the Siglec-10 extracellular domain.

The different structural fragments of the extracellular domain of Siglec-10 contained in the above-described Siglec-10 truncation (e.g., between the D1 region and the D2 region) are preferably linked by an original Linker (Linker) between the D1 region and the D2 region of the Siglec-10 protein, or by a Linker (Linker) which is a Linker conventional in the art, e.g., (G4S) or a mutant thereof₃. The N terminal or the C terminal of the Siglec-10 truncation preferably carries 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. Reference herein to a mutant of a linker or signal peptide is to a mutant which retains the function of the linker or signal peptide prior to the mutation.

"mutations" in the Siglec-10 truncation mutants described herein preferably occur at sialic acid binding sites, N-linked glycosylation sites, and/or cysteine sites. The mutants can be obtained by conventional mutation means in the field, such as substitution, addition or deletion of one or more amino acids; the mutant of the present invention is preferably obtained by substituting a single amino acid or a plurality of amino acids in the truncation. The substitutions preferably occur at one or more 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 truncation-Fc fusion protein 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 present invention is conceptual in that although it may not actually be present, antibody engineering may be performed based on the amino acid sequence of the desired Fc region variant to produce a polypeptide or fusion protein comprising this sequence or DNA encoding the amino acid sequence of the desired Fc region variant.

In another embodiment, the Fc region can be an Fc region variant. As used herein, "Fc region variant" refers to an Fc region obtained by modification of one or more amino acid residues of an Fc native amino acid sequence. Methods of modification are well known to those skilled in the art and include, but are not limited to, site-directed mutagenesis of a DNA sequence encoding an Fc region, for example using PCR mutagenesis and cassette mutagenesis to prepare variants of the Fc region. For example, one or more amino acid residues of the Fc region may be deleted in order to enhance binding to FcR. For example, in one embodiment, amino acid inserted Fc region variants can be made 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) can be introduced near one or more sites of the Fc region identified as affecting FcR binding. "nearby" means 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-crystal structure of a polypeptide comprising an FcR binding region (e.g., the extracellular domain of a target FcR) with an Fc region into which amino acid residues are to be inserted may be evaluated to relate to variants of the Fc region having, for example, enhanced FcR binding. Such insertions are typically placed in loops in the Fc region.

In one embodiment, Fc region variants can be prepared that mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of human effector cells and/or bind Fc γ receptors (fcyr) with greater affinity than recombinant proteins containing a native Fc region by introducing appropriate amino acid sequence modifications in 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, Fc region variants may include substitutions of 2, 3, 4, 5 or more amino acid residues, as in the specific FcR binding site identified.

The native Fc region is preferably a human Fc region, for example the native sequence of an Fc region of human IgG1(a or non-a isotype), IgG2, IgG3 or IgG 4.

The invention also provides a nucleic acid molecule encoding a Siglec-10 truncation as described above, a Siglec-10 truncation-Fc fusion protein as described above.

The present invention also provides an expression vector comprising a nucleic acid molecule as described above, preferably pcDNA3.4 (available from Thermo Fisher).

The present invention also provides a host cell comprising an expression vector as described above, preferably Expi293F (available from Thermo Fisher).

The invention also provides a Siglec-10 truncation as described above, and a method for preparing the Siglec-10 truncation-Fc fusion protein as described above, wherein the preparation method comprises expression by using the host cell as described above.

The invention also provides an application of the Siglec-10 truncation as described above and the Siglec-10 truncation-Fc fusion protein as described above in preparing a medicament for treating diseases related to high expression of CD 24.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the Siglec-10 truncated body-Fc fusion protein not only retains the binding activity with CD24, but also has higher expression level, is easy to prepare medicaments, has low preparation cost and has potential clinical application value.

Drawings

FIG. 1 is a schematic structural diagram of a Siglec-10 truncation-Fc fusion protein of the present invention.

FIG. 2 is a SDS-PAGE result of Siglec-10 truncation-Fc fusion proteins prepared according to the present invention.

FIG. 3 is a graph of the binding assay of Siglec-10 truncation-Fc fusion proteins of the present invention to highly expressed human CD24 cells.

Examples

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 Siglec-10 truncation-Fc fusion proteins and preparation thereof

Aiming at the structural characteristics of the human Siglec-10 protein and the binding mode of the human Siglec-10 protein and CD24, a Siglec-10 truncation-Fc fusion protein is designed, which consists of a structural fragment (such as D1 region, D2 region, etc.) in the extracellular domain of human Siglec-10 and Fc, such as a fusion protein composed of the D1 region in Siglec-10 and Fc (hereinafter, Siglec-10(D1) -Fc), the sequence is shown as SEQ ID NO:1, a fusion protein composed of the region D1 and the region D2 in Siglec-10 and Fc (hereinafter referred to as Siglec-10(D1+ D2) -Fc), the sequence is shown as SEQ ID NO:2, and a fusion protein consisting of the D1 region in Siglec-10 and Fc, wherein the cysteine at position 36 in the D1 region in the Siglec-10is mutated into serine (hereinafter, Siglec-10(D1m) -Fc), and the sequence is shown as SEQ ID NO:3, respectively.

Fusion protein sequences 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(D1m) -Fc amino acid sequence (SEQ ID NO:3)

Human Siglec-10 extracellular domain amino acid sequence (SEQ ID NO:4)

Underlined amino acids are the signal peptide sequence; the amino acid marked by the wavy line is the sequence of the human Siglec-10D 1 region; the amino acids marked by double underlining are the sequence of the human Siglec-10D 2 region; the amino acid shown in bold italics is the amino acid sequence after the 36 th mutation in the extracellular domain of human Siglec-10; the dashed line indicates the amino acid Fc sequence and the shaded amino acid is the linker (linker).

The encoding fragments of the fusion proteins are connected with an expression vector and then transferred into a host cell line (such as Expi293F) for expression, the inventor unexpectedly finds that most Siglec-10 truncation-Fc fusion proteins have low expression level and even can not be expressed by detection, and unexpectedly finds that the expression level of Siglec-10(D1m) -Fc reaches 2-3mg/L while the binding activity with CD24 is maintained, and the detail is shown in Table 1.

TABLE 1 expression level and purification efficiency of three fusion proteins in Expi293F system

Fusion proteins	Expression of	Yield (mg/L)	Purification (one-step purification)
				Siglec-10(D1)-Fc	Absence of expression	NA	NA
Siglec-10(D1+D2)-Fc	Low expression	<1	80％
				Siglec-10(D1m)-Fc	Has an expression	2-3	80％

The preparation method of the Siglec-10 truncated fusion protein comprises the following steps:

the nucleotide sequence expressing the Siglec-10 truncation-Fc fusion protein was cloned into an expression vector containing a Leader sequence, and the fusion protein was synthesized by PCR technique. Wherein, a related signal peptide sequence is designed at the N-terminal of the mammalian expression structure to ensure signal transduction and processing related to secretion. The expression vector comprises: a mammalian expression vector expressing a 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, it is a schematic structural diagram of Siglec-10 truncation-Fc fusion protein of the present invention. The fusion protein is prepared through standard molecular biology overlapping PCR to synthesize DNA segment, which is cloned into expression vector pcDNA3.4. Expression was then carried out in Expi293F cells. Finally, protein G affinity chromatography was used to purify the fusion protein, which was concentrated in an ultrafiltration tube and replaced into DPBS buffer ph 7.4. The specific process is as follows:

(1) expression of Siglec-10 truncation-Fc fusion proteins in mammalian cells

Plasmid transient transformation of the protein of interest was performed on the Expi293F cell line (purchased from ThermoFisher, cat # A14527). The fusion protein was purified using a GE protein purification column. Specifically, Expi293F cells were cultured in Expi293^TMExpression Medium (purchased from ThermoFisher, cat. No. A1435101) with Expifactamine^TM293 transfection kit (purchased from Invitrogen, A14525) into plasmid DNA. After 1 week, the supernatant was harvested and purified. The method comprises the following specific steps:

A. one day prior to transfection, Expi293F cells were passaged and fresh medium was added to a density of 2-3X 10⁶Cells/ml were cultured in a shaker at 120 rpm, 37 ℃ and 8% carbon dioxide.

B. On the day of transfection, the cell density was diluted to 2.0X 10 with fresh medium⁶Cells/ml, transfection was performed at a rate of 1. mu.g Expifeacmine transfection reagent per 1 ml of cells. After transfection, the cells were cultured in a shaker at a rotation speed of 120 rpm, 37 ℃ and 8% carbon dioxide

C. 20 hours after transfection, the feed was added proportionally.

D. And (5) transfecting for 5-7 days, detecting the cell viability, and if the viability is less than 60%, placing the cells in a centrifugal tube for 10000 r/min for centrifugation, and harvesting the supernatant.

(2) The specific steps of protein purification are as follows:

first, protein G affinity chromatography was used. The supernatant was diluted with binding buffer (PBS, pH7.4) and loaded onto a Protein G column (GE Healthcare) at a flow rate of 1-5 mL/min. Then, the column was washed with 5-10 column volumes of binding buffer (PBS, pH7.4), and the protein was eluted with an eluent (0.1M sodium citrate, pH3.0) 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 (10kD) ultrafiltration tube and the buffer was switched to PBS at pH 7.4. The purity of each protein was identified by SDS-PAGE, and the protein concentration was determined by A280.

Example 2 binding of Siglec-10(D1m) -Fc fusion protein to CD24 at the cellular level

The affinity of Siglec-10(D1m) -Fc fusion protein and CD24 was determined by FACS techniques.

The specific method comprises the following steps: the fusion protein S10- (D1m) -Fc with a certain concentration (40ug/mL) was mixed with 2X 10⁵A high-expression human CD24 CHOK1 cell (purchased from Biotech, Conn. Bo., Beijing) was incubated in 100uL (2% BSA in PBS) and after standing at 4 ℃ for 1 hour, the supernatant was centrifuged and washed twice with buffer. Anti-human IgG fluorescent secondary antibody (purchased from Sigma) was then added, and after standing at 4 degrees for 1 hour, the supernatant was discarded by centrifugation and the cells were washed twice with buffer. The antibody-labeled cells were subjected to flow cytometry (CantoII, BD bioscience) to obtain data analysis as shown in FIG. 3. As shown in fig. 3, Siglec-10(D1m) -Fc fusion protein retained binding activity to CD24 at the cellular level.

Claims

1. A Siglec-10 truncation-Fc fusion protein capable of blocking the interaction of CD24 with Siglec-10, wherein the Siglec-10 truncation may be a native Siglec-10 truncation or a mutant thereof.

2. The Siglec-10 truncation-Fc fusion protein of claim 1, wherein the Siglec-10 truncation is preferably a human Sigec-10 truncation; more preferably, the Siglec-10 truncation comprises part or all of the amino acid sequence of the extracellular domain of human Siglec-10; further preferably, the Siglec-10 truncation comprises the D1 region and/or the D2 region of the extracellular domain of human Siglec-10.

3. The Siglec-10 truncation-Fc fusion protein of claim 2, wherein the Siglec-10 truncation has a mutation at a sialic acid binding site, an N-linked glycosylation site, and/or a cysteine site; preferably, the mutation occurs at one or more amino acid residue positions 21F, 36C, 41C, 100N, 101C, 119R, 128Y, 164C and 173C of the sequence set forth in accession number Q96LC 7.

4. The Siglec-10 truncation-Fc fusion protein of claim 3, wherein the Siglec-10 truncation comprises the D1 region of the extracellular domain of human Siglec-10, and there are one or more amino acid mutations in the D1 region; preferably, the Siglec-10 truncation comprises the D1 region of the extracellular domain of human Siglec-10, and there is a mutation at 36C of the sequence shown by accession number Q96LC 7; more preferably, the 36C mutation is a cysteine to serine mutation.

5. A Siglec-10 truncation, wherein the Siglec-10 truncation is the Siglec-10 truncation of the Siglec-10 truncation-Fc fusion protein of any one of claims 1 to 4.

6. A nucleic acid molecule encoding a Siglec-10 truncation-Fc fusion protein of any one of claims 1 to 4 or a Siglec-10 truncation of claim 5.

7. An expression vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the expression vector of claim 7.

9. The method of producing a Siglec-10 truncation-Fc fusion protein of any one of claims 1 to 4 or a Siglec-10 truncation of claim 5, comprising the step of expressing the fusion protein using the host cell of claim 8.

10. Use of a Siglec-10 truncation-Fc fusion protein of any one of claims 1 to 4 or a Siglec-10 truncation of claim 5 for the preparation of a medicament for the treatment of a disease associated with high expression of CD 24.