CN107964041B - DMIC with high stability and high affinity and its preparation method - Google Patents

Abstract

The present invention provides a truncated MICA (MHC class I-related chain A) with high stability and high affinity, that is, DMIC molecule, its preparation method and application. The hydrophobic core region of the DMIC molecule of the present invention is mutated, resulting in an increase in its stability and affinity.

Description

DMIC with high stability and high affinity and its preparation method

technical field

The invention relates to the field of biotechnology, and more specifically relates to a soluble DMIC with a mutation in the hydrophobic core region, high stability and high affinity. The present invention also relates to the preparation method and application of the DMIC.

Background technique

NKG2D (Natural Killer Group 2D) is an activating receptor mainly expressed on the surface of NK cells, CD8+αβT cells, γδ+T cells and some CD4+T cells. MICA (MHC classⅠ-related chain A) is one of the ligands of human NKG2D, which is highly expressed on the surface of a variety of tumors, such as melanoma, glioma, and colon cancer, kidney cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer Cancer and other cancer cells, but no or low expression in normal cells and mature tissues. Studies have shown that the interaction between NKG2D and MICA plays an important role in tumor immune surveillance. Bauer's research shows that the interaction between NKG2D and MICA can directly activate NK cells to kill tumor cells, and at the same time, as a costimulatory molecule of TCR, activate CD8+αβT cells and γδ+T cells. Glioma cells overexpressing MICA can enhance the activity of NK cells and T cells in vitro, and at the same time, the growth of glioma overexpressing MICA in nude mice and VMDK mice was also significantly delayed. NKG2D-mediated cytotoxicity of NK cells is closely related to the density of MICA expressed on the tumor surface.

The MICA on the surface of the tumor, that is, the MICA on the membrane, plays the role of tumor immune supervision and anti-tumor, while the free and soluble MICA (sMICA) will damage the immune response of the body and promote the immune escape of the tumor. Studies have shown that MICA on the tumor surface will fall off from the tumor surface under the action of endoplasmic reticulum protease 5 (Erp5) and metalloproteinases (MPs), forming free sMICA. sMICA can down-regulate the expression of NKG2D on the surface of CD8+αβT cells, NK cells, γδ+T cells and CD3+CD56+NKT cells, thereby achieving immune escape of tumors. Because MICA participates in both tumor immune surveillance and tumor immune escape, its dual identity makes MICA a potential target for immunotherapy. In-depth research on MICA can better exert its anti-tumor effect. Kellner et al. made fusion protein between MICA and anti-tumor surface antigen antibody, which can activate NK cells to kill tumor cells.

Contents of the invention

The purpose of the present invention is to provide a truncated MICA (ie DMIC molecule) with high stability and high affinity.

Another object of the present invention is to provide the preparation method and application of the high-stability and high-affinity DMIC molecule.

In the first aspect of the present invention, a truncated MICA is provided, the truncated MICA includes the α1α2 domain of the mutated MICA, and the "mutation" includes one or more amino acid sequences of the α1α2 domain of the native MICA A surface-exposed hydrophobic amino acid residue was mutated into a hydrophilic amino acid residue.

In another preferred example, the amino acid sequence of the α1α2 domain of native MICA is shown in SEQ ID NO.1.

In another preferred example, the "hydrophobic amino acid residue exposed on the surface" is selected from the group consisting of 13L, 15W, 24L, 51A, 54V, 88L, 94I, 96V, 130M, 141M, and 179L, wherein the amino acid Residue numbering adopts the numbering shown in SEQ ID NO.1.

In another preferred example, the stability of the truncated MICA is higher than that of natural MICA, preferably, the Tm value of the truncated MICA is greater than or equal to 36°C.

In another preferred example, the affinity of the truncated MICA to NKG2D is at least 2 times; preferably at least 3 times; more preferably at least 5 times; most preferably at least 10 times that of the natural MICA molecule and NKG2D.

In another preferred example, the truncated MICA comprises one or more mutated amino acid residues selected from the group consisting of 13S, 15Q, 24Q, 51G, 54T, 88S, 94T, 96S, 130V, 141Q and 179P, wherein the numbering of amino acid residues adopts the numbering shown in SEQID NO.1.

In another preferred example, the truncated MICA has at least 80% homology with the amino acid sequence shown in SEQ ID NO.1, preferably at least 90% homology.

In another preferred example, the truncated MICA has at least 85% homology with the amino acid sequence shown in SEQ ID NO.3, preferably has at least 90% homology, more preferably has At least 95% homology, most preferably at least 98% homology.

In another preferred example, the "mutation" also includes one or more (preferably 2-30, more preferably 2-25) amino acid residues in the hydrophobic core of the natural MICA α1α2 domain mutation .

In another preferred example, the "amino acid residues in the hydrophobic core of the α1α2 domain of native MICA" are selected from the group consisting of 9N, 10L, 14S, 16D, 25T, 35L, 39R, 49Q, 51A, 52E, 54T, 70N, 109Q, 121Q, 128W, 147L, 150D, 153K, 155K, 158Y, 159H, 162H, and 178V, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO.1.

In another preferred example, the "amino acid residues in the hydrophobic core of the α1α2 domain of native MICA" include 178V and/or 162H, wherein the amino acid residues are numbered according to SEQ ID NO.1.

In another preferred example, the "amino acid residues in the hydrophobic core of the α1α2 domain of natural MICA" include 158Y and/or 147L, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO.1.

In another preferred example, the "amino acid residues in the hydrophobic core of the α1α2 domain of natural MICA" also includes 121Q and/or 109Q, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO.1.

In another preferred example, the "amino acid residues in the hydrophobic core of the α1α2 domain of native MICA" also includes 128W, wherein the amino acid residues are numbered using the numbering shown in SEQ ID NO.1.

In another preferred example, the "amino acid residues in the hydrophobic core of the α1α2 domain of native MICA" also include 35L, 10L, and/or 9N, wherein the numbering of amino acid residues is shown in SEQ ID NO.1 serial number.

In another preferred example, the "amino acid residues in the hydrophobic core of the α1α2 domain of natural MICA" also include 14S, 25T, 49Q, 51A, 52E, and/or 54T, wherein the numbering of amino acid residues uses SEQ ID The number shown in NO.1.

In another preferred example, the truncated MICA further comprises one or more mutated amino acid residues selected from the following group: 9D, 10F, 14M or 14I, 16N, 25A, 35I, 39T, 49D, 51E , 52N, 54Q, 70W, 109I or 109H, 121G, 121M, 121W or 121N, 128F, 147W, 150V, 153E, 155D, 158W or 158F, 159D, 162R, 178I or 178M, wherein the amino acid residue numbering adopts SEQ ID NO .1 number shown.

In another preferred example, the truncated MICA further comprises mutated amino acid residues: 162R, and/or 178I or 178M, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO.1.

In another preferred example, the truncated MICA further comprises mutated amino acid residues: 158W or 158F, and/or 147W, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO.1.

In another preferred example, the truncated MICA further comprises mutated amino acid residues: 121G, 121M, 121W or 121N, and/or 109I or 109H, wherein the numbering of the amino acid residues is shown in SEQ ID NO.1 number.

In another preferred example, the truncated MICA further comprises a mutated amino acid residue: 128F, wherein the numbering of the amino acid residues adopts the numbering shown in SEQ ID NO.1.

In another preferred example, the truncated MICA further comprises mutated amino acid residues: 35I, 10F, and/or 9D, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO.1.

In another preferred example, the truncated MICA further comprises mutated amino acid residues: 14M or 14I, 25A, 49D, 51E, 52N, and/or 54Q, wherein the amino acid residue numbering uses SEQ ID NO.1 number shown.

In another preferred example, the truncated MICA further comprises mutated amino acid residues: 16N, 39T, 70W, 150V, 153E, 155D, and/or 159D, wherein the amino acid residue numbering uses SEQ ID NO.1 number shown.

In another preferred example, the truncated MICA has an amino acid sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11 , SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO .29 and SEQ ID NO.31.

In another preferred embodiment, the truncated MICA is soluble.

The second aspect of the present invention provides a fusion protein having the truncated MICA described in the first aspect of the present invention.

In another preferred example, the fusion protein has an optional tag sequence that assists expression and/or purification.

In another preferred example, the fusion protein further includes antibody sequences and/or TCR sequences.

The third aspect of the present invention provides a nucleic acid molecule comprising the polynucleotide sequence encoding the truncated MICA described in the first aspect of the present invention or its complementary sequence.

The fourth aspect of the present invention provides a vector containing the nucleic acid molecule described in the third aspect of the present invention.

The fifth aspect of the present invention provides a host cell containing the vector of the fourth aspect of the present invention or the exogenous nucleic acid molecule of the third aspect of the present invention integrated in the chromosome.

The sixth aspect of the present invention provides a conjugate, which includes

(a) the truncated MICA of claim 1; and

(b) A conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.

In another preferred embodiment, the coupling moiety is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer X-ray tomography) contrast agents, or capable of producing detectable Products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, precursors Drug-activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin), or nanoparticles in any form, etc.

The seventh aspect of the present invention provides a method for preparing the truncated MICA described in the first aspect of the present invention, characterized in that it comprises the steps of:

1) cultivating the host cell described in the fifth aspect of the present invention, thereby expressing the truncated MICA described in the first aspect of the present invention;

2) Isolating or purifying the truncated MICA described in the first aspect of the present invention.

The eighth aspect of the present invention provides the use of the truncated MICA described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, and the conjugate described in the sixth aspect of the present invention, for the preparation of drugs or reagents;

The medicine is used to treat tumor or virus infection;

The reagents are used to detect NKG2D.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows a schematic diagram of the structure of the hydrophobic core region of the DMIC molecule.

Figures 2a and 2b are the amino acid sequence and nucleotide sequence (SEQ ID NO.1 and 2) corresponding to the α1α2 domain of the MICA molecule, respectively.

Figures 3a and 3b are the amino acid sequence and nucleotide sequence (SEQ ID NO.3 and 4) of wild-type DMIC, respectively. Compared with the sequence corresponding to the α1α2 domain of the MICA molecule, the hydrophobic amino acid residues exposed on the surface have undergone mutations , the mutated amino acid residues are shown in bold and underlined.

Figures 4a and 4b are the amino acid sequence and nucleotide sequence (SEQ ID NO.5 and 6) of the DMIC mutant B7, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 5a and 5b are the amino acid sequence and nucleotide sequence (SEQ ID NO.7 and 8) of the DMIC mutant strain C3, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 6a and 6b are the amino acid sequence and nucleotide sequence (SEQ ID NO.9 and 10) of the DMIC mutant strain C5, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 7a and 7b are the amino acid sequence and nucleotide sequence (SEQ ID NO.11 and 12) of the DMIC mutant strain D16, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 8a and 8b are the amino acid sequence and nucleotide sequence (SEQ ID NO.13 and 14) of the DMIC mutant strain M1, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 9a and 9b are the amino acid sequence and nucleotide sequence (SEQ ID NO.15 and 16) of the DMIC mutant strain M1a, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 10a and 10b are the amino acid sequence and nucleotide sequence (SEQ ID NO.17 and 18) of the DMIC mutant strain M2a, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 11a and 11b are the amino acid sequence and nucleotide sequence (SEQ ID NO.19 and 20) of the DMIC mutant strain M2b, respectively. Compared with wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 12a and 12b are the amino acid sequence and nucleotide sequence (SEQ ID NO.21 and 22) of the DMIC mutant strain C3H7, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 13a and 13b are the amino acid sequence and nucleotide sequence (SEQ ID NO.23 and 24) of the DMIC mutant strain C5H7, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 14a and 14b are the amino acid sequence and nucleotide sequence (SEQ ID NO.25 and 26) of the DMIC mutant strain D16H7, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 15a and 15b are the amino acid sequence and nucleotide sequence (SEQ ID NO.27 and 28) of the DMIC mutant strain M1aH7, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 16a and 16b are the amino acid sequence and nucleotide sequence (SEQ ID NO.29 and 30) of the DMIC mutant strain M2aH7, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figures 17a and 17b are the amino acid sequence and nucleotide sequence (SEQ ID NO.31 and 32) of the DMIC mutant strain M2bH7, respectively. Compared with the wild-type DMIC, the mutated amino acid residues are shown in bold and underlined.

Figure 18 is an elution profile of protein purification. (a) MICA, (b) wild-type DMIC, (c) H7, (d) mutant B7, (e) mutant C3, (f) mutant C5, (g) mutant D16, (h) mutant M1, (i) mutant M1a, (j) mutant M2a, (k) mutant M2b, (l) mutant C3H7, (m) mutant C5H7, (n) mutant D16H7, (o) mutant M1aH7 , (p) mutant M2aH7, (q) mutant M2bH7.

Figure 19 is the Non-reducing SDS-PAGE gel image of the purified protein. Lane 1: Molecular weight marker, Lane 2: Wild-type DMIC, Lane 3: Mutant B7, Lane 4: Mutant C3, Lane 5: Mutant C5, Lane 6: Mutant D16, Lane 7: Mutant M1, Lane 8 : mutant M1a, lane 9: H7, lane 10: mutant C3H7, lane 11: mutant C5H7, lane 12: mutant D16H7, lane 13: mutant M1aH7, lane 14: molecular weight marker, lane 15: mutant M2a , lane 16: mutant M2b, lane 17: mutant M2aH7, lane 18: mutant M2bH7, lane 19: MICA, lane 20: molecular weight markers.

Figure 20 is the data of the interaction between purified MICA protein, DMIC protein and NKG2D detected by Biacore SPR.

Fig. 21 is a graph showing the interaction between purified MICA protein, DMIC protein and NKG2D detected by Biacore SPR. (a) MICA, (b) wild-type DMIC, (c) H7, (d) mutant B7, (e) mutant C3, (f) mutant C5, (g) mutant D16, (h) mutant M1, (i) mutant M1a, (j) mutant M2a, (k) mutant M2b, (l) mutant C3H7, (m) mutant C5H7, (n) mutant D16H7, (o) mutant M1aH7 , (p) mutant M2aH7, (q) mutant M2bH7.

Figure 22 shows the Tm value and dH value of the purified protein detected by DSC.

Figure 23 is a graph of DSC raw curves for MICA and wild-type DMIC.

Fig. 24 is a DSC fitting curve of DMIC high stability and high affinity mutants and H7.

Detailed ways

After extensive and in-depth research, the present inventors found that mutants with high stability and high affinity can be obtained by targetedly mutating the hydrophobic core region of the protein, and completed the present invention on this basis.

The present inventors removed the α3 domain (amino acids 182-275) of MICA to generate a new molecule DMIC (SEQ ID NO.1 and 2, ie truncated MICA). Wild-type DMICs (DMIC-WT, SEQ ID NO. 3 and 4) were formed by replacing surface-exposed hydrophobic amino acids with hydrophilic amino acids. By introducing random mutations into the hydrophobic core of DMIC-WT, using directed molecular evolution and phage display technology, mutants that are more stable than DMIC-WT were isolated. The mutant obtained in the present invention not only has greatly improved stability than DMIC-WT, but also has a binding affinity to NKG2D that is 10-2600 times higher than that of DMIC-WT to NKG2D. The mutation sites on the stability mutants were transplanted into DMIC mutant H7 (mutation sites include 16N, 39T, 70W, 150V, 153E, 155D, 159D, wherein the numbering of amino acid residues is shown in SEQ ID NO.3 No.), compared with H7, the stability of the mutant transplanted with the stability mutation site was further enhanced.

Specifically, the inventors learned from the analysis of the protein crystal structure (PDB ID: 1HYR) of the complex between MICA and NKG2D that MICA (NCBI Accession Number: 1HYR_C) contains three structural domains, namely α1, α2 and α3, and α3 Not involved in the combination with NKG2D. Considering that proteins with smaller molecular weights are beneficial for subsequent research and development (such as making fusion proteins, etc.), the inventors removed the α3 domain (amino acids 182-275) of MICA to form a truncated MICA, and further Surface-exposed hydrophobic amino acid residues were replaced with hydrophilic amino acid residues (L13S; W15Q; L24Q; A51G; V54T; L88S; I94T; V96S; M130V; M141Q; L179P), resulting in a MICA-only α1α2 structure domain (amino acids 1-181), namely wild-type DMIC (DMIC-WT, SEQ ID NO. 3 and 4).

After inclusion body expression and protein renaturation in vitro, the signal of the combination of DMIC-WT and NKG2D could not be detected by Elisa, but the signal of the combination of DMIC-WT and NKG2D could be detected by Biacore SPR with higher sensitivity. This phenomenon indicates that DMIC -When WT was refolded in vitro, the proportion of protein folded into the correct conformation was small, so the signal of its combination with NKG2D could not be detected by Elisa. This is probably due to the poor thermal stability of DMIC-WT, resulting in poor renaturation efficiency in vitro. Poor stability and poor renaturation efficiency will inevitably affect the follow-up research of the protein, especially the functional research. For example, after making a fusion protein with an antibody or TCR, renaturation will be more difficult. Therefore, it is very important to optimize the stability of DMIC and improve its expression and renaturation efficiency.

The protein structure has a common feature, that is, there is a hydrophobic core inside the molecule, which is composed of tightly packed hydrophobic side chains. The embedded hydrophobic side chains shield them from water molecules, which is the original driving force for the folding of polypeptide chains. The main factor that stabilizes the three-dimensional structure of proteins. In order to improve the thermal stability of DMIC protein and improve its renaturation efficiency in vitro, the inventors introduced random mutations into the hydrophobic core region of DMIC-WT, constructed a phage library, and screened the library for stability using phage display technology. After obtaining the mutant strain, it was found that not only the stability of the obtained mutant was greatly improved, but also the affinity was increased by 10-2600 times. Furthermore, the inventors used DMIC-WT as a template to introduce seven mutation sites (D16N, R39T, N70W, D150V, K153E, K155D, H159D) to generate a new DMIC mutant named H7. Then, the mutation sites of the stability mutants screened from the phage library were transplanted into H7, and the results showed that, compared with H7, the stability of the mutants transplanted with the stability mutation sites was further improved. The invention provides a DMIC molecule with high stability and high affinity, which provides a solid foundation for further research on the function of the DMIC molecule (such as a fusion protein).

protein and its preparation

In the present invention, "truncated MICA" and "DMIC" are used interchangeably, referring to a protein derived from MICA including the α1α2 domain of MICA, and the protein contains mutations that improve affinity and stability. The present invention also provides fusion proteins comprising said truncated MICA, such as isolated fusion proteins. The protein of the present invention may be a monomer or a multimer (such as a dimer) formed from the monomer. Furthermore, it is to be understood that the term also includes active fragments and derivatives of DMIC or fusion proteins thereof.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state.

As used herein, "isolated fusion protein" refers to a fusion protein that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify fusion proteins using standard protein purification techniques. Essentially pure proteins yield a single major band on non-reducing polyacrylamide gels.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.

The present invention also relates to variants of the above polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the encoded polypeptide.

As used herein, the term "primer" refers to a general term for oligonucleotides that, when paired with a template, can be used as a starting point to synthesize a DNA chain complementary to the template under the action of DNA polymerase. Primers can be natural RNA, DNA, or any form of natural nucleotides. Primers can even be non-natural nucleotides such as LNA or ZNA, etc. A primer is "substantially" (or "essentially") complementary to a particular sequence on one strand of the template. A primer must be sufficiently complementary to one strand of the template to initiate extension, but the sequence of the primer does not have to be perfectly complementary to that of the template. For example, adding a non-complementary sequence to the 5' end of a primer whose 3' end is complementary to the template, such a primer is still substantially complementary to the template. As long as there is a sufficiently long primer that can fully bind to the template, non-completely complementary primers can also form a primer-template complex with the template, thereby performing amplification.

The full-length nucleotide sequence of the protein of the present invention or its fusion protein or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the published relevant nucleotide sequences, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as Template, amplified to obtain the relevant sequence. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the gene of the present invention. Primers for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering with the vector or fusion protein coding sequence of the present invention, and a method for producing the protein of the present invention through recombinant technology.

The polynucleotide sequences of the present invention can be used to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally speaking, there are the following steps:

(1) Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2) host cells cultured in a suitable medium;

(3) Separation and purification of protein from culture medium or cells.

Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: bacterial cells of Escherichia coli, Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, NSO, COS7, or 293 cells, etc.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The protein in the above method may be expressed inside the cell, or on the cell membrane, or secreted outside the cell. Proteins can be isolated and purified by various separation methods by taking advantage of their physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

As used herein, the term "truncated MICA" also includes variant forms having the above activities. These variant forms include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and/or substitutions, and additions or substitutions at the C-terminal and/or N-terminal Deletion of one or several (usually within 3, preferably within 2, more preferably within 1) amino acids. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. Furthermore, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (eg, cyclic peptides).

The present invention also includes active fragments, derivatives and analogs of the above-mentioned proteins. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention can be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more A polypeptide with substituent groups in amino acid residues, or (iii) a polypeptide formed by fusing an antigenic peptide to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a fusion protein fused to a leader sequence, a secretory sequence, or a tag sequence such as 6His). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

A preferred class of active derivatives refers to the formation of at most 3, preferably at most 2, and more preferably at most 1 amino acids replaced by amino acids with similar or similar properties compared with the amino acid sequence of formula Ia or formula Ib peptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.

Table A

initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

The invention also provides analogs of the truncated MICA proteins of the invention. The difference between these analogs and the polypeptide shown in the specific sequence of the present invention may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of polypeptides during synthesis and processing or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

The polypeptide (fusion protein) of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base. These salts include, but are not limited to, those formed with the following acids: hydrochloric, hydrobromic, sulfuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, succinic, oxalic, fumaric, maleic, acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as in the form of esters, carbamates or other conventional "prodrugs".

The fusion protein provided by the present invention may optionally contain a peptide linker, and the truncated MICA according to the present invention and other polypeptide elements (such as antibody, TCR, etc.) are connected through a peptide bond or a peptide linker. Peptide linker size and complexity may affect protein activity. In general, the peptide linker should be of sufficient length and flexibility to ensure that the two proteins being linked have sufficient degrees of freedom in space to function. At the same time, the influence of the formation of α-helix or β-sheet in the peptide linker on the stability of the fusion protein is avoided.

The length of the connecting peptide is generally 0-10 amino acids, preferably 1-5 amino acids.

Pharmaceutical composition and method of administration

The present invention also provides a composition, which contains: (i) the truncated MICA protein of the present invention or a polynucleotide encoding the truncated MICA protein of the present invention, and (ii) a pharmaceutically acceptable carrier, or excipient.

In the present invention, the term "comprising" means that various components can be applied together or present in the composition of the present invention. Accordingly, the terms "consisting essentially of" and "consisting of" are included in the term "comprising".

Compositions of the invention include pharmaceutical compositions.

The pharmaceutical composition of the present invention can be prepared into various conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.

Generally, the fusion protein of the present invention can be prepared in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably, the pH is about 6-8.

As used herein, the term "effective amount" or "effective dose" refers to the amount that can produce functions or activities on humans and/or animals and can be accepted by humans and/or animals, such as 0.001-99wt%; preferably 0.01-95wt%; more preferably, 0.1-90wt%.

As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation and allergic reactions), ie, has a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical composition of the present invention contains a safe and effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably produced under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparations of the present invention can also be made into sustained-release preparations.

The effective amount of the truncated MICA protein of the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. The selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: pharmacokinetic parameters of the fusion protein of the present invention such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the body weight of the patient, the immune status of the patient, the dose way etc. For tuberculosis patients, generally, when the fusion protein of the present invention is administered at a dose of about 0.5 mg-5 mg/kg animal body weight (preferably 2 mg-4 mg/kg animal body weight) per day, satisfactory effects can be obtained. For example, several divided doses may be administered daily or the dose may be proportionally reduced as the exigencies of the therapeutic situation dictate.

Preparation

One method of producing the DMIC molecule of the present invention is to select high stability, high affinity DMIC mutants from a diverse library of phage particles displaying the DMIC molecule.

Mutations may be performed using any suitable method, including but not limited to polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation-independent cloning (LIC) methods. These methods are detailed in many standard molecular biology textbooks. More details on polymerase chain reaction (PCR) mutagenesis and restriction enzyme based cloning can be found in Sambrook and Russell, (2001) Molecular Cloning - A Laboratory Manual (Third Edition) CSHL publishing house. More information on the LIC method can be found in (Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6).

The DMIC molecules of the invention can be chemically synthesized, or recombinant. Correspondingly, the DMIC molecules of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.

The polynucleotides of the present invention can be used to express or produce recombinant DMICs by conventional recombinant DNA techniques. Generally speaking, there are the following steps:

1) Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the DMIC of the present invention, or with a recombinant expression vector containing the polynucleotide;

2) culturing host cells in a suitable medium;

3) Separating and purifying the DMIC of the present invention from culture medium or cells.

DMIC can be expressed in the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Uses of the DMIC of the present invention

High stability of the present invention, high-affinity DMIC molecular use include the following points:

1) It is used to study the interaction between DMIC molecules and NKG2D, such as the effect of the affinity of DMIC molecules and NKG2D on the activation of NK cells or T cells;

2) Make a fusion protein with an antibody to prepare a drug for treating tumor or viral infection;

3) Make a fusion protein with T cell receptor (TCR) to prepare drugs for treating tumors or viral infections;

4) It can be used to immunize animals to prepare anti-DMIC or anti-MICA antibodies;

5) It can be coupled with fluorescein, and the expression of NKG2D on the cell surface can be detected by flow cytometry.

The main advantages of the present invention include:

1) DMIC molecular stability of the present invention is high;

2) DMIC molecular affinity of the present invention is high;

3) The DMIC molecules with high stability and high affinity can be screened efficiently and easily.

The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, such as Sambrook and Russell et al., Molecular Cloning: Laboratory Manual (Molecular Cloning-A Laboratory Manual) (Third Edition) (2001) CSHL Publishing House conditions as stated, or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

The construction of embodiment 1 wild-type DMIC and the optimization of sequence

According to the crystal structure of MICA and NKG2D complex (PDB ID: 1HYR) (Li et al., (2001), Nature Immunology (Nat Immunol) 2 (5): 443-451), the MICA (NCBI Accession Number: 1HYR_C) The α3 domain (amino acids 182-275) is removed, and the α1α2 domain (amino acids 1-181) is retained to form primary DMIC molecules (SEQ ID NO.1 and 2). Then, according to the crystal structure, the hydrophobic amino acid residues exposed on the surface were replaced with hydrophilic residues, the substitutions were L13S, W15Q, L24Q, A51G, V54T, L88S, I94T, V96S, M130V, M141Q, L179P, thus obtaining wild type DMIC (DMIC-WT, SEQ ID NO. 3 and 4). The amino acid sequence of DMIC-WT was optimized into a nucleotide sequence suitable for expression in Escherichia coli, and the gene was cloned into the PUC57 vector (Genscript) after gene synthesis.

Embodiment 2 DMIC-WT sequence is cloned into the expression plasmid based on pET-28a

DMIC-WT was cloned into the pET-28a expression plasmid (purchased from Novagen) by standard methods described in Molecular Cloning a Laboratory Manual (3rd edition, Sambrook and Russell). The DNA sequence encoding DMIC-WT cut with NcoI and NotI was ligated into the pET-28a vector cut with NcoI and NotI. The ligated plasmid was transformed into conventional competent Escherichia coli (Escherichia coli) strain BL21(DE3) cells (purchased from Vazyme), and inoculated on LB agar plates containing 50 μg/mL kanamycin. After culturing overnight at 37°C, a single colony was picked and cultured overnight at 37°C with shaking at 250 rpm in 5 ml LB medium containing 50 μg/mL kanamycin. Plasmids were extracted from the overnight culture solution and then sequenced.

Expression, renaturation and purification of embodiment 3DMIC-WT

The Escherichia coli strain BL21(DE3) containing the DMIC-WT expression plasmid obtained in Example 2 was inoculated in 5 ml LB medium containing 50 μg/mL kanamycin, and cultured overnight at 37° C. with shaking at 250 rpm. On the second day, transfer to 400ml LB medium containing 50μg/mL kanamycin at a volume ratio of 1:100, shake and culture at 250rpm at 37°C until the OD600 is 0.4-0.6 (about 2-3h), and add a final concentration of 0.7 Expression was induced by mM IPTG for 4 hours. Cells were harvested by centrifugation at 5,000rpm for 20min. The cell pellet was lysed with Bugbuster MasterMix (Merck), and the inclusion body pellet was recovered by centrifugation at 6,000 g for 15 min. The inclusion bodies were then washed three times with 10-fold diluted Bugbuster solution, and finally the inclusion bodies were dissolved in 6M guanidine hydrochloride buffer. After quantification by the BCA method, it was divided into 12 mg tubes and stored at -80°C.

Thaw 12 mg of DMIC-WT inclusion bodies, slowly drop into 200 ml of refolding buffer: 100 mM Tris.Cl, pH 8.5, 400 mM L-arginine, 2 mM EDTA, and the final concentrations of redox pairs are 1 mM and 10 mM, respectively. The solution was slowly stirred at 4°C for 10 minutes, put into a dialysis bag with a molecular cut-off of 3.5kD, and dialyzed with 5L of pure water at 4-8°C for renaturation overnight. On the second day, renaturation was dialyzed with 10mM Tris.Cl pH 8.5 buffer solution for 8 hours, and then replaced with the same fresh buffer solution and dialyzed once overnight.

Centrifuge the dialyzed refolded protein solution DMIC-WT and put it on an anion exchange column QFF (5ml, GE), use the GEAKTA protein purification system to elute with a linear gradient of 1M NaCl prepared with 10mM Tris.Cl pH 8.5, and collect the elution peaks , run SDS-PAGE gel analysis. After the component comprising DMIC-WT was diluted 10 times with 10mM Tris.Cl pH 8.5, the anion-exchange column QFF was applied again, and the 1M NaCl linear gradient elution (Fig. 18(b)) prepared with 10mM Tris.Cl pH 8.5 was run SDS-PAGE gel analysis and identification (relative molecular mass about 22kD, Figure 19).

Example 4 produces high stability and high affinity DMIC mutants

Random mutations were introduced into the DMIC-WT hydrophobic core, and four phage mutation libraries were constructed, and the libraries were panned by phage display technology. The above-mentioned hydrophobic core library construction and screening methods can refer to the construction and screening methods of the high-affinity TCR phage library described by Li et al. ((2005) Nature Biotech 23(3):349-354). When constructing the 4 DMIC hydrophobic core mutation libraries, the designed primers are shown in the following table (Table 1-4).

Primers designed when table 1 constructs DMIC hydrophobic core mutant library 1

Primers designed when table 2 constructs DMIC hydrophobic core mutant library 2

Primers designed when table 3 constructs DMIC hydrophobic core mutant library 3

Primers designed when table 4 constructs DMIC hydrophobic core mutant library 4

The degenerate base used in the present invention, as well known to those skilled in the art, the base type that can represent respectively is as follows: B=C or G or T; D=A or G or T; H=A or C or T; K=G or T; M=A or C; N=A or C or G or T; R=A or G; S=C or G; V=A or C or G; W=A or T; Y=C or T.

Optimization of screening conditions: the inventors first displayed DMIC-WT on the surface of filamentous phage, and detected the binding of DMIC-WT and NKG2D by phage ELISA (enzyme-linked immunosorbent assay) to obtain the interaction strength. In order to obtain optimal screening conditions, the inventors displayed DMIC-WT under the following 12 conditions: 26°C (IPTG concentrations were 0mM, 0.2mM, 0.5mM, 1mM), 30°C (IPTG concentrations were 0mM , 0.2mM, 0.5mM, 1mM), 37°C (IPTG concentrations were 0mM, 0.2mM, 0.5mM, 1mM), and then the combination of DMIC-WT and NKG2D was detected by phage Elisa, and the result was at 37°C, 1mM IPTG In the presence of conditions, DMIC-WT exhibited the worst effect. In order to allow those mutants whose stability is comparable to DMIC-WT or even worse than DMIC-WT to be directly eliminated during the expression process, and to retain mutants with better stability than DMIC-WT, that is, in order to reduce the expression of the background, the inventors The condition of 37° C. and the presence of 1 mM IPTG was selected as the expression condition of the subsequent mutant library. In order to further reduce the screening background, the DMIC mutant library displayed on the surface of the phage was first treated in a water bath at 65°C for 1 h in the presence of 0.02% SDS, cooled on ice for 15 min, and then combined with NKG2D for screening. Therefore, in order to eliminate the mutants with poor stability during the phage display process and screen the more stable mutants, the screening conditions were determined as follows: 1. Display the DMIC mutant library at 37°C; 2. Add 1mM DMIC when displaying the DMIC library IPTG; 3. Before screening, incubate the phages displaying DMIC mutants at 60-65° C. for 60 min in the presence of 0.02% SDS.

It was identified by sequencing that the hydrophobic cores of more than 50 DMIC mutants screened by the above method were all mutated. Then, after phage Elisa identification and combination of mutation sites, 8 mutant strains were finally selected for further identification. The 8 mutant strains were B7, C3, C5, D16, M1, M1a, M2a, and M2b. Among them, B7, C3, C5, and D16 are mutant strains directly screened out, and M1, M1a, M2a, and M2b are mutant strains after combining mutation sites.

Phage ELISA protocol:

1. Inoculate DMIC-WT and each mutant into 5 mL 2xTY (100 μg/mL ampicillin, 2% glucose) respectively, culture at 250 rpm/min, 37° C. overnight.

2. Respectively transfer 50 μL of overnight cultured bacterial solution to 5 mL of fresh 2xTY (100 μg/mL ampicillin, 2% glucose), culture at 250 rpm/min at 37°C until OD600=0.4; use 5 μL (6.5x 10 ¹⁰ ) of KM13 helper phage Infection, rest at 37°C for 30min, shake at 250rpm/min at 37°C for 30min, centrifuge and resuspend the pellet in 30mL 2xTY (100μg/mL ampicillin, 50μg/mL kanamycin, 0.1% glucose, 1mM IPTG), 250rpm/ Incubate overnight at 37°C.

3. Coat the immunosorbent plate (NUNC) with 10 μg/mL streptavidin (PBS, pH=7.4), 100 μL per well, overnight at 4°C.

4. Collect the supernatant of the overnight culture by centrifugation, precipitate the phage in the supernatant with 1/4 volume ratio of PEG/NaCl, place on ice for 1 hour, collect the precipitate by centrifugation, and resuspend in 3 mL PBS.

5. After washing the plate 3 times with 0.1% PBST, add 300 μL 3% Marvel-PBS to each well, block at 37°C for 1 hour; wash the plate 3 times with PBST, add 100 μL 10ug/mL biotinylated NKG2D to each well, and place at room temperature for 1 hour ; wash the plate 3 times, add 100 μL phage sample to each well (10 μL PEG-precipitated sample was incubated with 3% Marvel-PBS for 1 hour at room temperature), and place at room temperature for 1 hour; after washing the plate 3 times, add 100 μL anti-M13-HRP to each well Conjugate (GE Healthcare) (1:5000 diluted in 3% Marvel-PBS) was placed at room temperature for 1 hour; the plate was washed 5 times, 100 μL TMB was added to each well, and after standing for 5 minutes, 100 μL 1M sulfuric acid was added to each well to stop. Read the value of OD 450nm with a microplate reader.

Example 5 Analysis of binding activity of DMIC-WT and each mutant protein to NKG2D

Using the method described in Example 3, wild-type MICA, DMIC-WT and mutants B7, C3, C5, D16, M1, M1a, M2a, M2b were expressed, refolded, purified and then run on SDS-PAGE gel.

The binding activity of MICA, DMIC-WT and mutants B7, C3, C5, D16, M1, M1a, M2a, M2b to NKG2D was detected using BIAcore T200 real-time analysis system. Add the anti-streptavidin antibody to the coupling buffer (10mM sodium acetate buffer, pH 4.77), then pass the antibody through the CM5 chip activated with EDC and NHS in advance, so that the antibody is immobilized on the chip surface, and finally use The hydrochloric acid solution of ethanolamine blocked the unreacted activated surface to complete the coupling process, and the coupling level was about 15,000 RU.

Let a low concentration of streptavidin flow over the surface of the antibody-coated chip, then flow biotinylated NKG2D through the detection channel, and the other channel as a reference channel, and then 0.05mM biotin at 10μL/min The flow rate flowed through the chip for 2 minutes to block the remaining binding sites of streptavidin. Using a single-cycle kinetic analysis method to determine its affinity, MICA, DMIC-WT and mutants B7, C3, C5, D16, M1, M1a, M2a, M2b were treated with HEPES-EP buffer (10mM HEPES, 150mMNaCl, 3mM EDTA, 0.005% P20, pH 7.4) was diluted into several different concentrations, and flowed over the surface of the chip sequentially at a flow rate of 30 μL/min. The binding time of each injection was 120s, and it was allowed to dissociate for 600s after the last injection. The chip was regenerated with 10 mM Gly-HCl, pH 1.75, after each run. Kinetic parameters were calculated using BIAcore Evaluation software.

The expression, refolding and purification process of NKG2D used in this example is the same as that of DMIC-WT in Example 3. The biotinylation process is as follows:

a.Biotinylation

Concentrate the purified NKG2D protein with a Millipore ultrafiltration tube, and replace the buffer with 10mM Tris.Cl pH 8.0, then add biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50μM D-Biotin, 100μg/ml For BirA enzyme (GST-BirA), the mixture was incubated overnight at 20° C. with 80 rpm shaking, and SDS-PAGE was used to detect whether the biotinylation was complete.

b. Purification of the biotinylated complex

The biotinylated NKG2D protein was concentrated to 0.5ml with Millipore ultrafiltration tube, and the biotinylated NKG2D was purified by gel filtration chromatography. Using AKTA protein purification system (GE), the Superdex75 16/20 column was equilibrated with PBS ( GE), load 0.5ml concentrated biotinylated NKG2D protein, and continue to equilibrate with PBS at a flow rate of 0.5ml/min. Biotinylated NKG2D protein elutes as a single peak at about 10 ml. The protein-containing fractions were combined, concentrated with Millipore ultrafiltration tubes, and the protein concentration was determined by BCA method. The biotinylated NKG2D protein was stored at -80°C.

Through the process described in this example, it is detected that the KD value of MICA binding to NKG2D protein is 5.62E-07M; the KD value of DMIC-WT binding to NKG2D protein is 1.479E-07M; the KD value of B7 binding to NKG2D protein is 1.177E -09M; the KD value of C3 binding to NKG2D protein is 1.056E-10M; the KD value of C5 binding to NKG2D protein is 5.489E-11M; the KD value of D16 binding to NKG2D protein is 6.840E-11M; the binding value of M1 to NKG2D protein The KD value of M1a and NKG2D protein binding is 6.740E-11M; the KD value of M1a binding to NKG2D protein is 1.751E-10M; the KD value of M2a binding to NKG2D protein is 2.137E-10M; the KD value of M2b binding to NKG2D protein is 4.435E-10M (Figure 20). Figure 21 is a curve of the binding of each protein to NKG2D detected by Biacore SPR. The results showed that the affinity of each mutant to NKG2D was increased by 10-2600 times compared with DMIC-WT.

Embodiment 6DMIC-WT and the stability test of each mutant protein

Using the method described in Example 3, wild-type MICA, DMIC-WT and mutants B7, C3, C5, D16, M1, M1a, M2a, M2b were expressed, renatured, purified (Figure 18), and run SDS- PAGE gel identification (Figure 19).

The Tm value and dH value of the above-mentioned purified protein MICA, DMIC-WT and each mutant were measured by a differential scanning calorimeter (Nano DSC) of TA (waters) company in the United States. The scanning range is 10-100°C, the heating rate is 1°C/min, the sample concentration is 0.5mg/mL, and the sample loading volume is 900μL. Among them, the Tm value is obtained by fitting the fitting model TwostateScaled of the analysis software Nanoanalyze.

Fig. 19 is the SDS-PAGE gel picture of the proteins MICA, DMIC-WT and the mutants obtained after two times of anion exchange column purification as described in Example 3.

Fig. 22 is the DSC data of protein MICA, DMIC-WT and each mutant. The results showed that MICA and DMIC-WT obtained the correct conformation after renaturation and purification. The protein content was very small, and there was no obvious protein unfolding endothermic peak. The Tm value and dH value could not be obtained by using the analysis software Nanoanalyze, but the hydrophobic core mutation The Tm and dH values of the mutants are as follows: the Tm value of B7 is 38.62°C, and the dH is 430.34kJ/mol; the Tm value of C3 is 45.78°C, and the dH is 449.20kJ/mol; the Tm value of C5 is 44.38°C, dH is 484.33kJ/mol; D16 has double peaks, Tm values are 43.50°C and 61.16°C, dH is 596.70kJ/mol and 375.40kJ/mol; Tm value of M1 is 40.81°C, dH is 382.62kJ/mol; M1a The Tm value of M2a is 41.60°C and dH is 415.10kJ/mol; the Tm value of M2a is 43.48°C and dH is 324.74kJ/mol; the Tm value of M2b is 43.61°C and dH is 354.87kJ/mol. Figure 23 is the DSC curves of MICA and DMIC-WT, and Figure 24 is the DSC fitting curves of each mutant. It shows that the mutants B7, C3, C5, D16, M1, M1a, M2a, M2b are more resistant to unfolding, more resistant to improper or undesired folding and have significantly improved thermal stability compared to DMIC-WT.

The transplantation of embodiment 7DMIC mutation site

The mutation sites of the highly stable mutants C3, C5, D16, M1a, M2a, and M2b screened in Example 4 were respectively transplanted into the DMIC mutant H7 (compared with DMIC-WT, containing 7 mutation sites: D16N, R39T, N70W, D150V, K153E, K155D, H159D), constructed 6 new mutants, namely C3H7, C5H7, D16H7, M1aH7, M2aH7, M2bH7, and performed protein expression on 6 molecules, renaturation, Purification, Biacore SPR affinity detection, DSC stability determination.

H7, C3H7, C5H7, D16H7, M1aH7, M2aH7, M2bH7 were expressed, renatured, purified ( FIG. 18 ), and identified by SDS-PAGE gel ( FIG. 19 ) using the method described in Example 3. At the same time, the expression level and protein renaturation yield were calculated. Wherein, the expression amount is the yield of the inclusion body after 1L Escherichia coli induced expression and purification. The calculation formula of protein renaturation yield is as follows: protein renaturation yield (%)=100*purified protein amount (mg)/refolding used inclusion body amount (mg). The expression amount and protein renaturation yield mentioned in the present invention are all calculated according to the above calculation method, unless otherwise specified. The following table (Table 5) shows the protein expression and protein renaturation yield before and after H7 was introduced into the stable hydrophobic core.

Table 5 DMIC protein expression and renaturation yield

protein name Expression (mg/L) Protein refolding yield (%) H7 406 14.7 C3H7 607 41 C5H7 476 34 D16H7 420 28.6 M1aH7 657 28.5 M2aH7 443 17.5 wxya 473 16.6

As can be seen from the data in the above table, from the perspective of protein expression, the expression levels of C3H7, C5H7, D16H7, M1aH7, M2aH7, and M2bH7 are higher than those of H7. In terms of renaturation yield, the renaturation yields of M2aH7 and M2bH7 are slightly higher than those of H7, while the renaturation yields of C3H7, C5H7, D16H7 and M1aH7 are much higher than those of H7.

Using the method described in Example 5, Biacore SPR was used to detect the binding activity of H7, C3H7, C5H7, D16H7, M1aH7, M2aH7, M2bH7 to NKG2D. The KD value of H7 binding to NKG2D protein is 3.556E-09M; the KD value of C3H7 binding to NKG2D protein is 2.872E-09M; the KD value of C5H7 binding to NKG2D protein is 1.736E-09M; the KD value of D16H7 binding to NKG2D protein The KD value of M1aH7 binding to NKG2D protein is 1.478E-08M; the KD value of M1aH7 binding to NKG2D protein is 4.264E-09M; the KD value of M2aH7 binding to NKG2D protein is 1.967E-09M; the KD value of M2bH7 binding to NKG2D protein is 5.121E-09M (Figure 20 ). Fig. 21 is the binding curve of each protein and NKG2D detected by Biacore SPR. The above data show that, except that the affinity of D16H7 to NKG2D is lower than that of H7 to NKG2D, the affinity of C3H7, C5H7, M1aH7, M2aH7, M2bH7 to NKG2D is basically the same as that of H7 to NKG2D.

Using the method described in Example 6, the thermal stability of H7, C3H7, C5H7, D16H7, M1aH7, M2aH7, and M2bH7 was detected by DSC. As shown in Figure 22, the Tm value of H7 is 48.40°C and dH is 335kJ/mol; the Tm value of C3H7 is 55.10°C and dH is 270kJ/mol; the Tm value of C5H7 is 53.90°C and dH is 233kJ/mol; the Tm value of D16H7 is 50.2°C, dH is 304kJ/mol; the Tm value of M1aH7 is 50.4°C, and dH is 367kJ/mol; M2aH7 has double peaks, Tm values are 55.57°C and 84.84°C, and dH is 216.39kJ/mol and 269.92kJ/mol ; M2bH7 has double peaks, Tm values are 57.04 ℃ and 85.49 ℃, dH are 223.28kJ/mol and 252.67kJ/mol. Figure 24 is the DSC fitting curve of each protein in DMIC. The above data show that the Tm values of C3H7, C5H7, D16H7, M1aH7, M2aH7, and M2bH7 are all higher than those of H7.

Through the comparative analysis of protein expression, protein renaturation yield and Tm value, it can be seen that C3H7, C5H7, D16H7, M1aH7, M2aH7, M2bH7 optimized by hydrophobic core have higher protein expression than H7 without modified hydrophobic core , stronger renaturation ability, more resistance to unfolding, more resistance to improper or unwanted folding, higher protein renaturation yield and significantly improved thermal stability. Therefore, C3H7, C5H7, D16H7, M1aH7, M2aH7, M2bH7 have significantly improved stability compared with H7.

The stability and affinity of the DMIC mutants screened by the present invention are significantly higher than those of DMIC-WT. At the same time, transplanting the mutation sites of DMIC mutants to H7 can also enhance the stability. The high-stability, high-affinity DMIC mutants screened by the present invention are used for subsequent research on the function of DMIC (such as the effect of the affinity of DMIC and NKG2D on the activation of NK cells or T cells), and the development of DMIC-related drugs (such as with antibodies or TCR is made into a fusion protein to prepare medicines for treating tumors or viral infections, etc.) or reagents, which provides a firm foundation.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (15)

1. A truncated MICA, characterized in that the truncated MICA comprises a mutated α1α2 domain of MICA, said "mutation" comprising a mutation of one or more surface-exposed hydrophobic amino acid residues in the amino acid sequence of the α1α2 domain of natural MICA to hydrophilic amino acid residues;

The "surface-exposed hydrophobic amino acid residues" are selected from the group consisting of: 13L, 15W, 24L, 51A, 54V, 88L, 94I, 96V, 130M, 141M, and 179L, wherein the amino acid residue numbers are as shown in SEQ ID No.1, and the amino acid sequence of the truncated MICA is as follows: SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13, SEQ ID No.15, SEQ ID No.17, SEQ ID No.19, SEQ ID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No.27, SEQ ID No.29 or SEQ ID No.31.

2. The truncated MICA of claim 1, wherein the truncated MICA has a stability that is greater than that of the native MICA.

3. The truncated MICA of claim 1, wherein the truncated MICA has an affinity for NKG2D that is at least 2-fold greater than the affinity of the native MICA molecule for NKG 2D.

4. The truncated MICA of claim 1, wherein the truncated MICA is soluble.

5. A fusion protein comprising the truncated MICA of claim 1.

6. The fusion protein of claim 5, further comprising an antibody sequence and/or a TCR sequence.

7. A nucleic acid molecule comprising a polynucleotide sequence encoding a truncated MICA as described in claim 1.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising the vector of claim 8 or the nucleic acid molecule of claim 7 integrated into a chromosome.

10. A conjugate, wherein the conjugate comprises

(a) The truncated MICA of claim 1; and

(b) A coupling moiety selected from the group consisting of: detectable labels, drugs, toxins.

11. The conjugate of claim 10, wherein the conjugate moiety comprises a cytokine, or an enzyme.

12. The conjugate of claim 10, wherein the detectable label comprises a radionuclide.

13. A method of making the truncated MICA of claim 1, comprising the steps of:

1) Culturing the host cell of claim 9 so as to express the truncated MICA of claim 1;

2) Purifying the truncated MICA of claim 1.

14. Use of the truncated MICA of claim 1, the fusion protein of claim 5, the conjugate of claim 10 for the preparation of a reagent;

the reagent is used for detecting NKG2D.

15. Use of a fusion protein formed by fusion of a truncated MICA with an antibody or TCR according to claim 1, for the manufacture of a medicament for the treatment of a tumour or a viral infection.