JP4263158B2 - HLA-E chimera molecule - Google Patents

Description

  The present invention relates to HLA-E chimeric molecules. More specifically, an HLA-E chimeric molecule that confers resistance to cytotoxic activity of human NK cells to non-human mammalian cells, a base sequence encoding the chimeric molecule, and a non-human transformed with the base sequence It relates to mammalian cells and non-human mammals.

Organ transplantation is a very useful treatment. Organ transplantation includes allogeneic transplantation and xenotransplantation, and each has advantages and disadvantages. The allotransplantation from human to human has the advantage of being established as a medical treatment, but has the disadvantage that the number of donors is limited. On the other hand, xenotransplantation from non-human mammals (for example, pigs) to humans has advantages such as being able to supply a large number of grafts, but hyperacute rejection (HAR) and acute vascular rejection (AVR) It has the disadvantage of producing a rejection reaction peculiar to xenotransplantation.
Therefore, for the purpose of overcoming rejection specific to xenotransplantation, a method for expressing a human complement regulatory factor in a non-human mammal (for example, Patent Document 1), which does not exist in primates or humans but exists in non-human mammals To reduce the non-reducing terminal Galα1,3Gal sequence (hereinafter referred to as α-Gal antigen) (for example, Patent Document 2), α-1,3 galactosyltransferase involved in the production of α-Gal antigen A method of knocking out a gene (for example, Non-Patent Documents 1 and 2) has been developed.

Japanese Patent Laid-Open No. 11-239430 Japanese Patent Laid-Open No. 2002-291372 Science 2002, 295, 1089 Nat. Biotechnol. 2002, 20, 251

When a non-human mammal graft is transplanted into a human, even if HAR can be overcome, the latter antibody (such as anti-α-Gal antibody), complement, platelets, and natural killer (NK) cells It attaches to cells and activates them. Activated cells release various cytokines, release heparin, create gaps between adjacent cells, expose basement membrane collagen, induce blood coagulation, block blood vessels, Necroses human grafts (Non-patent Document 3). Such rejection is called acute vascular rejection AVR, but a method for efficiently suppressing the cytotoxic activity of NK cells, which is one of the causes of AVR, has not been developed.

Xenotransplantation 1998, 5, 169

NK cells adhere to target cells via two types of receptors. That is, a killer cell activation receptor that induces cytotoxic activity and a killer cell inhibitory receptor that recognizes its own MHC class I molecule and suppresses cytotoxic activity. When the signal from the former exceeds the signal from the latter, the target cell is necrotized, but when the signal from the latter exceeds the signal from the former, the target cell is not necrotized.
Since human cells express HLA class I molecules (HLA-A, -B, -C, -E, -F, -G), human cells are not damaged by human NK cells. On the other hand, since non-human mammalian cells do not express human HLA class I molecules, they are damaged by human NK cells. Thus, a method has been developed in which non-human mammalian cells are transformed with human HLA-A, HLA-B, HLA-C or HLA-G genes to avoid cytotoxicity caused by human NK cells (Patent Document 3). However, since HLA-A, HLA-B and HLA-C are polymorphic and there are 175, 344 and 90 alleles, respectively, it is necessary to prepare non-human mammalian cells that can handle each HLA. Was not practical.
Therefore, paying attention to the fact that HLA-E and HLA-G are not polymorphic, the use of HLA-E and HLA-G was attempted. As a result, it is relatively easy to express HLA-G on the surface of non-human mammalian cells, but the cytotoxic activity of human NK cells is low, and conversely, HLA-E is expressed on the surface of non-human mammalian cells. It was not easy to express, but it was found that the cytostatic activity by human NK cells is high (Non-patent Document 4).
For the purpose of improving the HLA-E expression levels of the non-human mammalian cell surface, HLA-E, beta ₂ microglobulin and leader peptide of HLA-A2 (Val-Met- Ala-Pro-Arg-Thr-Leu-Val- Attempts have also been made to use a base sequence encoding Leu) or a base sequence encoding a leader peptide of HLA-G (Val-Met-Ala-Pro-Arg-Thr-Leu-Phe-Leu). However, the HLA-E expression level and NK cell cytotoxicity inhibitory activity of these transformants were not sufficient (Non-patent Document 5).

Japanese National Patent Publication No. 11-510698 Transplantation Proceedings 2000, 32, 939 Transplantation 2002, 73, 1582

The present invention has been made to solve the above-mentioned problems existing in the prior art, and the present inventors have provided an HLA-E chimeric molecule that confers resistance to cytotoxic activity by human NK cells to non-human mammalian cells. Have studied
(1) an HLA-E chimeric molecule in which all or part of the α2 domain of the HLA-E molecule is replaced with all or part of the α2 domain of the HLA-G1 molecule,
(2) An HLA-E chimeric molecule in which the signal peptide (SP) of the HLA-E molecule is replaced with a modified SP obtained by modifying a part of the SP of the HLA-G1 molecule together with (1) above, or
(3) Along with (2) above, HLA-E in which a part of the amino acid sequence of α1 domain and α2 domain of HLA-E molecule is replaced with a part of the amino acid sequence of α1 domain and α2 domain of HLA-G1 molecule, respectively. Chimeric molecule,
When the non-human mammalian cells were transformed using the nucleotide sequences encoding each of these, the expression level of the HLA-E chimeric molecule increased and the resistance to cytotoxic activity by human NK cells increased. As a result, the present invention has been completed.
That is, the present invention relates to an HLA-E chimeric molecule that confers resistance to cytotoxic activity of human NK cells to non-human mammalian cells, a base sequence encoding them, and a non-human mammal transformed with the base sequence Cells and non-human mammals are provided.
The modified SP refers to a sequence in which one or two or more amino acids in the amino acid sequence of SP are substituted or deleted, or one or two or more amino acids are added. For example, the SP (HLA-G1 molecule SP ( SP (SEQ ID NO: 11), which is a modified SP (Met Ala Val Met Ala Pro Arg Thr Leu Val Leu Leu Leu Ser Gly Ala Leu Thr Leu Thr Glu Thr Trp Ala) is exemplified.
In addition, the amino acid sequences of the signal peptide (SP), α1 domain, α2 domain, α3 domain and transmembrane (TM) domain of the HLA-E molecule are shown in SEQ ID NOs: 1 to 5, respectively, and their base sequences are shown in SEQ ID NOs: Shown in 6-10.
The amino acid sequences of the SP, α1 domain, α2 domain, α3 domain, and TM domain of the HLA-G1 molecule are shown in SEQ ID NOs: 11 to 15, respectively, and their base sequences are shown in SEQ ID NOs: 16 to 20, respectively.

The gist of the present invention for solving the above-mentioned problems is an HLA-E chimera molecule that confers resistance to cytotoxic activity by human NK cells to non-human mammalian cells, and a base sequence encoding them, and more specifically As an example, a chimeric molecule having the following properties and a base sequence encoding it are shown.
(1) It has a modified SP, and amino acid number 91-182 of the α2 domain of the HLA-E molecule (number from the N-terminal of the α1 domain; the same applies hereinafter) is amino acid number 91 of the α2 domain of the HLA-G1 molecule. An HLA-E chimeric molecule which is an HLA-E molecule except that it is substituted with -182, and a base sequence encoding it. In the chimeric molecule, the amino acid sequences of SP, α1 domain, α2 domain, α3 domain, and TM domain are shown in SEQ ID NOs: 21 to 25, respectively, and their base sequences are shown in SEQ ID NOs: 26 to 30, respectively.
(2) HLA except that it has a modified SP and the amino acid number 137-182 in the second half of the α2 domain of the HLA-E molecule is replaced with amino acid number 137-182 in the second half of the α2 domain of the HLA-G1 molecule. -An HLA-E chimeric molecule which is an E molecule and a base sequence encoding it. In the chimeric molecule, the amino acid sequences of SP, α1 domain, α2 domain, α3 domain and TM domain are shown in SEQ ID NOs: 31 to 35, respectively, and their base sequences are shown in SEQ ID NOs: 36 to 40, respectively.
(3) It has a modified SP, and amino acid numbers 137-150 in the first half of the α2 domain of the HLA-E molecule are the amino acids in the first half of the α2 domain of the HLA-G1 molecule. The HLA-E chimeric molecule which is an HLA-E molecule except for substitution to No. 137-150 and the base sequence encoding it. In the chimeric molecule, the amino acid sequences of SP, α1 domain, α2 domain, α3 domain and TM domain are shown in SEQ ID NOs: 41 to 45, respectively, and their base sequences are shown in SEQ ID NOs: 46 to 50, respectively.
(4) An HLA-E chimeric molecule which is an HLA-E molecule except that it has an SP of the HLA-E molecule or a modified SP, and amino acid number 147 of the α2 domain of the HLA-E molecule is replaced with cysteine. The base sequence to encode. In this chimeric molecule, the amino acid sequences of the SP, α1 domain, α2 domain, α3 domain and TM domain of the chimeric molecule having SP of HLA-E molecule are shown in SEQ ID NOs: 51 to 55, respectively, and the base sequences thereof are respectively arranged. The amino acid sequences of SP, α1 domain, α2 domain, α3 domain and TM domain of the chimeric molecule having the modified SP are shown in SEQ ID NOs: 61 to 65, respectively, and the base sequences thereof are respectively arranged. Shown as numbers 66-70;
(5) HLA-E molecule has SP or modified SP, amino acid number 11 of α1 domain of HLA-E molecule is substituted with alanine, and amino acid number 147 of α2 domain is substituted with cysteine Other than the above, the HLA-E chimera molecule which is an HLA-E molecule and the base sequence encoding it. In the chimeric molecule, the amino acid sequences of the SP, α1 domain, α2 domain, α3 domain and TM domain of the chimeric molecule having SP of HLA-E molecule are shown in SEQ ID NOs: 71 to 75, respectively, and the base sequences thereof are respectively arranged. Nos. 76 to 80, and amino acid sequences of SP, α1 domain, α2 domain, α3 domain, and TM domain of chimeric molecules having modified SP are shown in SEQ ID NOs: 81 to 85, respectively, and their base sequences are respectively arranged. Number 86-90; and
(6) Resistant to the cytotoxic activity of human NK cells produced by transformation with one of the nucleotide sequences encoding the HLA-E chimeric molecule of any one of (1) to (5) above Non-human mammalian cell or non-human mammal.

  Since the HLA-E chimeric molecule of the present invention is efficiently expressed in non-human mammalian cells, it can confer resistance to cytotoxic activity by human NK cells to non-human mammalian cells. Therefore, the HLA-E chimeric molecule of the present invention is effective in preventing the occurrence of cell damage and acute vascular rejection (AVR) by human NK cells that occur when xenotransplantation of non-human mammalian cells, tissues, and organs to humans. .

Human HLA class I molecules signal peptide (SP), [alpha] 1 domain, [alpha] 2 domain consists α3 domain and transmembrane (TM) domains, take it to the beta ₂ microglobulin (β _{2 m)} was arranged structure.
Further, human HLA class I molecules present antigens in a three-dimensional structure by sandwiching an oligopeptide derived from a signal peptide (SP) in a groove formed by an α1 domain and an α2 domain.
As described above, it is relatively easy to transform non-human mammalian cells using the HLA-G gene, but the ability to suppress human NK cytotoxic activity is low. Conversely, if non-human mammalian cells are transformed with the HLA-E gene, a higher ability to suppress human NK cytotoxic activity can be obtained, but transformation itself is not easy. Therefore, in order to confer resistance to the cytotoxic activity of human NK cells to non-human mammalian cells, a base encoding an HLA-E chimera molecule in which the intramolecular domains of HLA-G1 and HLA-E molecules are interchanged. A sequence was prepared, a non-human mammalian cell line was transformed, and an increase / decrease in expression intensity was analyzed using an anti-HLA antibody (B9.12.1, Cosmo Bio), and intensive studies were conducted. As shown in Examples below, the expression level of HLA-E in non-human mammalian cells transformed with a base sequence encoding the following HLA-E chimeric molecule is increased, and the cytotoxicity of human NK cells is increased. It has been found that the resistance increases.

(1) Replacing the SP (SEQ ID NO: 1) of the HLA-E molecule with the SP (SEQ ID NO: 21; the modified SP used in Examples below) modified from the SP (SEQ ID NO: 11) of the HLA-G1 molecule And an HLA-E chimeric molecule (see SEQ ID NOs: 21 to 30) in which the α2 domain (amino acid numbers 91-182) of the HLA-E molecule is replaced with the α2 domain (amino acid numbers 91-182) of the HLA-G1 molecule,
(2) The SP of the HLA-E molecule is replaced with the modified SP, and the latter half of the α2 domain (amino acid number 137-182) of the HLA-E molecule is replaced with the latter half of the α2 domain of the HLA-G1 molecule (amino acid number 137). -182) HLA-E chimeric molecule (see SEQ ID NOS: 31-40),
(3) The SP of the HLA-E molecule is replaced with the modified SP, and the first half (amino acid number 137-150) of the second half of the α2 domain of the HLA-E molecule is replaced with the α2 domain of the HLA-G1 molecule. HLA-E chimeric molecule (see SEQ ID NOs: 41 to 50) substituted in the first half (amino acid numbers 137-150) of the second half,
(4) Replacing the SP of HLA-E molecule with the above modified SP or without replacing serine of amino acid number 147 of α2 domain of HLA-E molecule with cysteine of amino acid number 147 of α2 domain of HLA-G1 molecule HLA-E chimeric molecules (see SEQ ID NO: 51-60 and SEQ ID NO: 61-70), and
(5) Substituting or not replacing the SP of the HLA-E molecule with the above modified SP, the serine of amino acid number 11 of the α1 domain of the HLA-E molecule and the serine of amino acid number 147 of the same α2 domain are each HLA-G1 HLA-E chimeric molecule in which alanine at amino acid number 11 of α1 and cysteine at amino acid number 147 of α2 are substituted (see SEQ ID NOs: 71 to 80 and SEQ ID NOs: 81 to 90).

From the base sequence encoding any one of the HLA-E chimeric molecules described in (1) to (5) above, a gene promoter (for example, β-actin promoter, pMCP promoter, etc.) and / or other expression regulatory sequences A non-human mammalian cell having resistance to the cytotoxic activity of human NK cells can be prepared.
In addition, if the transgene is injected into a fertilized egg of a non-human mammal by the microinjection method, a non-human transgenic mammal comprising cells, tissues, and organs having resistance to the cytotoxic activity of human NK cells can be prepared. it can. The non-human transgenic mammal in the present invention is not particularly limited as long as it is a mammal other than a human, and examples thereof include pigs, mice, rats, hamsters, cows, horses, sheep, rabbits, dogs, cats, etc. Considering xenotransplantation, pigs suitable as donors are preferred.
Furthermore, if the nuclear transfer method is applied using a non-human mammalian cell resistant to the cytotoxic activity of human NK cells as a donor cell, a non-human mammal cell consisting of cells, tissues and organs resistant to the cytotoxic activity of human NK cells. Human cloned mammals can be prepared. As a method for producing these non-human transgenic mammals or non-human cloned mammals, known methods and optimum conditions may be appropriately selected.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these examples.
Example 1
Expression of various HLA-E chimeric molecules in non-human mammalian cells (1)
A base sequence encoding an amino acid sequence having the structure shown in Table 1 was introduced into an expression vector pCXN (having chicken β-actin promoter and CMV enhancer). Next, each transgene was introduced into CHO cells, and FACS analysis was performed using an anti-HLA antibody (Pan-Class I antibody, B9.12.1, Cosmo Bio) to determine the relative value of the expression level. The results are shown in Table 1. In addition, operations such as production of an expression vector and transformation were performed according to a conventional method of gene recombination technology (the same applies hereinafter).

  From the results shown in Table 1, the SP sequence of the HLA-E molecule was replaced with a modified SP (SEQ ID NO: 21) similar to the SP of HLA-G1, and the α2 domain (amino acid numbers 91-182) of the HLA-E molecule , HLA-G1 corresponding to the second half of the α2 domain of the HLA-E molecule (amino acid number 137-182) or the first half of the second half of the α2 domain of the HLA-E molecule (amino acid number 137-150) By substituting the amino acid sequence of the molecule, it was confirmed that the HLA-E chimeric molecule was efficiently expressed on CHO cells.

Example 2
Expression of various HLA-E chimeric molecules in non-human mammalian cells (2)
A base sequence encoding an amino acid sequence having the structure described in Table 2 was introduced into pCXN as an expression vector. Next, each transgene was introduced into CHO cells, and FACS analysis was performed using an anti-HLA antibody. The results are shown in Table 2.

  From the results shown in Table 2, the HLA-E chimeric molecule in which the serine at amino acid number 147 of the α2 domain of the HLA-E molecule was replaced with the cysteine at amino acid number 147 of the α2 domain of the HLA-G1 molecule was efficiently expressed on CHO cells. HLA-E chimeric molecule in which the SP sequence of the HLA-E molecule was confirmed to be replaced with a modified SP (SEQ ID NO: 21) and the amino acid sequence number 147 of the α2 domain of the HLA-E molecule was replaced with cysteine Was more efficiently expressed on CHO cells. It was confirmed that the HLA-E chimeric molecule was more efficiently expressed on CHO cells by replacing the β-position hydroxyl group (—OH) of serine with the cysteine thiol group (—SH).

Example 3
Resistance to cytotoxic activity of human NK cells of non-human mammalian cells expressing HLA-E chimeric molecules A base sequence encoding an amino acid sequence consisting of the composition described in Table 3 was introduced into pCXN as an expression vector. Each transgene was then introduced into porcine vascular endothelial cells (SEC) to produce each stable cell line. Human NK-like cells (YT) are allowed to act on each transformed SEC cell at a ratio of 5: 1 (37 ° C, 4 hours), and lactate dehydrogenase (LDH) released from SEC is used as an indicator for human NK cell cells The cytotoxic activity was measured and the relative value of the cytotoxic rate was determined. The results are shown in Table 3.

  From the results shown in Table 3, the HLA-E chimeric molecule in which the SP of the HLA-E molecule is replaced with the modified SP, the SP of the HLA-E molecule is replaced with the modified SP, and the amino acid number of the α2 domain of the HLA-E molecule Porcine vascular endothelium transformed with a base sequence encoding any one of HLA-E chimeric molecule in which 147 is replaced with cysteine and HLA-E chimeric molecule in which amino acid number 11 of the α1 domain is substituted with alanine in addition to the above The cells were confirmed to suppress the cytotoxicity caused by human NK cells.

Claims (3)

An HLA-E chimeric molecule having the following amino acid sequence:
(1) The amino acid sequence according to SEQ ID NO: 1 of the HLA-E molecule is replaced with the amino acid sequence according to SEQ ID NO: 21, and the amino acid number (α1 domain) of the amino acid sequence of the α2 domain (SEQ ID NO: 3) of the HLA-E molecule An amino acid sequence obtained by substituting 147 serine with cysteine;
(2) The amino acid sequence according to SEQ ID NO: 1 of the HLA-E molecule is replaced with the amino acid sequence according to SEQ ID NO: 21, and the serine of amino acid number 11 of the amino acid sequence of the α1 domain of the HLA-E molecule (SEQ ID NO: 2) Is an amino acid sequence in which is substituted with alanine and serine of amino acid number 147 in the amino acid sequence of the α2 domain of the HLA-E molecule (SEQ ID NO: 3) is replaced with cysteine.   A gene encoding each HLA-E chimeric molecule according to claim 1. A non-human mammalian cell or a non-human mammal transformed with the gene according to claim 2.