KR20090020275A - Biograft composition for treatment of uterine cancer comprising CD3 positive T immune cells derived from umbilical cord blood - Google Patents

Abstract

The present invention provides a biograft composition for the treatment of uterine cancer comprising CD3 positive T immune cells (CD3 + T lymphocytes) derived from umbilical cord blood and a pharmaceutically acceptable carrier. The present invention also provides a method for separating the CD3-positive T immune cells.

According to the present invention, when grafted CD3 positive T immune cells derived from umbilical cord blood, cervical cancer can be effectively treated. In particular, the umbilical cord blood-derived CD3-positive T immune cells recognize HPV, which is a cancer antigen of uterine cancer, and specifically attack only uterine cancer cells without attacking normal cells, thereby effectively treating uterine cancer. In addition, the cord blood-derived CD3-positive T immune cells have a very low graft-versus-host reaction (GVH), which is an immune side effect that may occur due to transplantation, and therefore, almost no immune rejection reactions in vivo. It is possible to transplant patients.

Description

Composition for in vivo transplantation for the treatment of human cervical cancer, comprising CD3 + T lymphocytes derived from umbilical cord blood}

The present invention relates to a biograft composition for the treatment of uterine cancer, including CD3 positive T immune cells (CD3 + T lymphocytes) derived from umbilical cord blood. The present invention also relates to a method for isolating CD3-positive T immune cells derived from the cord blood.

Cancer mortality accounts for the second largest human mortality rate. The methods that have been used for most cancer treatment methods rely on the administration of anticancer drugs, irradiation and surgical procedures. In the early stages of cancer, such a method can be treated alone or in combination, but has a very low therapeutic effect in the case of cancer which has metastasized and recurred to other tissues in late stages or through blood.

Recently, research on immune cell therapy techniques for treating cancer by re-transplanting a patient through in vitro mass culture method using the peripheral blood of the patient has attracted attention. In other words, a technique for removing cancer cells by cytotoxic T cells, which are cancer cell-specific in mass cultured immune cells, has been developed by proliferating a large amount of immune cells extracted from the patient's blood in vitro and administering them to the patient (Adoptive transfer of tumor-reactive Melan-A-specific CTL clones in melanoma patients is followed by increased frequencies of additional Melan-A-specific T cells.Vignard V, Lemercier B, Lim A, Pandolfino MC, Guilloux Y, Khammari A, Rabu C, Echasserieau K, Lang F, Gougeon ML, Dreno B, Jotereau F, Labarriere NJ Immunol. 2005, 175: 4797-805).

Adoptive cellular immunotherapy involves the use of immune cell activating factors, such as IL-2, in vitro for NK cells, T cells, or Lymphokine-activated killer (LAK) cells, which are representative immune cells present in the peripheral blood of a patient. By mass culturing at large, most attempts have been made to proliferate cells with the ability to recognize cancer cells to treat cancer patients through autografting. However, these treatments are often performed without verification of the presence of cancer cell specific immune cells, and it is difficult to expect satisfactory therapeutic effects according to clinical applications.

Rosenberg et al., In vitro in vitro culture using tumor-infiltrating lymphocytes (TILs), which are immune cells present in tumor tissues, have been applied to patients, and malignant skin cancer tumors have been reduced, thereby enhancing the reactivity of tumor-specific immune cells. has reported a method of bars (Interleukin 2 expanded tumor-infiltrating lymphocytes in human renal cell cancer:.. isolation, characterization, and antitumor activity Belldegrun a, Muul LM, Rosenberg SA cancer Res . 1988. 48 (1): 206-14).

In addition, dendritic cells with excellent antigen-presenting ability to present cancer cell-specific antigens can be isolated and cultured in vitro and added to the patient with cancer cell lysates or by adding cancer-specific antigen proteins to the patient. Vaccination of Japanese patients with advanced melanoma with peptide, tumor lysate or both peptide and tumor lysate-pulsed mature, monocyte-derived dendritic cells. N, Asai J, Ueda E, Takenaka H, Katoh N, Kishimoto SJ Dermatol. 2006. 33: 462-72.Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma: results of a clinical phase I / II trial.Yamanaka R , Homma J, Yajima N, Tsuchiya N, Sano M, Kobayashi T, Yoshida S, Abe T, Narita M, Takahashi M, Tanaka R. Clin Cancer Res. 2005, 11: 4160-7).

Recently, a method of treating cancer using Graft-versus-Tumor (GVT) has been developed. HLA is a method for removing residual cancer cells by inducing GVH (Graft-versus-Host) response by collecting blood from genetically different donors and administering T cells and NK cells in the blood to cancer patients (A phase 1 trial of donor lymphocyte infusions expanded and activated ex vivo via CD3 / CD28 costimulation.Porter DL, Levine BL, Bunin N, Stadtmauer EA, Luger SM, Goldstein S, Loren A, Phillips J, Nasta S, Perl A, Schuster S, Tsai D, Sohal A, Veloso E, Emerson S, June CH. Blood. 2006, 107: 1325-31.Allogeneic hematopoietic stem cell transplantation for metastatic breast cancer.Bishop MR.Haematologica. 2004, 89: 599-605 ). This is expected as a cell therapy technology for new cancer treatment because it can induce a very high immune response compared to the method using autoimmune cells.

On the other hand, conventional cancer treatment method using immune cells, that is, when transplanted by separating the immune cells from the peripheral blood, because the proliferation ability of the transplanted immune cells is low, long-term continuous cancer treatment is difficult. In addition, the cancer treatment method using immune cells isolated from peripheral blood, because of the graft-versus-host GVH (Graft-verse-Host), cells derived from patients in vitro ( in vitro) were incubated and then there is again implanted in the patient, in a test tube (in In vitro ), the cultured cells have a limited cell proliferation capacity as cells in the terminal stage of cell division, and therefore it is difficult to expect a long-term therapeutic effect of immune cells even when transplanted into a patient.

The inventors of the present invention not only have excellent hematopoietic cell regeneration ability, but also major histocompatibility antigens when the monocytes are separated from umbilical cord blood which can be collected without causing any special harm to the human body and applied to uterine cancer. It has been found that treatment of uterine cancer can be effectively treated by showing very low GVHD even in patients with inconsistent (HLA), and the patent application was completed on October 24, 2006 (Korean Patent Application No. 10-2006 -0103627).

The present inventors conducted various studies to clarify the molecular mechanism of uterine cancer treatment of the umbilical cord blood-derived monocytes, and selectively induced CD3-positive T-immune cells when injecting umbilical cord blood-derived monocytes into congenital immunodeficiency mice. We established an animal model that differentiates and proliferates, and found that CD3-positive T-immune cells obtained from these animal models have an excellent effect of treating uterine cancer by specifically acting on HPV, a gene causing uterine cancer. This is very surprising given that CD3-positive T immune cells derived from umbilical cord blood are known to be functionally immature T immune cells compared to CD3-positive T immune cells derived from normal peripheral blood.

Accordingly, an object of the present invention is to provide a biograft composition for the treatment of uterine cancer comprising the CD3-positive T immune cells derived from the cord blood.

Another object of the present invention is to provide a method for isolating CD3 positive T immune cells derived from cord blood using immunodeficient mice.

According to one aspect of the invention, there is provided a biograft composition for the treatment of uterine cancer comprising CD3 positive T immune cells (CD3 + T lymphocytes) derived from umbilical cord blood and a pharmaceutically acceptable carrier.

The uterine cancer includes cervical cancer caused by Papilloma Virus (HPV) infection. The CD3-positive T immune cells are preferably differentiated from monocytes derived from umbilical cord blood.

In addition, CD3-positive T immune cells differentiated from umbilical cord blood-derived monocytes (mononuclear cells), (a) injecting umbilical cord blood-derived monocytes (mononuclear cells) up to 1 week of age after immunodeficient mice; And (b) isolating CD3-positive T immune cells from blood obtained from peripheral blood or hematopoietic tissue of the mouse 2 to 8 weeks after the injection, or (a) monocytes derived from umbilical cord blood Injecting the cells (mononuclear cells) into immunodeficient mice of 1 week or less after birth; (b) injecting human uterine cancer cells into the mouse 2 to 8 weeks after the injection; And (c) separating CD3 positive T immune cells from blood obtained from peripheral blood or hematopoietic tissue of the mouse about 2 weeks after the human uterine cancer cell injection.

As the immunodeficiency mouse, a non-obese diabetic severe combined immunodeficiency (NOD-SCID) mouse may be preferably used, and the infusion of the monocytes may be intraperitoneally administered, more preferably in a dose of 1 × 10 ⁷ to 10 × 10 ⁷ . By intraperitoneal administration. In addition, the human uterine cancer cell infusion may be performed by implanting human uterine cancer cells into the subcutaneous tissue of the back of the immunodeficient mouse at a dose of 1 × 10 ⁶ to 5 × 10 ⁶ . The hematopoietic tissue comprises the thymus, spleen, or bone marrow of the mouse.

According to another aspect of the present invention, the method comprises the steps of: (a) injecting mononuclear cells derived from cord blood into immunodeficient mice of 1 week or less; And (b) isolating CD3-positive T immune cells from cord blood from blood obtained from peripheral blood or hematopoietic tissue of the mouse 2 to 8 weeks after the injection. .

According to another aspect of the invention, the method comprises the steps of: (a) injecting mononuclear cells derived from umbilical cord blood into immunodeficient mice up to 1 week of age; (b) injecting human uterine cancer cells into the mouse 2 to 8 weeks after the injection; And (c) separating CD3 positive T immune cells from cord blood from blood obtained from peripheral blood or hematopoietic tissue of the mouse about 2 weeks after the injection of human uterine cancer cells. Is provided.

In the separation method, as the immunodeficient mouse, a non-obese diabetic severe combined immunodeficiency (NOD-SCID) mouse may be preferably used, and the infusion of the monocytes may be intraperitoneally administered, more preferably 1 × 10 ^7- . It may be carried out by intraperitoneal administration at a dose of 10 × 10 ⁷ . In addition, the human uterine cancer cell infusion may be performed by implanting human uterine cancer cells into the subcutaneous tissue of the back of the immunodeficient mouse at a dose of 1 × 10 ⁶ to 5 × 10 ⁶ . The hematopoietic tissue comprises the thymus, spleen, or bone marrow of the mouse.

According to the present invention, when grafted CD3 positive T immune cells derived from umbilical cord blood, cervical cancer can be effectively treated. In particular, CD3-positive T immune cells derived from umbilical cord blood function as uterine cancer specific immune cells and have tumor therapeutic efficacy through direct cancer cell attack. That is, the umbilical cord blood-derived CD3-positive T immune cells recognize HPV, a cancer antigen of uterine cancer, and specifically attack only uterine cancer cells without attacking normal cells to induce cell death by effectively treating uterine cancer. In addition, the cord blood-derived CD3-positive T immune cells have a very low graft-versus-host reaction (GVH), which is an immune side effect that may occur due to transplantation, and therefore, almost no immune rejection reactions in vivo. It is possible to transplant patients.

The present invention includes biograft compositions for the treatment of uterine cancer comprising CD3 positive T immune cells (CD3 + T lymphocytes) derived from umbilical cord blood and a pharmaceutically acceptable carrier.

The inventors have revealed that the umbilical cord blood-derived monocytes have excellent efficacy for treating uterine cancer, and thus can be used in a biograft composition for treating uterine cancer. The monocytes are CD34 + hematopoietic stem cells and progenitor cells, CD3 + T immune cells, It has been disclosed to include CD19 + B immune cells (Korean Patent Application No. 10-2006-0103627, Patent Application of October 24, 2006).

11-15% of CD3-positive T-immune cells are present in cord blood. CD3-positive T-immune cells in cord blood are mostly CD45RA-positive immature cells compared to CD3-positive T-immune cells in peripheral blood. (CD3 positive T immune cells present in peripheral blood: 45%, CD3 positive T immune cells present in umbilical cord blood: 88%). On the other hand, CD3 positive T immune cells present in peripheral blood mostly express CD45RO, an activation marker (68% CD3 positive T immune cells present in peripheral blood: CD3 positive T immune cells present in umbilical cord blood: 32%). ). (Comparision of cord blood and adult blood lymphocyte normal ranges: a possible explanation for decreased severity of graft versus host disease after cord blood transplantation.Beck R, Lam-Po-Tang PJ Immunol. Cell Biol. 1994; 72-440-444, Flow cytometric characterization of human umbilical cord blood lymphocytes: immunophenotypic features.D'Arena G, Musto P, Cascavilla N, Di Giorqio G, Fusilli S, Zendoli F, Carotenuto M. Haematologica. 1998; 83: 197-203). In addition, peripheral blood and umbilical cord blood are stimulated with PHA (Pycohematoagglutin) or ConA (Concanavalin A) and PMA (Phorbol-12-myristate 13-acetate), respectively. Express CD40L, but only about 30% of cord blood expresses CD40L. In case of stimulating peripheral blood and umbilical cord blood with anti-CD3 antibody, another activator of T immune cells, no significant difference was found in the expression of TCRα and P56 ^lck , but IL-2, IL-3, IFN-γ The expression of mRNA is lower in umbilical cord blood than in peripheral blood. (Diminished expression of CD40 ligand by activated neonatal T cells.Nonoyama S, Penix LA, Edwards CP, Lewis DB, Ito S, Aruffo A, Wilson CB, Ochs HD.J Clin Invest. 1995. 95 (1): 66-75 , Functional characteristics of cord blood T lymphocytes after lectin and anti-CD3 stimulation.Differences in the way T cells express activation molecules and proliferate.Lucivero G, Dalla Mora L, Bresciano E, Loria MP, Pezone L, Mancino D. Int J Clin Lab Res. 1996, 26: 255-261).

That is, T immune cells present in umbilical cord blood are mainly composed of immature cells as compared to T immune cells present in peripheral blood or bone marrow. Thus, T immune cells, NK cells, dendritic cells, It has been recognized that its function is inferior to that of Lymphokine-activated killer (LAK) cells. However, it has now been discovered by the present invention that CD3-positive T immune cells derived from umbilical cord blood function as uterine cancer specific immune cells and have excellent tumor therapeutic efficacy through direct cancer cell attack. That is, the umbilical cord blood-derived CD3-positive T immune cells specifically recognize HPV, which is a cancer antigen of uterine cancer, thereby effectively treating uterine cancer by specifically attacking only uterine cancer cells and inducing cell death without attacking normal cells.

In addition, the cord blood-derived CD3-positive T-immune cells have very low graft-versus-host reaction (GVHD), which is an immune side effect that may be caused by transplantation. .

In the present specification, the term "cervical cancer" is a tumor occurring in the uterus, endometrial cancer known to be caused by hormones, genetic factors, etc., and mainly human papilloma virus (HPV) infection. Cervical cancer known to be caused by. Of course, the "uterine cancer" includes cervical cytology, vaginal magnification, histological examination, resection and cystoscopy, S colonoscopy, jugular vein angiography (IVP), computed tomography (CT) and magnetic resonance imaging (MRI). It should be understood that all forms of uterine cancer identified through various tests, such as proton emission tomography (PET). The pharmaceutical composition according to the present invention can be preferably applied to the prevention and / or treatment of cervical cancer caused by human Papilloma Virus (HPV) infection, the HPV is known such as HPV-16 or HPV-18 Includes all types of HPV.

The CD3-positive T immune cells are preferably differentiated from monocytes derived from umbilical cord blood. The cord blood-derived monocyte cells can be isolated from cord blood according to known methods, for example, moidfied Ficoll-Hypaque separation method, 3% gelatin separation method, Ficoll-Hypaque separation method, etc. (Kim et al., Optimal umbilical cord blood processing: Basic study for the establishment of cord blood bank.Korean Journal of Hematopoietic Stem Cell Transplantation, 2000.5: 61-68. In addition, an anticoagulant may be added to the cord blood, and monocyte cells may be separated by a Ficoll-Paque density gradient centrifugation method.

In vitro differentiation of the cord blood derived monocytes into CD3 positive T immune cells is described in Ross NL et al. Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro.Blood. 2005. 105: 1431-1439 Since most of them are obtained as immature T cells expressing both CD4 and CD8 molecules, they may be cells that do not have normal immune function. Therefore, in order to utilize the differentiation method, it may be necessary to further induce differentiation into mature immune cells.

The present inventors conducted various tests to avoid the above-mentioned problems and obtain CD3 positive T immune cells derived from mature cord blood. Surprisingly, when neonatal immunodeficient mice were injected with monocytes derived from cord blood, different immune It was found that cells, ie, B immune cells, NK cells, and myeloid cells, were not produced and only CD3 positive T immune cells could be selectively obtained.

Accordingly, the CD3-positive T immune cells differentiated from the mononuclear cells derived from the cord blood may include (a) injecting the mononuclear cells derived from the cord blood into immunodeficient mice 1 week or younger; And (b) isolating CD3 positive T immune cells from blood obtained from peripheral blood or hematopoietic tissue of the mouse 2 to 8 weeks after the injection.

In particular, as can be seen in the following examples, CD3-positive T immune cells isolated from mice exhibiting proliferation inhibition against uterine cancer cells by injecting umbilical cord blood-derived monocyte cells and human uterine cancer cells into immunodeficient mice were treated with superior uterine cancer. It was found to be effective.

Therefore, more preferably, the CD3-positive T immune cells differentiated from the umbilical cord blood-derived monocytes (a) are injected into the immunodeficient mice at 1 week of age or shorter after the umbilical cord blood-derived monocytes (mononuclear cells). step; (b) injecting human uterine cancer cells into the mouse 2 to 8 weeks after the injection; And (c) isolating CD3 positive T immune cells from blood obtained from peripheral blood or hematopoietic tissue of said mouse about 2 weeks after said human uterine cancer cell injection, preferably about 3 to 4 weeks. Can be obtained by

As the immunodeficiency mouse, a mouse without immune function can be used without limitation, and for example, a non-obese diabetic severe combined immunodeficiency (NOD-SCID) mouse can be preferably used. The immunodeficient mice are preferably neonatal mice, for example, newborn mice of 1 week or less. Infusion of the monocyte cells may be carried out by intraperitoneal administration, preferably by intraperitoneal administration of 1 × 10 ⁷ to 10 × 10 ⁷ cells, more preferably about 1 × 10 ⁷ cells. However, the dose of the monocyte cells is not particularly limited and may be appropriately selected as necessary.

The human uterine cancer cells can use cancer cells derived from uterine cancer patients without limitation, and for example, Caski cells (Korea Cell Line Bank, Cat. No. 21550), which is a uterine cancer cell line developed by HPV 16, can be used. In addition, the person uterine cancer cell implantation From the uterine cancer cells to ^{1 X 10 6 ~ 5 X 10} 6 cells, preferably, be carried out by a dose of about 2 X 10 ⁶ cells implanted in the subcutaneous tissue, such as the immune-deficient mice However, the dose is not greatly limited.

Separating the CD3-positive T immune cells is isolated from blood obtained from peripheral blood or hematopoietic tissue of a mouse injected with human uterine cancer cells, the hematopoietic tissue comprising the thymus, spleen, or bone marrow of the mouse. Isolation of CD3-positive T immune cells from blood can be performed by adding anti-CD3 antibodies and inducing antigen-antibody binding using a MACS (Magnetic cell sorter) or a flow cytometer (FACSvantage).

The compositions of the present invention can be administered in parenteral forms, including subcutaneous injection, and can be administered, for example, directly by injection or intravenous injection into cancer-forming tissue sites. Compositions for parenteral administration (e.g. injections) of the compositions according to the invention may be infused into a living body by dispersing and / or dissolving in pharmaceutically acceptable carriers such as sterile purified water, buffers of about pH 7, or physiological saline. And, if necessary, include conventional additives such as preservatives, stabilizers and the like.

The composition of the present invention is 1 X 10 ⁷ ~ 10 X 10 ⁷ cells / kg, preferably 1 X 10 ⁷ ~ 2 X 10 in the cord blood-derived CD3-positive T immune cells to the average adult patients (about 60kg) for the treatment of uterine cancer ^It may be administered at a dose of ⁷ cells / kg, more preferably about 2 X 10 ⁷ cells / kg, the dose may be changed depending on the type and condition of the disease. For a typical adult patient, each unit dosage form may comprise about 1 × 10 ⁷ cord blood derived CD3 positive T immune cells together with a suitable pharmaceutically acceptable carrier.

As described above, when mononuclear cells derived from umbilical cord blood are injected into neonatal immunodeficient mice according to the present invention, other immune cells, that is, B immune cells, NK cells, and myeloid system cells are not produced and are CD3-positive. It was newly discovered that only T immune cells of can be obtained selectively.

Therefore, the present invention comprises the steps of (a) injecting mononuclear cells (mononuclear cells) derived from umbilical cord blood into immunodeficient mice of 1 week or less; And (b) isolating CD3-positive T immune cells from umbilical cord blood, comprising: separating CD3-positive T immune cells from blood obtained from peripheral blood or hematopoietic tissue of the mouse 2 to 8 weeks after the injection. .

In particular, as can be seen in the following examples, CD3-positive T immune cells isolated from mice exhibiting proliferation inhibition against uterine cancer cells by injecting umbilical cord blood-derived monocyte cells and human uterine cancer cells into immunodeficient mice were treated with superior uterine cancer. It was found to be effective.

Therefore, the present invention comprises the steps of (a) injecting mononuclear cells (mononuclear cells) derived from umbilical cord blood into immunodeficient mice of 1 week or less; (b) injecting human uterine cancer cells into the mouse 2 to 8 weeks after the injection; And (c) isolating CD3 positive T immune cells from blood obtained from peripheral blood or hematopoietic tissue of said mouse about 2 weeks after said human uterine cancer cell injection, preferably about 3 to 4 weeks. Isolation method of CD3-positive T immune cells.

In the separation method, the immunodeficiency mouse can be used without a mouse having no immune function, for example, a non-Obese Diabetic Severe Combined Immunodeficiency (NOD-SCID) mouse can be preferably used. The immunodeficient mice are preferably neonatal mice, for example, newborn mice of 1 week or less. Infusion of the monocyte cells may be carried out by intraperitoneal administration, preferably by intraperitoneal administration of 1 × 10 ⁷ to 10 × 10 ⁷ cells, more preferably about 1 × 10 ⁷ cells.

The human uterine cancer cells can be used without limitation cancer cells derived from uterine cancer patients, for example, Caski cells (Korea Cell Line Bank, Cat. No. 21550) can be used. In addition, the person uterine cancer cell implantation From the uterine cancer cells to ^{1 X 10 6 ~ 5 X 10} 6 cells, preferably, be carried out by a dose of about 2 X 10 ⁶ cells implanted in the subcutaneous tissue, such as the immune-deficient mice have.

Separating the CD3-positive T immune cells is isolated from blood obtained from peripheral blood or hematopoietic tissue of a mouse injected with human uterine cancer cells, the hematopoietic tissue comprising the thymus, spleen, or bone marrow of the mouse. Isolation of CD3-positive T immune cells from blood can be performed by adding an anti-CD3 antibody and inducing antigen-antibody binding using a magnetic cell sorter (MACS) or a flow cytometer (FACSvantage).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example .

Human umbilical cord blood-derived monocytes were transplanted into a congenital immunodeficiency mouse (NOD-SCID mouse) to construct an animal model in which cord immune-derived T immune cells were differentiated from hematopoietic tissues, and blood was collected from the hematopoietic tissues. Caski cells (Korean Cell Line Bank, Cat. NO. 21550), which are cancer cells derived from uterine cancer of humans caused by HPV (16) infection, were injected into the subcutaneous tissues of mice that confirmed the presence of cord immune-derived T immune cells. The size and weight of uterine cancer tumors formed in the subcutaneous tissue of mice injected with Caski cells were measured, and the presence of cord blood-derived T immune cells in the formed uterine cancer tumor tissues was analyzed using immunohistostaining and flow cytometry. The presence of HPV cancer antigen-specific T-immune cells, the uterine cancer-causing gene, was analyzed by tetramer. The T-immune cells obtained from animal models were tested for uterine cancer cell removal by incubation with uterine cancer cells in vitro.

Example  1. Culture of Human Uterine Cancer Cells

Caski cells derived from human uterine cancer patients (Korea Cell Line Bank, Cat. NO. 21550) were treated with 10% fetal bovine serum (FBS; fetal bovine serum, Jeil Biotech Services), 0.25 M HEPES (N-2-hydroxyethyl -Rosewell Park Memorial Institue, supplemented with piperazine-N'-2-ethanesulfonic acid (N-2-hydroxyethyl-piperazine-N'-2-ethane-sulfonic acid), and 1% penicillin and streptomycin Gibco-BRL, Korea).

Example  2. Isolation of Umbilical Cord Blood-Derived Monocytes and Development of Experimental Animal Models for Umbilical Cord Blood-Derived T Immune Cell Proliferation

Umbilical cord blood to which anticoagulant (heparin) was added was layered into 50 ml Falcon Tubes injected with 20 ml of Ficoll-Paque solution (Amersham Biosciences AB, Sweden) and centrifuged at 2000 rpm for 20 minutes at room temperature. The monocyte portion of the separated intermediate layer was collected and diluted by adding two volumes of phosphate buffered saline (PBS), followed by washing by centrifugation at room temperature for 5 minutes.

Peripheral blood and hematopoietic tissues at 4 to 8 weeks of age after injection of the obtained cord blood-derived monocytes (1 × 10 ⁷ ) into the abdominal cavity of NOD-SCID (Non-Obese Diabetic Severe Combined Immunodeficiency) mice, one week before birth. (Bone marrow, spleen, thymus), anti-CD3 antibody (T immune cell), anti-CD19 antibody (B immune cell), anti-CD56 antibody (NK cell), anti-CD33 antibody (Myeloid cells), and anti-CD45 antibody (antibody against CD45 expressed in all kinds of monocyte hematopoietic cells), followed by immunostaining for 30 minutes, and PBS (D-phosphate buffered saline) was added, followed by 1500 Centrifugation for 5 minutes at rpm removed the antibody-antigen binding. After washing, flow cytometry (FACSvantage, BD, USA) was used to examine the presence of immune cells in peripheral blood and hematopoietic tissue.

1 is a result of analyzing the immune cells in the peripheral blood and hematopoietic tissue of the mouse transplanted with the umbilical cord blood-derived monocytes by flow cytometry and immunohistostaining. Figure 1a is a result of analyzing the presence of immune cells in the blood after collecting the peripheral blood of the mouse as a result of the presence of only CD3-positive T immune cells and other immune cells B cells, NK cells, bone marrow system Indicates no cell present. FIG. 1B is a result of analyzing cord blood-derived immune cells in bone marrow, spleen, and thymus, which are the hematopoietic tissues of the mouse, derived from human umbilical cord blood in bone marrow, spleen, and thymus tissue. Only CD3 positive T immune cells are present and B immune cells, NK cells, and myeloid cells are absent. Figure 1 c is an antibody that specifically binds to CD4 positive T cells (helper T cells, hT cells) and CD8 positive T cells (CTL) induced differentiation in cord blood stem cells to immunize the spleen and thymus tissue As a result of tissue staining, CD4 positive T cells (helper T cells) and CD8 positive T immune cells (CTL) cells derived from umbilical cord blood were present in high proportion in blood tissue. From the results of FIG. 1, it can be seen that only CD3-positive T immune cells (hT cells and CTL) are present in the hematopoietic tissues including the blood of the mouse.

Figure 2 shows the expression of CD3, a function regulatory molecule of T immune cells, by analyzing the characteristics of T immune cells in the blood collected from the thymus and spleen tissue of the mouse transplanted monocytes derived from cord blood with hCD4 antibody and hCD8 antibody This is the result of analysis. Figure 2a and b of the blood cells collected from the mouse thymus tissue and spleen tissue, respectively, immunostained CD4 positive by immunostaining with anti-CD4 antibody, anti-CD8 antibody, anti-CD3 antibody human T immune cell specific antibodies As a result of analyzing CD3 expression patterns in T cells and CD8 positive T cells, CD4 positive and CD8 positive T cell populations, it was found that each group normally expressed CD3 which plays an important role in the activation of T immune cells. have. 2c shows normal expression and expression of TCRαβ (T cell receptor) molecule, which is a functional molecule of CD3 T immune cells in spleen tissue.

Example  3. In Vivo Transplantation of Uterine Cancer Tumor Cells and Evaluation of Anticancer Activity by Immune Cells in Umbilical Cord Blood-Derived T Immune Cell Proliferation

6-8 weeks old NOD-SCID mice (Group 1, n = 7) and in the same manner as in Example 2 cord blood-derived monocytes were injected into the abdominal cavity before 1 week of age, and then peripheral blood was collected and analyzed. Results Caski cells obtained by culturing in Example 1 in 6-8 week old NOD-SCID mice (group 2, n = 7) in which proliferation of umbilical cord blood-derived T immune cells were confirmed in vivo was 2 × per mouse. 10 ⁶ doses were dispensed into physiological saline and cell transplantation was performed with 1 ml syringe into subcutaneous tissues such as mice.

Figure 3 shows the tumor suppression effect by the uterine cancer tumor and umbilical cord blood-derived T immune cells formed in the mouse subcutaneous tissue 6-7 weeks after implantation of Caski cells, uterine cancer cell line into the subcutaneous tissue. Figure 3a shows a picture of a tumor formed by transplanting Caski cells in mice transplanted with only Caski cells into subcutaneous tissue and mice with cord blood-derived T immune cells (CTN: cord blood derived T cell engrafted NOD-SCID mice). Figure 3 b shows the size of the uterine cancer formed in the subcutaneous tissue after 6-7 weeks by vernier calipers, Figure 3 c is a uterine cancer tumor collected by lethal 6-7 weeks after transplantation It shows that the weight of is measured. As can be seen from the results of FIG. 3, the tumor size was significantly reduced in mice with umbilical cord blood-derived T immune cells (52 ± 23.7) compared to mice implanted with only uterine cancer cells (715 ± 312.9). High tumor weight was significantly decreased (P <0.05) in mice with cord blood-derived T immune cells (0.1 ± 0.05) compared to tumor weight in mice implanted with uterine cancer cells only (1.3 ± 0.3).

4 is a umbilical cord blood present in peripheral blood and spleen, liver and bone marrow tissue of CTN mice after subcutaneous transplantation of Caski cells into cord blood derived T cell engrafted NOD-SCID mice (CTN). Derived T immune cells were measured by flow cytometry. Figure 4a is a result of analyzing the characteristics of the immune cells present in the peripheral blood collected at the time of death to measure the size and weight of the uterine cancer 6-7 weeks after uterine cancer cell transplantation, CD4-positive in CD3-positive T immune cells T cells (hT cells) and CD8 positive T cells (CTL cells) are present. 4B shows that CD3 positive T cells, CD4 positive T cells, and CD8 positive T cells also exist in the liver, spleen and bone marrow.

FIG. 5 shows a tumor fragment observed in tumor tissue after subcutaneous transplantation of Caski cells into a cord blood derived T cell engrafted NOD-SCID mouse (CTN). Analysis of the presence and activation of T immune cells; And cell death of uterine cancer cells induced by umbilical cord blood-derived T-immune cells with TUNEL to analyze specific uterine cancer immune cell therapeutic effects by T-immune cells. 5a shows human CD4 positive T cells (hT cells) and CD8 positive T cells (CTL) infiltrated into uterine cancer tissues. 5B shows the result of analyzing the expression of CD45RO, a CD8 positive T cell functional activation marker, in uterine cancer tumor tissue, and it can be seen that most of the CD8 positive T cells express CD45RO. 5c shows the results of TUNEL analysis of uterine cancer tumor tissue formed in the subcutaneous tissue of NOD-SCID mice, and d is the result of TUNEL analysis of uterine cancer tissue formed in NOD-SCID mouse subcutaneous tissue in which cord blood-derived T immune cells were expanded. The results are shown.

From the results of FIGS. 4 and 5, the T-immune cells derived from cord blood penetrate the tumor tissue of uterine cancer cells to actively inhibit tumor formation, and the T-immune cells specifically induce cell death of uterine cancer cells. It can be seen. Thus, the results directly show that cord blood-derived T immune cells have efficacy as immune cell therapy for the treatment of uterine cancer.

Example  4. In vitro with cord blood-derived T immune cells ( in in vitro Evaluation of Efficacy in Uterine Cancer

In each group of mice treated according to Example 3, 6-7 weeks after uterine cancer cell transplantation, T-immune cells present in mice whose growth inhibition and treatment efficacy of uterine cancer tumors were confirmed were collected in vitro ( In vitro specific immune responses to cervical cancer cells were verified.

In the spleen tissues of mice whose umbilical cord blood-derived T-immune-proliferated TN cells were observed through subcutaneous transplantation of uterine cancer cells, T-immune-derived T-immune cells were immunostained with hCD3 antibody, followed by MACS (cell separator). Magnetic cell sorter) was used to isolate only T immune cells. After the coculture with the isolated T-immune cells using Caski cells, which are human uterine cancer cells, and A549, the human lung cancer cell line, as a control group, specific immune responses against uterine cancer cells were removed by T-immune immune responses. Reaction). All cells harvested in vitro after 3 days of co-culture were analyzed by flow cytometry to examine the rate of cell death induced by the immune response (see FIG. 6).

As can be seen from the results of FIG. 6, umbilical cord blood-derived T immune cells with uterine cancer tumor suppression efficacy in mice can induce specific cell death for the same uterine cancer tumor cells in vitro. . These results indicate that the activation of specific T immune cells against uterine cancer cells is induced in mouse in vivo.

Example  5. Evaluation of Uterine Cancer Treatment Efficacy of Umbilical Cord Blood-derived T Immune Cells by Uterine Cancer Specific Immune Cells

NOD-SCID mice 6-8 weeks of age (group 1, n = 7); In the same manner as in Example 2, NOD-SCID mice whose proliferation of umbilical cord blood-derived T-immune cells were confirmed in vivo after injecting umbilical cord blood-derived monocytes into the abdominal cavity and injecting them into the abdominal cavity. 2nd group, 1 ^st CTN, n = 5); And injecting cord blood-derived mononuclear cells into the abdominal cavity before 1 week of age in the same manner as in Example 2, and then collecting peripheral blood to analyze NOD-SCID mice whose proliferation of cord blood-derived T immune cells was confirmed in vivo. In human uterine cancer-derived cell line, Caski cells were dispensed in physiological saline at a dose of 2 x 10 ⁶ per mouse, and cell transplantation was performed with 1 ml syringe to subcutaneous tissues such as mice. the NOD-SCID mice (group 3, 2 ^nd CTN, n = 6) was used to evaluate the efficacy of uterine cancer treatment on uterine cancer specific immune cells.

In the spleen tissues of NOD-SCID mice of the 2nd and 3rd groups, T-immune-derived T-immune cells were immunostained with hCD3 antibody, and then only T-immune cells were isolated by MACS (Magnetic cell sorter). Analysis of cells isolated by MACS using flow cytometry (FACSvantage) confirmed that the hCD3-positive T-immune cells had a high purity of more than 95%.

Human uterine cancer cell line Caski cells were implanted with 2 x 10 ⁶ cells per 6-8 weeks old NOD-SCID mice into the subcutaneous tissue to form a uterine cancer tumor for 4 weeks, and then a second group of uterine cancer tumor tissues of the mouse (1 ^st CTN) and Group 3 (2 ^nd 1 x 10 ⁶ cells per mouse were injected into the cord blood-derived CD3 positive T-immune cells of 95% purity or higher extracted from CTN), and the size of the uterine cancer tumor was measured every week. The efficacy of treatment of uterine cancer cells by CD3-positive T immune cells was observed.

Fig. 7A shows uterine cancer tumors obtained by implanting human uterine cancer cell line Caski cells to form uterine cancer tumors and injecting cord blood-derived CD3 positive T immune cells from each group. The tumor marked Caski in Fig. 7a shows uterine cancer tumors extracted after 8 weeks after implantation of only Caski cells, which are human uterine cancer cell lines, into the subcutaneous tissue in the first group of mice; Caski + 1 ^st CTN is a pre-NOD for hCD3-positive T immune cells (hCD3-positive T immune cells with purity greater than 95% isolated by MACS) isolated from spleen tissue 6-7 weeks after transplanting human cord blood, a second group of mice. Uterine cancer cell line 2 x 10 ⁶ Caski cells were implanted in the subcutaneous tissue of SCID mice into tumor tissue of mice that formed uterine cancer for 4 weeks, followed by uterine cancer tumors harvested from the subcutaneous tissue 4 weeks later; Caski + 2 ^nd CTN is a human uterine cancer cell line in the subcutaneous tissue of 6-7 week old NOD-SCID mice whose human umbilical cells were confirmed by peripheral blood analysis after human cord blood, which is a third group of mice, was transplanted by intraperitoneal administration. HCD3-positive T-immune cells isolated from spleen tissues of mice whose uterine cancer tumor suppression efficacy was observed 8 weeks after transplantation of 2 × 10 ⁶ Caski cells (hCD3-positive T-immune cells with purity greater than 95% isolated by MACS) 2 x 10 ⁶ Caski cells, which are uterine cancer cell lines, were previously transplanted into NOD-SCID mice to tumor tissues of mice that formed uterine cancer for 4 weeks to show uterine cancer tumors collected from the subcutaneous tissue after 4 weeks. will be.

7 b shows the results of observing the size change of uterine cancer tumors formed in the subcutaneous tissues weekly using the NOD-SCID mouse models of the three test groups, compared to the mice transplanted with only Caski cells. 1 ^st Better tumor suppression of uterine cancer was observed in CTN mice and Caski + 2 ^nd CTN showed significantly higher uterine cancer suppression effect than two mice.

FIG. 7C shows the weight of uterine cancer tumors present in the subcutaneous tissue after 4 weeks of transplantation of CD3-positive T immune cells into uterine cancer tumor tissues in the NOD-SCID mouse models of the three test groups. As a result, Caski + 1 ^st compared to mice transplanted with only Caski cells Better tumor suppression of uterine cancer was observed in CTN mice and Caski + 2 ^nd CTN showed significantly higher uterine cancer suppression effect than two mice.

8 a is Caski + 2 ^st HCD3-positive T immune cells isolated from spleen tissue 6-7 weeks after transplanting human cord blood from CTN group 2 mice (hCD3-positive T immune cells with purity greater than 95% isolated by MACS) In the subcutaneous tissue of NOD-SCID mice, 2 x 10 ⁶ Caski cells, which are uterine cancer cell lines, were transplanted into tumor tissues of mice that formed uterine cancer for 4 weeks, and then 4 weeks later, from uterine cancer tumors collected from the subcutaneous tissue. Tissue sections were prepared and tissue immunostained with hCD8-FITC and hCD4-PE antibodies. FIG. 8B shows 6 to 7 that human T immune cells are present through peripheral blood analysis after transplanting human umbilical cord blood derived from a third group of mice, which are Caski + 2 ^nd CTN, by intraperitoneal administration. HCD3-positive T-immune cells (MACS) were isolated from spleen tissues of mice whose human uterine cancer cell line 2 x 10 ⁶ Caski cells were transplanted into subcutaneous tissues of old NOD-SCID mice after 8 weeks. HCD3-positive T immune cells with a purity of 95% or higher) were transplanted into 2 x 10 ⁶ Caski cells, which are uterine cancer cell lines, in tumor tissues of mice in which uterine cancer was formed for 4 weeks. 4 weeks later, tissue sections were obtained from uterine cancer tumors obtained from subcutaneous tissues and tissue immunostained with hCD8-FITC antibodies and hCD4-PE antibodies, which are human T immune cell-specific antibodies. 8 c shows the results of observing the expression of CD45RO, an activation marker of CD8 positive T immune cells, using the uterine cancer tumor tissue section in b.

As can be seen from the results of a and b in FIG. 8, Caski + 1 ^st Caski + 2 ^nd compared to hCD4 positive and hCD8 positive T immune cells in uterine cancer tissues in CTN mice It can be seen that hCD4 positive and hCD8 positive T immune cells present in CTN mouse uterine cancer tumor tissues are present at an extremely high rate. As also shown in c of FIG. 8, Caski + 2 ^nd The hCD8 positive T immune cells, cord blood-derived cytotoxic T lymphocytes (CTLs) present in CTN mouse uterine cancer tumor tissues, account for the majority of the cells expressing the activation marker hCD45RO. Caski + 1 ^st Caski + 2 ^nd compared to T immune cells present in CTN The T immune cells in the spleen of CTN mice can be seen that there are a lot of uterine cancer cell-specific T immune cells capable of recognizing uterine cancer cells, and the immune cells express the activation markers of normal T immune cells. Can be.

8 d is the three groups (Caski, Caski + 1 ^st CTN, Caski + 2 ^nd After uterine cancer tissue extracted from NOD-SCID mouse subcutaneous tissue of CTN) was made into a single cell, T immune cells present in the uterine cancer tissue were mutated to E7 antigen (YMLDLQPETT) of HPV (Human Papilloma Virus), a gene causing uterine cancer. Using HPV-A2 Tetramer (ORPEGEN Pharma GmbH. Heidelberg, Germany) combined with HLA-A2 protein, a major tissue-conjugated antigen, hCD8 positive T immune cells, CTL cells with TCRαβ that recognize HPV E7 antigens, were applied to uterine cancer tissues. It is the result of measuring by the flow cytometer the existence.

As can be seen in FIG. 8d, no hCD8 positive cells were observed in uterine cancer tissues implanted with Caski alone, and HPV cancer antigen E7 at a rate of 1.5 ± 0.4% in uterine cancer tumor tissues of Caski + 1 ^st CTN. the T cells of the immune hCD8-positive with a TCRαβ were present to recognize, Caski + 2 ^nd In the uterine cancer tumor tissue of CTN, hCD8 positive T immune cells with TCRαβ that recognizes E7, an HPV cancer antigen, are present at a rate of 4.3 ± 0.6%. These results indicate that cord-positive CD8-positive T-immune cells specifically recognize uterine cancer tumors formed in subcutaneous tissue of NOD-SCID mice, inhibit the formation of uterine cancer tumors and apoptosis the uterine cancer cells. Able to know.

As can be seen from the results of FIGS. 6 to 8, the cord blood-derived CD3-positive T immune cells have specific tumor removal efficacy against uterine cancer tumors in vivo in an animal model of uterine cancer, in particular, analysis using HPV-tetramer. As can be seen from the results, it can be seen that the CD8 positive T immune cells derived from cord blood have immune cells that specifically recognize the E7 antigen of HPV, a cancer antigen of uterine cancer.

FIG. 1 shows the results of analysis of immune cells in peripheral blood and hematopoietic tissue of mice transplanted with umbilical cord blood-derived monocytes by flow cytometry and immunohistostaining. Figure 1a shows the result of analyzing the presence of immune cells present in the blood after collecting the peripheral blood of the mouse. Figure 1 b shows the results of analyzing the cord blood-derived immune cells present in the hematopoietic tissue bone marrow, spleen, thymus of the mouse. Figure 1 c is an antibody that specifically binds to CD4 positive T cells (helper T cells, hT cells) and CD8 positive T cells (CTL) induced differentiation in cord blood stem cells to immunize the spleen and thymus tissue The result of analysis by tissue staining is shown.

FIG. 2 shows the expression of CD3, a function regulatory molecule of T immune cells, by analyzing the characteristics of T immune cells in blood collected from the thymus and spleen tissue of mice transplanted with monocytes derived from cord blood. The result of the analysis. Figure 2a and b of the blood cells collected from the mouse thymus tissue and spleen tissue, respectively, immunostained CD4 positive by immunostaining with anti-CD4 antibody, anti-CD8 antibody, anti-CD3 antibody human T immune cell specific antibodies As a result of analyzing CD3 expression patterns in T cells and CD8 positive T cells, CD4 positive and CD8 positive T cell populations, it was found that each group normally expressed CD3 which plays an important role in the activation of T immune cells. have. 2c shows normal expression and expression of TCRαβ (T cell receptor) molecule, which is a functional molecule of CD3 T immune cells in spleen tissue.

Figure 3 shows the tumor suppression effect by the uterine cancer tumor and umbilical cord blood-derived T immune cells formed in the mouse subcutaneous tissue 6-7 weeks after implantation of Caski cells, uterine cancer cell line into the subcutaneous tissue. Figure 3a shows a picture of a tumor formed by transplanting Caski cells in mice transplanted with only Caski cells into subcutaneous tissue and mice with cord blood-derived T immune cells (CTN: cord blood derived T cell engrafted NOD-SCID mice). Figure 3 b shows the size of the uterine cancer formed in the subcutaneous tissue after 6-7 weeks by vernier calipers, Figure 3 c is a uterine cancer tumor collected by lethal 6-7 weeks after transplantation It shows that the weight of is measured.

4 is a umbilical cord blood present in peripheral blood and spleen, liver and bone marrow tissue of CTN mice after subcutaneous transplantation of Caski cells into cord blood derived T cell engrafted NOD-SCID mice (CTN). Derived T immune cells were measured by flow cytometry. Figure 4a is a result of analyzing the characteristics of the immune cells present in the peripheral blood collected at the time of death to measure the size and weight of the uterine cancer 6-7 weeks after uterine cancer cell transplantation, CD4-positive in CD3-positive T immune cells T cells (hT cells) and CD8 positive T cells (CTL cells) are present. 4B shows that CD3 positive T cells, CD4 positive T cells, and CD8 positive T cells also exist in the liver, spleen and bone marrow.

FIG. 5 shows a tumor fragment observed after subcutaneous transplantation of Caski cells into a cord blood derived T cell engrafted NOD-SCID mouse (CTN) -containing mouse. Analysis of the presence and activation of T immune cells; And cell death of uterine cancer cells induced by umbilical cord blood-derived T-immune cells with TUNEL to analyze specific uterine cancer immune cell therapeutic effects by T-immune cells. 5a shows human CD4 positive T cells (hT cells) and CD8 positive T cells (CTL) infiltrated into uterine cancer tissues. 5B shows the result of analyzing the expression of CD45RO, a CD8 positive T cell functional activation marker, in uterine cancer tumor tissue, and it can be seen that most of the CD8 positive T cells express CD45RO. 5c shows the results of TUNEL analysis of uterine cancer tumor tissue formed in the subcutaneous tissue of NOD-SCID mice, and d is the result of TUNEL analysis of uterine cancer tissue formed in NOD-SCID mouse subcutaneous tissue in which cord blood-derived T immune cells were expanded. The results are shown.

6 shows the efficacy of tumor suppression of uterine cancer in vitro of cord blood-derived T-immune cells obtained from the spleen and liver tissue of mice in which CTN mice transplanted with cord blood stem cells were implanted subcutaneously and uterine cancer tumor suppression ability was observed. The analysis results are shown.

Fig. 7A shows uterine cancer tumors obtained by implanting human uterine cancer cell line Caski cells to form a uterine cancer tumor, followed by injection of cord blood-derived CD3 positive T immune cells from each group. The tumor marked Caski in Fig. 7a shows uterine cancer tumors extracted after 8 weeks after implantation of only Caski cells, which are human uterine cancer cell lines, into the subcutaneous tissue in the first group of mice; Caski + 1 ^st CTN is a pre-NOD for hCD3-positive T immune cells (hCD3-positive T immune cells with purity greater than 95% isolated by MACS) isolated from spleen tissue 6-7 weeks after transplanting human cord blood, a second group of mice. Uterine cancer cell line 2 x 10 ⁶ Caski cells were implanted in the subcutaneous tissue of SCID mice into tumor tissue of mice that formed uterine cancer for 4 weeks, followed by uterine cancer tumors harvested from the subcutaneous tissue 4 weeks later; Caski + 2 ^nd CTN is a human uterine cancer cell line in the subcutaneous tissue of 6-7 week old NOD-SCID mice whose human umbilical cells were confirmed by peripheral blood analysis after human cord blood, which is a third group of mice, was transplanted by intraperitoneal administration. HCD3-positive T-immune cells isolated from spleen tissues of mice whose uterine cancer tumor suppression efficacy was observed 8 weeks after transplantation of 2 × 10 ⁶ Caski cells (hCD3-positive T-immune cells with a purity greater than 95% isolated by MACS) 2 x 10 ⁶ Caski cells, which are uterine cancer cell lines, were previously transplanted into NOD-SCID mice to tumor tissues of mice that formed uterine cancer for 4 weeks to show uterine cancer tumors collected from the subcutaneous tissue after 4 weeks. will be. Figure 7b shows the results of observing the change in the size of the uterine cancer tumors formed in the subcutaneous tissue weekly using the NOD-SCID mouse model of the three test groups, Figure 7c is a view of the three test groups In the NOD-SCID mouse model, CD3-positive T-immune cells were transplanted into uterine cancer tumor tissue, and 4 weeks later, mice of each group were killed and the weight of uterine cancer tumors present in the subcutaneous tissue was measured.

8 a is Caski + 2 ^st HCD3-positive T immune cells isolated from spleen tissue 6-7 weeks after transplanting human cord blood from CTN group 2 mice (hCD3-positive T immune cells with purity greater than 95% isolated by MACS) In the subcutaneous tissue of NOD-SCID mice, 2 x 10 ⁶ Caski cells, which are uterine cancer cell lines, were transplanted into tumor tissues of mice that formed uterine cancer for 4 weeks, and then 4 weeks later, from uterine cancer tumors collected from the subcutaneous tissue. Tissue sections were prepared and tissue immunostained with hCD8-FITC and hCD4-PE antibodies. FIG. 8B shows 6 to 7 that human T immune cells are present through peripheral blood analysis after transplanting human umbilical cord blood derived from a third group of mice, which are Caski + 2 ^nd CTN, by intraperitoneal administration. HCD3-positive T-immune cells (MACS) were isolated from spleen tissues of mice whose human uterine cancer cell line 2 x 10 ⁶ Caski cells were transplanted into subcutaneous tissues of old NOD-SCID mice after 8 weeks. HCD3-positive T immune cells with a purity of 95% or higher) were transplanted into 2 x 10 ⁶ Caski cells, which are uterine cancer cell lines, in tumor tissues of mice in which uterine cancer was formed for 4 weeks. 4 weeks later, tissue sections were obtained from uterine cancer tumors obtained from subcutaneous tissues and tissue immunostained with hCD8-FITC antibodies and hCD4-PE antibodies, which are human T immune cell-specific antibodies. 8 c shows the results of observing the expression of CD45RO, an activation marker of CD8 positive T immune cells, using the uterine cancer tumor tissue section in b. 8 d is the three groups (Caski, Caski + 1 ^st CTN, Caski + 2 ^nd After uterine cancer tissue extracted from NOD-SCID mouse subcutaneous tissue of CTN) was made into a single cell, T immune cells present in the uterine cancer tissue were transferred to E7 antigen (YMLDLQPETT) of HPV (Human Papilloma Virus), a gene that causes uterine cancer. Using HPV-A2 Tetramer (ORPEGEN Pharma GmbH. Heidelberg, Germany) bound to HLA-A2 protein, a major human tissue conjugate antigen, hCD8 positive T immune cells, CTL cells with TCRαβ that recognize HPV E7 antigen It is the result of measuring by the flow cytometer whether it exists in the.

Claims (17)

Biograft composition for the treatment of uterine cancer comprising CD3 positive T immune cells (CD3 + T lymphocytes) derived from umbilical cord blood and a pharmaceutically acceptable carrier. The composition of claim 1, wherein the CD3-positive T immune cells are differentiated from monocytes derived from umbilical cord blood. 3. The method of claim 2, wherein the CD3-positive T immune cells are (a) injecting mononuclear cells derived from umbilical cord blood into immunodeficient mice of 1 week or less; And (b) isolating CD3 positive T immune cells from blood obtained from the peripheral blood or hematopoietic tissue of the mouse 2 to 8 weeks after the injection. 3. The method of claim 2, wherein the CD3-positive T immune cells are (a) injecting mononuclear cells derived from umbilical cord blood into immunodeficient mice of 1 week or less; (b) injecting human uterine cancer cells into the mouse 2 to 8 weeks after the injection; And (c) isolating CD3 positive T immune cells from blood obtained from peripheral blood or hematopoietic tissue of said mouse about 2 weeks after said human uterine cancer cell injection. The composition of claim 3 or 4, wherein the immunodeficient mouse is a NO-Obese Diabetic Severe Combined Immunodeficiency (NOD-SCID) mouse. The composition according to claim 3 or 4, wherein the infusion of monocytes is performed by intraperitoneal administration. The composition of claim 6, wherein the infusion of monocytes is performed by intraperitoneal administration at a dose of 1 × 10 ⁷ to 10 × 10 ⁷ . The composition according to claim 4, wherein the injection of human uterine cancer cells is performed by transplanting human uterine cancer cells into the subcutaneous tissue of the back of the immunodeficient mouse at a dose of 1 x 10 ⁶ to 5 x 10 ⁶ . The composition of claim 3 or 4, wherein the hematopoietic tissue is thymus, spleen, or bone marrow of the mouse. The composition according to any one of claims 1 to 4, wherein the uterine cancer is cervical cancer caused by Papilloma virus (HPV) infection. (a) injecting mononuclear cells derived from umbilical cord blood into immunodeficient mice up to 1 week of age; And (b) separating CD3 positive T immune cells from cord blood from blood obtained from peripheral blood or hematopoietic tissue of the mouse 2 to 8 weeks after the injection. (a) injecting mononuclear cells derived from umbilical cord blood into immunodeficient mice up to 1 week of age; (b) injecting human uterine cancer cells into the mouse 2 to 8 weeks after the injection; And (c) separating CD3-positive T immune cells from cord blood from blood obtained from peripheral blood or hematopoietic tissue of the mouse about 2 weeks after the injection of human uterine cancer cells. The method of claim 11 or 12, wherein the immunodeficient mouse is a NOD-SCID (Non-Obese Diabetic Severe Combined Immunodeficiency) mouse. The method of claim 11 or 12, wherein the infusion of monocytes is performed by intraperitoneal administration. 15. The method of claim 14, wherein the infusion of monocytes is performed by intraperitoneal administration at a dose of 1 × 10 ⁷ to 10 × 10 ⁷ . The method of claim 12, wherein the injection of human uterine cancer cells is performed by transplanting human uterine cancer cells into subcutaneous tissue of the back of the immunodeficient mouse at a dose of 1 × 10 ⁶ to 5 × 10 ⁶ . The method of claim 11 or 12, wherein the hematopoietic tissue is thymus, spleen, or bone marrow of the mouse.

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