JP3267783B2 - CD34 binding factor and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a 14 kD molecular weight compound having a function of specifically binding to the CD34 antigen expressed on human hematopoietic stem cells, acting on hematopoietic stem cells, and promoting and maintaining proliferation of hematopoietic stem cells. And a protein having the name CD34Lp14 and a method for producing the same.

[0002] The present protein has a function of promoting and maintaining the proliferation of hematopoietic stem cells, and is used for hematopoietic tumors such as leukemia, allogeneic / autologous bone marrow transplantation, cytopenia associated with radiotherapy / chemotherapy for cancer patients, and regeneration. It has use as a diagnostic agent for aplastic anemia and the like, and a therapeutic agent.

[0003]

2. Description of the Related Art The CD34 antigen has a molecular weight of 105 to 120.
The KD glycoprotein is expressed in about 1 to 3% of bone marrow mononuclear cells, and is also expressed in a part of vascular endothelial cells and progenitor cells of stromal cells constituting a hematopoietic microenvironment.

Hematopoietic stem cells that are said to express the CD34 antigen have the ability to produce mature blood cells such as erythrocytes, granulocytes, megakaryocytes, platelets, T lymphocytes, B lymphocytes (multipotent). Cells that have the ability to self-renew as well as to have Therefore, purifying hematopoietic stem cells is not only indispensable for investigating the hematopoietic mechanism and its regulatory factors, but also important for the diagnosis and treatment progression of hematopoietic diseases. It is being applied.

[0005] The inclusion of hematopoietic stem cells in CD34-positive cells was proved by the experiments described below. CD34-positive cells were separated using an anti-CD34 antibody column, and 3.2-4.4 × 10 ⁶ / kg of CD34-positive cells were transplanted into the irradiated baboon. As a result, the baboon's hematopoietic system was restored. . On the other hand, recovery of the hematopoietic system was not observed even when 10-fold amount of CD34 negative cells was transplanted. 37% of CD34-positive cells formed colonies in vitro by the single cell culture method using FACS, and 1% of the colonies formed.
Form mixed colonies.

[0006] In clinical practice, hematopoietic stem cells are used
Purification using antibody 34 and autologous transplantation to patients after radiotherapy or chemotherapy have begun to be performed, and it has been reported that hematopoietic recovery was observed by this autologous transplantation. In addition, as a pretreatment of transplanted cells in autologous transplantation, an effect can be exhibited in reducing malignant cells by collecting normal hematopoietic stem cells using an anti-CD34 antibody (positive selection). This autologous hematopoietic stem cell transplantation will be important as supportive therapy for high-dose chemotherapy and radiation therapy in the future.

However, in any case, the amount cannot be increased merely by purifying hematopoietic stem cells (CD34-positive cells) using an anti-CD34 antibody. If the number of hematopoietic stem cells, which are very small in bone marrow and peripheral blood, can be increased, the amount of hematopoietic stem cells collected can be reduced, and the burden on patients can be reduced.

[0008] However, the growth factors reported so far have a specific function of proliferating hematopoietic stem cells.
It is hard to say that it is enough. For example, Stem cell
Although the factor functions as a growth factor for progenitor cells, its effect on uncultivated hematopoietic stem cells having self-renewal ability is unclear.

[0009] As described above, there are no known human hematopoietic stem cell growth factors that are sufficiently satisfactory. Also,
The present invention relates to a CD34 binding factor having a molecular weight of 14 kD, but no examples have been known in which such a ligand has been successfully isolated. Nonetheless, it has been shown that such a ligand has a function of acting on hematopoietic stem cells. No confirmed example is known at all, and the present invention is the first.

[0010]

In such prior art, conventionally, it has been considered as a marker for hematopoietic stem cells,
The results of a report on the function of the CD34 antigen, whose function was unknown, were suggested to be involved in some kind of signal transduction and involved in the proliferation of hematopoietic stem cells. That is, it was shown that the serine residue of the CD34 antigen was phosphorylated by TPA treatment, and colony formation was suppressed when an anti-CD34 antibody was added to a methylcellulose culture system of bone marrow mononuclear cells or CD34-positive cells.

Thus, it is expected that the binding factor (ligand) to the CD34 antigen will be involved in the proliferation of hematopoietic stem cells through the binding to the CD34 antigen. That is, C
It is possible that the D34 antigen-binding factor is a hematopoietic stem cell growth factor, and the present inventors paid their attention to this point for the first time.

On the other hand, as a protein binding to the CD34 antigen, L-Selectin which is known as an adhesion factor is used.
Has already been reported. However, the CD34 antigen in this case is not expressed on hematopoietic stem cells, and L-
Selectin has a molecular weight of 90 kD, and it has not been clarified whether or not it is expressed in stromal cells constituting the hematopoietic microenvironment necessary for the proliferation of hematopoietic stem cells, or whether it acts on hematopoietic stem cells.

The present invention has been made in the current state of the art, and according to the above-mentioned point of view, conventional separation has not yet been confirmed, not to mention separation.
The purpose of the present invention is not only to isolate the D34 antigen-binding factor, but also to confirm the function of regulating the proliferation of hematopoietic stem cells, and to utilize the function effectively.

That is, an object of the present invention is to provide a protein having a function of specifically binding to the CD34 antigen expressed on human hematopoietic stem cells, exerting an effect on hematopoietic stem cells, and promoting and maintaining the proliferation of hematopoietic stem cells. It is intended for that purpose.

[0015]

Means for Solving the Problems The present invention has been made to achieve the above-mentioned object, and relates to a protein having a function of promoting and maintaining the proliferation of hematopoietic stem cells expressing the CD34 antigen. is there.

[0016] Therefore, the present inventors first, human CD3
4 antigen cDNA was converted to human immunoglobulin (Ig) G Fc
The cDNA was ligated to the region and incorporated into an animal cell expression vector, which was transduced into Balb / 3T3 to prepare a transduced cell (transfectant) (CD34-Ig / 3T3). Next, this cell line is cultured in a large amount, and the CD34-Ig chimeric protein is purified from the culture supernatant using an anti-CD34 monoclonal antibody. Using the CD34-Ig chimeric protein thus obtained, a normal fibroblast cell line derived from human fetal lung (H
FL-III, RIKEN, Cell Development Bank, Cell
No. RCB0523) was successfully isolated and purified from a lysate of a protein having a molecular weight of 14 kD, and furthermore, a protein (adhesion factor) that binds to the CD34 antigen.
Is known as a previously unknown substance because it is completely different from L-selectin known in the art in all aspects of molecular weight, cell origin, and action, and was identified as CD34Lp14.
The present invention was completed as a result of further study.

According to the present invention, in addition to stromal cells such as fibroblasts and endothelial cells, cells containing a CD34-binding factor are cultured, and the resulting culture is subjected to the above-mentioned chimeric protein, mono (poly) clonal antibody, and the like. By treating with a substance that specifically binds to the CD34 binding factor,
D34 binding factor can be produced efficiently.

The protein having a molecular weight of 14 kD, which is a CD34-binding factor of the present invention, specifically binds to the CD34 antigen and exerts an effect on hematopoietic stem cells. In addition to maintaining and promoting proliferation and differentiation, various adjustments are also possible.

The thus obtained protein having a molecular weight of 14 kD is subjected to a test for examining the function of CD34-positive cells including hematopoietic stem cells and a test for examining the function of stromal cells expressing the protein. Can be.

Furthermore, hematopoietic tumors such as leukemia,
It can be used as a diagnostic or therapeutic agent for allogeneic / autologous bone marrow transplantation, cytopenia associated with radiation therapy / chemotherapy for cancer patients, aplastic anemia and the like.

The present protein is administered in various forms. Examples of the administration form include tablets, capsules, granules,
Oral administration by powders, syrups and the like or parenteral administration by injections (intravenous, intramuscular, subcutaneous), drops, suppositories and the like can be mentioned. These various preparations are commonly used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents, and the like, in accordance with the usual methods for the main drug. It can be formulated using known adjuvants. The amount used varies depending on symptoms, age, body weight, administration method, dosage form, etc., but usually about 0.1 mg to 1,000 mg once for an adult.
Can be administered. In addition, since this protein is derived from human cells, it is naturally safe, and
No acute toxicity was observed when 0 mg / kg body weight was orally administered.

[0022]

The CD34 antigen is expressed on about 1 to 3% of bone marrow nucleated cells, and hematopoietic stem cells and various blood cell progenitor cells are included in this fraction. This fraction is separated and various cytokines,
After culturing in vitro in the presence of a colony stimulating factor or the like, it is possible to examine the function of hematopoietic stem cells and various hematopoietic progenitor cells for proliferation by a test for examining colony forming ability in a semi-solid medium. By such a test, a protein having a molecular weight of 14 kD provided by the present invention, C
It can be shown that D34Lp14 binds to CD34 antigen expressed on hematopoietic stem cells and has a function of maintaining and promoting the proliferation.

[0023]

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

[0024]

Example 1: Immunoprecipitation Immunoprecipitation was performed using the CD34-Ig chimeric protein.

The method will be described below. A normal fibroblast cell line derived from human fetal lung (HFL-III, RIKEN, Cell Development Bank, Cell No. RCB0523) was spread on the plate, and was heated to 37 ° C. until it reached 80% confluence.
Incubate with Medium without methionine and cysteine (DME
After washing with (-Met, -Cys)), a medium for ³⁵ S radiolabeling (Trans- ³⁵ S) was added, and the mixture was cultured at 37 ° C for 16 to 18 hours.
Cultured for hours. After washing the cells, Lysis Buffer
r (0.1% Triton X-100, 10 mM T
risHCl, 0.15 M NaCl, 2 mM PMS
F, 1 mM EDTA) and dissolve well. After leaving it on ice for 60 minutes, it was transferred to Tube and centrifuged (15000 rpm).
m, 5 min), and the supernatant was collected and used as Lysate.
Non-specific binding substances in Lysate were replaced with human Ig-
Fc fraction-Protein G-Seph
After removing using arose 4Fast Flow (Pharmacia) (4 ° C., 3 hours), Prote
inG-Sepharose 4Fast Flow
(Pharmacia) to remove non-specific binding substances (4
C., 2 hours), and centrifuged (15000 rpm, 1 min) to separate the supernatant.

Reaction with CD34-Ig chimeric protein (4
C., 16-18 hours), after which Protein G-Seph
arose was added, and the mixture was slowly rotated at 4 ° C. for 3 hours. After washing Protein G-Sepharose,
SDS sample buffer (0.1M TrisHCl
(PH 6.8), 0.16% glycerol, 3.2% S
DS, 0.002% Bromophenol Blue
e, 0.08% 2-Mercaptoethanol)
And boil for 10 minutes, centrifuge (15000 rpm, 1m
in) to obtain a sample for electrophoresis. This is SDS
After electrophoresis by PAGE, the gel was dried, subjected to autoradiography, and detected. As a result, the CD34-Ig chimeric protein had a molecular weight of 14 k
It specifically bound to the protein D, CD34Lp14.
(Fig. 1)

[0027]

Example 2 Immunoprecipitation in Various Cell Lines Using the method described in Example 1, a protein with a molecular weight of 14 kD, CD34Lp1, which is a CD34 antigen-binding factor in various cell lines.
4 was examined. As a result, a normal fibroblast cell line derived from human fetal lung (HFL-I, RIKEN, Cell Development Bank,
Cell No. RCB0521) and human normal umbilical vein endothelial cells (HUV-ECl, RIKEN, Cell Development Bank, Cell No. RCB0696). (Fig. 2)

[0028]

Example 3: Resistance to trypsin treatment After trypsin was allowed to act on a culture plate, the cells were removed from the plate, washed well, and subjected to immunoprecipitation using the method described in Example 1 to obtain CD34 antigen. It was examined whether CD34Lp14, a protein having a molecular weight of 14 kD, which is a binding factor, was affected by trypsin treatment, and whether it was a protein bound to cells. As a result, the presence of CD34Lp14 was confirmed even after trypsinization. These results suggested that the protein was (1) a trypsin-resistant protein and (2) a cell-binding protein. (Fig. 3)

[0029]

According to the present invention, a 14 kD protein that specifically binds to the CD34 antigen has been isolated for the first time. This protein has the function of acting on hematopoietic stem cells and regulating the proliferation of the cells. Therefore, the present protein is used, for example, as a hematopoietic stem cell growth factor to prevent various blood system diseases,
Used for therapeutic agents.

[Brief description of the drawings]

FIG. 1. CD34-Ig against lysate of a normal fibroblast cell line derived from human fetal lung biosynthesized and labeled with ³⁵ S-methionine.
Fig. 3 is a drawing in which immunoprecipitation was performed using a chimeric protein.

FIG. 2 shows a normal fibroblast cell line derived from human fetal lung (HFL-I, RIKEN, Cell Development Bank, Cell No. RCB0521) and human normal umbilical vein endothelial cells (Human umbilical cord vein endothelial cells) in the same manner as in FIG. It is the figure which performed immunoprecipitation about HUV-ECl, RIKEN, Cell Development Bank, CellNo.RCB0696).

FIG. 3. After trypsin is acted on a culture plate,
FIG. 2 is a drawing in which immunoprecipitation was performed in the same manner as in FIG. 1 of Example 1.

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Kaoru Ishii 566-6 Shimofujisawa, Iruma-shi, Saitama 102 Happiras Musashi Fujisawa 102 (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 14/00-14 / 825 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. Stromal cells are cultured, and the resulting culture is treated with a CD34 antigen protein column to obtain CD34.
A protein CD that specifically binds to an antigen and has the following properties:
A method for producing a protein CD34Lp14, comprising isolating and collecting 34Lp14. (1) SDS-polyacrylamide gel electrophoresis shows a molecular weight of 14 kD. (2) It specifically binds to the CD34 antigen expressed on hematopoietic stem cells and has a function as a regulator that regulates proliferation and differentiation of hematopoietic stem cells. (3) HFL-III cells can be cultured and produced from the resulting culture. 2. A protein CD34Lp having the following properties:
14. (1) SDS-polyacrylamide gel electrophoresis shows a molecular weight of 14 kD. (2) It specifically binds to the CD34 antigen expressed on hematopoietic stem cells and has a function as a regulator that regulates proliferation and differentiation of hematopoietic stem cells. (3) HFL-III cells can be cultured and produced from the resulting culture.