KR20230001726A - A method for mass production of immune inhibitory T cells - Google Patents
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Abstract
Description
본 발명은 면역억제 효과를 나타내는 T 세포를 체외에서 대량 생산하기 위한 방법에 관한 것으로, 보다 구체적으로는 G-CSF(granulocyte colony stimulating factor)를 포함하는, CD3+, PD1+ 및 Tim3+의 면역 표현형을 갖는 T 세포를 대량 생산하기 위한 방법에 관한 것이다.The present invention relates to a method for mass-producing T cells exhibiting immunosuppressive effects in vitro, and more specifically, T cells having immunophenotypes of CD3+, PD1+ and Tim3+, including granulocyte colony stimulating factor (G-CSF). It relates to a method for mass-producing cells.
이식편대숙주병(graft-versus-host disease, GvHD)은 이식 후 공여자와 환자의 조직적합원(histocompativility antigens, HLA)이 불일치하여 발생하는 동종 면역반응으로, 빈도는 10%부터 80%까지 나타나며, 사망률이 높은 중증 GvHD(grade 3~4)의 경우도 약 20% 환자에서 발생하는 것으로 알려져 있다. 이식편대 숙주병의 치료를 위해 스테로이드 치료부터 면역글로불린, 탈리도마이드(thalidomide), 사이클로스포린(cyclosporine)등의 면역억제제를 다양하게 사용하고 있으나, 스테로이드에 반응이 없는 경우 대부분의 환자는 감염으로 사망하거나, 장기기능이 손상되어 삶의 질이 저하되는 문제점이 대두되고 있다. 이에 면역억제를 위한 세포 치료제의 개발이 시급하다.Graft-versus-host disease (GvHD) is an allogeneic immune response caused by a mismatch between donor and patient histocompatibility antigens (HLA) after transplantation, with a frequency ranging from 10% to 80%. Severe GvHD (grade 3-4) with a high mortality rate is also known to occur in about 20% of patients. Various immunosuppressants such as immunoglobulin, thalidomide, and cyclosporine are used for the treatment of graft-versus-host disease, but most patients die from infection or long-term The problem of deteriorating quality of life due to impaired function has emerged. Therefore, the development of cell therapy for immunosuppression is urgently needed.
면역억제용 세포 치료제에 대한 시대적 요구가 증가됨에 따라, 면역반응을 일방적으로 억제하는 면역억제제보다는 세포 간의 균형을 교정하면서 동종면역반응을 조절할 수 있는 면역세포치료가 가장 이상적으로 평가되고 있다. 하지만, 면역 억제용 세포치료제인 MSC(Mesenchymal Stem Cells)는 세포 수의 확보를 위해 여러 단계의 배양 과정이 필요하며, 이 과정에서 발생하는 활성산소로 인해 세포기능이 손상된다.As the demand for cell therapy for immunosuppression increases, immune cell therapy that can control the alloimmune response while correcting the balance between cells is evaluated as the most ideal, rather than immunosuppressive agents that unilaterally suppress the immune response. However, MSC (Mesenchymal Stem Cells), a cell therapy for immunosuppression, requires a multi-step culture process to secure the number of cells, and cell function is damaged due to active oxygen generated during this process.
한편, 항원 침입 신호는 항원 제시 세포(antigene presenting cells)가 T 세포의 TCR과 공동 자극 인자(co-stimulatory molecule)를 통해 신호를 전달하여 면역 반응이 일어난다. 면역반응이 종료되면 T 세포는 central memory cell로 분화되는 반면, 지속적인 자극이 있는 상태에서는 T 세포가 PD-1을 비롯한, Tim3, LAG-3, TIGIT 등과 같은 공동 억제 인자(co-inhibitory molecule)가 발현하게 되고, 이렇게 표면항원이 변환된 T 세포는 면역반응이 과다하게 일어나는 것을 차단하고 tolerance 상태를 유지하게 된다. 따라서, 표면항원이 변환된 T 세포를 유도하여 면역반응을 억제시킬 수 있는 세포치료제를 개발 할 수 있다면 여러 면역 질환에서 다양한 용도로 유용하게 사용 될 수 있을 것이다. DNA 절단(cleavage)으로 T 세포 표면항원을 선택적으로 불활성화시키는 연구가 진행 중이긴 하나(한국 공개특허 10-2016-0029017), 비-동종반응성(non-alloreactive) 면역 억제 관한 연구가 대부분일 뿐, 동종면역반응을 억제시키는 T 세포 및 이의 용도에 대해서는 아직까지 연구가 미미한 실정이다.On the other hand, the antigen invasion signal is transmitted by antigen presenting cells through the TCR of T cells and co-stimulatory molecules, and an immune response occurs. When the immune response is terminated, T cells differentiate into central memory cells, whereas under continuous stimulation, T cells produce co-inhibitory molecules such as PD-1, Tim3, LAG-3, and TIGIT. expression, and the surface antigen-converted T cells block excessive immune responses and maintain a tolerance state. Therefore, if a cell therapy agent capable of suppressing the immune response by inducing surface antigen-transformed T cells can be developed, it will be useful for various purposes in various immune diseases. Although studies on selective inactivation of T cell surface antigens by DNA cleavage are in progress (Korean Patent Publication No. 10-2016-0029017), most studies are only on non-alloreactive immunosuppression, Studies on T cells and their use for suppressing alloimmune responses are still insignificant.
상기와 같은 문제점을 해결하기 위하여, 본 발명자들은 G-CSF를 피하 주사한 개체의 말초 혈액 조혈모세포에서 단핵구를 분리하고, 분리된 단핵구에서 CD3+ 세포 분획을 분류한 뒤 저용량의 IL-2와 함께 배양함으로써 T 세포 표면에 공동 억제 인자(co-inhibitory molecule)의 발현이 증가된 세포를 대량수집하는 방법을 규명하였다.In order to solve the above problems, the present inventors isolated monocytes from peripheral blood hematopoietic stem cells of subjects injected subcutaneously with G-CSF, sorted a CD3+ cell fraction from the isolated monocytes, and cultured them with a low dose of IL-2. By doing so, a method for collecting large amounts of cells with increased expression of co-inhibitory molecules on the surface of T cells was identified.
이에, 본 발명은 G-CSF(granulocyte colony stimulating factor)를 포함하는, CD3+, PD1+ 및 Tim3+의 면역 표현형을 갖는 T 세포의 대량 생산용 배지 조성물을 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a medium composition for mass production of T cells having immunophenotypes of CD3+, PD1+ and Tim3+, including granulocyte colony stimulating factor (G-CSF).
또한, 본 발명은 하기의 단계를 포함하는, CD3+, PD1+ 및 Tim3+의 면역 표현형을 갖는 T 세포의 대량생산 방법을 제공하는 것을 다른 목적으로 한다.Another object of the present invention is to provide a method for mass-producing T cells having CD3+, PD1+ and Tim3+ immune phenotypes, which includes the following steps.
(a) G-CSF(granulocyte colony stimulating factor)로부터 말초 혈액 조혈모세포(Peripheral blood stem cell, PBSC)를 수집하는 단계;(a) collecting peripheral blood stem cells (PBSC) from granulocyte colony stimulating factor (G-CSF);
(b) 상기 말초 혈액 조혈모세포(PBSC)로부터 단핵구를 분리하는 단계; (b) separating monocytes from the peripheral blood hematopoietic stem cells (PBSC);
(c) 분리된 단핵구에서 CD3+ 세포 분획을 분류하는 단계; 및 (c) sorting the CD3+ cell fraction from the isolated monocytes; and
(d) IL-2(interukin-2)가 포함된 무혈청 배지에서 상기 CD3+ 세포 분획을 배양하는 단계를 포함하는 단계.(d) culturing the CD3+ cell fraction in a serum-free medium containing interukin-2 (IL-2).
또한, 본 발명은 상기 방법을 이용하여 대량 생산되며, CD3+, PD1+ 및 Tim3+의 면역 표현형을 갖는 T 세포가 10% 이상 포함된 T 세포 집단을 제공하는 것을 또 다른 목적으로 한다.In addition, another object of the present invention is to provide a T cell population that is mass-produced using the above method and contains 10% or more of T cells having immunophenotypes of CD3+, PD1+ and Tim3+.
또한, 본 발명은 상기 T 세포 집단을 포함하는 면역억제용 조성물을 제공하는 것을 또 다른 목적으로 한다.In addition, another object of the present invention is to provide a composition for immunosuppression comprising the T cell population.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 목적을 달성하기 위하여 본 발명은 G-CSF(granulocyte colony stimulating factor)를 포함하는, CD3+, PD1+ 및 Tim3+의 면역 표현형을 갖는 T 세포의 대량 생산용 배지 조성물을 제공한다.In order to achieve the above object, the present invention provides a medium composition for mass production of T cells having immunophenotypes of CD3+, PD1+ and Tim3+, including granulocyte colony stimulating factor (G-CSF).
또한, 본 발명은 하기의 단계를 포함하는, CD3+, PD1+ 및 Tim3+의 면역 표현형을 갖는 T 세포의 대량생산 방법을 제공한다.In addition, the present invention provides a method for mass-producing T cells having CD3+, PD1+ and Tim3+ immune phenotypes, comprising the following steps.
(a) G-CSF (granulocyte colony stimulating factor)로부터 말초 혈액 조혈모세포(Peripheral blood stem cell, PBSC)를 수집하는 단계;(a) collecting peripheral blood stem cells (PBSC) from granulocyte colony stimulating factor (G-CSF);
(b) 상기 말초 혈액 조혈모세포(PBSC)로부터 단핵구를 분리하는 단계; (b) separating monocytes from the peripheral blood hematopoietic stem cells (PBSC);
(c) 분리된 단핵구에서 CD3+ 세포 분획을 분류하는 단계; 및 (c) sorting the CD3+ cell fraction from the isolated monocytes; and
(d) IL-2(interukin-2)가 포함된 무혈청 배지에서 상기 CD3+ 세포 분획을 배양하는 단계를 포함하는 단계.(d) culturing the CD3+ cell fraction in a serum-free medium containing interukin-2 (IL-2).
본 발명의 일구현예로, 상기 T 세포는 Lag3+ 및 TIGIT+에서 선택되는 하나 이상의 면역 표현형을 더 포함할 수 있다.In one embodiment of the present invention, the T cells may further include one or more immunophenotypes selected from Lag3+ and TIGIT+.
본 발명의 다른 구현예로, 상기 (b) 단계에서 단핵구를 분리하는 단계는 혈액 성분 분리기(COBE spectra)를 이용하여 수행될 수 있다.In another embodiment of the present invention, the separating of monocytes in step (b) may be performed using a blood component separator (COBE spectra).
본 발명의 또 다른 구현예로, 상기 (c) 단계에서 CD3+ 세포 분획을 분류하는 단계는 자기 세포분리기(MACS magnetic) 또는 MS 컬럼(MS column)을 이용하여 수행될 수 있다.In another embodiment of the present invention, the sorting of the CD3+ cell fraction in step (c) may be performed using a MACS magnetic or MS column.
본 발명의 또 다른 구현예로, 상기 (d) 단계에서 배양은 3 내지 7일 동안 수행될 수 있다.In another embodiment of the present invention, the culturing in step (d) may be performed for 3 to 7 days.
또한, 본 발명은 상기 방법을 이용하여 대량 생산되며, CD3+, PD1+ 및 Tim3+의 면역 표현형을 갖는 T 세포가 10% 이상 포함된 T 세포 집단을 제공한다.In addition, the present invention provides a T cell population that is mass-produced using the above method and contains 10% or more of T cells having CD3+, PD1+, and Tim3+ immunophenotypes.
본 발명의 일구현예로, 상기 T 세포는 Lag3+ 및 TIGIT+에서 선택되는 하나 이상의 면역 표현형을 더 포함할 수 있다.In one embodiment of the present invention, the T cells may further include one or more immunophenotypes selected from Lag3+ and TIGIT+.
또한, 본 발명은 상기 T 세포 집단을 포함하는 면역억제용 조성물을 제공한다.In addition, the present invention provides a composition for immunosuppression comprising the T cell population.
본 발명에 의하여 면역억제 효과가 있는 CD3+, PD1+ 및 Tim3+의 면역 표현형을 포함하는 세포를 대량으로 생산할 수 있으며, 상기 세포는 동종 면역 반응 억제 효과를 가지므로, 동종 면역 억제를 위한 세포치료제로 널리 활용될 수 있을 것으로 기대된다.According to the present invention, cells containing CD3+, PD1+, and Tim3+ immune phenotypes with immunosuppressive effects can be mass-produced, and since the cells have an alloimmune response suppression effect, they are widely used as cell therapy agents for alloimmune suppression. hopefully it can be
도 1은 G-CSF mobilize PBSC CD3+ 림프구를 배양하지 않거나(PBSC T 세포), IL-2(50U/mL)와 함께 5일간 배양(IL-2(+) PBSC T 세포)하거나, IL-2 처리 없이 5일간 배양(IL-2(-) PBSC T 세포)하고 각 세포를 분석한 것으로, 도 1a는 각 세포 표면에서 co-inhibitory molecule의 발현 정도를 확인한 것이고, 도 1b는 각 세포의 세포 분획을 비교한 결과를 나타낸 것이고, 도 1c는 IL-2(+) PBSC T 세포와 IL-2(-) PBSC T 세포를 각각 CD14+ 동종 단핵구(mononuclear cells; MNCs)와 혼합하고 면역반응(mixed lymphocyte reaction, MLR)을 유도한 결과를 나타낸 것이다.
도 2는 CD3+PD1+Tim3+ 세포의 면역억제 효과를 분석한 것으로, 도 2a는 CD3+PD1+ 세포 분획 결과 및 PD1+세포를 제거한 후 PD1- 세포만을 이용하여 면역반응(mixed lymphocyte reaction, MLR)을 유도한 결과를 나타낸 것이고, 도 2b는 CD3+PD1+Tim3+ 세포 분획 결과 및 Tim3+ 세포를 제거 한 후 면역반응(mixed lymphocyte reaction, MLR)을 유도한 결과를 나타낸 것이다.
도 3은 CD3+PD1+Tim3+Lag3+TIGIT+ 세포의 면역억제 효과를 분석한 것으로, 도 3a는 CD3+PD1+Tim3+Lag3+ 세포 분획 결과를 나타낸 것이고, 도 3b는 CD3+PD1+Tim3+TIGIT+ 세포 및 CD3+PD1+Tim3+TIGIT- 세포 분획 결과와 TIGIT 발현 여부에 따라 면역반응(mixed lymphocyte reaction, MLR)을 유도한 결과를 나타낸 것이고, 도 3c는 배양 전의 PBSC T 세포, 배양후의 IL-2(+) PBSC T 세포 및 IL-2(-) PBSC T 세포에서 도 3b에서 동종면역반응 억제효과가 탁월한 CD3+PD1+Tim3+TIGIT+ 세포 수 (1x103 CD3+ 세포당)를 비교한 결과를 나타낸 것이다.Figure 1 shows G-CSF mobilize PBSC CD3+ lymphocytes not cultured (PBSC T cells), cultured with IL-2 (50 U/mL) for 5 days (IL-2(+) PBSC T cells), or treated with IL-2 Each cell was cultured for 5 days (IL-2(-) PBSC T cells) and analyzed. Figure 1a confirms the expression level of the co-inhibitory molecule on the surface of each cell, and Figure 1b shows the cell fraction of each cell. 1c shows the results of comparison, and FIG. 1c shows that IL-2(+) PBSC T cells and IL-2(-) PBSC T cells were mixed with CD14+ allogeneic monocytes (MNCs), respectively, and the immune response (mixed lymphocyte reaction, It shows the result of inducing MLR).
FIG. 2 analyzes the immunosuppressive effect of CD3+PD1+Tim3+ cells, and FIG. 2a shows the result of CD3+PD1+ cell fractionation and the induction of an immune response (mixed lymphocyte reaction, MLR) using only PD1- cells after removing PD1+ cells. 2b shows the results of CD3+PD1+Tim3+ cell fractionation and the induction of a mixed lymphocyte reaction (MLR) after removing Tim3+ cells.
Figure 3 analyzes the immunosuppressive effect of CD3+PD1+Tim3+Lag3+TIGIT+ cells, Figure 3a shows the results of CD3+PD1+Tim3+Lag3+ cell division, and Figure 3b shows CD3+PD1+Tim3+TIGIT+ cells. and CD3+PD1+Tim3+TIGIT- cell fractionation results and results of inducing an immune response (mixed lymphocyte reaction, MLR) according to TIGIT expression, and FIG. 3c shows PBSC T cells before culture and IL-2 after culture ( +) PBSC T cells and IL-2(-) PBSC T cells in Fig. 3b shows the result of comparing the number of CD3+PD1+Tim3+TIGIT+ cells (per 1x10 3 CD3+ cells) with excellent alloimmune response suppression effect.
본 발명자들은 G-CSF를 피하 주사한 개체의 말초 혈액 조혈모세포에서 단핵구를 분리하고, 분리된 단핵구에서 CD3+ 세포 분획을 분류한 뒤 저용량의 IL-2 (50U/mL)와 함께 배양함으로써 T 세포 표면에 공동 억제 인자(co-inhibitory molecule)의 발현이 증가된 세포를 대량수집하는 방법을 규명하였다.The present inventors isolated monocytes from peripheral blood hematopoietic stem cells of subjects injected subcutaneously with G-CSF, sorted a CD3+ cell fraction from the isolated monocytes, and cultured them with a low dose of IL-2 (50 U/mL) on the T cell surface. A method for collecting large amounts of cells with increased expression of co-inhibitory molecules was identified.
이에, G-CSF(granulocyte colony stimulating factor)를 포함하는, CD3+, PD1+ 및 Tim3+의 면역 표현형을 갖는 T 세포의 대량 생산용 배지 조성물을 제공한다.Accordingly, a medium composition for mass production of T cells having immunophenotypes of CD3+, PD1+ and Tim3+ containing G-CSF (granulocyte colony stimulating factor) is provided.
상기 배지 조성물에서 상기 T 세포는 Lag3+ 및 TIGIT+에서 선택되는 하나 이상의 면역 표현형을 더 포함할 수 있으나, 이에 제한되는 것은 아니다.In the medium composition, the T cells may further include one or more immunophenotypes selected from Lag3+ and TIGIT+, but are not limited thereto.
본 발명에서 사용되는 용어 “대량 생산”이란 치료용량의 세포 수만큼 세포를 확보하는 것을 의미하며, 본 발명에서는 동종 면역 반응을 억제하는 능력이 탁월한 충분한 수의 CD3+, PD1+ 및 Tim3+의 면역 표현형을 포함하는 세포를 생산하는 것을 의미한다.The term "mass production" used in the present invention means securing cells as many as the number of cells in a therapeutic dose, and in the present invention, a sufficient number of CD3+, PD1+, and Tim3+ immune phenotypes that are excellent in suppressing alloimmune responses are included. means to produce cells that
또한, 본 발명은 하기의 단계를 포함하는, CD3+, PD1+ 및 Tim3+의 면역 표현형을 갖는 T 세포의 대량생산 방법을 제공한다.In addition, the present invention provides a method for mass-producing T cells having CD3+, PD1+ and Tim3+ immune phenotypes, comprising the following steps.
(a) G-CSF(granulocyte colony stimulating factor)로부터 말초 혈액 조혈모세포(Peripheral blood stem cell, PBSC)를 수집하는 단계;(a) collecting peripheral blood stem cells (PBSC) from granulocyte colony stimulating factor (G-CSF);
(b) 상기 말초 혈액 조혈모세포(PBSC)로부터 단핵구를 분리하는 단계; (b) separating monocytes from the peripheral blood hematopoietic stem cells (PBSC);
(c) 분리된 단핵구에서 CD3+ 세포 분획을 분류하는 단계; 및 (c) sorting the CD3+ cell fraction from the isolated monocytes; and
(d) IL-2(interukin-2)가 포함된 무혈청 배지에서 상기 CD3+ 세포 분획을 배양하는 단계를 포함하는 단계.(d) culturing the CD3+ cell fraction in a serum-free medium containing interukin-2 (IL-2).
상기 방법에서 상기 T 세포는 Lag3+ 및 TIGIT+에서 선택되는 하나 이상의 면역 표현형을 더 포함할 수 있으나, 이에 제한되는 것은 아니다.In the method, the T cells may further include one or more immunophenotypes selected from Lag3+ and TIGIT+, but are not limited thereto.
상기 (b) 단계에서 단핵구를 분리하는 단계는 혈액 성분 분리기(COBE spectra)를 이용하여 수행될 수 있으나, 이에 제한되는 것은 아니다.Separating monocytes in step (b) may be performed using a blood component separator (COBE spectra), but is not limited thereto.
상기 (c) 단계에서 CD3+ 세포 분획을 분류하는 단계는 자기 세포분리기(MACS magnetic) 또는 MS 컬럼(MS column)을 이용하여 수행될 수 있으나, 이에 제한되는 것은 아니다.Sorting the CD3+ cell fraction in step (c) may be performed using a MACS magnetic or MS column, but is not limited thereto.
상기 (d) 단계에서 배양은 3 내지 7일 동안 배양하는 것일 수 있으며, 보다 바람직하게는 5일 동안 배양할 수 있으나, 이에 제한되는 것은 아니다.In step (d), the culture may be cultured for 3 to 7 days, and more preferably, cultured for 5 days, but is not limited thereto.
본 발명자들은 구체적인 실시예를 통해 본 발명의 세포가 동종면역반응을 억제하는데 효과적임을 확인하였다.The present inventors confirmed through specific examples that the cell of the present invention is effective in suppressing the alloimmune response.
본 발명의 일실시예에서는 IL-2(-) PBSC T 세포와 비교하여 PD1, Tim3 및 Lag3 등의 co-inhibitory molecule이 발현된 IL-2(+) PBSC T 세포의 동종면역반응을 확인한 결과, IL-2(+) PBSC T 세포의 경우, allo-MNCs에 대해 반응하여 증식하지 않는 세포의 비율이 31.5 %로 나타나 IL-2(-) PBSC T 세포(18.2%)에 비해 1.7배 증가되는 것을 확인하였다(실시예 2 참조).In one embodiment of the present invention, as a result of confirming the alloimmune response of IL-2(+) PBSC T cells expressing co-inhibitory molecules such as PD1, Tim3 and Lag3 compared to IL-2(-) PBSC T cells, In the case of IL-2(+) PBSC T cells, the proportion of cells that did not proliferate in response to allo-MNCs was 31.5%, indicating a 1.7-fold increase compared to IL-2(-) PBSC T cells (18.2%). confirmed (see Example 2).
본 발명의 다른 실시예에서는 CD3+PD1+ 세포 내에서 PD1 또는 Tim3를 제거 한 후 면역반응(mixed lymphocyte reaction, MLR)을 유도한 결과, 동종 면역반응 억제되는 효과가 소실되는 결과가 나타나, CD3+PD1+Tim3+ 세포 내에 면역반응을 억제하는 T 세포가 존재한다는 것을 확인하였다(실시예 3 참조).In another embodiment of the present invention, as a result of inducing a mixed lymphocyte reaction (MLR) after removing PD1 or Tim3 from CD3+PD1+ cells, the effect of suppressing the allogeneic immune response is lost, resulting in CD3+PD1 It was confirmed that T cells suppressing the immune response exist in +Tim3+ cells (see Example 3).
이에 더하여, 상기 CD3+PD1+Tim3+ 세포는 대부분이 Lag3+이었으며, TIGIT 발현 여부에 따라 동종면역반응을 확인한 결과, CD3+PD1+Tim3+Lag3+TIGIT+세포의 경우, allo-MNCs에 대해 반응하여 증식하지 않는 세포의 비율이 46.8±6.7%로 나타나 CD3+PD1+Tim3+Lag3+TIGIT- 세포(25,5±3.3%)에 비해 1.8배 증가되는 것을 확인하였다(실시예 4 참조).In addition, most of the CD3+PD1+Tim3+ cells were Lag3+, and as a result of confirming the alloimmune response depending on whether TIGIT was expressed, CD3+PD1+Tim3+Lag3+TIGIT+ cells did not proliferate in response to allo-MNCs. It was confirmed that the ratio of non-reactive cells was 46.8±6.7%, which was increased by 1.8 times compared to CD3+PD1+Tim3+Lag3+TIGIT- cells (25.5±3.3%) (see Example 4).
상기 실시예의 결과로부터 본 발명의 세포를 포함하는 조성물은 co-inhibitory molecule의 발현을 통해 동종면역억제를 위한 세포치료제로 활용될 수 있을 것으로 기대된다.From the results of the above examples, it is expected that the composition containing the cells of the present invention can be used as a cell therapy agent for alloimmunosuppression through the expression of co-inhibitory molecules.
본 발명의 다른 양태로써, 본 발명은 상기 방법을 이용하여 대량 수집되며, CD3+, PD1+ 및 Tim3+의 면역 표현형을 포함하는 세포가 10% 이상 포함된 세포 집단을 제공한다.As another aspect of the present invention, the present invention provides a cell population that is collected in large quantities using the above method and contains 10% or more of cells having immunophenotypes of CD3+, PD1+, and Tim3+.
상기 세포 집단에서 상기 T 세포는 Lag3+ 및 TIGIT+에서 선택되는 하나 이상의 면역 표현형을 더 포함할 수 있으나, 이에 제한되는 것은 아니다.The T cells in the cell population may further include one or more immunophenotypes selected from Lag3+ and TIGIT+, but are not limited thereto.
본 발명의 또 다른 양태로써, 본 발명은 상기 세포 집단을 포함하는 면역억제용 조성물을 제공한다.As another aspect of the present invention, the present invention provides a composition for immunosuppression comprising the cell population.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.
[실시예][Example]
실시예 1. 세포 수집 및 증식 방법Example 1. Cell collection and propagation method
말초 조혈모세포 공여자에게 G-CSF(neutrogin; 10㎍/kg)를 5일간 피하 주사한 뒤 COBE spectra를 이용하여 G-CSF mobilized peripheral blood stem cell(PBSC)을 수집한다. 이후 Ficoll-Plaque plus (GE healthcare, 17-1440-02)로 단핵구를 분리한 뒤 CD3 MicroBeads, human(miltenyibiotec. 130-050-101)dl 포함된 MACS magnetic, MS column(Miltenyibiotec. 130-042-201)을 이용하여, CD3+ 림프구를 sorting 한다. 이후, IL-2(50U/ml, human IL-2(peprotech, 200-02)가 포함된 무혈청 배지 15mL를 배양액으로 100π dish에 0.5X107개의 CD3+ 세포를 로딩하여 5일간 배양하였다.Peripheral hematopoietic stem cell donors are subcutaneously injected with G-CSF (neutrogin; 10 μg/kg) for 5 days, and then G-CSF mobilized peripheral blood stem cells (PBSC) are collected using COBE spectra. After that, monocytes were separated with Ficoll-Plaque plus (GE healthcare, 17-1440-02) and then MACS magnetic, MS column (Miltenyibiotec. 130-042-201) containing CD3 MicroBeads, human (miltenyibiotec. 130-050-101) dl ) to sort CD3+ lymphocytes. Thereafter, 0.5X10 7 CD3+ cells were loaded on a 100π dish with 15mL of serum-free medium containing IL-2 (50U/ml, human IL-2 (peprotech, 200-02)) as a culture medium and cultured for 5 days.
실시예 2. 증식된 세포의 표현형 및 면역 억제 효과 확인Example 2. Verification of phenotype and immunosuppressive effect of proliferated cells
G-CSF mobilize PBSC CD3+ 림프구를 배양하지 않거나(PBSC T 세포), IL-2(50U/mL)와 함께 5일간 배양(IL-2(+) PBSC T 세포)하거나, IL-2 처리 없이 5일간 배양(IL-2(-) PBSC T 세포)하고, 각 세포 표면에서 co-inhibitory molecule의 발현 정도를 확인하였다. G-CSF mobilize PBSC CD3+ lymphocytes without culture (PBSC T cells), with IL-2 (50 U/mL) for 5 days (IL-2(+) PBSC T cells), or without IL-2 treatment for 5 days After culturing (IL-2(-) PBSC T cells), the expression level of the co-inhibitory molecule on each cell surface was confirmed.
그 결과, 도 1a 및 1b에 나타낸 바와 같이 IL-2(50U/mL)와 함께 5일간 배양된 PBSC T 세포의 경우, IL-2를 처리하지 않고 배양된 PBSC T 세포 및 배양되지 않은 PBSC에 비해 LAG3, TIM3, TIGIT의 발현이 현저하게 증가되는 것을 확인하였다. As a result, as shown in Figures 1a and 1b, in the case of PBSC T cells cultured for 5 days with IL-2 (50 U / mL), compared to PBSC T cells cultured without IL-2 and uncultured PBSC. It was confirmed that the expression of LAG3, TIM3, and TIGIT was remarkably increased.
이에 더하여, 상기와 동일한 PBSC T 세포, IL-2(+) PBSC T 세포 및 IL-2(-) PBSC T 세포, 상기 세가지 세포를 CD14+ 동종 단핵구(mononuclear cells; MNCs)와 혼합하고 면역반응(mixed lymphocyte reaction, MLR)을 유도한 결과, 도 1c에 나타낸 바와 같이 IL-2(+) PBSC T 세포의 경우, allo-MNCs에 대해 반응하여 증식하지 않는 세포의 비율이 31.5 %로 나타나 IL-2(-) PBSC T 세포(18.2%)에 비해 현저히 증가되는 것을 확인하였다.In addition, PBSC T cells, IL-2(+) PBSC T cells and IL-2(-) PBSC T cells, the same as above, were mixed with CD14+ allogeneic mononuclear cells (MNCs) and immunoreacted (mixed). As a result of inducing the lymphocyte reaction (MLR), as shown in Figure 1c, in the case of IL-2(+) PBSC T cells, the proportion of cells that did not proliferate in response to allo-MNCs was 31.5%, indicating that IL-2 ( -) It was confirmed that it significantly increased compared to PBSC T cells (18.2%).
실시예 3. CD3+PD1+Tim3+ 세포의 면역억제 효과 확인Example 3. Confirmation of immunosuppressive effect of CD3+PD1+Tim3+ cells
G-CSF mobilize PBSC CD3+ 림프구를 IL-2(50U/mL)와 함께 5일간 배양(IL-2(+) PBSC 5day)한 세포에서 PD1+ 세포의 비율을 확인한 결과, 도 2a 좌측에 나타낸 바와 같이 PD1+ 세포의 비율은 29.0±9.8%로 나타났으며, CD3+PD1+ 세포 내에서 PD1+세포를 제거한 후 PD1- 세포만을 이용하여 면역반응(mixed lymphocyte reaction, MLR)을 유도한 결과, 도 2a 우측에 나타낸 바와 같이 거의 대부분의 세포가 allo-MNC에 반응하여 증식하는 것을 확인하였다. 상기 결과로부터, 면역반응을 억제하는 T 세포가 CD3+PD1+ 세포군 중에 존재하는 것을 확인하였다.As a result of confirming the ratio of PD1+ cells in cells cultured with G-CSF mobilized PBSC CD3+ lymphocytes with IL-2 (50 U/mL) for 5 days (IL-2(+) PBSC 5 days), as shown in the left side of Figure 2a, PD1+ The percentage of cells was 29.0 ± 9.8%, and as a result of inducing a mixed lymphocyte reaction (MLR) using only PD1- cells after removing PD1 + cells from CD3 + PD1 + cells, as shown in the right side of Figure 2a. As such, it was confirmed that most of the cells proliferated in response to allo-MNC. From the above results, it was confirmed that T cells suppressing the immune response were present in the CD3+PD1+ cell population.
또한, 도 2b 좌측에 나타낸 바와 같이 상기 CD3+PD1+ 세포 내에서 70% 이상이 Tim3 세포 표면 항원에 양성으로 나타났으며, 전체 IL-2(+) PBSC T 세포에서 CD3+PD1+Tim3+ 세포는 18.0±3.3%를 나타내는 것을 확인하였다.In addition, as shown on the left side of FIG. 2B, more than 70% of the CD3+PD1+ cells were positive for the Tim3 cell surface antigen, and CD3+PD1+Tim3+ cells out of total IL-2(+) PBSC T cells were 18.0 It was confirmed that it represents ±3.3%.
이에 더하여, Tim3 발현 여부가 면역억제에 영향을 미치는지 확인하기 위해, CD3+PD1+ 세포군에서 Tim3+ 세포를 제거 한 후 면역반응(mixed lymphocyte reaction, MLR)을 유도한 결과, CD3+PD1+ 세포군에서 Tim3+ 세포를 제거하는 경우 동종 면역반응이 억제되는 효과가 소실되는 결과를 확인하였다. 따라서, 상기 결과로부터 CD3+PD1+Tim3+ 세포 내에 면역반응을 억제하는 T 림프구가 존재한다는 것을 확인하였다.In addition, in order to determine whether the expression of Tim3 affects immunosuppression, Tim3+ cells were removed from the CD3+PD1+ cell group and then a mixed lymphocyte reaction (MLR) was induced. As a result, Tim3+ cells were isolated from the CD3+PD1+ cell group. In the case of removal, it was confirmed that the effect of suppressing the allogeneic immune response was lost. Therefore, it was confirmed from the above results that T lymphocytes suppressing the immune response exist in CD3+PD1+Tim3+ cells.
실시예 4. CD3+PD1+Tim3+Lag3+TIGIT+ 세포의 면역억제 효과 확인Example 4. Confirmation of immunosuppressive effect of CD3+PD1+Tim3+Lag3+TIGIT+ cells
상기 실시예 3에 개시된 CD3+PD1+Tim3+ 세포(18.0±3.3%)를 분석한 결과, 도 3a에 나타낸 바와 같이 96.2±3.3%로 대부분의 세포가 Lag3+을 나타냈으며, TIGIT의 경우 도 3b 좌측에 나타낸 바와 같이 CD3+PD1+Tim3+ 세포 중 28.9±5.4%가 TIGIT+를, CD3+PD1+Tim3+ 세포 중 63.7±7.8%가 TIGIT-를 나타내는 것을 확인하였다.As a result of analyzing the CD3+PD1+Tim3+ cells (18.0±3.3%) described in Example 3, most of the cells showed Lag3+ at 96.2±3.3%, as shown in FIG. 3a, and in the case of TIGIT, the left side of FIG. 3b As shown, it was confirmed that 28.9±5.4% of CD3+PD1+Tim3+ cells expressed TIGIT+ and 63.7±7.8% of CD3+PD1+Tim3+ cells expressed TIGIT−.
이에 더하여, 상기 CD3+PD1+Tim3+ 세포에서 TIGIT 발현 여부에 따라 면역반응(mixed lymphocyte reaction, MLR)을 유도한 결과, 도 3b 우측에 나타낸 바와 같이 CD3+PD1+Tim3+Lag3+TIGIT- 세포(25,5±3.3%)보다는 CD3+PD1+Tim3+Lag3+TIGIT+세포(46.8±6.7%)에서 면역억제 효과가 더 우수하게 나타나는 것을 확인하였다.In addition, as a result of inducing a mixed lymphocyte reaction (MLR) according to the expression of TIGIT in the CD3+PD1+Tim3+ cells, as shown on the right side of FIG. 3B, CD3+PD1+Tim3+Lag3+TIGIT- cells (25 It was confirmed that the immunosuppressive effect was more excellent in CD3+PD1+Tim3+Lag3+TIGIT+ cells (46.8±6.7%) than in ,5±3.3%).
나아가, 본 발명의 도 3c에 나타낸 바와 같이, 면역억제 효과가 우수한 CD3+PD1+Tim3+Lag3+TIGIT+세포 수는 PBSC 0 day (대조군)에 비해 IL-2(+) PBSC T 세포군내에서는 약 141배 증가된다. 이에 비해 IL-2(-) PBSC T 세포 군에서는 PBSC T 세포(대조군)에 비해 CD3+PD1+Tim3+Lag3+TIGIT-세포 수가 약 48배 증가되는 것을 확인하였다. Furthermore, as shown in FIG. 3c of the present invention, the number of CD3+PD1+Tim3+Lag3+TIGIT+ cells having excellent immunosuppressive effect was about 141 in the IL-2(+) PBSC T cell group compared to
또한, 본 발명에서 CD3+PD1+Tim3+Lag3+TIGIT+세포는 전제 CD3+ 세포 중 13.5±5.9%의 비율을 나타내었는데, 상기 결과는 PBSC를 한번 수집하고 배양하는 방법으로 면역억제 반응과 관련된 치료에 필요한 충분한 량의 CD3+PD1+Tim3+Lag3+TIGIT+세포 (총 11±3x108 CD3+PD1+Tim3+Lag3+TIGIT+ cells) 수를 확보할 수 있음을 알 수 있었다.In addition, in the present invention, CD3+PD1+Tim3+Lag3+TIGIT+ cells accounted for 13.5±5.9% of the total CD3+ cells, which is necessary for treatment related to immunosuppressive response by collecting and culturing PBSCs once. Sufficient amount of CD3+PD1+Tim3+Lag3+TIGIT+ cells (total 11±3x10 8 It was found that the number of CD3+PD1+Tim3+Lag3+TIGIT+ cells) could be secured.
상기 실시예 결과들로부터 CD3+ 세포분획을 IL-2+와 혼합하여 배양함으로써 동종 면역 반응을 억제하는 능력이 탁월한 충분한 수의 CD3+PD1+Tim3+Lag3+TIGIT+ 세포를 획득할 수 있으며, 상기 CD3+PD1+Tim3+Lag3+TIGIT+ 림프구는 co-inhibitory molecule 발현을 통해 동종면역억제를 위한 세포치료제로 활용될 수 있을 것으로 기대된다.From the results of the above examples, it is possible to obtain a sufficient number of CD3+PD1+Tim3+Lag3+TIGIT+ cells excellent in suppressing the alloimmune response by culturing the CD3+ cell fraction mixed with IL-2+, and the CD3+ PD1+Tim3+Lag3+TIGIT+ lymphocytes are expected to be used as cell therapy agents for alloimmunosuppression through the expression of co-inhibitory molecules.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.
Claims (10)
A medium composition for mass production of T cells having immunophenotypes of CD3+, PD1+ and Tim3+, including granulocyte colony stimulating factor (G-CSF).
상기 T 세포는 Lag3+ 및 TIGIT+에서 선택되는 하나 이상의 면역 표현형을 더 포함하는 것을 특징으로 하는, 조성물.
According to claim 1,
The composition characterized in that the T cells further comprise one or more immunophenotypes selected from Lag3+ and TIGIT+.
(a) G-CSF(granulocyte colony stimulating factor)로부터 말초 혈액 조혈모세포(Peripheral blood stem cell, PBSC)를 수집하는 단계;
(b) 상기 말초 혈액 조혈모세포(PBSC)로부터 단핵구를 분리하는 단계;
(c) 분리된 단핵구에서 CD3+ 세포 분획을 분류하는 단계; 및
(d) IL-2(interukin-2)가 포함된 무혈청 배지에서 상기 CD3+ 세포 분획을 배양하는 단계를 포함하는 단계.
A method for mass-producing T cells having immunophenotypes of CD3+, PD1+ and Tim3+, comprising the following steps.
(a) collecting peripheral blood stem cells (PBSC) from granulocyte colony stimulating factor (G-CSF);
(b) separating monocytes from the peripheral blood hematopoietic stem cells (PBSC);
(c) sorting the CD3+ cell fraction from the isolated monocytes; and
(d) culturing the CD3+ cell fraction in a serum-free medium containing interukin-2 (IL-2).
상기 T 세포는 Lag3+ 및 TIGIT+에서 선택되는 하나 이상의 면역 표현형을 더 포함하는 것을 특징으로 하는, 방법.
According to claim 3,
Characterized in that the T cell further comprises one or more immunophenotypes selected from Lag3+ and TIGIT+.
상기 (b) 단계에서 단핵구를 분리하는 단계는 혈액 성분 분리기(COBE spectra)를 이용하여 수행되는 것을 특징으로 하는, 방법.
According to claim 3,
The step of separating monocytes in step (b) is characterized in that carried out using a blood component separator (COBE spectra).
상기 (c) 단계에서 CD3+ 세포 분획을 분류하는 단계는 자기 세포분리기(MACS magnetic) 또는 MS 컬럼(MS column)을 이용하여 수행되는 것을 특징으로 하는, 방법.
According to claim 3,
The step of sorting the CD3+ cell fraction in step (c) is characterized in that carried out using a magnetic cell separator (MACS magnetic) or MS column (MS column), the method.
상기 (d) 단계에서 배양은 3 내지 7일 동안 수행되는 것을 특징으로 하는, 방법.
According to claim 3,
In step (d), the culturing is performed for 3 to 7 days.
A T cell population produced in large quantities by the method of claim 3 and containing at least 10% of T cells having immunophenotypes of CD3+, PD1+ and Tim3+.
상기 T 세포는 Lag3+ 및 TIGIT+에서 선택되는 하나 이상의 면역 표현형을 갖는 더 포함하는 것을 특징으로 하는, T 세포 집단.
According to claim 8,
Characterized in that the T cell population further comprises having one or more immunophenotypes selected from Lag3+ and TIGIT+.
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