JP2012077006A - Klrg1-positive immunocyte-affinity peptide, klrg1-positive immunocyte separation material and klrg1-positive immunocyte separation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new peptide as a ligand that has a low molecular weight, is inexpensive and has KLRG1 affinity and a KLRG1-positive immunocyte separation material that supports the peptide and selectively removes KLRG1-positive immunocytes in peripheral blood.SOLUTION: The KLRG1-positive immunocyte separation material includes a water-insoluble carrier to which a peptide containing an amino acid sequence of Asp-Trp-Val-Ile-Pro-Pro-Ile-Ser-Cys-Pro-Glu-Asn and having a total number of constituent amino acid residues of ≤80 is immobilized.

Description

The present invention relates to a peptide that recognizes KLRG1-positive immune cells. The present invention further relates to a KLRG1-positive immune cell separating material for selectively removing KLRG1-positive immune cells from a solution containing KLRG1-positive immune cells such as blood, body fluid, cell culture medium and treatment solutions thereof.

In recent years, the number of morbidity and mortality of breast cancer has increased remarkably in Japan. Up to now, breast cancer has tended to be treated by a method other than surgery depending on the expression status of estrogen receptor (ER) and progesterone receptor (PgR). Drug therapy such as hormone therapy and anticancer drug therapy is useful, and the response rate and survival rate have been improved. However, about 50% of human epidermal growth factor receptor-related 2 (HER2)-positive breast cancers were hormone receptor-negative and were excluded from hormone therapy. Recently, molecular-targeted therapies such as trastuzumab-based antibody therapy for HER2-positive breast cancer have been performed, and their usefulness has been demonstrated, and they are expected as a powerful therapeutic method in the future.

Breast cancer cells express various growth factors and their receptors to form a signal transduction system related to growth. The oncogene HER2 identified in 1984 is one of them, and is a member of the human epidermal growth factor receptor (EGFR, HER) family. The receptor is a transmembrane glycoprotein, and HER1, HER2, HER3, and HER4 are present due to structural similarity (Non-Patent Document 1). The EGF receptor family contributes to signal transduction through dimerization, and it is believed that the ligand-bound receptor contributes to signal transduction by forming a dimer with another receptor.

HER2/neu, which defines the HER2 receptor, is considered to be one of proto-oncogenes and exists on chromosome 17 (17q21.1) (Non-patent document 2). Amplification and overexpression of the HER2/neu gene have been observed in various cancers such as breast cancer, ovarian cancer, lung cancer, and gastric cancer. HER2-positive breast cancer accounts for 20% to 30% of all breast cancers. Expression of this gene is very weak in normal adult tissues. Its natural history is unique, with early recurrence in the absence of systemic therapy, and in many studies since HER2 amplification was reported to correlate with poor breast cancer prognosis in HER2-positive metastatic breast cancer. Is revealed to be bad.

In 1986, anti-HER2 monoclonal antibody was shown to inhibit the malignant phenotype of neu-transformed cells, leading to the development of trastuzumab targeting HER2. The murine monoclonal antibody mu4D5 against the extracellular domain of the HER2 receptor, which is the prototype of trastuzumab, has been confirmed to have a growth inhibitory action against HER2-positive tumor cell lines in vitro (Non-patent Document 3). Furthermore, antibody-dependent cell cytotoxicity (ADCC) of mouse splenocytes against HER2-positive human breast cancer cell line SKBR3 is enhanced by mouse monoclonal antibody, and ADCC activity is also antitumor in vivo. There is also a report that it contributes to the effect (Non-Patent Document 4). However, for clinical application, the appearance of human anti-mouse antibodies (HAMA) becomes a problem when using mouse antibodies as they are. Therefore, trastuzumab (trade name: Herceptin (registered trademark)) was prepared as a humanized antibody in which only the variable region, which is the antigen-binding site of the mouse monoclonal antibody against the extracellular domain of HER2 receptor, was transplanted to the constant region of human IgG1. ).

HER2 induces various signal transduction network including PI3K and MAPK, but trastuzumab inhibits this signal transduction pathway by binding to HER2 receptor, and induces cell cycle arrest, apoptosis, angiogenesis inhibition, etc. Then, it is considered that the tumor cell growth inhibitory effect is directly exhibited. In addition, inhibition of Src tyrosine kinase, accompanying activation of PTEN, and dephosphorylation of Akt have been reported. Furthermore, it has been shown that Fc receptors expressed in immune cells such as NK cells bind to the Fc portion of trastuzumab bound to tumor cells and exhibit a tumor cell killing effect (ADCC activity) (Non-Patent Document 1). 5 to 8). Two of these direct tumor cell growth inhibitory actions and ADCC activity are considered to be the main action mechanisms of trastuzumab.

Trastuzumab targets HER2, and its therapeutic effect greatly depends on the expression level of HER2 protein. It is known that the positive rate varies greatly depending on the measurement method and material, but in order to investigate overexpression of HER2 protein and DNA amplification, immunohistochemical method (IHC method) and fluorescence in situ hybridization( FISH) is widely practiced. In addition, trastuzumab has been shown to be highly effective when used in postoperative adjuvant therapy, as shown in several large-scale clinical trials including the HERA trial.

Various studies have shown that inhibition of HER2 by trastuzumab administration has a significant effect on the natural course of breast cancer. However, it has also been found that trastuzumab does not alter the natural course of all HER2-overexpressing breast cancers, and it is estimated that about 1/3 or less of HER2-overexpressing breast cancers respond to initial trastuzumab monotherapy. ing. Similarly, in the case of microscopic metastases, it is suggested that a significant proportion of tumors are resistant to trastuzumab. However, the mechanism of trastuzumab resistance has not been clarified. At present, the resistance mechanisms are: (1) Insufficient access of trastuzumab, so that inhibition of the extracellular region of HER2 is not sufficient, (2) reduction of HER2 expression, (3) downstream regulation of HER2 function Factor changes (p27 ^kip1 reduction, PTEN loss or inactivation, etc.), (4) Signal transduction by alternative pathways (insulin-like growth factor I receptor: IGF1R overexpression), ( 5) Possibility of decreased immunocompetence, especially decreased ADCC activity has been suggested.

The studies on immunocompetence and resistance mechanism mentioned in (5) above are not very active. In recent years, the function of NK cells, which is a factor involved in ADCC activity, which is one of the main action mechanisms of trastuzumab, has been clarified. An inhibitory receptor is expressed in NK cells, and one of them was identified as the lectin-like receptor killer cell lectin-like receptor G1 (KLRG1). Most of the ligands of inhibitory receptors revealed so far on NK cells are related to MHC class I molecules, but in 2006, by M. Ito et al., the ligand of KLRG1 is other than MHC class I molecule. It was reported.

KLRG1 was discovered as a functional molecule MAFA (mast cell function-associated antigen) expressed in the rat basophilic leukemia cell line RBL-2H3 (Non-patent document 9). This KLRG1 suppresses the degranulation reaction by Fc receptor stimulation by cross-linking KLRG1 with an anti-KLRG1 antibody in RBL-2H3 cells. From the cDNA cloning of rat KLRG1, it is found that it is a type II transmembrane protein homodimer having a C-type lectin-like structure in the extracellular region and an ITIM (immunoreceptor tyrosine-based inhibitory motif) in the intracellular region. Reported by Pecht et al. (Non-Patent Document 10). It is known that KLRG1 is expressed in a part of NK cells and T cells in humans and mice (Non-patent Documents 11 to 13), but is expressed in about 50% of peripheral blood NK cells in healthy people.

Trastuzumab treatment is expanding its application from recurrence treatment to postoperative adjuvant therapy for the purpose of preventing recurrence, and preoperative treatment, and further development to improve the curativeness of HER2-positive cancer is expected. It is expected that the number of patients treated with trastuzumab will greatly increase due to the expanded indication. However, at present, there are many trastuzumab ineffective groups among the target cases.

On the other hand, KLRG1 has been reported to bind to three classical cadherins (E-, N-, R-) that are ubiquitously expressed in vertebrates and mediate cell-cell adhesion. It has been suggested that the KLRG1 ligation reaction by E-, N-, R-cadherin regulates the cytotoxic activity of killer cells and prevents damage to the tissues expressing cadherin (Patent Document 14). In addition, the binding between these cadherins and KLRG1 has been investigated, and further, regarding E-cadherin, as a result of SPR measurement, KLRG1 is bound to the N-terminal immunoglobulin-like domain of E-cadherin, KLRG1-E- As a result of crystal structure analysis of cadherin complex, 9 amino acids of E-cadherin (3rd, 4th, 5th, 6th, 7th, 92nd, 93rd, 94th, and 95th residues) interact with KLRG1 and It has been reported that five amino acids (residues 4, 5, 7, 93 and 95) also contain 9 hydrogen bonds (Non-Patent Document 15). Furthermore, using an E-cadherin mutant in which any one of amino acids 2 to 6 from the N-terminal side of E-cadherin was replaced with alanine, the interaction with KLRG1 was analyzed by SPR and flow cytometry. It was reported that KLRG1 recognizes the N-terminal portion of residues 2 to 6 of E-cadherin as a result of evaluation by the KLRG1 reporter assay and cytotoxic activity measurement (Non-Patent Document 16).

Pinkas-Kramarski, R., I. Arloy and Y. Yarden (1997) ErbB receptors and EGF-like ligands: cell lineage determination and oncogenesis through combinatorial signaling. J Mammary Gland Biol Neoplasia, 2:97-107. Schechter, AL, DF Stern, L. Vaidyanathan, SJ Decker, JA Drebin, MI Greene and RA Weinberg (1984) The neu oncogene: an erb-B-related gene encoding a 185,000-Mr tumour antigen. Nature, 312:513- 516. Hudziak RM, Lewis GD, Winget M, Fendly BM, Shepard HM, Ullrich A (1989) p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor. Mol Cell Biol, 9(3):1165- 72. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, Gianni L, Baselga J, Bell R, Jackisch C, Cameron D, Dowsett M, Barrios CH, Steger G, Huang CS, Andersson M, Inbar M, Lichinitser M, Lang I, Nitz U, Iwata H, Thomssen C, Lohrisch C, Suter TM, Ruschoff J, Suto T, Greatorex V, Ward C, Straehle C, McFadden E, Dolci MS, Gelber RD; Herceptin Adjuvant (HERA) Trial Study Team (2005) Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer.N Engl J Med, 353(16):1659-72 Clynes RA, Towers TL, Presta LG, Ravetch JV (2000) Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med, 6(4):443-6. Izumi Y, Xu L, di Tomaso E, Fukumura D, Jain RK (2002) Tumour biology: herceptin acts as an anti-angiogenic cocktail. Nature, 416(6878):279-80 Gennari R, Menard S, Fagnoni F, Ponchio L, Scelsi M, Tagliabue E, Castiglioni F, Villani L, Magalotti C, Gibelli N, Oliviero B, Ballardini B, Da Prada G, Zambelli A, Costa A (2004) Pilot study of the mechanism of action of preoperative trastuzumab in patients with primary operable breast tumors overexpressing HER2. Clin Cancer Res., 10(17):5650-5 Barok, et al., (2007) Trastuzumab causes antibody-dependent cellular cytotoxicity-mediated growth inhibition of submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug resistance. Molecular Cancer Therapeutics, 6:2065-2072. Ortega Soto E, Pecht I (1988) A monoclonal antibody that inhibits secretion from rat basophilic leukemia cells and binds to a novel membrane component.J Immunol, 141(12):4324-32. Abramson J, Xu R, Pecht I (2002) An unusual inhibitory receptor--the mast cell function-associated antigen (MAFA). Mol Immunol, 38(16-18):1307-13. Hanke T, Corral L, Vance RE, Raulet DH (1998) 2F1 antigen, the mouse homolog of the rat "mast cell function-associated antigen", is a lectin-like type II transmembrane receptor expressed by natural killer cells.Eur J Immunol ., 28(12):4409-17 Butcher S, Arney KL, Cook GP (1998) MAFA-L, an ITIM-containing receptor encoded by the human NK cell gene complex and expressed by basophils and NK cells. Eur J Immunol., 28(11):3755-62 Blaser C, Kaufmann M, Pircher H (1998) Virus-activated CD8 T cells and lymphokine-activated NK cells express the mast cell function-associated antigen, an inhibitory C-type lectin. J Immunol., 161(12):6451- Four Masayuki Ito, Takuma Maruyama, Naotoshi Saito, Satoru Koganei, Kazuo Yamamoto, Naoki Matsumoto (2006) Killer Cell lectin-like receptor G1 bind three members of the classical cadherin family to inhibit NK cell cytotoxicity.J Exp Med., 203(2) :289-95 Li Y, Hofmann M, Wang Q, Teng L, Chlewicki LK, Pircher H, Mariuzza RA. (2009) Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition. Immunity., 17 31(1):35-46 Nakamura S, Kuroki K, Ohki I, Sasaki K, Kajikawa M, Maruyama T, Ito M, Kameda Y, Ikura M, Yamamoto K, Matsumoto N, Maenaka K. (2009) Molecular basis for E-cadherin recognition by killer cell lectin -like receptor G1 (KLRG1). J Biol Chem., 2;284(40):27327-35.

The present invention relates to a novel peptide having a low molecular weight and low cost as a ligand having an affinity for KLRG1, and a KLRG1 positive immune cell carrying the above peptide for selectively removing KLRG1 positive immune cells in peripheral blood. Providing a separating material was a problem to be solved.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that in a peptide consisting of an amino acid sequence of 1 to 8 residues, which contains the amino acid sequence of 2 to 7 residues at the N-terminus of E-cadherin, Although the ability to bind to KLRG1 cannot be expressed, a peptide in which the C-terminal side is further extended to the 12th residue has the ability to bind to KLRG1, and by immobilizing this peptide on the surface of a water-insoluble carrier, The present inventors have completed the present invention by finding the fact that KLRG1-positive immune cells can be selectively removed from blood.

That is, aspects of the invention include the following.
(1) A peptide containing the amino acid sequence of Asp-Trp-Val-Ile-Pro-Pro-Ile-Ser-Cys-Pro-Glu-Asn (SEQ ID NO: 1) and having a total number of constituent amino acid residues of 80 or less A KLRG1-positive immune cell separating material comprising a water-insoluble carrier to which is immobilized.
(2) The KLRG1-positive immune cell separating material according to (1), wherein the peptide containing the amino acid sequence of SEQ ID NO: 1 has an active amino acid residue for immobilizing on a water-insoluble carrier added to its C-terminal side.
(3) The KLRG1-positive immune cell separating material according to (2), wherein the active amino acid residue is Lys.
(4) The KLRG1-positive immune cell separating material according to (1), wherein the peptide of SEQ ID NO: 1 is immobilized on the water-insoluble carrier via a spacer.
(5) The KLRG1-positive immune cell separating material according to (4), wherein the spacer has an active amino acid residue added to the binding terminal side with the water-insoluble carrier.
(6) The KLRG1-positive immune cell separating material according to (4) or (5), wherein the active amino acid residue is Lys.
(7) The KLRG1-positive immune cell separating material according to any one of (1) to (6), wherein the water-insoluble carrier is a woven or non-woven fabric.
(8) The KLRG1-positive immune cell separating material according to any one of (1) to (7), wherein the total number of the constituent amino acid residues is 50 or less.
(9) The KLRG1-positive immune cell separating material according to any one of (1) to (7), wherein the total number of the constituent amino acid residues is 40 or less.
(10) The KLRG1-positive immune cell separating material according to any one of (1) to (7), wherein the total number of the constituent amino acid residues is 30 or less.
(11) A peptide consisting of the amino acid sequence of Asp-Trp-Val-Ile-Pro-Pro-Ile-Ser-Cys-Pro-Glu-Asn (SEQ ID NO: 1) is immobilized on the water-insoluble carrier via a spacer. A KLRG1-positive immune cell separating material.
(12) The amino acid sequence of Asp-Trp-Val-Ile-Pro-Pro-Ile-Ser-Cys-Pro-Glu-Asn (SEQ ID NO: 1) is included, and the total number of constituent amino acid residues is 80 or less. , KLRG1-positive immune cell affinity peptide.
(13) The KLRG1-positive immune cell affinity peptide according to (12), which has Lys at the C-terminal side of the amino acid sequence of SEQ ID NO: 1.
(14) The amino acid sequence of Asp-Trp-Val-Ile-Pro-Pro-Ile-Ser-Cys-Pro-Glu-Asn (SEQ ID NO: 1) is included, and the total number of constituent amino acid residues is 80 or less. A KLRG1-positive immune cell separating agent comprising a peptide.
(15) A method for separating KLRG1-positive immune cells, which comprises contacting a solution containing KLRG1-positive immune cells with the KLRG1-positive immune cell separating material according to any one of (1) to (11).

If the separating material of the present invention is used, for example, KLRG1-positive immune cells can be selectively removed from blood, body fluid, cell culture fluid, and treatment liquids thereof. Therefore, it can be expected that an effective anti-cancer effect can be obtained even in a patient who has not conventionally exhibited an anti-cancer effect even when an antibody against a cancer-specific membrane antigen expressed in epithelial cancer cells is administered.

Further, by using the peptide of the present invention, it is possible to inhibit the binding between KLRG1 and a ligand for KLRG1 (eg, E-cadherin) in KLRG1-positive immune cells, and block the inhibitory signal mediated by KLRG1. Therefore, an antibody having antibody-dependent cytotoxic activity against a cancer-specific membrane antigen expressed in epithelial cancer cells, and an epithelial cancer cell positive for the cancer-specific membrane antigen and positive for a ligand for KLRG1 It is expected that sufficient cytotoxic activity will be obtained even when the cell population containing mononuclear cells is brought into contact with the cell population in the past, even when the cytotoxic activity against epithelial cancer cells was not exerted.

Hereinafter, the present invention will be described in detail. The present invention is not limited to the following embodiments, and can be variously modified and implemented within the scope of the gist.

The KLRG1-positive immune cell separating material of the present invention selectively removes KLRG1-positive immune cells from a solution containing KLRG1-positive immune cells such as blood, body fluid, cell culture solution, and a solution obtained by treating these. A separation material capable of being, which comprises the amino acid sequence of Asp-Trp-Val-Ile-Pro-Pro-Ile-Ser-Cys-Pro-Glu-Asn (SEQ ID NO: 1), It is characterized in that peptides having a total number of 80 or less are immobilized on a water-insoluble carrier.

SEQ ID NO: 1 of the peptide used in the separation material of the present invention is the amino acid sequence of the 12th residue from the N-terminal of E-cadherin. In a separation material capable of selectively removing KLRG1-positive immune cells from a solution containing KLRG1-positive immune cells, the present inventors have focused on E-cadherin as a ligand that binds to KLRG1 on the surface of a water-insoluble carrier. Then, it was examined to cut out a part of E-cadherin protein as a peptide for designing. The present inventors have found that the amino acid sequences at the 2nd to 7th residues and at the 92nd to 95th residues of the N-terminal portion of E-cadherin are important in the interaction with KLRG1 from the previous literature (Non-Patent Documents 15 and 16). As a result of further studies, it was confirmed that when a peptide consisting of the amino acid sequence of the 1st to 8th residues, including the 2nd to 7th residues of the N-terminal part, was immobilized on a water-insoluble carrier, KLRG1-positive immunity It has no ability to bind to cells and cannot selectively remove KLRG1-positive immune cells from a solution containing KLRG1-positive immune cells, but the amino acid sequence of the peptide is further extended by 4 amino acids to the C-terminal side, and E-cadherin When a peptide containing SEQ ID NO: 1 at the N-terminal portion 1 to 12 residues was immobilized on a water-insoluble carrier, KLRG1-positive immunity was obtained even without a peptide containing an amino acid sequence at the N-terminal portion 92 to 95 residues. The present inventors have found that KLRG1-positive immune cells can be selectively removed from a solution that expresses a cell-binding ability and that contains KLRG1-positive immune cells, and completed the present invention.

In the case of a peptide consisting of the amino acid sequence of the 1st to 8th residues of the N-terminal portion of E-cadherin, the peptide cannot maintain a three-dimensional structure for binding and capturing KLRG1 on a water-insoluble carrier, and thus has a binding ability. However, in the case of a peptide containing 12 amino acids (SEQ ID NO: 1) which is the amino acid sequence of residues 1 to 12 of the N-terminal portion of E-cadherin, it can interact with KLRG1 on a water-insoluble carrier. It is presumed that it can have a three-dimensional structure and can exhibit binding and capturing ability.

Further, according to the present invention, the number of amino acids of the peptide on the surface of the water-insoluble carrier is 12 or more and 80 or less. On the surface of the water-insoluble carrier, SEQ ID NO: 1 may further extend amino acids to the C-terminal side and N-terminal side of the sequence, and the three-dimensional structure of SEQ ID NO: 1 is maintained even if the number of amino acids is increased. As the number increases, cells other than the KLRG1-positive immune cells are more likely to be nonspecifically adsorbed, so the number of amino acids is preferably 12 or more and 80 or less. Moreover, 12 or more and 50 or less are more preferable, 12 or more and 40 or less are more preferable, 12 or more and 30 or less are more preferable, 12 or more and 20 or less are especially preferable, 12 or more and 15 from the synthetic method or cost. The number of pieces or less is most preferable. The type of amino acid extended to the N-terminal side of the amino acid sequence of SEQ ID NO: 1 is not particularly limited, and any amino acid can be extended. The type of amino acid extended to the C-terminal side of the amino acid sequence of SEQ ID NO: 1 is not particularly limited, and any amino acid may be extended, or the amino acid at the 13th residue and after on the N-terminal side of E-cadherin. The sequence may be extended C-terminal to the amino acid sequence of SEQ ID NO:1.

In addition, the terminal of the peptide may be free, for example, the N-terminal may be blocked by acetylation and the C-terminal may be blocked by amidation, and in consideration of immobilization on a water-insoluble carrier, etc. A functional group may be introduced.

In the separating material of the present invention, the peptide containing the amino acid sequence of SEQ ID NO: 1 is more preferably added with an active amino acid residue for immobilizing it on a water-insoluble carrier at its C-terminal side. The active amino acid residue is an amino acid having an active group for binding to a water-insoluble carrier in its side chain, for example, lysine, arginine, cysteine, tryptophan, histidine, asparagine, aspartic acid, glutamine, glutamic acid, serine, threonine. , Tyrosine, and lysine and cysteine are preferable and lysine is more preferable because they are easily fixed to a water-insoluble carrier.

In the separation material of the present invention, the peptide of SEQ ID NO: 1 may be immobilized on a water-insoluble carrier via a spacer. The term “spacer” as used herein refers to a molecule that is located between a peptide containing the amino acid sequence of SEQ ID NO: 1 and a water-insoluble carrier and that can fix the peptide to the water-insoluble carrier with a space between them. means. The spacer prevents non-specific interaction between cells and the separation material due to the fluidity of the spacer and the excluded volume effect. With respect to the length of the spacer, a preferable result is obtained from one having no spacer to 2000 in terms of the number of atoms contained therein. Spacers that are too long have poor thermal stability and radiation resistance, and cause elution. The number of atoms contained therein is more preferably 1,000 or less, still more preferably 500 or less, and particularly preferably 200 or less, from the one having no spacer. The spacer may be introduced on the surface of the water-insoluble carrier or may be introduced on the peptide. In that case, the position at which the spacer is introduced is a position where the amino acid sequence of SEQ ID NO: 1 is not interrupted. The C-terminal side is more preferable than SEQ ID NO: 1. Specific materials for the spacer include, but are not limited to, polymeric materials such as polyethylene glycol and the like, polypeptides, and the like. In addition, in the spacer, a functional group may be introduced at the binding end with the water-insoluble carrier in consideration of immobilization on the water-insoluble carrier, or an active amino acid residue is preferably added. Active amino acid residues that can be added to the spacer include those mentioned herein above.

In the separation material of the present invention, the peptide of SEQ ID NO: 1 is immobilized on the water-insoluble carrier via a spacer. Specific examples of the embodiment include the case where the spacer is introduced into the peptide and the spacer is introduced into the water-insoluble carrier. Or a spacer is introduced into both the peptide and the water-insoluble carrier. That is, the peptide-spacer conjugate may be immobilized on a water-insoluble carrier, the peptide may be immobilized on a water-insoluble carrier having a spacer, or the peptide-spacer conjugate may have a spacer. It may be immobilized on a water-insoluble carrier. At this time, a functional group (an active amino acid residue or the like) for fixing the peptide to the water-insoluble carrier can be appropriately added to any one or more of the peptide, the spacer and the water-insoluble carrier.

The shape of the water-insoluble carrier that can be used in the present invention is preferably a surface area larger than the contact frequency in order to make good contact with a solution containing KLRG1-positive immune cells in the liquid layer. Examples include fibrous structures such as non-woven fabrics, fibrous, cotton-like, yarn-like, bundle-like, blind-like and woven fabrics, flat membranes, polymeric porous bodies such as sponges, and particles (such as magnetic particles). The structure is mentioned. In particular, woven fabrics and non-woven fabrics are preferable from the viewpoint of adsorbability of blood cells and handleability as a separating material, and among them, non-woven fabrics are most preferable because they can be contacted with cells at multiple points.

When a fibrous carrier such as a non-woven fabric is used as the shape of the water-insoluble carrier, the fiber diameter contributes to the cell adsorbing ability, and therefore it is important to use an effective average fiber diameter. The average fiber diameter of the present invention is determined by photographing the surface of the fibrous substrate with a scanning electron microscope and visually measuring 100 or more randomly distributed diameters of the yarns dispersed on the photographed surface. If the fiber diameter is too large, the adsorption amount and adsorption rate of KLRG1-positive immune cells will decrease, and if it is too small, non-specific adsorption of lymphocytes other than KLRG1-positive immune cells, monocytes, granulocytes, platelets, etc. The average fiber diameter is preferably 0.5 μm or more and 30 μm or less from the viewpoint of easily causing the above. More preferably, it is 1 μm or more and 20 μm or less. Most preferably, it is 2 μm or more and 10 μm or less. Further, the filament may be a monofilament or a multifilament, and may be a porous filament or an atypical filament.

When the water-insoluble carrier has a granular shape, it can be effectively used in any shape such as spherical or polygonal. Further, any surface can be usefully used as long as it has a smooth surface or unevenness and can selectively remove KLRG1-positive immune cells. The particle size is preferably 50 μm or more and 10 mm or less. If the particle size is less than 50 μm, it is difficult to prevent the particles from flowing out, which is not preferable. If the particle size is larger than 10 mm, a sufficient surface area tends not to be obtained, which is not preferable. The particle size is preferably 80 μm or more and 8 mm or less, and most preferably 100 μm or more and 6 mm or less.

Examples of the material of the water-insoluble carrier that can be used in the present invention include polyesters such as polyethylene terephthalate, polybutylene terephthalate and polyoxyethylene terephthalate, polyamides such as polyacrylonitrile, nylon 6, nylon 6,6, aromatic polyamide, polystyrene. And its derivatives, polyolefins such as polyethylene, polypropylene and polybutene, polymer compounds obtained by polymerizing methacrylic acid ester derivatives such as methyl methacrylate and ethyl methacrylate, and acrylic acid ester derivatives such as methyl acrylate and ethyl acrylate. Examples of the polymer compound include synthetic polymers such as polytrifluorochloroethylene, polyvinyl formal, polysulfone, polyurethane, polyvinyl acetal, and polycarbonate, and regenerated fibers such as cellulose and/or its derivatives. These may be used alone, or may be used by blending or alloying the above materials. Preferable examples include polyolefin such as polyethylene and polypropylene, polystyrene such as styrene-butadiene copolymer, and the like, from the viewpoint of moldability of fibers and easy introduction of active groups. In particular, polyethylene and polypropylene are recommended in terms of less eluate. Further, a material whose surface is modified with a hydrophilic polymer material by a method such as coating or radiation grafting for the purpose of imparting hydrophilicity to these is also useful.

As the method for allowing the peptide of the present invention to be present on the surface of the water-insoluble carrier, any known method such as covalent bond, ionic bond, physical adsorption, embedding, or precipitation insolubilization on the carrier surface can be used, but ligand elution From the viewpoint of properties, it is preferable to use by fixing and insolubilizing by covalent bond. Therefore, a well-known method of introducing an active group into a carrier, which is usually used for immobilized enzymes and affinity chromatography, is favorably used. For example, a method of using a carrier having a carboxyl group and converting the carboxyl group to N-hydroxysuccinimide to convert to a succinimideoxycarbonyl group, and reacting the peptide at the amino group portion ( Active ester method), a method of using a carrier having an amino group or a carboxyl group, and subjecting the amino group or the carboxyl group of the carrier to the carboxyl group or the amino group of the peptide in the presence of a condensation reagent such as dicyclohexylcarbodiimide to bond them. (Condensation method), a method in which a carrier and a peptide are crosslinked and bonded using a compound having two or more functional groups such as glutaraldehyde (carrier crosslinking method), a carrier having a hydroxyl group is used, and cyan bromide or the like is used. A method in which cyanogen halide is allowed to act on a carrier to react and bond at the amino group portion of a peptide or protein, a method of using a carrier having a thiol group and a reaction at the thiol group portion of a peptide or protein, and epichlorohydride A method in which an epoxide such as phosphorus is allowed to act on the carrier to react by binding at the amino group portion or hydroxyl group portion of the peptide, and an α-acetaminohalogen group is introduced onto the carrier surface using N-hydroxymethylhaloacetamide or the like. Method of reacting at the thiol group of peptide or protein, grafting a vinyl compound such as glycidyl methacrylate to a water-insoluble carrier that has generated radicals by irradiation with γ-rays or electron beams and introducing an epoxy group into the peptide or peptide Examples thereof include a method of reacting at the amino group or thiol group of the protein, but the method is not limited thereto. As a method for directly synthesizing a peptide on a water-insoluble carrier, a binding group that is not cleaved even under the condition of removing a protective group is added to the water-insoluble carrier, and the peptide is immobilized on the water-insoluble carrier as a solid phase of the following peptide. Examples include a method of synthesizing by a synthetic method and removing the protective group from all the functional groups to which the protective group is added.

The amount of the peptide immobilized on the water-insoluble carrier is not particularly limited, but in order to effectively express the function of the peptide, it is preferably 0.01 nmol/m ² or more and 100 μmol/m ² or less per surface area of the water-insoluble carrier. preferably at 0.1 nmol / m ² or more 10 .mu.mol / m ² or less, further preferably 1.0 nmol / m ² or more 1.0 [mu] mol / m ² or less.

In the present invention, the type of KLRG1-positive immune cells is not particularly limited, and examples thereof include KLRG1-positive NK cells and KLRG1-positive T cells.

In the present invention, when the solution containing KLRG1-positive immune cells is blood, an anticoagulant can be added to blood for the purpose of anticoagulating blood. Examples of the anticoagulant include, but are not particularly limited to, compounds having anticoagulant activity, heparin, low molecular weight heparin, nafamostat mesylate, gabexate mesylate, argatroban, sodium citrate and the like are preferred examples. Heparin or sodium citrate is preferably used.

Peptide synthesis is performed by a method usually used in peptide synthesis, for example, a solid phase method or a liquid phase method, but the solid phase method is preferable because the operation is simple. By further purifying the synthesized peptide, a highly pure peptide can be obtained. For purification of the peptide, a method generally used for purification of organic compounds can be used. In particular, purification by column chromatography is preferable because efficient purification can be performed. The spacer can be introduced by a known synthesis method. For example, in peptide synthesis by the solid phase method, a spacer molecule having an amino group and a carboxyl group may be introduced in the same manner as an amino acid, or by a liquid phase method. You may.

As described above, the separation material of the present invention requires expensive ligands such as antibodies and proteins from solutions containing KLRG1-positive immune cells such as blood, body fluids, cell culture fluids, and treatment solutions thereof. Since it is possible to selectively separate KLRG1-positive immune cells, it is effective as a KLRG1-positive immune cell separator for extracorporeal circulation for the purpose of selective cell removal by filling a container having at least an inlet and an outlet. Can be used for.

The KLRG1-positive immune cell separating material of the present invention is, for example, KLRG1-positive in the peripheral blood of a living body of epithelial cancer in which the cancer-specific membrane antigen expressed in epithelial cancer cells is positive and the ligand for KLRG1 is positive. A step (a) of selectively reducing immune cells in vitro, and an antibody against the cancer-specific membrane antigen expressed in the epithelial cancer cells, the antibody having antibody-dependent cytotoxic activity in the organism; The method for treating a living body of epithelial cancer, which comprises the step (b) of administering the contained cancer therapeutic agent, can be used in the step (a). In the step (a), preferably, a KLRG1-positive immune cell of the present invention, which is a cell separator that extracorporeally circulates peripheral blood of a living body and has a higher affinity for a KLRG1-positive immune cell than a KLRG1-negative immune cell. KLRG1-positive immune cells can be selectively reduced through the separating material. The cancer-specific membrane antigen expressed in the epithelial cancer cells is preferably HER2, and the antibody against the cancer-specific membrane antigen expressed in the epithelial cancer cells is preferably trastuzumab.

In another embodiment, immune cells taken out from the patient's body are grown by cell culture treatment, and the immune cells are brought into contact with the separating material of the present invention to selectively remove KLRG1-positive immune cells. Then, the liquid to be treated from which the KLRG1-positive immune cells have been selectively removed can be returned to the patient again.

In addition, since the peptide of the present invention has an affinity for KLRG1-positive immune cells, for example, a fluorescent substance such as FITC is introduced, KLRG1-positive immune cells are labeled, and KLRG1-positive immune cells are detected using a flow cytometer. It can be effectively used for detection and analysis of KLRG1 or KLRG1-positive immune cells as an ELISA method instead of an antibody or protein after being used for analysis or immobilized on a microplate. In addition, it can be expected that these peptides can be effectively used as drugs and carrier substances for drugs against diseases in which KLRG1-positive immune cells are involved. For example, the binding of KLRG1 in a KLRG1-positive immune cell to a ligand for KLRG1 (eg, E-cadherin) can be inhibited, and the inhibitory signal mediated by KLRG1 can be blocked. Therefore, an antibody having antibody-dependent cytotoxic activity against a cancer-specific membrane antigen expressed in epithelial cancer cells, and an epithelial cancer cell positive for the cancer-specific membrane antigen and positive for a ligand for KLRG1 It is expected that sufficient cytotoxic activity will be obtained even when the cell population containing mononuclear cells is brought into contact with the cell population in the past, even when the cytotoxic activity against epithelial cancer cells was not exerted.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

<Preparation of water-insoluble carrier>
Polypropylene non-woven fabric (hereinafter abbreviated as PP non-woven fabric, P080HW-00F manufactured by TAPYRUS Co., Ltd., weight 80 g/m ² , average fiber diameter 5.1 μm) 0.158 m ² (area) is enclosed in a low oxygen permeability bag together with an oxygen scavenger. After sufficiently removing oxygen, it was irradiated with 100 kGy γ-rays at −78° C. 50 mL of glycidyl methacrylate (GMA) was dissolved in 450 mL of methanol, and nitrogen was passed through at 40° C. for 60 minutes for deoxidation treatment. The above-mentioned non-woven fabric was quickly put in a pressure-resistant glass container, and after the pressure was reduced, the above-mentioned solution was drawn in and reacted at 40°C for 15 minutes. After the reaction, the unwoven fabric taken out was washed with dimethylformamide and methanol and vacuum dried at 35° C. overnight to obtain a PP nonwoven fabric in which GMA was graft-polymerized (hereinafter, abbreviated as GMA nonwoven fabric). The graft ratio calculated from the increase in weight before irradiation with γ rays was 99%. Here, the graft ratio (%) is a value represented by the following formula.
Graft ratio (%)=[(weight of nonwoven fabric after grafting-weight of nonwoven fabric before grafting)/(weight of nonwoven fabric before grafting)]×100

Next, 10 g of the GMA non-woven fabric was immersed in ethanol and then washed with water for injection to make it hydrophilic, then immersed in 200 ml of 25 wt% ammonia water, and allowed to stand at 25° C. for 4 hours. After the reaction, the non-woven fabric taken out was washed with water for injection until the washing water became neutral, and vacuum dried overnight at 35° C. to obtain an aminated GMA non-woven fabric (hereinafter, referred to as GMA(NH3) non-woven fabric). Abbreviated).

Next, 1.0 g of GMA (NH3) non-woven fabric was immersed in 12 ml of polyethylene glycol diglycidyl ether (Nagase Chemtex Co., Ltd., Denacol EX-821, n=4, hereinafter abbreviated as EX-821), and a reaction vessel. The whole was kept at 25° C. for 22 hours with gentle shaking. After that, the taken out non-woven fabric is washed 10 times with water for injection and vacuum dried at 40° C. overnight to form a non-woven fabric in which EX-821 is introduced into the GMA(NH3) non-woven fabric (hereinafter, abbreviated as EX-821 non-woven fabric) Got

<Preparation of peptide>
The peptides listed in Table 1 were synthesized by the Fmoc solid phase method. The peptide chain was extended using p-alkoxybenzyl alcohol resin as a carrier. For peptides modified with biotin at the N-terminus (peptides 1 and 7), after extension of the peptide chain, deprotection of Fmoc at the N-terminus was followed by attachment of D(+)-biotin to the N-terminus in the same manner as the extension of the peptide chain. .. The peptides in which the side chain of lysine was modified with biotin (peptides 2 and 8) were peptide-extended using Fmoc-Lys(biotin)-OH. The N-terminal acetylated peptides (peptides 3, 4, 5, 6, 9, and 10) were acetylated by reacting with acetic anhydride after deprotecting the N-terminal Fmoc after peptide chain extension. Unmodified N-terminal peptides (peptides 2, 8) deprotected N-terminal Fmoc. Peptide 6 was repeatedly used three times using 8-(Fmoc-amino)-3,6-dioxaoctanoic acid (Peptides International, Inc., Fmoc-mini-PEG) instead of Fmoc-(protected) amino acid as a spacer. It was extended and introduced. Next, after deresinating and deprotecting to obtain a crude peptide, purification was carried out by reverse phase high performance liquid chromatography, and the obtained purified peptide was identified by confirming its molecular weight by MALDI TOF-MS.

<Preparation of cell separation material>
Eight pieces obtained by cutting the EX-821 non-woven fabric obtained above into circles having a diameter of 0.68 cm were immersed in ethanol, and subsequently, a phosphate buffer solution containing no calcium or magnesium (hereinafter abbreviated as PBS(−)). ), it was made hydrophilic. Further, the peptides shown in Table 1 were dissolved in PBS(-) to prepare a 200 nmol/mL peptide solution. Eight hydrophilic EX-821 non-woven fabrics were immersed in 0.8 ml of the peptide solution and allowed to stand at 37° C. for 40 hours to obtain a non-woven fabric on which peptides were immobilized (hereinafter abbreviated as peptide-immobilized non-woven fabric). Next, the peptide-immobilized nonwoven fabric is washed with PBS(-), immersed in 0.8 ml of 0.2% polyoxyethylene sorbitan monolaurate/PBS(-) solution, and allowed to stand at room temperature for 2.5 hours, and then PBS. The cells were washed with (-) to obtain a cell separation material.

<Preparation of cell separator>
A container having an inlet and an outlet and having a volume of about 1 ml was filled with the above-mentioned four cell separation materials and PBS(-) as a filling solution to obtain a cell separator.

Examples 1-6, Comparative Examples 1-4
<Evaluation of selective removal performance of KLRG1-positive NK cells>
4 ml of physiological saline was passed through the column inlet of the cell separator using a syringe pump at a flow rate of 1.0 ml/min for priming. Subsequently, 2.0 ml of human fresh blood (blood:ACD-A solution=8:1) to which ACD-A (acid citrate dextrose solution-A) solution was added as an anticoagulant was flowed from the column inlet of the cell separator. The liquid was passed at 0.2 ml/min, and the treated blood was collected from the column outlet.

The number of lymphocytes in blood before and after the treatment with the cell separator was measured using a microcell counter (XT-1800i manufactured by Sysmex Corporation). In addition, each of these bloods was diluted 1:1 with PBS(-) and overlaid on Ficoll-Paque PLUS (manufactured by GE Healthcare), and centrifuged at 400 xg for 30 minutes to collect a mononuclear cell suspension. did. This mononuclear cell suspension was directly or indirectly fluorescently labeled with anti-human CD3 antibody, anti-human CD56 antibody, anti-human KLRG1 antibody, and a flow cytometer (BECTON DICKINSON, FACS CantoII) was used. The number of NK cells in lymphocytes and the KLRG1 positive rate of NK cells were measured.

The KLRG1-positive NK cell removal rate and the KLRG1-negative NK cell removal rate were calculated from the number of NK cells in the blood before and after the treatment with a cell separator and the KLRG1-positive rate of the NK cells, and the ratio (=KLRG1-positive NK cell removal rate) was calculated. /KLKL1-negative NK cell removal rate), the selective removal performance of KLRG1-positive NK cells was evaluated. The results are shown in Table 2.

Reference example 1
As the water-insoluble carrier, a GMA nonwoven fabric was used instead of the EX-821 nonwoven fabric, and instead of the peptide, recombinant human E-cadherin (Recombinant Human E-Cadherin/Fc Chimera, manufactured by R&D Systems Inc.) was immobilized on the water-insoluble carrier. As in Example 1 except for the above, the ability of the cell separating material to selectively remove KLRG1-positive NK cells was evaluated. The results are shown in Table 2.

Examples 7 and 8, Comparative Examples 5 and 6
<Evaluation of peptide binding ability to KLRG1-positive lymphocytes and KLRG1-negative lymphocytes>
A mononuclear cell suspension was prepared from the fresh human blood to which the ACD-A solution was added as an anticoagulant by using the above method. The mononuclear cell suspension was subjected to Fc receptor blocking treatment using an Fc receptor blocking agent (ClearBack (Human Fc receptor blocking reagent) manufactured by MBL).

A mouse anti-human KLRG1 polyclonal antibody (manufactured by Abnova Corporation, KLRG1 purified MaxPab mouse polyclonal antibody (B01P)) using a mononuclear cell suspension as a primary antibody, and a FITC-labeled goat anti-mouse IgG (γC) antibody as a secondary antibody. (Manufactured by Biotech) was used to distinguish between KLRG1-positive lymphocytes and KLRG1-negative lymphocytes. Then, after reacting with the biotin-labeled peptide of Table 3, it was labeled with PE Streptavidin (manufactured by BD Biosciences), and the peptide-positive rate to KLRG1-positive lymphocytes and the peptide-positive rate to KLRG1-negative lymphocytes were measured using a flow cytometer. evaluated. The results are shown in Table 3.

In order to confirm the present evaluation system, a hamster anti-mouse KLRG1 monoclonal antibody (manufactured by eBioscience, Biotin anti-mouse KLRG1/MAFA) that cross-reacts with human KLRG1 was used instead of the biotin-labeled peptide, and the same manner was used. The ability of KLRG1 monoclonal antibody to bind to KLRG1-positive lymphocytes and KLRG1-negative lymphocytes was evaluated. As a result, the positive rate of KLRG1 monoclonal antibody to KLRG1-positive lymphocytes was 96.5%, and the positive rate of KLRG1 monoclonal antibody to KLRG1-negative lymphocytes was 4.2%. It was confirmed that the peptide binding ability to spheres and KLRG1-negative lymphocytes can be evaluated.

Claims (15)

A peptide containing the amino acid sequence of Asp-Trp-Val-Ile-Pro-Pro-Ile-Ser-Cys-Pro-Glu-Asn (SEQ ID NO: 1) and having a total number of constituent amino acid residues of 80 or less is immobilized. A material for separating KLRG1-positive immune cells, which comprises a water-insoluble carrier. The KLRG1-positive immune cell separating material according to claim 1, wherein an active amino acid residue for fixing to a water-insoluble carrier is added to the C-terminal side of the peptide containing the amino acid sequence of SEQ ID NO: 1. The KLRG1-positive immune cell separating material according to claim 2, wherein the active amino acid residue is Lys. The KLRG1-positive immune cell separating material according to claim 1, wherein the peptide of SEQ ID NO: 1 is immobilized on the water-insoluble carrier via a spacer. The KLRG1-positive immune cell separating material according to claim 4, wherein the spacer has an active amino acid residue added to the binding terminal side with the water-insoluble carrier. The KLRG1-positive immune cell separating material according to claim 4 or 5, wherein the active amino acid residue is Lys. The KLRG1-positive immune cell separating material according to any one of claims 1 to 6, wherein the water-insoluble carrier is a woven fabric or a non-woven fabric. The KLRG1-positive immune cell separating material according to any one of claims 1 to 7, wherein the total number of the constituent amino acid residues is 50 or less. The KLRG1-positive immune cell separating material according to any one of claims 1 to 7, wherein the total number of the constituent amino acid residues is 40 or less. The KLRG1-positive immune cell separating material according to any one of claims 1 to 7, wherein the total number of the constituent amino acid residues is 30 or less. KLRG1 in which a peptide consisting of the amino acid sequence of Asp-Trp-Val-Ile-Pro-Pro-Ile-Ser-Cys-Pro-Glu-Asn (SEQ ID NO: 1) is immobilized on the water-insoluble carrier via a spacer. Positive immune cell separation material. KLRG1-positive, containing the amino acid sequence of Asp-Trp-Val-Ile-Pro-Pro-Ile-Ser-Cys-Pro-Glu-Asn (SEQ ID NO: 1), and having a total number of constituent amino acid residues of 80 or less Immune cell affinity peptide. The KLRG1-positive immune cell affinity peptide according to claim 12, which has Lys at the C-terminal side of the amino acid sequence of SEQ ID NO: 1. Includes a peptide containing the amino acid sequence of Asp-Trp-Val-Ile-Pro-Pro-Ile-Ser-Cys-Pro-Glu-Asn (SEQ ID NO: 1) and having a total number of constituent amino acid residues of 80 or less. , A KLRG1-positive immune cell separating agent. A method for separating KLRG1-positive immune cells, which comprises contacting a solution containing KLRG1-positive immune cells with the KLRG1-positive immune cell separating material according to any one of claims 1 to 11.