WO2017061784A1 - 암-고환 항원을 포함하는 한국 난치성 다발골수종 예방 또는 치료용 세포 면역치료제 조성물 및 그 제조방법 - Google Patents

암-고환 항원을 포함하는 한국 난치성 다발골수종 예방 또는 치료용 세포 면역치료제 조성물 및 그 제조방법 Download PDF

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WO2017061784A1
WO2017061784A1 PCT/KR2016/011178 KR2016011178W WO2017061784A1 WO 2017061784 A1 WO2017061784 A1 WO 2017061784A1 KR 2016011178 W KR2016011178 W KR 2016011178W WO 2017061784 A1 WO2017061784 A1 WO 2017061784A1
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seq
cancer
multiple myeloma
cells
primer
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이제중
최누리
이현주
정성훈
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Vaxcell-Bio Inc
Industry Foundation of Chonnam National University
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Industry Foundation of Chonnam National University
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    • A—HUMAN NECESSITIES
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • NAME OF THE INVENTION Cell-immune therapeutic compositions for preventing or treating Korean intractable multiple myeloma, including cancer-testis antigens
  • the present invention relates to a composition for the treatment or prophylaxis of cancer-inflammatory multiple myeloma cancers in Korea, including testicular antigens. More specifically, dendritic cells carrying the antigens and cancer-derived testicular antigens. Cytotoxic T lymphocytes
  • the present invention relates to a composition for the prevention and treatment of multiple myeloma, which is included as an active ingredient, and a method for producing the same.
  • the present invention provides a safe and non-neoplastic tumor-immunotherapy of multiple myeloma and increases the tumor-specific immune response.
  • a composition comprising cancer-testis antigens for prevention and treatment that can be effectively treated, and using the above composition to screen tumor antigen candidate proteins overexpressed from malignant multiple myeloma cells through comparative analysis of expression from cells. It was intended to provide a quantitative method.
  • the present invention is cancer-testis antigens CTAG1B, SSX2, SSX4, SSX5, MAGEA3, MAGEC1,
  • the present invention also provides a method for preparing malignant multiple myeloma cells; b) identifying the purity of malignant multiple myeloma cells; c) judging whether the cancer-testis antigen is overexpressed from the malignant multiple myeloma cells harvested with high purity by quantitative analysis using a primer of cancer-testicular antigen; and d)
  • the present invention relates to a method for preparing a cell-immune therapeutic composition for preventing or treating multiple myeloma, including a dianine compound prepared by recombinant-tumor antigen protein and appropriately mounted on dendritic cells.
  • FIG. 6 is a quantitative analysis of the expression of cancer-testis antigen CTAG2 (LAGE-1).
  • FIG. 7 shows target cell recognition responses using cancer-testis antigen-specific cytotoxic T lymphocyte cells derived from cancer-testicular antigen-recombinant protein appropriately loaded on dendritic cells or induced therefrom.
  • the present invention is cancer-testis antigens CTAG1B, SSX2, SSX4, SSX5, MAGEA3, MAGEC1,
  • the present invention relates to a composition for preventing or treating multiple myeloma including the cancer-testis antigen-specific cytotoxic T lymphocyte cells as an active ingredient.
  • TA tumor-associated antigen
  • Dendritic cell therapy peptide vaccines, DNA vaccines, etc.
  • TA-specific treatments are distinguished.
  • Dendritic cells are the most potent antigen-presenting cells that induce tumor antigen-specific immune responses
  • the cancer immunotherapy method using the dendritic cells is isolated from monocytes from the peripheral blood of cancer patients, differentiated and matured into cytokines in vitro, and detects tumor antigen (TA) and administers it to the patient, and then TA-specific. Inducing the immune response can be a powerful anti-cancer treatment.
  • Multiple myeloma is a type of hematologic cancer caused by malignant proliferation of plasma cells.
  • the most ideal tumor antigen for cancer immunotherapy is not expressed in normal cells but has a specific expression only in cancer cells.
  • CTAG1B is SEQ ID NO: 1
  • SSX2 is SEQ ID NO: 2
  • SSX4 is SEQ ID NO: 3
  • SSX5 is SEQ ID NO: 4
  • MAGEA3 is SEQ ID NO: 5
  • MAGEC1 is SEQ ID NO: 6
  • MAGEC2 is SEQ ID NO: 7
  • BAGE1 can have an amino acid sequence of SEQ ID NO: 8, CTAG2 of SEQ ID NO: 9 and SPA17 of SEQ ID NO: 10.
  • the present invention also provides a method for preparing malignant multiple myeloma cells; b) identifying the purity of malignant multiple myeloma cells; c) judging overexpression from the malignant multiple myeloma cells harvested with high purity by quantitative analysis using a primer of cancer-testicular antigen; and d) determining the cancer-testis antigen.
  • the present invention relates to a method for producing a cell-immune therapeutic composition for preventing or treating multiple myeloma, comprising the step of preparing the recombinant-tumor antigen protein and mounting it on the dendritic cells as appropriate.
  • the purity of the malignant myeloma cells may be 80% or more, although not limited thereto.
  • the purity of the malignant myeloma cells is higher than 90% and preferably higher than 95%. .
  • the gene encoding the CTAG1B protein may include a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12;
  • the gene encoding SSX2 protein includes a forward primer of SEQ ID NO: 13, a reverse primer of SEQ ID NO: 14;
  • the gene encoding SSX4 protein includes a forward primer of SEQ ID NO: 15, a reverse primer of SEQ ID NO: 16;
  • Genes encoding the SSX5 protein include a forward primer of SEQ ID NO: 17, a reverse primer of SEQ ID NO: 18;
  • Genes encoding the MAGEA3 protein include a forward primer of SEQ ID NO: 19, a reverse primer of SEQ ID NO: 20;
  • the gene encoding the MAGEC1 protein is the forward direction of SEQ ID NO: 21.
  • Genes encoding the MAGEC2 protein include a forward primer of SEQ ID NO: 23, a reverse primer of SEQ ID NO: 24;
  • Genes encoding BAGE1 protein include a forward primer of SEQ ID NO: 25 and a reverse primer of SEQ ID NO: 26;
  • the gene encoding the CTAG2 protein can be amplified using the forward primer of SEQ ID NO: 27, the reverse primer of SEQ ID NO: 28; and the gene encoding the SPA17 protein, using the forward primer of SEQ ID NO: 29 and the reverse primer of SEQ ID NO: 30.
  • the quantitative analysis in step c) is not limited, but may be characterized by measuring expression by real-time quantitative polymerase chain reaction.
  • It can be used as a control to measure the ratio of expression to the control.
  • MACS magnetic activated cell
  • a separation kit was used to determine the purity of the malignant tumor cell phenotype.
  • Human plasma cell isolation kit II Miltenyi Biotec
  • CD 138 microbeads Miltenyi Biotec
  • the non-pollasma cell removal process is omitted and positive selection is performed using CD138 microbeads.
  • Monoclonal antibodies of BD were used to confirm the cell phenotype of positively isolated plasma cells using the above two methods.
  • TITC was treated with 5 x 10 4 to 10 5 cells in 1% serum-containing PBS (phosphate-buffered saline; Life Technologies) complete solution 200 (1 per iL) for 30 minutes at 4 ° C, followed by 1 Wash twice with PBS containing% serum Expression of CD19, CD38, CD56 and CDB8 was analyzed using BD's flow cytometer (FACSCalibur, BD).
  • the standard cell phenotype of malignant multiple myeloma cells is CD19 Low, CD22 negative, CD34 negative, CD56 positive, and CD 138 positive.
  • the expression of malignant multiple myeloma cells is shown in FIG. 1.
  • RNA of high purity was isolated from Rneasy mini kit (Qiagen) from samples obtained from patients with multiple myeloma, and cDNA was synthesized using Primerscript 1st strand cDNA synthesis kit (Takara).
  • SYBR Green PCR Master mix (QUANTI tech) and primers of SEQ ID NOS: 11 to 30 for the expression of 10 cancer-testis antigens in Table 2 were used.
  • the T Forward and R: Reverse real-time quantitative polymerase chain reactions were performed under conditions of 30 seconds at 95 ° C, 30 seconds at 52 0 C, and 30 seconds at 72 ° C.
  • beta-actin was used to measure expression.
  • Quantitative expression analysis was performed using Roger-gene 6 software of the RG-3000 (Roger-gene) instrument.
  • -Quantitative expression of 10 cancers compared to actin-testicular antigen was shown using GraphPad Prism 5 software. Results for some of the two antigens are shown in Figures 2-6. As can be seen from the results of FIGS.
  • the ratio of the quantitative expression of the target mRNA and the beta-actin was compared, and in the case of multiple myeloma patients, the ratio of the expression in the CD138 positive representing the malignant multiple myeloid cells was It is confirmed that the increase is remarkable, and from this, it is possible to clearly select the normal person and the patient.
  • CTLs cytotoxic T lymphocytes
  • the dendritic cells used in the present invention were obtained with consent from multiple myeloma patients.
  • Mononuclear cells were obtained through centrifugation using lymphoprep (Ficoll-Hypaque) from peripheral blood, and Percoll density gradient black CDI4 positive cells were obtained using magnetic beads.
  • the obtained monocytes were collected in 6-well plates at a density of 2 x 10 6 cells.
  • 3-5 quantitative recombinant cancer-testis antigens selected and prepared from malignant multiple myeloma cells using the primers of Table 2 above were 0.5 glmi per 2 ⁇ 10 5 to 3 ⁇ 10 5 cells. Treatment was performed at a concentration of 10 / ⁇ for 2 hours in a 37 ° C0 2 incubator and co-cultured with CD3 or CD8 positive T lymphocytes to induce cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • the protocol was used according to the protocol (BD TM ELISPOT human IFN- ⁇ set, cat no.551849, BD).
  • Malignant cells were used as target cells at a 1:10 or 1:20 ratio and co-cultured in a 96-well plate coated with a human interferon-gamma antibody for 24 hours.
  • the number of spots of interferon-gamma was identified.
  • the number of spots obtained immediately means the number of cancers expressed in target cells-cancers that recognize testicular antigens-testicular antigen specific cytotoxic T lymphocyte cells.
  • the degree of secretion of interferon gamma was confirmed by specifically recognizing other 3 ⁇ 4 cells by the prepared cytotoxic T lymphocyte cells.
  • K562 cell line is a cell line that is dependent on NK cells and kills RPMT8226.
  • the un-CTL is caused by dendritic cells not loaded with cancer-testis antigens described above.
  • Induced cytotoxic T lymphocytes and antigen-nonspecific responses can be identified from the blue graph of FIG.
  • SSX2-, SSX4-, MAGEA3- and MAGEC2-CTL are cancer-testicular antigens
  • the interval of the spots in the graph of Figure 7 is cancer-testicular antigen-specific cytotoxic T lymphocytes
  • the intensity of the reaction is indicative of that the greater the distance between the blue and red graphs, the stronger the antigen-specific response.
  • MHC major histocompatibility complex
  • NL-normal, MM-multimyeloma patients PB- peripheral blood mononuclear cells, BB cells, BM-myeloid mononuclear cells, IgA, -immunoglobulin A, IgG-immunoglobulin G, lamda-lamda light chain, kappa- kappa light chain, CD138-CD138 negative multiple myeloma cells, CD138 + -CD138 positive multiple myeloma cells, MACS-magnectic activated cell separation.

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Abstract

본 발명은 암-고환 항원을 포함하는 한국 난치성 다발골수종 암 면역치료 또는 예방용 조성물에 관한 것으로, 상기 항원을 탑재한 수지상세포 및 이로부터 유도되는 암-고환 항원 특이적 세포독성 τ 림프구세포를 유효성분으로 포함하는 다발골수종 예방또는 치료용 세포 면역치료제 조성물을 이용함으로써 암 면역 치료에 있어 강력한 종양 특이적 세포독성 면역 반응을 유도하는 동시에 안전하고 부작용이 없는 암 치료용 백신으로 유용하게 활용할 수 있을 것으로 기대된다.

Description

명세서
발명의명칭:암 -고환항원을포함하는한국난치성다발골수종 예방또는치료용세포면역치료제조성물및그제조방법 기술분야
[1] 본발명은암 -고환항원을포함하는한국난치성다발골수종암면역치료또는 예방용조성물에관한것으로，보다상세하게는상기항원을탑재한수지상세포 및이로부터유도되는암 -고환항원특이적세포독성 T림프구세포를
유효성분으로포함하는다발골수종예방또는치료용세포면역치료제조성물 및이의제조방법에관한것이다.
배경기술
[2] 다발골수종 (multiple myeloma)은형질세포의악성증식에의해발생하는
혈액암의 일종으로국내에서는악성림프종과급성 백혈병다음으로많이 발생하는질병이다.최근 30년간국내에서 30배정도로발병률이증가하였으며， 현재 5,000명정도의환자가있는것으로추정된다.이러한환자의급격한 증가로인해향후사회적인문제가될수있는질병이다.
[3] 21세기들어다발골수종에 대하여탈리도마이드,보르테조밉 및
렌날리도마이드등과같은표적치료제가임상에도입되면서긍정적인치료 결과를얻은환자또한증가하였으나,여전히평균생존률이 5년정도로짧으며 대부분의환자들이사망에이르고있디■.또한상기표적치료제는가격이 비싸고，심각한부작용을초래함으로인해지속적인치료를유지하는데 어려움이 있다.따라서치료효과가좋고사용에따른부작용이적은새로운 치료법이필요하다.
[4] 암의치료를위해사용되고있는일반적방법의한계극복을위해다양한치료 방법에 대한연구가시도되었으며 이중근래들어암특이적치료효과를보이고 부작용이없는암면역치료 (cancer immunotherapy)가주목받고있다.상기 방법은암세포만을선택적으로사멸시키고부작용이거의 없는치료법으로 알려져 있다 (2nd Novel Immunotherapeutics Summit 2013 Jan 30-Feb 1).
[5] 현재다발골수종자체를이용하여 암면역치료제의종양항원으로이용하고 있으나,다수의환자에서치료를위한다발골수종세포를골수로부터 얻기에는 어려움이 있다.따라서，다발골수종에서면역세포를이용한암면역치료제 개발에 있어서의최대난제는다발골수종에특이성을가지는종양항원의 개발이며.이에대한발굴이 절실한상황이다.
발명의상세한설명
기술적과제
[6] 상기문제점의해결을위해본발명에서는다발골수종의세포면역치료법으로 안전하고부작용이 없으면서종양특이적인면역반응을증가시켜해당종양을 효과적으로치료할수있는예방및치료를위한암 -고환항원을포함하는 조성물을제공하고자하였으며，상기조성물을이용하여，세포로부터발현량의 비교분석을통해악성다발골수종세포로부터과발현되는종양항원후보 단백질을선별하는정량적방법을제공하고자하였다.
과제해결수단
[7] 본발명은암 -고환항원인 CTAG1B, SSX2, SSX4, SSX5, MAGEA3, MAGEC1,
MAGEC2, BAGE1, CTAG2및 SPA17로이루어진군으로부터선택되는어느 하나이상의단백질;상기단백질의항원성펩티드가탑재된수지상세포;또는 상기암 -고환항원-특이적세포독성 T림프구세포를유효성분으로포함하는 다발골수종예방또는치료용조성물에관한것이다.
[8] 또한본발명은 a)악성다발골수종세포를준비하는단계 ; b)악성다발골수종 세포의순도를확인하는단계; c)높은순도로수확된악성다발골수종 세포로부터암 -고환항원의프라이머를이용한정량분석을통해상기 세포로부터과발현여부를판단하는단계 ;및 d)상기 암 -고환항원을
재조합-종양항원단백질로제조하여수지상세포에적절히탑재하는딘계;를 포함하는다발골수종예방또는치료용세포면역치료제조성물의 제조방법에 관한것이다.
발명의효과
[9] 본발명을통한암 -고환항원단백질또는이의항원성펩티드를포함하는 조성물을이용함으로써암면역치료에 있어강력한종양특이적세포독성면역 반웅을유도하는동시에안전하고부작용이 없는암치료용백신으로유용하게 활용할수있을것으로기대된다.
도면의간단한설명
[10] 도 1은한국난치성다발골수종환자의골수단핵세포에서악성종양세포의 분리및세포표현형을확인한결과이며，
[11] 도 2는암-고환항원 SSX2의발현을정량적으로분석한결과이며，
[12] 도 3은암 -고환항원 SSX42의발현을정량적으로분석한결과이며,
[13] 도 4는암 -고환항원 MAGE-A3의발현을정량적으로분석한결과이며,
[14] 도 5는암 -고환항원 MAGE-C2의발현을정량적으로분석한결과이며,
[15] 도 6은암 -고환항원 CTAG2(LAGE-1 )의발현을정량적으로분석한결과이며 ,
[16] 도 7은암 -고환항원재조합단백질을수지상세포에적절히탑재하는단계 또는이로부터유도되는암 -고환항원특이적세포독성 T림프구세포를이용한 타겟세포인식반응을나타낸결과이다.
발명의실시를위한최선의형태
[17] 본발명은암 -고환항원인 CTAG1B, SSX2, SSX4, SSX5, MAGEA3, MAGEC1,
MAGEC2, BAGE1 , CTAG2및 SPA17로이루어진군으로부터선택되는어느 하나이상의단백질;상기단백질의항원성펩티드가탑재된수지상세포;또는 상기암 -고환항원-특이적세포독성 T림프구세포를유효성분으로포함하는 다발골수종예방또는치료용조성물에관한것이다.
[18] 암면역치료법은종양 -특이항원 (tumor-associated antigen, TA)-특이적
치료법 (수지상세포치료,펩티드백신， DNA백신등)과 TA-비특이적인 치료법으로구분할수있다.수지상세포 (DC)는가장강력한항원제시세포로 종양항원 -특이적면역반응을유도하여강력한항암면역반웅을유도할수 있다.상기수지상세포를이용한암면역치료법은암환자의말초혈액으로부터 단구를분리하여시험관내에서사이토카인으로분화및성숙시킨후,종양항원 (TA)을탐재하여환자에게투여후 TA-특이적면역반웅을유도하는것으로 강력한항암치료효과를나타낼수있다.앞에서언급한바와같이
다발골수종 (multiple myeloma)은형질세포의악성증식에의해발생하는 혈액암의 일종이다.암면역치료에가장이상적인종양항원은정상세포에서는 발현되지않고,암세포에서만특이적으로발현되는특징을가지며，
항원성 (immunogeneciy)을갖는것이메우중요한데,이와같은조건을충족하는 단백질이 암 -고환힝원 (cancer-testis antigen)이다.
[19] 본발명에서제한되지는않으나상기 CTAG1B는서열번호 1， SSX2는서열번호 2， SSX4는서열번호 3, SSX5는서열번호 4， MAGEA3은서열번호 5, MAGEC1은 서열번호 6， MAGEC2는서열번호 7， BAGE1은서열번호 8, CTAG2는서열번호 9 및 SPA17은서열번호 10의아미노산서열을갖을수있다.
[20]
[21] 또한본발명은 a)악성다발골수종세포를준비하는단계; b)악성다발골수종 세포의순도를확인하는단계; c)높은순도로수확된악성다발골수종 세포로부터 암 -고환항원의프라이머를이용한정량분석을통해상기 세포로부터과발현여부를판단하는단계;및 d)상기 암 -고환항원을
재조합-종양항원단백질로제조하여수지상세포에적절히탑재하는단계;를 포함하는다발골수종예방또는치료용세포면역치료제조성물의제조방법에 관한것이다.
[22] 본발명에서상기악성다발골수종세포의순도는제한되지는않지만 80% 이상일수있으며，보다좋게는 90%이상,바람직하게는 95%이상고순도를 가질수록실험결과의정확도를높이는데있어바람직하다.
[23] 본발명의상기 c)단계의프라이머는 CTAG1B단백질을코딩하는유전자는 서열번호 11의정방향프라이머，서열번호 12의 역방향프라이머; SSX2 단백질을코딩하는유전자는서열번호 13의 정방향프라이머，서열번호 14의 역방향프라이머； SSX4단백질을코딩하는유전자는서열번호 15의정방향 프라이머，서열번호 16의 역방향프라이머; SSX5단백질을코딩하는유전자는 서열번호 17의 정방향프라이머,서열번호 18의 역방향프라이머; MAGEA3 단백질을코딩하는유전자는서열번호 19의정방향프라이머,서열번호 20의 역방향프라이머; MAGEC1단백질을코딩하는유전자는서열번호 21의 정방향 프라이머，서열번호 22의 역방향프라이머; MAGEC2단백질을코딩하는 유전자는서열번호 23의정방향프라이머,서열번호 24의 역방향프라이머; BAGE1단 질을코딩하는유전자는서열번호 25의 정방향프라이머，서열번호 26의 역방향프라이머; CTAG2단백질을코딩하는유전자는서열번호 27의 정방향프라이머，서열번호 28의 역방향프라이머；및 SPA17단백질을코딩하는 유전자는서열번호 29의정방향프라이머，서열번호 30의 역방향프라이머를 사용하여증폭할수있다.
[24] 본발명에서상기 c)단계에서의정량분석은제한되지는않으나실시간-정량적 중합효소연쇄반웅 (real-time quantitative polymerase chain reaction)으로발현량을 측정하는것을특징으로할수있다.
[25] 본발명에서상기정량분석은제한되지는않으나베타-액틴 (beta-actin)을
대조군으로사용하여대조군과의발현량의비율을측정할수있다.
[26]
[27] 이하，실시예를통하여본발명을보다상세히설명한다.그러나이들실시예는 본발명에 대한이해를돕기위한것일뿐，어떤의미로든본발명의 범위가이들 예로만한정되는것은아니다.이 때，사용되는기술용어 및과학용어에 있어서 특별한다른정의가없다면,본발명이속하는기술분야에서통상의지식을 가진자가일반적으로이해하고있는의미를가지며，하기의설명또는첨부 도면에서불필요하게본발명의요지를흐릴수있는공지기능및구성에대한 설명은생략한다.
[28]
[29] [실시예 1]한국난치성다발골수종환자의골수단핵세포에서악성
종양세포의분리및세포표현형확인
[30] 악성종양세포의순수한분리를위하여 MACS(magnetic activated cell
separation)법을사용하였다.악성종양세포의세포표현형의순도확인을위해 분리키트로 human plasma cell isolation kit II(Miltenyi Biotec)또는 CD 138 microbeads(Miltenyi Biotec)을이용하였다.
[31] 상기두가지방법은아래와같이 약간의차이점이 있다.
[32] 상기키트를이용하여먼저골수-단핵세포로부터 non-플라즈마세포를
제거하고 CD138 microbeads를이용하여 CD138양성의플라즈마세포를양성 분리 (positive selection)하며 , CD138 microbeads를이용한분리는상기
non-폴라즈마세포제거과정을생략하고 CD138 microbeads를이용하여양성 분리 (positive selection)하게된다.
[33] 상기두가지방법을이용하여양성분리된플라즈마세포 (plasma cell)의세포 표현형을확인하기위하여， BD사의단클론성항체 (CD19-PE-Cy5, CD38-PE, CD56-PE-Cy5, CD138-TITC)를 5 x 104 ~ 105개의세포를 1%혈청이포함된 PBS(phosphate-buffered saline; Life Technologies)완층용액 200 (iL당 1 를 처리하여 4 °C에서 30분간반웅시킨후， 1%혈청이포함된 PBS로 2회세척하고 CD19, CD38, CD56및 CDB8의발현을 BD사의유세포분석기 (FACSCalibur, BD)를이용하여분석하였다.
[34] 악성다발골수종세포의표준세포표현형은 CD19 Low, CD22음성， CD34 음성, CD56양성및 CD 138양성인것등이악성다발골수종세포에해당하는데 그발현정도를확인하여도 1에도시하였다.
[35]
[36] [실시예 2]실시간-정량적중합효소연쇄반응 (real-time qPCR)을이용한암 -고환 항원의정량적발현비교
[37] 하기표 1의정상인과다발골수종환자로부터얻은시료로부터 Rneasy mini kit(Qiagen)를이용하여고순도의 total RNA를분리하고 Primerscript 1st strand cDNA synthesis kit(Takara)를이용하여 cDNA를합성하였다.
[38] [Table 1]
Figure imgf000007_0001
[39] *약어에대한설명은하기부호의설명항목참고
[40]
[41] 합성된 cDNA각시료를주형 (template)으로하여실시간-정량적중합효소
연쇄반웅 (real-time quantitative polymerase chain reaction)을위해 SYBR Green PCR Master mix(QUANTI tech)와표 2의 10개암 -고환항원의발현을위한서열번호 11내지 30의프라이머를이용하였다.
[42] [43] [Table 2]
Figure imgf000008_0001
T: Forward (정방향)， R: Reverse (역방향) 실시간-정량적중합효소연쇄반웅은 95 °C에서 30초， 52 0C에서 30초, 72 °C에서 30초의조건으로진행하였다.상기 10개의항원에 대한대조군으로는 베타 -액틴 (beta-actin)을사용하여발현량을측정하였다.정량적발현량의분석은 RG-3000(Roger-gene)기기의 Roger-gene 6소프트웨어를이용하였으며，대조군인 베타-액틴과비교한 10가지암 -고환항원의정량적발현을 GraphPad Prism 5 소프트웨어를이용하여도시하였다.이증일부항원에대한결과는도 2-도 6에 도시하였다. [47] 도 2-도 6의결과에서확인할수있는바와같이목적 mRNA와베타-액틴의 정량적발현량의비를비교한결과，다발골수종환자의경우악성다발골수성 세포를나타내는 CD138양성에서의발현량의비가현저히증가함을확인할수 있으며,이로부터정상인과환자를명확하게선별할수있음을확인하였다.
[48]
[49] [실시예 3]수지상세포의제조,악성다발골수종세포로부터선별，제조된
재조합암 -고환항원을수지상세포에탑재및암 -고환항원특이적세포독성 T 림프구세포 (cytotoxic T lymphocytes; CTLs)유도
[50] 본발명에사용된수지상세포는동의서를얻은다발골수종환자의
말초혈액으로부터 lymphoprep(Ficoll-Hypaque)을이용한원심분리를통해 단핵세포 (mononuclear cells)를획득하였고,이를다시퍼콜 (Percoll)밀도구배 흑은 CDI4양성세포를마그네틱비드 (magnetic bead)를이용하여
단구세포 (monocytes)를수득하였다.
[51] 상기수득된단구세포를 6-well플레이트에 2 x 106cells의밀도로
분주 (seeding)하고， 50 ng/mL재조합인간 GM-CSF와 20 ng/mL재조합인간 IL-4을 2일간격으로총 6일간처리하여미성숙수지상세포로분화시킨다음， (주)박샐바이오에서개발된수지상세포 (VaxDC)표준제조프로토콜 (SOP)에따라, VaxDC칵테일을 2일간처리 및배양하여성숙수지상세포를수득하였다.
[52] 악성다발골수종세포로부터상기표 2의프라이머를이용한정량분석을통해, 선별및제조된재조합암 -고환항원 3-5개를수지상세포에 2 X 105내지 3 X 105 세포당 0.5 glmi내지 10 / ^의농도로처리하여 37 °C C02배양기에서 2시간 동안반웅시키고 CD3또는 CD8양성 T림프구세포와함께공동배양하여세포 독성 T림프구세포 (cytotoxic T lymphocytes; CTLs)를유도하였다.
[53] 공동배양 3일후， 5 ng/mL재조합인간 IL-2와 10 ng/mL IL-7을첨가하여세포 독성 T림프구세포를중식시켰다.이후 7일간격으로,증식된세포독성 T 림프구세포를상기항원이탑재된수지상세포로 2회 이상자극하고공동 배양하여 5일후,반웅이유도된세포독성 T림프구세포를수득하였다.
[54] 이를통해암 -고환항원을과발현하는다발골수종세포와다발골수종환자의 악성세포를타겟세포로하여，세포독성 T림프구세포와공동배양하여암 -고환 항원-특이적세포독성반웅및그살상능을확인하였다.
[55]
[56] [실시예 4]인간인터페론 -감마 ELISPOT기법을이용한암 -고환항원특이적 세포독성 T림프구세포의살상능
[57] 본실험에사용된인간인터페론 -감마 ELISPOT kit(BD)는제조자의
프로토콜 (BD™ ELISPOT human IFN-η set, cat no.551849, BD)에 따라사용하였다.
[58] 암 -고환항원이과발현되는다발골수종세포주와다발골수종환자의
악성세포를 1:10또는 1:20비율로타겟세포로사용하여인간인터페론 -감마 항체가코팅된 96-well플레이트에서 24시간공동배양한다음,발색된인간 인터페론-감마의스팟 (spots)의수를확인하였다.얻어진스팟의수는곧타켓 세포에발현된암 -고환항원을인식하는암 -고환항원특이적세포독성 T 림프구세포의수를의미한다.
[59] 각각의선별제조된암 -고환항원이제시된수지상세포로부터특이적으로
제조된세포독성 T림프구세포에의해타¾세포를특이적으로인식하여 인터페론감마를분비하는정도를확인하였다.
[60]
[61] 실험결과는아래와같다.
[62] K562세포주는 NK세포에의존적으로살상되는세포주이고, RPMT8226과
U266세포주는암 -고환항원을과발현하는다발골수종세포주 (n=2)이며， P3, P4,
P5및 P6은암 -고환항원을과발현하는다발골수종환자 (n=4)의
다발골수종세포이다.
[63] un-CTL은상기 기술된암 -고환항원이탑재되지않은수지상세포에의해
유도된세포독성 T림프구세포이며도 7의청색그래프로부터 항원-비특이적 반응을확인할수있다.
[64] SSX2-, SSX4-, MAGEA3-및 MAGEC2-CTL은암 -고환항원이탑재된
수지상세포에의해유도된암 -고환항원-특이적세포독성 T림프구세포 (도 7의 적색그래프)이다.
[65] 도 7의그래프에서스팟의간격은암 -고환항원 -특이적세포독성 T림프구
반응의강도를의미하는데，파란색과빨간색그래프의간격이크게벌어질수록 항원-특이적반응의세기가강함을나타내는것이다.
[66] 도 7의결과에서확인할수있듯이,암 -고환항원으로유도된세포독성 T
림프구세포의경우，암-고환항원을과발현하는타겟세포에반웅하여 인터페론 감마를분비하는스팟이특이적이고강하게생성되었으며，이러한반웅은 MHC(major histocompatibility complex)분자 I의단클론항체에의해특이적으로 저해되는것으로부터， MHC분자 I매개에의한항원-특이적인반응임을 확인하여본발명을완성하였다.
[67]
[68] NL-정상인， MM-다발골수종환자, PB-말초혈액단핵세포， B-B세포， BM-골수 단핵세포， IgA, -면역글로블린 A, IgG-면역글로블린 G, lamda-lamda light chain, kappa-kappa light chain, CD138-CD138음성의다발골수종세포， CD138+-CD138 양성의다발골수종세포， MACS-magnectic activated cell separation.
서열목록 Free Text
[69] 서열목록은별도의파일에기재하였다.

Claims

청구범위
[청구항 1] 암 -고환항원인 CTAGIB, SSX2, SSX4, SSX5, MAGEA3, MAGEC1 ,
MAGEC2, BAGEl, CTAG2및 SPA17로이루어진군으로부터 선택되는어느하나이상의단백질;상기단백질의항원성 펩티드가탑재된수지상세포;또는상기 암 -고환항원 -특이적 세포독성 T림프구세포를유효성분으로포함하는다발골수종 예방또는치료용세포면역치료제조성물.
[청구항 2] 제 1항에 있어서,
상기 CTAGIB는서열번호 1， SSX2는서열번호 2, SSX4는 서열번호 3， SSX5는서열번호 4， MAGEA3은서열번호 5，
MAGEC1은서열번호 6， MAGEC2는서열번호 7， BAGE1은 서열번호 8, CTAG2는서열번호 9및 SPA17은서열번호 10의 아미노산서열을갖는것을특징으로하는조성물.
[청구항 3] a)악성다발골수종세포를준비하는단계；
b)상기악성다발골수종세포의순도를확인하는단계 ;
c)높은순도로수확된악성다발골수종세포로부터암 -고환 항원의프라이머를이용한정량분석을통해상기세포로부터 과발현여부를판단하는단계;및
d)상기 암 -고환항원을재조합-종양항원단백질로제조하여 수지상세포에 적절히탑재하는단계；
를포함하는다발골수종예방또는치료용세포면역치료제 조성물의 제조방법.
[청구항 4] 제 3항에 있어서,
상기 c)단계의프라이머는
CTAGIB단백질을코딩하는유전자는서열번호 U의 정방향 프라이머,서열번호 12의 역방향프라이머;
SSX2단백질을코딩하는유전자는서열번호 13의정방향 프라이머,서열번호 14의 역방향프라이머;
SSX4단백질을코딩하는유전자는서열번호 15의정방향 프라이머，서열번호 16의 역방향프라이머;
SSX5단백질을코딩하는유전자는서열번호 17의정방향 프라이머,서열번호 18의 역방향프라이머;
MAGEA3단백질을코딩하는유전자는서열번호 19의 정방향 프라이머,서열번호 20의 역방향프라이머;
MAGEC1단백질올코딩하는유전자는서열번호 21의정방향 프라이머，서열번호 22의 역방향프라이머;
MAGEC2단백질을코딩하는유전자는서열번호 23의 정방향 프라이머 ,서열번호 24의 역방향프라이머；
BAGE1단백질을코딩하는유전자는서열번호 25의정방향 프라이머,서열번호 26의 역방향프라이머;
CTAG2단백질을코딩하는유전자는서열번호 27의 정방향 프라이머,서열번호 28의 역방향프라이머;및
SPA17단백질을코딩하는유전자는서열번호 29의정방향 프라이머,서열번호 30의 역방향프라이머를사용하여증폭시키는 것을특징으로하는제조방법.
[청구항 5] 제 3항에 있어서,
상기 c)단계에서의 정량분석은실시간-정량적중합효소 연쇄반웅 (real-time quantitative polymerase chain reaction)으로 발현량을측정하는것을특징으로하는제조방법 .
[청구항 6] 제 5항에 있어서,
상기정량분석은베타 -액틴 (beta-actin)을대조군으로사용하여 대조군과의발현량의 비율을측정하는것을특징으로하는 제조방법.
PCT/KR2016/011178 2015-10-06 2016-10-06 암-고환 항원을 포함하는 한국 난치성 다발골수종 예방 또는 치료용 세포 면역치료제 조성물 및 그 제조방법 Ceased WO2017061784A1 (ko)

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US20130096025A1 (en) * 2010-05-27 2013-04-18 Erasmus University Medical Center Rotterdam Molecular classification of multiple myeloma

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