JP2008174466A - Carrier for idiotype antigen and idiotype vaccine using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method, a reagent (carrier), a vaccine, and the like, each for efficiently making cytotoxic lymphocytes to recognize a cancer-specific antigen in a cancer-specific immunotherapy, especially to provide a method, a reagent (carrier), a vaccine, and the like, each for enabling that the antigen can especially efficiently be taken into antigen-exhibiting cells and simultaneously activate the antigen-exhibiting cells, even when an immunization globulin massively existing in the body is used as a cancer-specific antigen. <P>SOLUTION: An idiotype vaccine comprises a composite material which comprises an idiotype antigen and a carrier comprising urate crystals. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention provides a carrier for specific antigens for immune stimulation, more specifically, for efficiently inducing cytotoxic T cells (hereinafter sometimes referred to as CTL) targeting cancer cells. It relates to a carrier for a specific antigen. The present invention also relates to an antigen complex using these carriers, an immunostimulatory composition (vaccine) containing the same, a method for producing the vaccine, and the like.

  In recent years, tumor immunotherapy has been developed through the discovery of tumor-specific antigens and the use of dendritic cells. Tumor-specific cellular immunity using cancer antigen protein (cancer vaccine) can be applied to cancer treatment and recurrence prevention. Tumor-specific cellular immunity consists of (1) preparation of specific antigen (cancer antigen protein), (2) antigen sensitization to antigen-presenting cells, and (3) education and proliferation of CTL by antigen-presenting cells. It is evoked through.

  When a cancer cell has a unique antigen, the antigen protein is decomposed into a peptide in the tumor cell, a part forms a complex with MHC class I, and is presented on the surface of the cancer cell. CTL present in peripheral blood binds to the MHC class I complex of cancer cells via the T cell antigen receptor on the cell surface, and actively attacks when the cancer cells are recognized as non-self. To do. Therefore, CTL can be efficiently induced by using a cancer-specific antigen as presented on a class I molecule as a cancer vaccine.

  Once a cancer-specific antigen has been prepared, the challenge for successful cancer-specific immunotherapy is how to make the cancer-specific antigen recognized by cytotoxic lymphocytes. Dendritic cells (hereinafter sometimes referred to as DC) are cells having the most powerful antigen-presenting function in the living body, presenting the antigens they have taken up to naive T cells, and antigen-specific CTL proliferation activity. And has the ability to function specific immunity. It is known that when dendritic cells are sensitized with an antigen and mixed with lymphocytes, CTLs specific to tumors are induced most efficiently and sensitization efficiency is increased. Therefore, dendritic cells are used as cells that serve as a control tower for tumor-specific cellular immunotherapy using cancer vaccines.

  Multiple myeloma is one of the hematopoietic malignancies similar to leukemia and malignant lymphoma, and is a disease that develops when cancer cells are differentiated and matured from lymphocytes, which are a type of white blood cell. Plasma cells produce a protein (antibody = immunoglobulin) that has the effect of eliminating foreign substances such as viruses and bacteria that have entered the body. An immunoglobulin is divided into a Fab region (ab fraction; Fab) portion including a variable region and a constant region (Fc) portion, and the Fab variable region is an idiotype antigen specific to the target antigen. Since plasma cells produce only one type of immunoglobulin, the variable region portion of cancerous plasma cells (myeloma cells) can be used as a tumor-specific antigen.

  Already, the present inventors excised the IgG variable region from the culture supernatant of the multiple myeloma cell line IM-9 to form an idiotype antigen, which was derived from monocytes of volunteers matched with IM-9 cells and HLA-A. In an experimental system co-cultured with lymphocytes obtained from the same volunteer after sensitizing the resulting dendritic cells, it was proved that the proliferated lymphocytes had cytotoxicity against the myeloma cell line IM-9 (non-patented). References 1 and 2). That is, in multiple myeloma, there is a possibility that tumor-specific cytotoxic T cells can be efficiently induced by sensitizing dendritic cells with an immunoglobulin Fab region that can be purified relatively easily as an antigen. It has been proven.

  However, since immunoglobulin is present in a large amount in the patient, it is expected that it is difficult to sensitize as a cancer antigen. Therefore, even when immunoglobulins are used as antigens, tumors can be more efficiently developed by developing a method that allows antigens to be specifically taken into antigen-presenting cells and simultaneously activate antigen-presenting cells. It is desired that specific cellular immunity be elicited.

  Sodium urate (MSU) crystals are substances that form in the human body due to precipitation of uric acid that is excessively present in body fluids in the body and cause a specific joint inflammation state called gout attack. The MSU crystal has a long needle shape or a saddle shape of 2 to 20 μm, and the crystal surface is irregular, and has a surface structure in which space filling is poor and the flammability is strong. The surface of the crystal has a negative charge, and adhesion of immunoglobulins and lipoproteins centering on IgG is observed, and IgG is said to be bound to the MSU crystal at the positively charged Fab portion (Non-patent Document) 3).

  In recent years, it has come to be considered that MSU crystals work as an important substance of local immune reaction. For example, Shi et al. Show that uric acid released from locally injured cells acts on dendritic cells as an intrinsic danger signal, and that adjuvant activity is seen when pre-formed MSU crystals are injected in vivo. (Non-Patent Document 4). In addition, Hu et al. Reported that immune rejection increased depending on the uric acid concentration and that xenograft rejection was promoted by subcutaneous administration of MSU crystals (Non-patent Document 5).

However, there is no description of pre-binding MSU crystals to specific antigens.
Ippei Sakamaki, Takanori Ueda, Yumie Takasawa, Hiromichi Iwasaki, Kunihiro Inai, Hiroshi Tsuya: “Induction of allogenic CTL against myeloma cell line using Fab as an antigen”. 65th Annual Meeting of the Japanese Society of Hematology, 45th Annual Meeting of the Japanese Society of Clinical Hematology, 2003.8. Clinical Blood 44 (8), 874, 2003, 8. Sakamaki, I., Inai, K., Takazawa, Y., Tsutani, H., * Ueda, T .: Clonal expansion of TCR V beta repertoires in CD8 and CD4 T cells in response to idiotype protein. American Association for Cancer Research 95th Annual Meeting, Abstract. 45, 294-295, 2004, 3. Kozin F. et al., J Lab Clin Med. 1980 Shi et al., Nature 425: 516-521 (2003) Hu et al., Cancer Res. 64: 5059-5062 (2004)

  An object of the present invention is to provide a method, a reagent (carrier), a vaccine and the like for efficiently recognizing a cancer-specific antigen to cytotoxic lymphocytes in cancer-specific immunotherapy. In particular, even when immunoglobulin present in large amounts in the body is used as a cancer-specific antigen, the antigen is specifically and efficiently taken into the antigen-presenting cell and simultaneously activates the antigen-presenting cell. It is an object to provide a method and a reagent (carrier), a vaccine, and the like that make it possible.

  The present inventors have found that the above object can be achieved by using MSU crystals as an antigen carrier, and have completed the present invention.

That is, according to the present invention,
[1] An idiotype vaccine comprising a complex comprising an idiotype antigen and a carrier comprising urate crystals;
[2] The idiotype vaccine according to [1], wherein the idiotype antigen is an immunoglobulin produced by multiple myeloma cells or a part thereof including a variable region;
[3] The idiotype vaccine according to the above [1] or [2], wherein the urate crystals are those obtained by ultrasonically treating needle-like crystals;
[4] A method for producing the idiotype vaccine according to any one of [1] to [3] above, wherein the idiotype antigen and urate crystals are allowed to stand together to leave the idiotype antigen and Forming a complex with urate crystals;
[5] A method comprising stimulating dendritic cells using the idiotype vaccine according to any one of [1] to [3], and stimulating cytotoxic T cells using the dendritic cells. A method of promoting an immune response of specific cytotoxic T cells;
[6] A carrier for producing the idiotype vaccine according to any one of [1] to [3] above, which is a carrier made of sodium urate crystals.

  The idiotypic antigen carrier of the present invention can efficiently take up an idiotype antigen into dendritic cells. Further, by using the idiotype antigen carrier of the present invention, dendritic cells can be activated simultaneously with the promotion of antigen uptake. That is, the carrier of the present invention allows immature dendritic cells with excellent antigen uptake ability to take up antigens, and at the same time, mature dendrites with excellent antigen presentation ability without using various conventionally used mature substances. This is very advantageous because it allows maturation into cells. As a result, the idiotype vaccine using the idiotype antigen carrier of the present invention is very effective and is expected to increase the possibility of successful tumor-specific immunotherapy. Further, since the idiotype antigen carrier of the present invention is based on a substance that can exist in the living body, there is no risk of causing unpredictable side effects, and the safety is high. Therefore, a complex obtained by binding a tumor-derived idiotype antigen to the carrier of the present invention can be used as a cancer vaccine (anticancer agent).

  The carrier of the present invention consists of urate crystals. Urate crystals are known in various shapes, but any shape may be used, and crystals may be produced by any known method. In general, a solution containing uric acid is prepared by dissolving uric acid that is hardly soluble in water by alkali or heating, and crystals are precipitated by standing and cooling.

  Smaller crystal sizes are advantageous for cellular uptake. For example, the crystal is preferably smaller in size than the dendritic cell so as to be easily phagocytosed, for example, having a diameter of 10 μm or less. In order to obtain fine crystals, once produced crystals may be crushed by ultrasonic treatment or the like. In this case, from the viewpoint of easy crushing, it is preferable to use only acicular crystals or crystals mainly containing acicular crystals.

  Particularly preferred as urate are sodium urate (MSU) and sodium hydrogen urate, and most preferred is monosodium urate monohydrate. The urate salt in vivo such as in gout nodules is sodium hydrogen urate, and further contains 1 molecule of crystal water to form monosodium urate monohydrate (Non-patent Document 3). MSU crystals, together with calcium pyrophosphate crystals and hydroxyapatite, are causative substances for crystal-induced arthritis, and are the crystals with the strongest inflammatory effect. In addition, it is most preferable as a carrier from the viewpoints of safety to living bodies and ease of production.

  In addition, the carrier of the present invention may be appropriately modified or modified as long as it retains functions such as protein binding ability and dendritic cell sensitization ability.

Urate crystals are known to be negatively charged. Thus, the carrier of the present invention has the physical property of electrically binding to a positively charged immunoglobulin Fab moiety. The idiotype vaccine of the present invention comprises a complex composed of the carrier comprising the urate crystals of the present invention and the idiotype antigen bound in this manner. The idiotype antigen contained in the idiotype vaccine of the present invention may be any protein or polypeptide containing at least a variable region, for example, whole immunoglobulin or a portion containing a variable region, such as Fab, F (ab ′) _2. , Light chain, heavy chain, and the Fab portion of the heavy chain. For vaccine use, IgG is preferably IgG, but in some cases, other immunoglobulin or the like or a portion derived therefrom may be used. The preparation of such antibodies and antibody fragments are well known to those skilled in the art.

  The idiotype vaccine of the present invention can be applied for therapeutic purposes to tumors that produce monoclonal proteins typified by multiple myeloma. Therefore, an idiotype antigen can be obtained from blood of a patient such as multiple myeloma.

  These complexes can be obtained by binding the carrier of the present invention to the idiotype antigen. The binding between the carrier and the antigen can be easily caused only by mixing a suitable amount of both and allowing to stand. For example, a complex is formed by mixing urate crystals: Fab in a phosphate buffered saline in a weight ratio of about 0.1: 1 to 4: 1, and allowing to stand at 4 ° C. to room temperature for 1 to 48 hours. Is done. Optimum complex formation conditions under specific conditions can be appropriately determined by the method described below.

  The idiotype vaccine of the present invention can be administered directly to a patient. The administration route is not particularly limited, but a parenteral route is preferable, generally intradermal administration, and preferably intradermally in the vicinity of lymph nodes. Therefore, the idiotype vaccine of the present invention includes a complex of the idiotype antigen and the carrier of the present invention, as well as sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium hydroxide, A buffering agent such as hydrochloric acid and a pH adjusting reagent may be added, and a soothing agent such as benzyl alcohol, lactose hydrate, D-sorbitol, L-glutamine as necessary depending on the administration route and dosage form. Ingredients known in the pharmaceutical field such as stabilizers such as gelatin and inactivators such as phenol and formalin may be included. Moreover, you may combine with another anticancer agent etc.

  The dose varies depending on the patient's condition, administration route, dosing schedule, etc., but generally, the antigen amount is about 1.0 to 20.0 μg / kg body weight, and the carrier amount is 2.0 to 40.0 μg / kg. It is about kg body weight (3.0 to 60.0 μg / kg body weight as the amount of the complex of antigen and carrier). Since the dosage of such urate crystals is very small, it corresponds to about 20,000 to 1/1000 of the amount of MSU crystals in gout nodules found in the skin of gout patients, and gout that can be recognized visually. Gout attacks that do not make nodules and are not seen in the skin in the first place are not likely to be triggered. Moreover, even if all dissolved and moved into the blood, the serum uric acid level is increased by 0.0025 to 0.05 mg / dl, and it is considered that administration of the carrier promotes deposition of urate crystals in the tissue. Absent. Therefore, it is considered that there is almost no possibility of side effects due to urate crystals.

  Moreover, the idiotype vaccine of the present invention produces dendritic cells targeting cancer cells by sensitizing dendritic cells with the idiotype vaccine of the present invention outside the body without direct administration, Further, the dendritic cells can be used to induce specific CTLs. Tumor treatment can also be performed by providing such a dendritic cell or CTL to a patient.

1. Preparation of sodium urate (MSU) crystal carrier (1)
Sodium urate crystals were prepared as follows based on the method of MaCarty et al. (Lancet 2: 682, 1962).

  To 336 mg of uric acid (Nacalai Tesque, Kyoto), 40 mL of distilled water and 0.080 mg of sodium hydroxide were added and stirred. Distilled water was further added to make 80 mL, and the mixture was heated to boiling to completely dissolve uric acid. After heating, the solution volume was adjusted again to 80 mL (uric acid concentration 420 mg / dL), and sterilized by filtration when the temperature reached 45 ° C. under natural cooling (trade name “Sterilcup-GP Filter Unit”, Nippon Millipore Corporation ),Tokyo). The filtrate was quickly dispensed into 1.5 mL sterilized microtubes and stored at 4 ° C. for over 5 days to maximize the formation of needle crystals. During this time, the decrease in uric acid concentration in the solution was monitored over time using the uricase peroxidase method. The result is shown in FIG.

  The generated sodium urate (MSU) crystals were crushed to 10 μm or less immediately before binding to idiotype antigens to facilitate phagocytosis by dendritic cells. Using a closed ultrasonic cell crusher (Bioraptor UCW200TM, Tojo Electric Co., Ltd., Yokohama), treatment at 200W is performed once for 30 seconds. The deposited microtube was processed to make the MSU crystal in the microtube fine.

  The progress of crushing in this operation was observed with a microscope (magnification 100 times). The results are shown in FIG. Clean fragmentation was performed in 30 seconds of crushing (Panel D), but in 60 seconds (Panel E), the fragment size was likely to be larger than 30 seconds. The crushing conditions were typically used for 2 seconds (4 cycles of 30 seconds on, 30 seconds off; panel F).

  After flushing the crushed solution at 7,000 × g, the precipitated crystals were washed with 0.1M phosphate buffered saline (pH 7.0) and finally settled in the same solution. An antigen carrier was used.

2. Preparation of MSU crystal support (2)
100 mg of uric acid was added to 80 mL of 0.1 M phosphate buffered saline (pH 7.4) and heated to boiling to completely dissolve uric acid. The heating was terminated, the amount of the solution was adjusted again to 80 mL, and the solution was sterilized by filtration when the temperature reached 45 ° C. under natural cooling (Stericup-GP filter unit, Nippon Millipore Corporation, Tokyo). The filtrate was quickly dispensed into 1.5 mL sterilized Eppendorf tubes and stored at 4 ° C. for over 5 days to maximize the formation of needle crystals.

  Thereafter, an idiotype antigen carrier was prepared in the same manner as described above.

3. Preparation of idiotype antigen 0.5-1.0 mL of IgG multiple myeloma patient serum (equivalent to 40 mg IgG) was collected and diluted with 0.1 M phosphate buffered saline (pH 7.4). Were injected onto a Protein G column (trade name “Hitrap protein G 1 mL”, Amersham Pharmacia Biotech Co., Ltd., Tokyo) previously adsorbed and adsorbed. Further, 0.1M phosphate buffered saline (pH 7.0) was injected to wash out non-adsorbed components from the column. 3 mL of 1M glycine-hydrochloric acid buffer (pH 2.7) was injected to elute the adsorbed IgG, and 1M Tris-hydrochloric acid buffer (pH 9.0) was added to the eluate to bring the pH to about 7.4. It was adjusted.

  The eluted IgG was digested with 0.25 mg / mL papain (MERCK, Darmstadt, Germany) in a mixture of 0.12 mg / mL cysteine and 2 mM sodium EDTA (pH 7.4) at 37 ° C. for 12 hours. The IgG was cleaved into the Fab part and the Fc part. The reaction was then stopped with 10.8 mM iodoacetamide.

  It is injected into a protein A column (trade name “Hitrap protein A 5 mL”, Amersham Pharmacia Biotech Co., Ltd., Tokyo) to adsorb the Fc portion and undigested IgG, and further 0.1 M phosphate buffered saline (pH 7. 4) was injected to collect the Fab portion which is a non-adsorbing component. Next, after centrifugal concentration (trade name “Amicon Ultra-4 Centrifugal Filter Unit”, Nippon Millipore Co., Ltd., Tokyo), 0.1M phosphate buffered saline (pH 7.4) was used as a dialysis solution (dialysis membrane ( Dialyzed for 90 minutes by Drop Dialysis method using Millipore 0.025μM VSWP 25mm, Nippon Millipore Co., Ltd., Tokyo), and further sterilized syringe pressure type filter unit (trade name “Mirex-GV”, 0.22μm, PVDF And sterilized by filtration through Nippon Millipore Corporation, Tokyo) to obtain an idiotype antigen solution.

4). Formation of complex (1)
Using a solution containing a certain amount of idiotype antigen (Fab), the relationship between the amount of MSU crystal carrier and the amount of adsorbed Fab protein was examined.

  100 μL of 0.1 M phosphate buffered saline containing 100 μg of the idiotype antigen (Fab) prepared in 3 above is added to the microtube containing 0 to 360 μg of the MSU crystal carrier prepared in 1 above at 4 ° C. It was allowed to stand for 24 hours. After flushing this solution at 7,000 × g, the protein concentration of the supernatant was measured by the pyrogallol red method (trade name “Micro TP-Test”, Wako Pure Chemical Industries, Ltd., Tokyo).

  The results are shown in FIG. It was estimated that the amount of protein in the buffer solution decreased as the amount of added MSU crystal carrier increased, and the MSU crystal carrier adsorbed Fab protein. Moreover, since it is estimated from calculation that about 50 μg of Fab protein is adsorbed with 100 μg of MSU crystal carrier, in 100 μL of 0.1 M phosphate buffered saline, the amount is doubled with respect to 100 μg of MSU crystal carrier. It was considered that by adding Fab protein, the MSU crystal carrier can secure the adsorption of Fab protein of 50 μg or more.

5. Formation of complex (2)
In order to confirm the state of binding, 400 μ1 of the idiotype antigen carrier suspension prepared in the same manner as in 1 above (corresponding to 1.4 mg of MSU crystal; but subjected to ultrasonic treatment for 30 seconds) was labeled with the mouse FITC label. 100 μL of IgG • F (ab ′) ₂ (Dako Japan, Kyoto), or mixed with mouse FITC-labeled IgG • Fab prepared from mouse FITC-labeled IgG • F (ab ′) ₂ by the same method described in 3 above. It left still at 4 degreeC for 1 hour. After washing three times with dilute hydrochloric acid solution (pH 6.5 to 7.0) and obtaining a transmission image of the MSU crystal under an optical microscope, a fluorescence microscope image with an excitation wavelength of 488 nm was observed.

The additive-free MSU crystal was colorless and transparent under a fluorescence microscope with only the outline being distinguishable (results not shown). On the other hand, MSU crystals mixed with mouse FITC-labeled IgG • Fab or mouse FITC-labeled IgG • F (ab ′) ₂ appeared to shine green under a fluorescence microscope. In FIG. 4, panels A and B show the results of binding of the MSU crystal carrier and mouse FITC-labeled IgG • Fab, and panels E and F show the results of binding of mouse FITC-labeled IgG • F (ab ′) ₂ . Panels A and E are optical microscope images, and panels B and F are combined images of a fluorescence microscope image and an optical microscope image. Further, when the MSU crystal was previously mixed with fetal calf serum and then mixed with the MSU crystal carrier and mouse FITC-labeled IgG • Fab, the green glow under the fluorescence microscope was attenuated.

6). Preparation of immature dendritic cells from monocytes Heparin blood was collected from a healthy donor, and monocytes were removed using “RosetteSep Human Monocyte Enrichment Cocktail” (trade name; StemCell Technologies Inc, Vancouver, Canada). GM-CSF (PEPRO TECH INC, Rocky Hill, NJ) (100 ng / mL) and IL-4 (PEPRO TECH EC, London, UK) (100 ng / mL) were added to these monocytes (peripheral blood monocytes). Immature dendritic cells were prepared by culturing in RPMI 1640 medium containing 10% AB serum at 37 ° C. and 5% CO ₂ for 6 days.

7). Maturation of dendritic cells by urate crystals 20 μg of the idiotype antigen (Fab) prepared in 3 above and 20 μg of the MSU crystal carrier prepared in 1 above were allowed to stand at 4 ° C. for 3 hours to form a complex. Thereafter, the cells were cultured with immature dendritic cells (1 × 10 ⁵ cells / well) in 100 μL of RPMI1640 culture medium containing 10% FBS for 48 hours. Similarly, only immature dendritic cells were cultured for 48 hours. For these dendritic cells, the expression intensity of CD83, which is an index of maturation, was measured by flow cytometry (EPICS) (Coulter-Immunotec) using PE-labeled anti-CD83 antibody (PE COULTER; Coulter-Immunotec, Krefeld, Germany). , Krefeld, Germany) under single color conditions.

  The results are shown in FIG. The CD83 expression intensity (panel B) of the dendritic cells co-cultured with the complex of the urate crystal carrier and the antigen in advance (panel B) was clearly higher than the CD83 expression intensity (panel A) of those cultured only with dendritic cells. Therefore, it was confirmed that the complex of the urate crystal carrier and the antigen promotes the maturation of immature dendritic cells.

  Although dendritic cells are excellent in antigen uptake when immature, mature dendritic cells are considered to be superior in antigen-presenting ability and ability to stimulate T cells. In order to promote maturation of dendritic cells, the use of cytokines such as TNF-α and IL-6, prostaglandin E2, and picibanil has been reported, but a complex of urate crystal carrier and antigen is used. Thus, it was found that dendritic cells can be matured at the same time as antigen protein uptake, and that the ability to present antigen can be increased without the use of a maturation substance as described above.

8). Autologous T cell proliferation test As in 6 above, 20 μg of the patient-derived IgG Fab prepared in 3 above and 20 μg of the MSU crystal carrier were allowed to stand at 4 ° C. for 3 hours, and then 1 × 10 ⁵ immature trees. Co-cultured with dendritic cells for 48 hours. In addition, the cells were cultured with immature dendritic cells for 48 hours without adding any of Fab protein alone or urate crystals alone. The respective mixing 1 × 10 ⁵ cells of T cells to 1 × 10 ⁴ cells dendritic cells of, were cultured for 5 days at 100 [mu] L / well.

These dendritic cells were washed twice with cold PBS and then collected from the same healthy donor using “RosetteSep Human T cell Enrichment Cocktail” (trade name; StemCell Technologies Inc, Vancouver, Canada) (1 × 10 ⁶ / mL CD3 positive cells> 95%) and a T cell: dendritic cell number ratio of 10: 1 (ie 1 × 10 ⁵ for 1 × 10 ⁴ dendritic cells). In a 96-well flat-bottom plate, the cells were cultured in a 10% AB serum-containing AIM-V medium (SIGMA) for 5 days so that the final volume was 100 μL. The proliferation of T cells was measured using “CellTiter 96 Non-Radioactive Cell Proliferation Assay” (trade name: Promega, WI, USA). The growth ratio (Stimulation Index; SI) was calculated as the measured value of each well of T cells co-cultured with dendritic cells / measured value of T cells not stimulated with dendritic cells. Each data is shown as the mean ± standard deviation of the results of 3 healthy subjects.

  The results are shown in FIG. Proliferation of T cells stimulated with dendritic cells co-cultured with a complex of urate crystal carrier and Fab protein (column A; SI = 2.44 ± 0.15) was obtained by dendritic cells co-cultured with Fab alone It was significantly higher than the proliferation of T cells stimulated with (column C; SI = 1.73 ± 0.24). Proliferation of T cells stimulated with dendritic cells co-cultured only with urate crystals (column B; SI = 2.27 ± 0.23) is the proliferation of T cells stimulated only with dendritic cells (column D; SI = 1.29 ± 0.13).

9. Cytotoxicity test Monocytes were extracted from healthy donors positive for HLA-A2 using “RosetteSep Human Monocyte Enrichment Cocktail” (StemCell Technologies Inc), GM-CSF (100 ng / mL), IL-4 (100 ng / mL) Was cultured for 6 days at 37 ° C. and 5% CO ₂ in a 10% AB serum-containing RPMI 1640 culture solution to which immature dendritic cells were prepared.

Monoclonal IgG secreted by the same cell line was purified from the culture supernatant of HLA-A2-positive IgG-producing myeloma cell line IM-9 in the same manner as above 3, and then the Fab region was excised and idiotype antigen (Fab Protein). Dendritic cells (A) co-cultured for 48 hours after binding the Fab protein and the urate crystal carrier prepared in 1 or 2 above, FBS (bovine serum) used in the co-culture, and urate crystal carrier Dendritic cells that have been bound and co-cultured with Fab (a model in which Fab is not co-cultured with DC without binding uric acid crystal carrier) (B), dendritic cells that have been co-cultured with Fab alone for 48 hours (C), Using only dendritic cells (D), T cells collected from the same donor were stimulated in the same manner as described above. After culturing for 7 days, the cells were similarly stimulated with dendritic cells. After further culturing for 7 days, CD8 positive cells were taken out using “MACS” (trade name; Miltenyi Biotec, Bergisch Glanbach, Germany). CD8 positive cells: 5: 1 tumor cells (ie, 1 × 10 ⁴ tumor cells to 1 × 10 ⁵ CD8 positive cells) were mixed and cultured in 100 μL for 4 hours, then “CytoTox 96” ( Cytotoxicity of T cells against IM-9 cells was measured with a registered trademark Non-Radioactive Cytotoxicity Assay (Promega, WI, USA).

  The results are shown in FIG. The cytotoxicity to IM-9 of CD8 positive cells induced by dendritic cells co-cultured with a complex of urate crystal carrier and Fab protein was significantly higher than others. In particular, cytotoxicity to IM-9 of CD8 positive cells induced by dendritic cells co-cultured with a complex of urate crystal carrier and Fab protein compared to those co-cultured without binding uric acid crystal carrier and Fab The sex was significantly higher, and it was shown that the antigen protein and uric acid crystals were previously bound to efficiently induce CTL. Therefore, it was found that the idiotype vaccine of the present invention efficiently induces CTL targeting tumor cells.

FIG. 1 shows the generation of MSU crystals from solution over time. The vertical axis represents the uric acid concentration (mg / dL) in the solution, and the horizontal axis represents time (hr). FIG. 2 is a photograph (optical microscope, 100 × magnification) showing the crushing of MSU crystals. Panel A is a microscopic image of a sample that has been subjected to ultrasonic treatment for the time indicated at the lower left of the panel before panels A to B. FIG. 3 is a graph showing the relationship between the amount of MSU crystal carrier and the amount of adsorbed Fab protein (change in protein concentration in Fab solution due to addition of MSU crystal) using a solution containing a certain amount of idiotype antigen (Fab). It is. FIG. 4 is a photograph showing the binding of MSU crystal and Fab. Panels A, C, and E are optical microscope images, and panels B, D, and F are combined images of a fluorescence microscope image and an optical microscope image (magnification 100 times). Panels A and B show the binding between the MSU crystal carrier and mouse FITC-labeled IgG / Fab. Panels C and D show the MSU crystal carrier and mouse FITC-labeled IgG / Fab bound after mixing the MSU crystal with fetal bovine serum in advance. Panels E and F show the results of binding the MSU crystal carrier and mouse FITC-labeled IgG · F (ab ′) ₂ , respectively. The lower right bar of each panel is 50 μm. FIG. 5 is a diagram showing maturation of dendritic cells by MSU crystals. The left panel (A) is the control (no MSU crystal added), and the right panel is the result of MSU crystal addition (B) (dendritic cell CD83 expression level detected by flow cytometer). FIG. 6 is a diagram showing the autologous lymphocyte proliferation stimulating action of dendritic cells incorporating the idiotype vaccine of the present invention. A = idiotype vaccine of the present invention (complex of MSU crystal carrier and Fab), B = MSU crystal carrier only, C = Fab only, D = no additives (dendritic cells only) Induced T cells using cells. (N = 3) FIG. 7 is a graph showing cytotoxicity of CTL induced by the idiotype vaccine of the present invention against myeloid cell lines. A = Idiotype vaccine of the present invention, B = MSU crystal carrier previously blocked with FBS and Fab mixture (model co-cultured with DC without binding Fab and MSU crystal carrier), C = Fab only, D = Induced T cells using dendritic cells cultured without additives. (N = 3)

Claims (6)

  An idiotype vaccine comprising a complex comprising an idiotype antigen and a carrier comprising urate crystals.   The idiotype vaccine of claim 1, wherein the idiotype antigen is an immunoglobulin produced by multiple myeloma cells or a part thereof comprising a variable region.   The idiotype vaccine according to claim 1 or 2, wherein the urate crystals are those obtained by sonicating acicular crystals.   A method for producing the idiotype vaccine according to any one of claims 1 to 3, wherein the idiotype antigen and the urate crystals are combined by allowing the idiotype antigen and the urate crystals to stand together. A method comprising the step of forming a body.   A specific cytotoxicity characterized by stimulating dendritic cells using the idiotype vaccine according to any one of claims 1 to 3 and stimulating cytotoxic T cells using the dendritic cells. A method of promoting an immune response of sex T cells.   A carrier for producing the idiotype vaccine according to any one of claims 1 to 3, wherein the carrier comprises sodium urate crystals.

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