KR100482920B1 - Compositions for treating hepatocellular cancer - Google Patents
Compositions for treating hepatocellular cancer Download PDFInfo
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- KR100482920B1 KR100482920B1 KR10-2002-7010174A KR20027010174A KR100482920B1 KR 100482920 B1 KR100482920 B1 KR 100482920B1 KR 20027010174 A KR20027010174 A KR 20027010174A KR 100482920 B1 KR100482920 B1 KR 100482920B1
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- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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Abstract
본 발명은 포유동물의 암을 예방하거나 치료하기 위한 방법에 관한 것이며, 여기서 암세포는 세포 표면에서 알파 페토프로테인 분자의 적어도 일부분을 발현시키며, 상기 방법은 포유류에게 알파 페토프로테인 분자의 아미노산 서열의 적어도 일부분에 대한 면역 반응을 생성시키는 단계를 포함하고, 여기서 면역 반응은 암세포에 대한 알파 페토프로테인 펩티드 특이적인 T 림프구를 활성화시키는 것을 포함한다. 본 발명은 알파 페토프로테인의 서열의 적어도 일부분을 갖는 펩티드를 포함하는, 암을 예방하거나 치료하기 위한 조성물에 관한 것이다.The present invention relates to a method for preventing or treating cancer in a mammal, wherein the cancer cells express at least a portion of an alpha fetoprotein molecule at the cell surface, wherein the method comprises at least a portion of the amino acid sequence of the alpha fetoprotein molecule to the mammal. Generating an immune response against, wherein the immune response comprises activating alpha-fetoprotein peptide specific T lymphocytes against cancer cells. The present invention relates to a composition for preventing or treating cancer, comprising a peptide having at least a portion of the sequence of alpha fetoprotein.
Description
원발성 간암은 전세계 암 사망의 주요 원인이다. 간세포암(HCC)이 가장 보편적인 타입의 원발성 간암이며, 1년에 약 120만명의 환자가 전세계에서 발생한다. 남동아시아 및 사하라 이남 아프리카와 같은 일부 지역에서, 간세포암은 가장 보편적인 타입의 악성종양중 하나이다. 이들 질환의 높은 빈도는 이들 지역에서의 바이러스성 간염의 높은 발생과 관련되는 것 같다.Primary liver cancer is the leading cause of cancer deaths worldwide. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, with about 1.2 million patients a year occurring worldwide. In some regions, such as Southeast Asia and Sub-Saharan Africa, hepatocellular carcinoma is one of the most common types of malignancy. The high frequency of these diseases seems to be associated with the high incidence of viral hepatitis in these areas.
간세포암의 치료법은 현재 비전이성 질환에 걸린 개체에게 제한되어 있고 간이식의 존재 또는 부재하에 종양의 외과적인 절제와 관련되어 있다. 그러나 외과적 절제 및 이식 조차 절제후 재발로 인해 대부분의 종양을 치료하지 못한다. 치료를 위한 화학치료학적 접근법은 대개 비효과적이었다.Therapies for hepatocellular carcinoma are currently limited to individuals with non-metastatic disease and are associated with surgical resection of the tumor in the presence or absence of liver transplantation. However, even surgical resection and transplantation do not cure most tumors due to relapse after resection. Chemotherapy approaches for treatment have often been ineffective.
그러므로, 간세포암을 효과적으로 치료할 필요가 있다. 상기 치료는 이상적으로는 상기 질환이 가장 많이 발생하는 후진 개발도상국에 사용하기에 적합해야 한다. 또한, 상기 치료는 절제될 수 없는 종양 및 전이성 질환에 걸린 개체에 사용하기에 적절해야 한다.Therefore, there is a need to effectively treat hepatocellular carcinoma. The treatment should ideally be suitable for use in backward developing countries where the disease is most common. In addition, the treatment should be suitable for use in individuals with tumors and metastatic disease that cannot be resected.
발명의 요약Summary of the Invention
하나의 구체예에서, 본 발명은 포유동물의 암을 예방하거나 치료하는 방법에 관한 것이며, 여기서 암세포는 세포 표면에 알파 페토프로테인 분자의 적어도 일부분을 발현시킨다. 상기 방법은 포유동물에게 알파 페토프로테인 분자의 아미노산 서열의 적어도 일부분에 대한 면역 반응을 생성시키는 단계를 포함하며, 여기서 면역 반응은 암세포에 대한 알파 페토프로테인 펩티드 특이적인 T 림프구를 활성화시키는 것을 포함한다. 하나의 구체예에서, 알파 페토프로테인 펩티드 특이적인 T 림프구는 세포독성 T 림프구이다. 바람직한 구체예에서, 알파 페토프로테인 분자는 SEQ ID NO:2이다. 특히 바람직한 구체예에서, 알파 페토프로테인 분자는 SEQ ID NO:2의 잔기 137 내지 145, SEQ ID NO:2의 잔기 158 내지 166, SEQ ID NO:2의 잔기 325 내지 334 및 SEQ ID NO:2의 잔기 542 내지 550으로 구성된 군으로부터 선택된다. 하나의 구체예에서, 암은 간세포암이다. 또 다른 구체예에서, 포유동물은 사람이다.In one embodiment, the invention is directed to a method of preventing or treating cancer in a mammal, wherein the cancer cells express at least a portion of an alpha fetoprotein molecule on the cell surface. The method includes generating an immune response to at least a portion of the amino acid sequence of the alpha fetoprotein molecule in a mammal, wherein the immune response comprises activating alpha fetoprotein peptide specific T lymphocytes against cancer cells. In one embodiment, the alpha fetoprotein peptide specific T lymphocytes are cytotoxic T lymphocytes. In a preferred embodiment, the alpha fetoprotein molecule is SEQ ID NO: 2. In a particularly preferred embodiment, the alpha fetoprotein molecule comprises residues 137-145 of SEQ ID NO: 2, residues 158-166 of SEQ ID NO: 2, residues 325-334 of SEQ ID NO: 2 and SEQ ID NO: 2 Selected from the group consisting of residues 542-550. In one embodiment, the cancer is hepatocellular carcinoma. In another embodiment, the mammal is a human.
바람직한 구체예에서, 면역 반응을 일으키는 단계는 알파 페토프로테인 아미노산 서열의 적어도 일부분을 포함하는 펩티드를 포함하는 하나 이상의 조성물을 포유동물에게 투여하는 것을 포함한다. 특히 바람직한 구체예에서, 펩티드는 SEQ ID NO:2의 잔기 137 내지 145, SEQ ID NO:2의 잔기 158 내지 166, SEQ ID NO:2의 잔기 325 내지 334 및 SEQ ID NO:2의 잔기 542 내지 550으로 구성된 군으로부터 선택된다. 또 다른 바람직한 구체예에서, 펩티드는 SEQ ID NO:2의 잔기 1 내지 9, SEQ ID NO:2의 잔기 178 내지 186, SEQ ID NO:2의 잔기 218 내지 226, SEQ ID NO:2의 잔기 235 내지 243, SEQ ID NO:2의 잔기 306 내지 315, SEQ ID NO:2의 잔기 485 내지 493, SEQ ID NO:2의 잔기 492 내지 500, SEQ ID NO:2의 잔기 507 내지 516, SEQ ID NO:2의 잔기 547 내지 556 및 SEQ ID NO:2의 잔기 555 내지 563으로 구성된 군으로부터 선택된다.In a preferred embodiment, the step of generating an immune response comprises administering to the mammal at least one composition comprising a peptide comprising at least a portion of an alpha fetoprotein amino acid sequence. In a particularly preferred embodiment, the peptide comprises residues 137 to 145 of SEQ ID NO: 2, residues 158 to 166 of SEQ ID NO: 2, residues 325 to 334 of SEQ ID NO: 2 and residues 542 to SEQ ID NO: 2 It is selected from the group consisting of 550. In another preferred embodiment, the peptide comprises residues 1-9 of SEQ ID NO: 2, residues 178-186 of SEQ ID NO: 2, residues 218-226 of SEQ ID NO: 2, residue 235 of SEQ ID NO: 2 To 243, residues 306 to 315 of SEQ ID NO: 2, residues 485 to 493 of SEQ ID NO: 2, residues 492 to 500 of SEQ ID NO: 2, residues 507 to 516 of SEQ ID NO: 2, SEQ ID NO Residues 547 to 556 of: 2 and residues 555 to 563 of SEQ ID NO: 2.
또 다른 바람직한 구체예에서, 면역 반응을 일으키는 단계는 SEQ ID NO:2의 아미노산 서열의 적어도 일부분을 형성하는 하나 이상의 펩티드로 펄싱된 수상 세포를 포함하는 하나 이상의 조성물을 포유동물에게 투여하는 것을 포함한다. 또 다른 바람직한 구체예에서, 면역 반응을 일으키는 단계는 알파 페토프로테인을 엔코딩하는 재조합 아데노바이러스 벡터로 형질도입된 수상 세포를 포함하는 하나 이상의 조성물을 포유동물에게 투여하는 것을 포함한다.In another preferred embodiment, the step of generating an immune response comprises administering to the mammal at least one composition comprising dendritic cells pulsed with one or more peptides forming at least a portion of the amino acid sequence of SEQ ID NO: 2 . In another preferred embodiment, the step of generating an immune response comprises administering to the mammal at least one composition comprising dendritic cells transduced with a recombinant adenovirus vector encoding alpha fetoprotein.
또 다른 구체예에서, 본 발명은 사람의 간세포암을 예방하거나 치료하는 방법에 관한 것이며, 여기서 암세포는 세포 표면에 알파 페토프로테인 분자의 적어도 일부분을 발현시키고, 상기 방법은 SEQ ID NO:2의 잔기 137 내지 145, SEQ ID NO:2의 잔기 158 내지 166 및 SEQ ID NO:2의 잔기 325 내지 334로 구성된 군으로부터 선택된 펩티드를 포함하는 하나 이상의 조성물을 사람에게 투여함으로써 SEQ ID NO:2의 아미노산 서열의 적어도 일부분에 대해 암세포에 대한 알파 페토프로테인 세포독성 T 림프구를 활성화시키는 단계를 포함한다.In another embodiment, the present invention relates to a method for preventing or treating human hepatocellular carcinoma, wherein the cancer cell expresses at least a portion of an alpha-fetoprotein molecule at the cell surface, wherein the method comprises a residue of SEQ ID NO: 2. Amino acid sequence of SEQ ID NO: 2 by administering to a human at least one composition comprising a peptide selected from the group consisting of 137-145, residues 158-166 of SEQ ID NO: 2 and residues 325-334 of SEQ ID NO: 2 Activating alpha-fetoprotein cytotoxic T lymphocytes against cancer cells for at least a portion of.
또 다른 구체예에서, 본 발명은 사람의 간세포암을 예방하거나 치료하는 방법에 관한 것이며, 여기서 암세포는 세포 표면에 알파 페토프로테인 분자의 적어도 일부분을 발현시키고, 상기 방법은 SEQ ID NO:2의 잔기 542 내지 550으로 구성된 군으로부터 선택된 펩티드를 포함하는 하나 이상의 조성물을 사람에게 투여함으로써 SEQ ID NO:2의 아미노산 서열의 적어도 일부분에 대해 암세포에 대한 알파 페토프로테인 세포독성 T 림프구를 활성화시키는 단계를 포함한다.In another embodiment, the present invention relates to a method for preventing or treating human hepatocellular carcinoma, wherein the cancer cell expresses at least a portion of an alpha-fetoprotein molecule at the cell surface, wherein the method comprises a residue of SEQ ID NO: 2. Activating alpha fetoprotein cytotoxic T lymphocytes against cancer cells on at least a portion of the amino acid sequence of SEQ ID NO: 2 by administering to the human at least one composition comprising a peptide selected from the group consisting of 542-550 .
또 다른 구체예에서, 본 발명은 사람의 간세포암을 예방하거나 치료하는 방법에 관한 것이며, 여기서 암세포는 세포 표면에 알파 페토프로테인 분자의 적어도 일부분을 발현시키고, 상기 방법은 SEQ ID NO:2의 잔기 1 내지 9, SEQ ID NO:2의 잔기 178 내지 186, SEQ ID NO:2의 잔기 218 내지 226, SEQ ID NO:2의 잔기 235 내지 243, SEQ ID NO:2의 잔기 306 내지 315, SEQ ID NO:2의 잔기 485 내지 493, SEQ ID NO:2의 잔기 492 내지 500, SEQ ID NO:2의 잔기 507 내지 516, SEQ ID NO:2의 잔기 547 내지 556 및 SEQ ID NO:2의 잔기 555 내지 563으로 구성된 군으로부터 선택된 펩티드를 포함하는 하나 이상의 조성물을 사람에게 투여함으로써 SEQ ID NO:2의 아미노산 서열의 적어도 일부분에 대해 암세포에 대한 알파 페토프로테인 세포독성 T 림프구를 활성화시키는 단계를 포함한다.In another embodiment, the present invention relates to a method for preventing or treating human hepatocellular carcinoma, wherein the cancer cell expresses at least a portion of an alpha-fetoprotein molecule at the cell surface, wherein the method comprises a residue of SEQ ID NO: 2. 1 to 9, residues 178 to 186 of SEQ ID NO: 2, residues 218 to 226 of SEQ ID NO: 2, residues 235 to 243 of SEQ ID NO: 2, residues 306 to 315 of SEQ ID NO: 2, SEQ ID Residues 485 to 493 of NO: 2, residues 492 to 500 of SEQ ID NO: 2, residues 507 to 516 of SEQ ID NO: 2, residues 547 to 556 of SEQ ID NO: 2 and residue 555 of SEQ ID NO: 2 Activating alpha fetoprotein cytotoxic T lymphocytes against cancer cells on at least a portion of the amino acid sequence of SEQ ID NO: 2 by administering to the human at least one composition comprising a peptide selected from the group consisting of 563.
또 다른 구체예에서, 본 발명은 사람의 간세포암을 예방하거나 치료하는 방법에 관한 것이며, 여기서 암세포는 세포 표면에 알파 페토프로테인 분자의 적어도 일부분을 발현시키고, 상기 방법은 SEQ ID NO:2의 아미노산 서열의 적어도 일부분을 형성하는 하나 이상의 펩티드로 펄싱된 수상 세포를 포함하는 하나 이상의 조성물을 사람에게 투여함으로써 SEQ ID NO:2의 아미노산 서열의 적어도 일부분에 대해 암세포에 대한 알파 페토프로테인 세포독성 T 림프구를 활성화시키는 단계를 포함한다. 상기 하나 이상의 펩티드는 SEQ ID NO:2의 잔기 137 내지 145, SEQ ID NO:2의 158 내지 166, SEQ ID NO:2의 325 내지 334 및 SEQ ID NO:2의 잔기 542 내지 550으로 구성된 군으로부터 선택된 하나 이상의 펩티드로 펄싱된 수상 세포로부터 선택된다.In another embodiment, the present invention relates to a method of preventing or treating human hepatocellular carcinoma, wherein the cancer cell expresses at least a portion of an alpha-fetoprotein molecule on the cell surface, wherein the method comprises an amino acid of SEQ ID NO: 2 Alpha petoprotein cytotoxic T lymphocytes against cancer cells were challenged against at least a portion of the amino acid sequence of SEQ ID NO: 2 by administering to the human at least one composition comprising dendritic cells pulsed with one or more peptides forming at least a portion of the sequence. Activating. Said at least one peptide is from the group consisting of residues 137-145 of SEQ ID NO: 2, 158-166 of SEQ ID NO: 2, 325-334 of SEQ ID NO: 2 and residues 542-550 of SEQ ID NO: 2 It is selected from dendritic cells pulsed with one or more peptides selected.
또 다른 구체예에서, 본 발명은 사람의 간세포암을 예방하거나 치료하는 방법에 관한 것이며, 여기서 암세포는 세포 표면에 알파 페토프로테인 분자의 적어도 일부분을 발현시키고, 상기 방법은 알파 페토프로테인을 엔코딩하는 재조합 아데노바이러스 벡터로 형질도입된 수상 세포를 포함하는 하나 이상의 조성물을 사람에게 투여함으로써 SEQ ID NO:2의 아미노산 서열의 적어도 일부분에 대해 암세포에 대한 알파 페토프로테인 세포독성 T 림프구를 활성화시키는 단계를 포함한다.In another embodiment, the present invention relates to a method for preventing or treating human hepatocellular carcinoma, wherein the cancer cell expresses at least a portion of an alpha fetoprotein molecule on the cell surface and the method encodes a recombinant to encode alpha fetoprotein. Activating alpha fetoprotein cytotoxic T lymphocytes against cancer cells against at least a portion of the amino acid sequence of SEQ ID NO: 2 by administering to the human at least one composition comprising dendritic cells transduced with an adenovirus vector. .
또 다른 구체예에서, 본 발명은 SEQ ID NO:2의 잔기 137 내지 145, SEQ ID NO:2의 잔기 158 내지 166 및 SEQ ID NO:2의 잔기 325 내지 334로 구성된 군으로부터 선택된, 암을 예방하거나 치료하기에 유용한 단리된 펩티드에 관한 것이다. 바람직한 구체예에서, 본 발명은 포유동물이 알파 페토프로테인에 대한 면역 반응을 일으키기에 충분한 양으로 SEQ ID NO:2의 잔기 137 내지 145, SEQ ID NO:2의 잔기 158 내지 166 및 SEQ ID NO:2의 잔기 325 내지 334로 구성된 군으로부터 선택된 하나 이상의 펩티드를 포함하는, 암을 예방하거나 치료하기 위한 조성물에 관한 것이다. 상기 조성물은 애쥬번트를 추가로 포함할 수 있다. 또 다른 구체예에서, 본 발명은 상기 펩티드중 하나 또는 상기 조성물중 하나를 사람에게 투여하는 단계를 포함하여, 사람의 암을 예방하거나 치료하는 방법에 관한 것이다.In another embodiment, the invention is a cancer prevention method selected from the group consisting of residues 137-145 of SEQ ID NO: 2, residues 158-166 of SEQ ID NO: 2 and residues 325-334 of SEQ ID NO: 2 To isolated peptides useful for treating or treating. In a preferred embodiment, the present invention provides a mammal sufficient to cause an immune response to alpha fetoprotein, residues 137 to 145 of SEQ ID NO: 2, residues 158 to 166 of SEQ ID NO: 2 and SEQ ID NO: A composition for preventing or treating cancer comprising at least one peptide selected from the group consisting of residues 325 to 334 of 2. The composition may further comprise an adjuvant. In another embodiment, the present invention relates to a method of preventing or treating cancer in a human, comprising administering one of the peptides or one of the compositions to a human.
본 발명은 또한 SEQ ID NO:2의 잔기 137 내지 145, SEQ ID NO:2의 잔기 158 내지 166, SEQ ID NO:2의 잔기 325 내지 334 및 SEQ ID NO:2의 잔기 542 내지 550으로 구성된 군으로부터 선택된 하나 이상의 펩티드를 포함하는, 암을 예방하거나 치료하기 위한 수단을 포함한다.The invention also comprises a group consisting of residues 137-145 of SEQ ID NO: 2, residues 158-166 of SEQ ID NO: 2, residues 325-334 of SEQ ID NO: 2 and residues 542-550 of SEQ ID NO: 2 Means for preventing or treating cancer, comprising one or more peptides selected from.
또 다른 구체예에서, 본 발명은 SEQ ID NO:2의 잔기 542 내지 550에 따른 서열을 갖는, 암을 예방하거나 치료하기에 유용한 단리된 펩티드에 관한 것이다. 바람직한 구체예에서, 본 발명은 SEQ ID NO:2의 잔기 542 내지 550에 따른 서열을 갖는 펩티드를 포함하는, 암을 예방하거나 치료하기 위한 조성물에 관한 것이다. 상기 조성물은 애쥬번트를 추가로 포함할 수 있다. 또 다른 구체예에서, 본 발명은 이러한 펩티드 또는 이들 조성물중 하나를 사람에게 투여하는 단계를 포함하여, 사람의 암을 예방하거나 치료하는 방법에 관한 것이다.In another embodiment, the invention relates to an isolated peptide having a sequence according to residues 542 to 550 of SEQ ID NO: 2, useful for preventing or treating cancer. In a preferred embodiment, the invention relates to a composition for preventing or treating cancer comprising a peptide having a sequence according to residues 542 to 550 of SEQ ID NO: 2. The composition may further comprise an adjuvant. In another embodiment, the invention relates to a method of preventing or treating cancer in a human, comprising administering such a peptide or one of these compositions to a human.
본 발명은 또한 SEQ ID NO:2의 잔기 542 내지 550에 따른 서열을 갖는 펩티드를 포함하는, 암을 예방하거나 치료하기 위한 수단을 포함한다.The invention also includes a means for preventing or treating cancer, comprising a peptide having a sequence according to residues 542 to 550 of SEQ ID NO: 2.
하나의 구체예에서, 본 발명은 간세포암을 치료하기 위해 단독 또는 혼합하여 사용될 수 있는 일군의 펩티드에 관한 것이다. 또 다른 구체예에서, 본 발명은 본 발명의 하나 이상의 펩티드를 단독 또는 혼합하여 투여함으로써 간세포암을 예방하거나 치료하는 방법, 또는 본 발명의 하나 이상의 펩티드를 포함하는 조성물에 관한 것이다. 또 다른 구체예에서, 본 발명은 본 발명의 하나 이상의 펩티드로 펄싱되거나 알파 페토프로테인을 엔코딩하는 재조합 아데노바이러스(AdV) 벡터로 형질도입된 수상 세포를 투여함으로써 간세포암을 예방하거나 치료하는 방법에 관한 것이다.In one embodiment, the invention relates to a group of peptides that can be used alone or in combination to treat hepatocellular carcinoma. In another embodiment, the invention relates to a method for preventing or treating hepatocellular carcinoma by administering one or more peptides of the invention alone or in combination, or a composition comprising one or more peptides of the invention. In another embodiment, the present invention relates to a method of preventing or treating hepatocellular carcinoma by administering a dendritic cell pulsed with one or more peptides of the invention or transduced with a recombinant adenovirus (AdV) vector encoding alpha fetoprotein. will be.
약 80%의 간세포암은 알파 페토프로테인 발현을 재활성화시킨다. 뮤린과 사람 둘 모두의 T 세포 레퍼토리는 MHC 클래스 I과 관련하여 AFP-유래된 펩티드 에피토프를 인식할 수 있다. 그러므로, 배 발생 동안 이러한 종양성태아성 단백질의 높은 혈장 레벨에 노출됨에도 불구하고 모든 AFP-특이적 T 세포가 면역계의 개체발생 동안 제거되는 것은 아니다.About 80% of hepatocellular carcinoma reactivates alpha fetoprotein expression. T cell repertoires of both murine and human can recognize AFP-derived peptide epitopes with respect to MHC class I. Therefore, not all AFP-specific T cells are eliminated during the development of the immune system, despite exposure to the high plasma levels of these oncofetal proteins during embryonic development.
본 발명은 HLA-A*0201과 관련하여 프리젠테이션되는 경우 사람 T 세포 레퍼토리에 의해 인식되는 사람 알파 페토프로테인, 즉 SEQ ID NO:2로부터 유래된 펩티드의 동일성의 결정과 관련있다. 하기 표 1에 요약된 바와 같이, 4가지 AFP-유래된 펩티드가 확인되었고, "도미넌트(dominant)" 펩티드로 칭하였다. 이들은 PLFQVPEPV, hAFP137-145, SEQ ID NO:2의 잔기 137 내지 145; FMNKFIYEI, hAFP158-166, SEQ ID NO:2의 잔기 158 내지 166; GLSPNLNRFL, hAFP325-334, SEQ ID NO:2의 잔기 325 내지 334; 및 GVALQTMKQ, hAFP542-550, SEQ ID NO:2의 잔기 542 내지 550이다. 각각은 한개 또는 두개의 앵커 잔기를 가지고 있다.The present invention relates to the determination of the identity of human alpha fetoproteins, ie peptides derived from SEQ ID NO: 2, which are recognized by the human T cell repertoire when presented in connection with HLA-A * 0201. As summarized in Table 1 below, four AFP-derived peptides have been identified and termed "dominant" peptides. These include PLFQVPEPV, hAFP 137-145, residues 137-145 of SEQ ID NO: 2; FMNKFIYEI, hAFP 158-166, residues 158-166 of SEQ ID NO: 2; GLSPNLNRFL, hAFP 325-334, residues 325-334 of SEQ ID NO: 2; And GVALQTMKQ, hAFP 542-550, residues 542-550 of SEQ ID NO: 2. Each has one or two anchor residues.
각각의 도미넌트 펩티드는 농도-의존성 클래스 I 결합 검정에서 T2 세포상에 HLA-A*0201을 안정화시켰다. 상기 펩티드는 오프-카이네틱스(off-kinetics) 검정에서 2 내지 6시간 동안 안정하였다. 또한, 각각의 도미넌트 펩티드는 몇가지 정상적인 HLA-A*0201 공여체로부터 시험관내에서 펩티드-특이적 T 세포를 유도시켰다. 중요하게는, 이들 hAFP 펩티드-특이적 T 세포는 또한 세포독성 검정 및 IFNg ELISPOT 검정 둘 모두에서 HLA-A*0201+/AFP 포지티브 종양 세포를 인식할 수 있었다. 이러한 정보를 요약하였다.Each dominant peptide stabilized HLA-A * 0201 on T2 cells in a concentration-dependent Class I binding assay. The peptides were stable for 2-6 hours in an off-kinetics assay. In addition, each dominant peptide induced peptide-specific T cells in vitro from several normal HLA-A * 0201 donors. Importantly, these hAFP peptide-specific T cells could also recognize HLA-A * 0201 + / AFP positive tumor cells in both cytotoxicity assays and IFNg ELISPOT assays. This information is summarized.
표 1Table 1
도미넌트 펩티드의 요약Summary of Dominant Peptides
본 발명에서 입증되는 바와 같이, 이들 T 세포의 활성화는 전문적인 항원 프리젠테이션 수상 세포에 의한 프리젠테이션을 포함하여, 면역자극과 관련하여 이들 도미넌트 펩티드를 프리젠테이션시킴으로써 달성될 수 있다. 알파 페토프로테인 cDNA, 즉 SEQ ID NO:1을 엔코딩하는 재조합 아데노바이러스(AdV) 벡터로 형질도입된 수상 세포는 MHC와 관련하여 4가지 도미넌트 펩티드 에피토프를 프로세싱하여 프리젠테이션시킬 것이고 AFP-특이적 T 세포 활성화를 유도할 것이다. 유사하게 면역화된 HLA-A*0201/Kb 마우스는 또한 사이토카인 방출 검정에서 AFP 펩티드-펄싱된 세포를 인식하였다. 또한, AFP 펩티드-자극된 사람 및 HLA-A*0201/Kb 마우스 T 세포 반응은 hAFP-조작된 표적을 인식하였고, 조금 덜한 정도로, 천연 AFP-발현 사람 간세포암 세포를 인식하였다. 마지막으로, 질량 스펙트로메트리를 사용하여 HLA-A*0201 + HCC 세포로부터 용리된 펩티드의 복합 혼합물로부터 3가지 이상의 AFP 에피토프를 확인하였다. 따라서, 이들 4가지 도미넌트 펩티드 각각이 면역원성이고 HLA-A*0201과 관련하여 천연적으로 프로세싱되어 프리젠테이션된다는 여러가지 증거가 제공된다.As demonstrated in the present invention, activation of these T cells can be accomplished by presenting these dominant peptides in connection with immunostimulation, including presentation by specialized antigen presentation dendritic cells. Dendritic cells transduced with alpha-fetoprotein cDNA, ie, recombinant adenovirus (AdV) vectors encoding SEQ ID NO: 1, will be processed and presented with four dominant peptide epitopes in connection with MHC and AFP-specific T cells. Will induce activation. Similarly immunized HLA-A * 0201 / K b mice also recognized AFP peptide-pulsed cells in cytokine release assays. In addition, AFP peptide-stimulated human and HLA-A * 0201 / K b mouse T cell responses recognized hAFP-engineered targets and, to a lesser extent, native AFP-expressing human hepatocellular carcinoma cells. Finally, mass spectrometry was used to identify three or more AFP epitopes from a complex mixture of peptides eluted from HLA-A * 0201 + HCC cells. Thus, various evidence is provided that each of these four dominant peptides is immunogenic and is naturally processed and presented in connection with HLA-A * 0201.
그 밖의 10가지 펩티드("서브도미넌트" 펩티드라 칭함)는 약하거나 덜한 재현성있는 반응을 가졌지만 하나 이상의 검정 타입에서 포지티브였다. 이들 10가지 펩티드는 MKWVESIFL, SEQ ID NO:2의 잔기 1 내지 9; ILLWAARYD, SEQ ID NO:2의 잔기 178 내지 186; LLNQHACAV, SEQ ID NO:2의 잔기 218 내지 226; FQAITVTKL, SEQ ID NO:2의 235 내지 243; TTLERGQCII, SEQ ID NO:2의 잔기 306 내지 315; CIRHEMTPV, SEQ ID NO:2의 잔기 485 내지 493; PVNPGVGQC, SEQ ID NO:2의 잔기 492 내지 500; NRRPCFSSLV, SEQ ID NO:2의 잔기 507 내지 516; TMKQEFLINL, SEQ ID NO:2의 잔기 547 내지 556 및 NLVKQKPQI, SEQ ID NO:2의 잔기 555 내지 563이다.The other ten peptides (called “subdominant” peptides) had weak or less reproducible responses but were positive in one or more assay types. These ten peptides include MKWVESIFL, residues 1-9 of SEQ ID NO: 2; ILLWAARYD, residues 178 to 186 of SEQ ID NO: 2; LLNQHACAV, residues 218-226 of SEQ ID NO: 2; FQAITVTKL, 235 to 243 of SEQ ID NO: 2; TTLERGQCII, residues 306 to 315 of SEQ ID NO: 2; CIRHEMTPV, residues 485 to 493 of SEQ ID NO: 2; PVNPGVGQC, residues 492-500 of SEQ ID NO: 2; NRRPCFSSLV, residues 507-516 of SEQ ID NO: 2; TMKQEFLINL, residues 547-556 of SEQ ID NO: 2 and residues 555-563 of NLVKQKPQI, SEQ ID NO: 2.
본 발명의 조성물 및 방법을 하나 이상의 도미넌트 펩티드, 즉, hAFP137-145, SEQ ID NO:2의 잔기 137 내지 145; hAFP158-166, SEQ ID NO:2의 잔기 158 내지 166; hAFP325-334, SEQ ID NO:2의 잔기 325-334; 및 hAFP542-550, SEQ ID NO:2의 잔기 542 내지 550를 사용한다는 견지에서 주로 설명하였지만, 하나 이상의 4가지 도미넌트 펩티드를 대신하거나 이와 함께 하나 이상의 10가지 서브도미넌트 펩티드를 사용하는 것은 본 발명의 범위내에 포함된다.The compositions and methods of the present invention may comprise one or more dominant peptides, ie, hAFP 137-145 , residues 137-145 of SEQ ID NO: 2; hAFP 158-166 , residues 158-166 of SEQ ID NO: 2; hAFP 325-334 , residues 325-334 of SEQ ID NO: 2; And hAFP 542-550 , residues 542-550 of SEQ ID NO: 2 have been described primarily, but the use of one or more ten subdominant peptides in place of or in combination with one or more four dominant peptides is It is included in the range.
도미넌트 펩티드 및 서브도미넌트 펩티드의 확인에 대해 더욱 상세하게 논의할 것이다. 우선, hAFP, SEQ ID NO:2로부터의 펩티드 서열들(Genbank 수탁 번호: J00077, J00076 및 V01514)은 HLA-A*0201에 잠재적으로 결합하는 것으로 확인되었다. 이들 펩티드는 길이가 9 내지 10개의 아미노산이고, 위치 2에 아미노산 이소류신, 류신 및 메티오닌이 존재하거나, 펩티드 길이에 따라 펩티드 위치 9 또는 10에 아미노산 이소류신, 류신 및 발린을 가질수 있거나 둘 모두일 수 있다. 이러한 74개 펩티드를 위스콘신 대학교 유전학 컴퓨터 그룹 프로그램 "파인드 패턴스(find patterns)"를 사용하여 확인하여 hAFP 서열, 즉 SEQ ID NO:2를 스크리닝하였다. 각각의 74개 펩티드를 표준 기술을 사용하여 합성하였다.The identification of dominant peptides and subdominant peptides will be discussed in more detail. First, peptide sequences (Genbank Accession No .: J00077, J00076 and V01514) from hAFP, SEQ ID NO: 2, were identified as potentially binding to HLA-A * 0201. These peptides are 9 to 10 amino acids in length and may have amino acids isoleucine, leucine and methionine at position 2, or may have amino acids isoleucine, leucine and valine at peptide positions 9 or 10, depending on peptide length, or both. These 74 peptides were identified using the University of Wisconsin Genetics Computer Group program “find patterns” to screen hAFP sequences, SEQ ID NO: 2. Each of the 74 peptides was synthesized using standard techniques.
각각의 74개 펩티드 후보를 HLA-A*0201 안정화 검정에서 T2 세포에 대한 농도 의존성 결합에 대해 시험하였다. 세포 표면 MHC 클래스 I 분자를 증가시키기 위해 전날밤 실온에서 인큐베이션시킨 T2(TAP 결핍) 세포를 0.1 mM 내지 100 mM의 펩티드 농도로 각 펩티드와 함께 밤새 인큐베이션시켰다. HLA-A*0201의 안정성을 세포를 안티-HLA-A2 항체(BB7.2) 및 염소 항마우스-FITC를 사용하여 염색시킨 후에 유동 세포계측법에 의해 검정하였다. Flu 매트릭스 펩티드(aa 58-66)(Flu)에 강력하게 결합한 HLA-A*0201을 포지티브 대조군으로서 사용하였다.Each 74 peptide candidates were tested for concentration dependent binding to T2 cells in the HLA-A * 0201 stabilization assay. T2 (TAP deficient) cells incubated overnight at room temperature to increase cell surface MHC class I molecules were incubated overnight with each peptide at a peptide concentration of 0.1 mM to 100 mM. Stability of HLA-A * 0201 was assayed by flow cytometry after staining cells with anti-HLA-A2 antibody (BB7.2) and goat antimouse-FITC. HLA-A * 0201 strongly bound to Flu matrix peptide (aa 58-66) (Flu) was used as a positive control.
다음으로, MHC-펩티드 복합체 안정성을 오프-카이네틱스 검정을 사용하여 결정하였다. HLA-A*0201 LCL을 온화한 pH3.2 시트레이트-포스페이트 산 완충액으로 제거하였다. 각 펩티드를 즉시 실온에서 3 ug/ml의 b2 마이크로글로불린의 존재하에 1시간 동안 200 mM에서 세포상에 펄싱시켰다. 초과 펩티드를 씻어내고 세포를 37℃에서 0, 2, 4 및 6시간 동안 인큐베이션시켰다. 각 시간지점이 끝날 때 세포를 세척하고 세포 표면 HLA-A2 발현에 대해 염색한 다음, 유량 세포계측법에 의해 분석하였다. 펩티드-MHC 클래스 I 복합체는 평균 형광 강도가, 제거되었지만 펩티드로 펄싱되지 않은 세포로부터 1.5배 이상 증가한 경우 안정하다고 생각되었다.Next, MHC-peptide complex stability was determined using an off-kinetics assay. HLA-A * 0201 LCL was removed with mild pH3.2 citrate-phosphate acid buffer. Each peptide was immediately pulsed on cells at 200 mM for 1 hour in the presence of 3 ug / ml b2 microglobulin at room temperature. Excess peptide was washed off and cells were incubated at 37 ° C. for 0, 2, 4 and 6 hours. At the end of each time point the cells were washed and stained for cell surface HLA-A2 expression and analyzed by flow cytometry. Peptide-MHC class I complexes were considered stable when the mean fluorescence intensity increased by more than 1.5-fold from cells that were removed but not pulsed with peptides.
그런 다음 4가지 도미넌트 펩티드에 대해 추가적인 면역학적 연구 및 물리화학적 연구를 수행하였다. 이들 연구는 (1) 펩티드 특이적이면서 인식된 네이티브 AFP를 발현 세포인 AFP 펩티드-특이적 사람 T 세포 배양물을 제조하기 위해 펩티드를 사용하고; 및 (2) 도미넌트 펩티드로 펄싱된 AFP 네가티브 세포 뿐만 아니라 AFP 포지티브 세포를 인식하는 AFP 특이적 사람 T 세포를 제조하기 위해 AdVhAFP-형질도입된 수상 세포를 사용하는 시험관내 연구; 및 (1) 펩티드 및 AFP 포지티브 세포를 인식하는 비세포(splenocyte)를 갖는, 펩티드로 면역화된 트랜스제닉 마우스; 및 (2) 도미넌트 펩티드로 펄싱된 AFP 네가티브 세포 뿐만 아니라 AFP 포지티브 세포를 인식하는 AdVhAFP/DC 면역화된 마우스를 사용하는 생체내 연구를 포함하였다. 이들 연구는 도미넌트 펩티드가 면역원성이고, AFP 자체가 면역원성이고, 도미넌트 펩티드가 천연적으로 프로세싱되어 AFP 포지티브 세포의 표면상에 프리젠테이션되고 AFP/DC 또는 도미넌트 펩티드 둘 모두가 사이토카인을 제조하고 AFP 포지티브 세포를 사멸시키는 AFP-특이적 T 세포를 생성시키기 위해 사용될 수 있음을 보여주었다. 또한, 질량 스펙트로스코피를 사용하여 AFP 포지티브 간세포암 세포의 표면으로부터 AFP 펩티드를 물리적으로 확인하였다.Further immunological and physicochemical studies were then performed on the four dominant peptides. These studies use (1) peptides to prepare AFP peptide-specific human T cell cultures that are peptide specific and recognized native AFP expressing cells; And (2) in vitro studies using AdVhAFP-transformed dendritic cells to prepare AFP specific human T cells recognizing AFP positive cells as well as AFP negative cells pulsed with dominant peptides; And (1) transgenic mice immunized with peptides having splenocytes that recognize peptides and AFP positive cells; And (2) in vivo studies using AdVhAFP / DC immunized mice recognizing AFP positive cells as well as AFP negative cells pulsed with dominant peptide. These studies show that the dominant peptide is immunogenic, the AFP itself is immunogenic, the dominant peptide is naturally processed and presented on the surface of the AFP positive cells, and both AFP / DC or dominant peptides produce cytokines and AFP It has been shown that it can be used to generate AFP-specific T cells that kill positive cells. Mass spectroscopy was also used to physically identify AFP peptides from the surface of AFP positive hepatocellular carcinoma cells.
우선, 나이브 HLA-A*0201 사람 T 세포 배양물의 반복적인 펩티드 자극을 수행하여 사람 T 세포 레퍼토리와 관련하여 펩티드 면역원성 및 AFP-트랜스펙션된 표적을 인식하는 펩티드-특이적 T 세포의 능력을 입증하였다. 벌크 T 세포 배양물을 각 도미넌트 AFP-유래된 펩티드(KLH, IL-7 및 IL-2로 보충됨)로 펄싱된 PBMC로부터 생성시키고 펩티드-펄싱된 표적 및 AFP-발현 표적 둘 모두를 인식하는 능력에 대해 3주 내지 7주의 확장을 시험하였다. 이 배양물은 특이적 펩티드-펄싱된 JY 세포의 인식시에 IFNg를 분비하는 능력에 의해 입증된 바와 같이 펩티드-특이적 T 세포를 확장시켰고 ELISPOT 검정에서 대조군 MART-127-35 펄싱된 JY는 그렇지 않았다. AFP 펩티드-특이적 벌크 T 세포는 또한 IFNg-생성 AFP-특이적 T 세포의 증가된 빈도에 의해 밝혀진 바와 같이 변형되지 않거나 AdVhAFP 없이 형질도입된 모세포와 비교하여 AFP 네가티브 안정하게 트랜스펙션되고 AdVhAFP-형질도입된 HLA-A*0201 흑색종 세포(M202) 둘 모두를 인식하였다. HLA-A*0201+, 천연적으로 AFP-발현 간세포암 세포주 HepG2(HLA-A2-/AFP 포지티브 HCC 라인 Hep3B과 비교하여)를 인식하는 능력을 평가하기 위해, 세포독성 및 ELISPOT 검정 둘 모두를 수행하였다.First, repeated peptide stimulation of naïve HLA-A * 0201 human T cell cultures was performed to assess peptide-specific T cells' ability to recognize peptide immunogenic and AFP-transfected targets with respect to human T cell repertoire. Proved. Ability to generate bulk T cell cultures from PBMC pulsed with each dominant AFP-derived peptide (supplemented with KLH, IL-7 and IL-2) and recognize both peptide-pulsed and AFP-expressing targets For 3 to 7 weeks of expansion. This culture expanded peptide-specific T cells as demonstrated by the ability to secrete IFNg upon recognition of specific peptide-pulsed JY cells and the control MART-1 27-35 pulsed JY in the ELISPOT assay It wasn't. AFP peptide-specific bulk T cells are also AFP negative stably transfected and AdVhAFP- compared to parent cells unmodified or transduced without AdVhAFP as revealed by the increased frequency of IFNg-producing AFP-specific T cells. Both transduced HLA-A * 0201 melanoma cells (M202) were recognized. To assess the ability to recognize HLA-A * 0201 +, a naturally occurring AFP-expressing hepatocellular carcinoma cell line HepG2 (compared to HLA-A2- / AFP positive HCC line Hep3B), both cytotoxicity and ELISPOT assays were performed. It was.
또한, CTL을 AdV 형질도입된 수상 세포로부터 생성시켰다. 요약하면, GM-CSF 및 IL-4와 함께 인큐베이션된 PBMC로부터 제조된 수상 세포를 2시간 동안 1,000의 다중감염도(moi)로 AdVhAFP 또는 AdVMART1으로 형질도입시켰다. 형질도입된 수상 세포를 세척하고, 조사시키고 1 내지 2 x 105 세포/ml로 플레이팅하여 CTL 생성을 위한 자극제로서 역할하도록 하였다. 자가 비-부착 세포를 마그네틱 비드 디플레션(magnetic bead depletion)에 의해 CD4, CD14, CD19 및 CD56+ 세포를 고갈시켜 CD8+ 부화된 반응 세포(군집 일반적으로 80% CD8+, 자료 제시안함)를 제조하였다. CD8+ 세포를 5% 자가 매질과 IL-7 10 내지 25 ng/ml 중에 형질도입된 수상 세포와 함께 2 x 106 세포/ml로 플레이팅하였다. 3 내지 4일 마다 IL-2 10U/ml을 배양물에 보충하였다. CD8+ CTL을 1 수상 세포 대 10 내지 20 CD8+ CTL의 비로 신선한 자가 AdV-형질도입된 수상 세포를 사용하여 매주 재자극시켰다. 대부분의 배양물은 일주일마다 기본으로 CD4+ 및 CD8+ 세포에 대한 표현형을 나타내었다. 각 도미넌트 펩티드는 몇몇 정상적인 HLA-A*0201 공여체로부터 시험관내에서 펩티드-특이적 T 세포를 유도시켰다.CTLs were also generated from AdV transduced dendritic cells. In summary, dendritic cells prepared from PBMCs incubated with GM-CSF and IL-4 were transduced with AdVhAFP or AdVMART1 at a multiple of 1,000 moi for 2 hours. Transduced dendritic cells were washed, irradiated and plated at 1-2 x 10 5 cells / ml to serve as stimulators for CTL production. Autologous non-adherent cells were depleted of CD4, CD14, CD19 and CD56 + cells by magnetic bead depletion to prepare CD8 + enriched reactive cells (cluster generally 80% CD8 +, data suggestion). CD8 + cells were plated at 2 × 10 6 cells / ml with 5% autologous medium and dendritic cells transduced in 10-25 ng / ml of IL-7. Every 3 to 4 days 10U / ml of IL-2 was supplemented to the culture. CD8 + CTLs were restimulated weekly using fresh autologous AdV-transduced dendritic cells at a ratio of 1 dendritic cells to 10-20 CD8 + CTLs. Most cultures showed phenotypes for CD4 + and CD8 + cells on a weekly basis. Each dominant peptide induced peptide-specific T cells in vitro from several normal HLA-A * 0201 donors.
AdVhAFP/DC 시험관내 자극된 사람 T 세포는 CTL 및 ELISPOT 검정 둘 모두에서 hAFP-트랜스펙션된 표적을 특이적으로 인식했기 때문에, 다음으로 4가지 도미넌트 펩티드가 AdVhAFP/DC 자극된 T 세포에 의해 특이적으로 인식되는 지를 결정하기 위해 이들을 연구하였다. 7일 내지 21일 배양시킨 후에, AdVhAFP/DC로 매주 자극된 CD8-부화된 T 세포를 hAFP 펩티드-특이적 IFNg 사이토카인 생성 세포의 세포독성 및 빈도 둘 모두에 대해 시험하였다. AdVhAFP/DC T 세포 배양물은 4가지 AFP 펩티드 각각에 의해 펄싱된 JY 세포에 대해 세포독성이었고, 이는 이들 펩티드에 대한 CTL이 정상 공여체의 말초혈로부터 확장될 수 있음을 시사한다. 자가 펩티드 펄싱된 LCL 또는 JY 세포로 재자극시킨 후에, 이들 벌크 배양물은 또한 AFP 펩티드에 특이적인 IFNg-분비 세포의 빈도가 MART-127-35에 비해 훨씬 높았으며, 이는 hAFP542-550에 추가적으로, 3가지 다른 도미넌트 펩티드가 또한 AdVhAFP-형질도입된 수상 세포에 의해 천연적으로 프로세싱되어 프리젠테이션됨을 시사한다.Since AdVhAFP / DC in vitro stimulated human T cells specifically recognized hAFP-transfected targets in both the CTL and ELISPOT assays, the next four dominant peptides were specific for AdVhAFP / DC stimulated T cells. We studied them to determine whether they were recognized as public. After 7 to 21 days of culture, weekly stimulated CD8-enriched T cells with AdVhAFP / DC were tested for both cytotoxicity and frequency of hAFP peptide-specific IFNg cytokine producing cells. AdVhAFP / DC T cell cultures were cytotoxic to JY cells pulsed by each of the four AFP peptides, suggesting that CTLs for these peptides can be expanded from the peripheral blood of normal donors. After restimulation with autologous peptide pulsed LCL or JY cells, these bulk cultures also had a much higher frequency of IFNg-secreting cells specific for AFP peptide compared to MART-1 27-35 , which resulted in hAFP 542-550 . In addition, it suggests that three other dominant peptides are also naturally processed and presented by AdVhAFP-transduced dendritic cells.
AdVhAFP/DC 자극된 T 세포 배양물은 또한 A*0201+/AFP 포지티브 간세포암 라인 HepG2를 인식하지만 A*0201-/AFP 포지티브 HCC 라인 Hep3B는 인식하지 않는 사이토카인-생성 세포의 빈도가 낮았다. Th1 사이토카인 IFNg 및 TNFa를 합성하는 T 림프구가 검출되었지만, Th2 사이토카인 IL-4는 검출되지 않았다. 또한 IL-10은 간세포암 라인이 T 세포 부재하에 플레이팅된 경우 검출되었으며, 이는 이러한 사이토카인의 생성이 종양 세포-유래되었음을 시사한다.AdVhAFP / DC stimulated T cell cultures also had a low frequency of cytokine-producing cells that recognized the A * 0201 + / AFP positive hepatocellular carcinoma line HepG2 but not the A * 0201- / AFP positive HCC line Hep3B. T lymphocytes synthesizing Th1 cytokines IFNg and TNFa were detected, but no Th2 cytokine IL-4 was detected. IL-10 was also detected when hepatocellular carcinoma lines were plated in the absence of T cells, suggesting that this cytokine production was tumor cell-derived.
하기와 같이 HLA-A*0201/Kb 트랜스제닉 마우스를 사용하여 74개의 펩티드를 스크리닝하고 이들 펩티드중 어떤 것이 면역원성이고, HLA-A*0201과 관련하여 천연적으로 프로세싱되어 프리젠테이션되는 지를 결정하였다. HLA-A*0201/Kb 트랜스제닉 암컷 마우스를 처음에 할란-스프라그 돌리(Harlan-Sprague Dawley; Indianapolis, IN US)로부터 구입하였고, 로스 앤젤레스에 소재한 캘리포니아 대학교의 방사선 종양학과의 동물 시설에 의해 현재 사육되고 있다.The 74 peptides were screened using HLA-A * 0201 / K b transgenic mice to determine which of these peptides were immunogenic and naturally processed and presented with respect to HLA-A * 0201 as follows. It was. HLA-A * 0201 / K b transgenic female mice were initially purchased from Harlan-Sprague Dawley (Indianapolis, IN US) and by animal facilities at the University of California, Radiation Oncology, Los Angeles. It is being raised now.
펩티드 면역화를 위해, 마우스에 100 ㎍ AFP 또는 완전 프로인트 애쥬번트중에 1:1로 에멀션화된 대조 펩티드를 피하적으로 주입하였다. 완전 프로인트 애쥬번트중에 에멀션화된 각 펩티드로 면역화시킨 후에, 배출된 림프절 세포는 hAFP로 안정하게 트랜스펙션된 세포의 인식시에 IFNg를 생성시켰다. 또한, 알파 페토프로테인 펩티드-특이적 T 세포는 AFP-발현 아데노바이러스(AdVhAFP)로 형질도입된 수상 세포로 면역화된 마우스의 비장에서 확인될 수 있었다. 따라서, 사익 4가지 도미넌트 펩티드는 클래스 I과 관련하여 천연적으로 프로세싱되고 프리젠테이션되며 면역원성이다.For peptide immunization, mice were injected subcutaneously with 1: 1 peptide emulsified in 100 μg AFP or complete Freund's adjuvant. After immunization with each peptide emulsified in complete Freund's adjuvant, drained lymph node cells produced IFNg upon recognition of cells stably transfected with hAFP. In addition, alpha-fetoprotein peptide-specific T cells could be identified in the spleens of mice immunized with dendritic cells transduced with AFP-expressing adenovirus (AdVhAFP). Thus, the four four dominant peptides are naturally processed and presented in connection with class I and are immunogenic.
다음으로, 이들 4가지 도미넌트 펩티드의 생체내 면역원성을 확인하였다. HLA-A*0201/Kb 마우스를 동계의(syngeneic) 수상 세포상에 펄싱된 각 도미넌트 펩티드로 면역화시켰다. IFNg 특이적 ELISPOT 검정을 면역화 도미넌트 펩티드(또는 MART-1 펩티드) 또는 주르카트(Jurkat)/AFP 또는 주르카트/MART 트랜스펙션된 세포주를 사용하여 시험관내에서 재자극된 비세포(splenocyte)를 사용하여 수행하였다. 각 hAFP 펩티드를 사용한 면역화 및 펩티드 또는 주르카트/AFP를 사용한 후속적인 재자극은 다수의 AFP-특이적 IFNg-생성 세포를 유도시켰다. PBS 주입된 마우스로부터의 림프구는 재자극에 상관없이 세포독성이나 IFNg 생성을 보여주지 않았다. MART-127-35 펩티드로 면역화된 마우스는 MART-1 특이적 반응을 생성시켰지만 AFP 펩티드 반응은 생성시키지 않았다.Next, the in vivo immunogenicity of these four dominant peptides was confirmed. HLA-A * 0201 / K b mice were immunized with each dominant peptide pulsed on syngeneic dendritic cells. IFNg specific ELISPOT assays use splenocytes re-stimulated in vitro using immunized dominant peptide (or MART-1 peptide) or Jurkat / AFP or Jurkat / MART transfected cell line It was performed by. Immunization with each hAFP peptide and subsequent restimulation with peptide or Jurkat / AFP induced multiple AFP-specific IFNg-producing cells. Lymphocytes from PBS injected mice showed no cytotoxicity or IFNg production regardless of restimulation. Mice immunized with MART-1 27-35 peptide generated a MART-1 specific response but no AFP peptide response.
그런 다음, 수상 세포를 GM-CSF에서의 차별화에 의해 골수 선조로부터 제조하였고 hAFP cDNA, 즉 SEQ ID NO:1을 포함하는 재조합 AdV 벡터(AdVhAFP)를 사용하여 형질도입시켰다. 시험관내에서 배양된 수상 세포를 100의 moi로 RPMI/2% FCS에서 형질도입시켰다. 형질도입을 37℃에서 2시간 동안 수행하였다. 그런 다음 수상 세포를 세척하고 주입을 위한 동물 당 0.2 ml PBS 당 5 x 105 수상 세포로 재현탁시켰다. 모든 경우에서 생존능력은 95%를 초과하였다. 면역화시킨지 2주 후에, 마우스로부터의 비세포를 hAFP(주르카트/AFP) 또는 MART-1(주르카트/MART)로 안정하게 트랜스펙션된 주르카트 세포로 재자극시켰다. AFP-특이적 IFNg-방출 대 MART-1 특이적 IFNg-방출의 빈도를 p<0.02인 3개의 독립 실험을 평균하여 ELISPOT에 의해 결정하였다. 세포독성을 5시간 51Cr-방출 검정에서 주르카트/AFP 및 주르카트/MART에 대해 검정하였다.Dendritic cells were then prepared from bone marrow progenitors by differentiation in GM-CSF and transduced using recombinant AdV vectors (AdVhAFP) containing hAFP cDNA, ie SEQ ID NO: 1. Dendritic cells cultured in vitro were transduced in RPMI / 2% FCS with 100 moi. Transduction was performed at 37 ° C. for 2 hours. The dendritic cells were then washed and resuspended with 5 x 10 5 dendritic cells per 0.2 ml PBS per animal for injection. In all cases, viability exceeded 95%. Two weeks after immunization, splenocytes from mice were restimulated with Jurkat cells stably transfected with hAFP (Jurkat / AFP) or MART-1 (Jurkat / MART). The frequency of AFP-specific IFNg-release versus MART-1 specific IFNg-release was determined by ELISPOT by averaging three independent experiments with p <0.02. Cytotoxicity was assayed for Jurkat / AFP and Jurkat / MART in a 5 hour 51 Cr-release assay.
다음으로, HPLC 및 질량 스펙트로메트리에 의한 확인을 HLA-A*0201 사람 간세포암 라인으로부터 용리된 도미넌트 펩티드상에서 수행하였다. 이들 분석의 결과의 요약을 하기 표 2에 기재하였다.Next, confirmation by HPLC and mass spectrometry was performed on dominant peptides eluted from the HLA-A * 0201 human hepatocellular carcinoma line. A summary of the results of these analyzes is shown in Table 2 below.
펩티드를 용리시키기 위해, HepG2 및 Hep3B 세포를 PBS로 3회 세척한 후에 pH 3.2인 시트레이트-포스페이트 완충액 5 ml과 함께 1분 동안 인큐베이션시켰다. 이 현탁액을 원심분리시키고(800x g, 5분) 무세포 상층액 500 ml 전부를 각 세포주에 대해 수집하였다. 이 재료를 동결건조시키고 -20℃에 보관하였다.To elute the peptide, HepG2 and Hep3B cells were washed three times with PBS and then incubated with 5 ml of citrate-phosphate buffer, pH 3.2, for 1 minute. This suspension was centrifuged (800 × g, 5 min) and all 500 ml of cell free supernatant were collected for each cell line. The material was lyophilized and stored at -20 ° C.
동결된 재료를 물/아세토니트릴/트리플루오로아세트산(W/A/TFA, 95/5/0.1 부피비) 30 ml중에 재용해시켰다. 이 용액을 W/A/TFA중에서 평형화된 분석 역상 HPLC 컬럼(키스톤 사이언티픽(Keystone Scientific) C18 베타실, 250 mm x 2 mm, 5 mm 입자 크기, 100 Å 포어 크기)상에 0.2 ml/분의 유속으로 펌핑하였다. 상기 컬럼을 아세토니트릴(시간/% 아세토니트릴 = 0/5, 5/5, 55/100, 60/100)중의 0.1% TFA의 증가하는 선형 구배를 사용하여 용리시켰다. 컬럼 용리물 흡수치를 215 nm 및 280 nm에서 모니터링하였고 1분 분획을 수집하였다. 면역자극 펩티드에 상응하는 아미노산 서열을 갖는 합성 펩티드의 체류 시간을 분리 컬럼상에 동일한 분리 구배를 사용하여 수득하였다.The frozen material was redissolved in 30 ml of water / acetonitrile / trifluoroacetic acid (W / A / TFA, 95/5 / 0.1 volume ratio). This solution was 0.2 ml / min on an analytical reversed phase HPLC column (Keystone Scientific C 18 betasil, 250 mm x 2 mm, 5 mm particle size, 100 mm pore size) equilibrated in W / A / TFA Pumped at a flow rate of. The column was eluted using an increasing linear gradient of 0.1% TFA in acetonitrile (times /% acetonitrile = 0/5, 5/5, 55/100, 60/100). Column eluate uptake was monitored at 215 nm and 280 nm and 1 minute fractions collected. Retention time of synthetic peptides having amino acid sequences corresponding to immunostimulatory peptides was obtained using the same separation gradient on a separation column.
MALDI-TOF 질량 스펙트로메트리를 수행하여 AFP 생성 간세포암 세포주 HepG2(HLA-A2+) 및 Hep3B(HLA-A2-)로부터 용리된 HPLC 분획화된 펩티드 산을 분석하였다. 장치(Voyager-REACTION PRODUCTS (PerSeptive Biosystems, Framingham, MA) Matrix Assisted Laser Desorption Ionization/Time-Of-Flight(MALDI-TOF))를 사용하여 질량 스펙트럼을 획득하였다. 상기 장치는 스테인레스강 표적을 사용하며, 그 위에 샘플이 증착되고 건조된다. 모든 스펙트럼을 인슐린을 사용하여 외부적으로 보정하였으며, 그 결과 질량 정확도는 전형적으로 ±0.1% 이내였다. 동결된 HPLC 분획들을 0.1% TFA를 함유한 70% 아세토니트릴 10 ㎕중에 재현탁시켰다. 이 재료 1 ㎕를 매트릭스 a-시아노-4-하이드록시신남산(Sigma, 70% ACN/0.1% TFA중에 10 mg/ml) 1㎕와 함께 점적하였다. 스펙트럼을 m/z 500-7000으로부터의 스캐닝에 의해 수득하였다.MALDI-TOF mass spectrometry was performed to analyze HPLC fractionated peptide acids eluted from AFP producing hepatocellular carcinoma cell lines HepG2 (HLA-A2 + ) and Hep3B (HLA-A2 − ). Mass spectra were acquired using a Voyager-REACTION PRODUCTS (PerSeptive Biosystems, Framingham, Mass.) Matrix Assisted Laser Desorption Ionization / Time-Of-Flight (MALDI-TOF). The apparatus uses a stainless steel target, on which samples are deposited and dried. All spectra were externally calibrated using insulin, with the result that the mass accuracy was typically within ± 0.1%. Frozen HPLC fractions were resuspended in 10 μl of 70% acetonitrile containing 0.1% TFA. 1 μl of this material was added dropwise with 1 μl of matrix a-cyano-4-hydroxycinnamic acid (Sigma, 10 mg / ml in 70% ACN / 0.1% TFA). Spectra were obtained by scanning from m / z 500-7000.
HPLC 분획의 MALDI 분석은 자주 소수의 우세한 시그널을 가졌지만, 거의 모든 분획이 700 내지 1500 Da의 질량의 20종의 상이한 펩티드를 함유함을 확립시켰다. 이러한 복합 혼합물 이외에, 피크는 hAFP158-166, SEQ ID NO:2의 wksrl 158 sowl 166; hAFP325-334, SEQ ID NO:2의 잔기 325 내지 334; 및 hAFP542-550, SEQ ID NO:2의 잔기 542 내지 550의 계산된 모노등방성의 양자화된 분자((M+H)+)에 상응하는 m/z 값을 가진 것으로 확인되었고, hAFP542-550, hAFP158-166 및 hAFP325-334는 HepG2 세포로부터 용리된 펩티드 푸울중에 존재한다. m/z 975.6의 펩티드는 HepG2 페티드 푸울로부터의 하나의 HPLC 분획에서 확인되었다. hAFP542-550의 계산된 (M+H)+는 975.5였고 hAFP542-550, SEQ ID NO:2의 잔기 542 내지 550에 상응하는 아미노산을 갖는 합성 펩티드의 체류 시간은 21.2 분이었다. 또한, 975.5±1의 m/z에서는 매트릭스를 단독으로 사용한 샘플 및 Hep3B 용리로부터의 HPLC 분획 18 내지 22에서 어떠한 시그널도 관찰되지 않았다. 유사하게, hAFP158-166, SEQ ID NO:2의 잔기 158 내지 166 및 hAFP325-334, SEQ ID NO:2의 잔기 325 내지 334의 계산된 (M+H)+에 사응하는 m/z를 갖는 피크가 표준 펩티드의 양상으로부터 예측된 HepG2로부터 유래된 적합한 분획에서 발견되었다. 이들 피크는 Hep3B로부터 용리된 펩티드 푸울중의 분획에서 부재했었다. 1152.2 m/z에서의 피크가 하나의 분획에서 관찰되었는데, 이는 hAFP325-334, SEQ ID NO:2의 잔기 325 내지 334의 나트륨 첨가생성물의 존재를 시사한다.MALDI analysis of HPLC fractions often had a few predominant signals, but established that almost all fractions contained 20 different peptides with a mass of 700-1500 Da. In addition to this complex mixture, the peaks are hAFP 158-166 , wksrl 158 sowl 166 of SEQ ID NO: 2; hAFP 325-334 , residues 325-334 of SEQ ID NO: 2; And the m / z values corresponding to the calculated monoisotropic quantized molecules ((M + H) + ) of hAFP 542-550 , residues 542-550 of SEQ ID NO: 2, hAFP 542-550 , hAFP 158-166 and hAFP 325-334 are present in peptide pools eluted from HepG2 cells. Peptides of m / z 975.6 were identified in one HPLC fraction from HepG2 peptide pools. Calculated (M + H) + 542-550 of hAFP is 975.5 was hAFP 542-550, SEQ ID NO: retention time of synthetic peptide having the amino acid corresponding to a residue of 2542 to 550 was 21.2 minutes. In addition, no signal was observed in the samples using the matrix alone and in HPLC fractions 18-22 from Hep3B elution at m / z of 975.5 ± 1. Similarly, m / z corresponding to the calculated (M + H) + of hAFP 158-166 , residues 158-166 of SEQ ID NO: 2 and residues 325-334 of hAFP 325-334 , SEQ ID NO: 2, A peak with was found in a suitable fraction derived from HepG2 predicted from the aspect of the standard peptide. These peaks were absent in the fraction in peptide pools eluted from Hep3B. A peak at 1152.2 m / z was observed in one fraction, suggesting the presence of the sodium adducts of residues 325-334 of hAFP 325-334 , SEQ ID NO: 2.
그러므로, 잠재적인 질량 후보물질은 HLA-A*0201 포지티브 HepG2 세포로부터 용리된 HPLC 분획화된 펩티드 푸울중의 4가지 펩티드중 3가지에 대해 확인되었지만 HLA-A*0201 네가티브 Hep3B 세포로부터는 확인되지 않았다. 확인된 3가지 펩티드에서, 피크는 점적 샘플의 반복된 스캐닝에서 관찰되었다. m/z 1020.9에서 폭넓은 피크는 HepG2 펩티드 푸울로부터의 하나의 분획에서 관찰되었으며 이는 이러한 물리화학적 분석에 의해 허용된 오차의 한계를 넘은 것이었다. 그러므로, HepG2 세포의 표면상에 hAFP137-145, SEQ ID NO:2의 잔기 137 내지 145의 존재를 입증하는 것은 불가능하였다.Therefore, potential mass candidates were identified for three of four peptides in HPLC fractionated peptide pools eluted from HLA-A * 0201 positive HepG2 cells but not from HLA-A * 0201 negative Hep3B cells. . In the three peptides identified, peaks were observed in repeated scanning of the drop sample. A broad peak at m / z 1020.9 was observed in one fraction from HepG2 peptide pools, which was beyond the limits of error allowed by this physicochemical analysis. Therefore, it was not possible to demonstrate the presence of hAFP 137-145 , residues 137-145 of SEQ ID NO: 2 on the surface of HepG2 cells.
면역학적으로 이들 분획에 도미넌트 펩티드의 존재를 확인하기 위해, HepG2 또는 Hep3B 세포로부터의 각 HPLC 분획 1 ml을 사용하여 ELISPOT 검정에서 AdVhAFP/DC 면역화된 뮤린 비세포를 재자극하였다. 200 내지 250 스팟/106 세포가 도미넌트 펩티드를 함유한 분획으로부터 관찰되었고, 100 내지 130 스팟/106 세포는 다른 분획으로부터 관찰되었고, 최대 50 스팟/106 세포는 Hep3B 분획으로부터 관찰되었다. 이것은 도미넌트 펩티드의 질량 스펙트로메트리 확인을 추가로 지지한다.To immunologically confirm the presence of dominant peptides in these fractions, 1 ml of each HPLC fraction from HepG2 or Hep3B cells was used to restimulate AdVhAFP / DC immunized murine splenocytes in an ELISPOT assay. 200-250 spot / 10 6 cells were observed from the fraction containing the dominant peptide, 100-130 spot / 10 6 cells were observed from the other fraction, and up to 50 spot / 10 6 cells were observed from the Hep3B fraction. This further supports mass spectrometry identification of the dominant peptide.
표 2TABLE 2
주석. 1. 펩티드 확인. 2. 대조 합성 펩티드의 전형적인 HPLC 지체 시간. 3. 예상된 질량/전하 측정치. 4. HepG2로부터의 산-용리된 펩티드에서 관찰된 질량/전하 측정치. 5. Hep3B로부터의 산-용리된 펩티드에서 관찰된 질량/전하 측정치. 6. HepG2로부터의 산-용리된 펩티드에서 관찰된 hAFP 펩티드 질량/전하 측정치의 분획(분). 7. IFNg ELISPOT에 의해 AdVhAFP/DC 프라이밍된 비세포를 재자극할 수 있는 펩티드를 함유하는 분획.Remark. 1. Peptide Identification. 2. Typical HPLC delay times for control synthetic peptides. 3. Expected mass / charge measurements. 4. Mass / charge measurements observed on acid-eluted peptides from HepG2. 5. Mass / charge measurements observed in acid-eluted peptides from Hep3B. 6. Fraction of minutes of hAFP peptide mass / charge measurements observed in acid-eluted peptides from HepG2. 7. Fractions containing peptides capable of restimulating AdVhAFP / DC primed splenocytes by IFNg ELISPOT.
실시예 IExample I
펩티드를 투여함으로써 간세포암을 예방하거나 치료하는 방법How to prevent or treat hepatocellular carcinoma by administering peptides
본 발명의 하나의 구체예에 따라, 간세포암에 걸린 환자를 예방하거나 치료하는 방법이 제공된다. 상기 방법은 AFP 포지티브 간세포암에 걸린 HLA-A*0201+와 같은 적합한 환자를 선택하는 것을 포함한다. 다음으로, 환자에게 본 발명의 하나 이상의 펩티드를 투여한다. 상기 펩티드는 충분한 양으로 투여되고, 바람직하게는, 상기 투여는 여러번 반복됨으로써 AFP에 대한 면역 반응을 일으켜 간세포암에 대한 면역 반응을 일으킨다.According to one embodiment of the present invention, a method of preventing or treating a patient with hepatocellular carcinoma is provided. The method involves selecting a suitable patient such as HLA-A * 0201 + with AFP positive hepatocellular carcinoma. Next, the patient is administered one or more peptides of the invention. The peptide is administered in a sufficient amount, and preferably, the administration is repeated several times resulting in an immune response to AFP resulting in an immune response against hepatocellular carcinoma.
바람직한 구체예에서, 펩티드는 hAFP137-145, SEQ ID NO:2의 잔기 137 내지 145; hAFP158-166, SEQ ID NO:2의 잔기 158 내지 166; hAFP325-334, SEQ ID NO:2의 잔기 325 내지 334; 및 hAFP542-550, SEQ ID NO:2의 잔기 542 내지 550의 혼합물이다. 또 다른 바람직한 구체예에서, 하나 이상의 펩티드가 2회 내지 5회 투여된다. 특히 바람직한 구체예에서, 펩티드는 3회 투여된다. 또 다른 바람직한 구체예에서, 하나 이상의 펩티드는 2주 간격으로 3회 투여된다.In a preferred embodiment, the peptide comprises hAFP 137-145 , residues 137-145 of SEQ ID NO: 2; hAFP 158-166 , residues 158-166 of SEQ ID NO: 2; hAFP 325-334 , residues 325-334 of SEQ ID NO: 2; And hAFP 542-550 , a mixture of residues 542-550 of SEQ ID NO: 2. In another preferred embodiment, one or more peptides are administered two to five times. In a particularly preferred embodiment, the peptide is administered three times. In another preferred embodiment, one or more peptides are administered three times at two week intervals.
바람직한 구체예에서, 상기 펩티드는 피내적으로 투여되지만, 본원의 명세서를 참고로 하여 당업자에게 이해되는 바와 같이 그 밖의 투여 경로도 적합하다.In a preferred embodiment, the peptide is administered intracutaneously, but other routes of administration are also suitable, as will be understood by those skilled in the art by reference to the specification herein.
바람직한 구체예에서, 하나 이상의 펩티드 각각은 4가지 펩티드가 혼합되는 경우, 이들이 몬타니드(Montanide) ISA-51 2 ml 전체에 에멀션화되어 투여되도록, 몬타니드 ISA-51 0.5 ml중에 에멀션화되어 투여된다. 상기 에멀션화된 펩티드 또는 펩티드들을 4개의 동일한 용량으로 나누고 각 용량을 개별적인 부위에 투여하였다. 바람직한 구체예에서, 하나 이상의 펩티드를 각각 약 50 ㎍ 내지 2000 ㎍의 용량으로 투여하였다. 바람직한 구체예에서, 하나 이상의 펩티드를 각각 약 100 ㎍ 내지 1000 ㎍의 용량으로 투여하였다. 특히 바람직한 구체예에서, 하나 이상의 펩티드를 약 500 ㎍의 용량으로 투여하였다. In a preferred embodiment, each of the one or more peptides is emulsified and administered in 0.5 ml of Montanide ISA-51 such that when four peptides are mixed, they are emulsified and administered throughout 2 ml of Montanide ISA-51. . The emulsified peptide or peptides were divided into four equal doses and each dose was administered to a separate site. In a preferred embodiment, one or more peptides are administered at a dose of about 50 μg-2000 μg each. In a preferred embodiment, one or more peptides are administered at a dose of about 100 μg to 1000 μg each. In a particularly preferred embodiment, one or more peptides are administered at a dose of about 500 μg.
본 발명의 방법 및 조성물을 AFP 포지티브/A2.1+ 간세포암에 걸린 몇몇 환자를 치료하기 위해 사용하였다. 각 환자를 본 방법에 따라 4가지 펩티드 hAFP137-145, SEQ ID NO:2의 잔기 137 내지 145; hAFP158-166, SEQ ID NO:2의 잔기 158 내지 166; hAFP325-334, SEQ ID NO:2의 잔기 325 내지 334; 및 hAFP542-550, SEQ ID NO:2의 잔기 542 내지 550으로 면역화시켰다. 상기 펩티드들을 몬타니드 ISA-51 0.5 ml중에 에멀션화시키고 전체 2 ml로 혼합하였다. 에멀션화된 펩티드를 4개의 동일한 용량으로 나누고 각 용량을 개별적인 부위에 투여하였다. 말초 T 세포 반응을 ELISPOT 및 테트라머 검정에 의해 측정하였다. 이들 시도는 4가지 AFP-유래된 펩티드가 면역화 이전에 혈청에서의 AFP 레벨이 극도로 높은 환자에서 조차도 생체내에서 면역원성임을 보여주었다.The methods and compositions of the present invention have been used to treat some patients with AFP positive / A2.1 + hepatocellular carcinoma. Each patient was treated with four peptides hAFP 137-145 , residues 137-145 of SEQ ID NO: 2; hAFP 158-166 , residues 158-166 of SEQ ID NO: 2; hAFP 325-334 , residues 325-334 of SEQ ID NO: 2; And hAFP 542-550 , residues 542-550 of SEQ ID NO: 2. The peptides were emulsified in 0.5 ml of Montanide ISA-51 and mixed to a total of 2 ml. The emulsified peptide was divided into four equal doses and each dose was administered to a separate site. Peripheral T cell responses were measured by ELISPOT and tetramer assays. These trials showed that the four AFP-derived peptides were immunogenic in vivo even in patients with extremely high AFP levels in serum prior to immunization.
AFP-A1으로 지정된 첫번째 환자는 재발성의 절제될 수 없는 AFP 포지티브/A2.1+ 간세포암에 걸려 있었다. 이 환자에게 2주 간격으로 몬타니드 ISA중의 각각의 4가지 펩티드 100 ㎍을 3회 면역화 투여하였다. 대등한 시험관내 PBMC 배양물을 또한 면역화 이전에 첫번째 환자의 혈액으로부터 확립시키고 각 펩티드를 사용하여 반복적으로 펄싱시켰다. ELISPOT에 의해 검사된 제 28일에 배양물의 모든 펩티드의 인식은 시험관내에서 명백하였고 테트라머에 의해서는 3가지 중 2가지, 즉 hAFP137, SEQ ID NO:2의 잔기 137 내지 145; 및 hAFP325-334, SEQ ID NO:2의 잔기 325 내지 334는 그러했지만 hAFP158-166, SEQ ID NO:2의 잔기 158 내지 166은 그러하지 않았다. AFP542-특이적 T 세포의 존재는 AFP542 펩티드 테트라머가 이들 반응물이 제조되는 경우 수월하게 폴딩될 수 없기 때문에 테트라머에 의해 평가될 수 없었다. 생체내에서 반응은 hAFP137-145, SEQ ID NO:2의 잔기 137 내지 145; hAFP158-166, SEQ ID NO:2의 잔기 158 내지 166; 및 hAFP542-550, SEQ ID NO:2의 잔기 542 내지 550를 명백하게 인식하였고; 제 2 및 제 3 면역화후에는 hAFP325-334, SEQ ID NO:2의 잔기 325 내지 334를 인식하는 경향을 띠는 것으로 나타났다.The first patient designated AFP-A1 suffered from recurrent non-resectable AFP positive / A2.1 + hepatocellular carcinoma. The patient was immunized three times with 100 μg of each of the four peptides in the Montanide ISA at two week intervals. Comparable in vitro PBMC cultures were also established from the blood of the first patient prior to immunization and repeatedly pulsed with each peptide. Recognition of all peptides in culture on day 28 examined by ELISPOT was evident in vitro and by tetramer two of three: hAFP 137 , residues 137-145 of SEQ ID NO: 2; And hAFP 325-334 , residues 325-334 of SEQ ID NO: 2, while hAFP 158-166 , residues 158-166 of SEQ ID NO: 2 did not. The presence of AFP 542 -specific T cells could not be assessed by tetramers because AFP 542 peptide tetramers could not be easily folded when these reactants were prepared. In vivo, the reactions include hAFP 137-145 , residues 137-145 of SEQ ID NO: 2; hAFP 158-166 , residues 158-166 of SEQ ID NO: 2; And hAFP 542-550 , residues 542-550 of SEQ ID NO: 2 were clearly recognized; After the second and third immunizations it was shown that there is a tendency to recognize hAFP 325-334 , residues 325-334 of SEQ ID NO: 2.
AFP-A2로 지정된 두번째 환자는 에탄올-유도된 간경변의 병력을 가진 70세의 백인 남성이었고, B형 간염 및 C형 간염에 대해 네가티브였다. 환자는 부풀어 있는 것으로 보였으며 좌간엽에 8 cm 덩어리가 있는 것으로 밝혀졌다. 프리젠테이션에서 환자의 AFP 레벨은 10,400 ng/ml이었다. 간의 생체검사에 의해 경변성 간에서 잘 구별된 간세포암이 드러났다. 환자에게 항진균제 FV-462를 사용하여 실험적 시도를 수행하였지만 질환은 진행되고 이독성을 나타냈다. 프리젠테이션 6개월 후에, 본 발명에 따른 프로토콜을 사용하여 4가지 펩티드 hAFP137-145, SEQ ID NO:2의 잔기 137 내지 145; hAFP158-166, SEQ ID NO:2의 잔기 158 내지 166; hAFP325-334, SEQ ID NO:2의 잔기 325 내지 334; 및 hAFP542-550, SEQ ID NO:2의 잔기 542 내지 550의 혼합물을 환자에게 수행하였다. 환자를 2주 간격으로 2회 면역화시켰다. 2회 면역화 이후의 테트라머 및 ELISPOT 데이터는 T 세포 반응이 모든 4 AFP 펩티드 에피토프에 대해 검출되었음을 보여주었다.The second patient, designated AFP-A2, was a 70-year-old white male with a history of ethanol-induced cirrhosis and was negative for hepatitis B and hepatitis C. The patient appeared to swell and was found to have an 8 cm mass in the left mesenchyme. The patient's AFP level was 10,400 ng / ml in the presentation. Liver biopsy revealed well-defined hepatocellular carcinoma in the cirrhosis of the liver. The patient performed experimental trials with antifungal FV-462, but the disease progressed and showed toxicity. Six months after the presentation, the four peptides hAFP 137-145 , residues 137-145 of SEQ ID NO: 2 using the protocol according to the invention; hAFP 158-166 , residues 158-166 of SEQ ID NO: 2; hAFP 325-334 , residues 325-334 of SEQ ID NO: 2; And a mixture of hAFP 542-550 , residues 542-550 of SEQ ID NO: 2 to the patient. Patients were immunized twice at two week intervals. Tetramer and ELISPOT data after two immunizations showed that T cell responses were detected for all 4 AFP peptide epitopes.
그러므로, 이러한 결과는 본 방법이 진행된 간세포암에 걸린 환자의 AFP-특이적 T 세포에 의해 검출할 수 있는 면역 반응을 생성시킴을 시사하였다.Therefore, these results suggested that the method produced an immune response detectable by AFP-specific T cells in patients with advanced hepatocellular carcinoma.
실시예 IIExample II
본 발명의 펩티드로 펄싱된 수상 세포를 투여함으로써 간세포암을 예방하거나 치료하는 방법A method for preventing or treating hepatocellular carcinoma by administering a dendritic cell pulsed with the peptide of the present invention
본 발명의 하나의 구체예에 따라, 간세포암에 걸린 환자를 예방하거나 치료하는 방법이 제공된다. 상기 방법은 AFP 포지티브 간세포암에 걸린 HLA-A*0201+ 환자와 같은 적합한 환자를 선택하는 것을 포함한다. 다음으로, 이 환자에게 본 발명의 하나 이상의 펩티드로 펄싱된 수상 세포를 투여한다. 수상 세포는 충분한 용량으로 투여되며, 바람직하게는, 투여는 AFP에 대한 면역 반응을 생성시키기 위해 여러번 반복되며, 이로써 간세포암에 대한 면역 반응이 생성된다.According to one embodiment of the present invention, a method of preventing or treating a patient with hepatocellular carcinoma is provided. The method includes selecting a suitable patient, such as a HLA-A * 0201 + patient with AFP positive hepatocellular carcinoma. This patient is then administered a dendritic cell pulsed with one or more peptides of the invention. The dendritic cells are administered in sufficient doses, preferably the administration is repeated several times to produce an immune response against AFP, thereby producing an immune response against hepatocellular carcinoma.
바람직한 구체예에서, 수상 세포는 hAFP137-145, SEQ ID NO:2의 잔기 137 내지 145; hAFP158-166, SEQ ID NO:2의 잔기 158 내지 166; hAFP325-334, SEQ ID NO:2의 잔기 325 내지 334; 및 hAFP542-550, SEQ ID NO:2의 잔기 542 내지 550의 혼합물로 펄싱된다. 또 다른 바람직한 구체예에서, 수상 세포는 2회 내지 5회 투여된다. 특히 바람직한 구체예에서, 수상 세포는 3회 투여된다. 또 다른 바람직한 구체예에서, 수상 세포는 2주 간격으로 3회 투여된다.In a preferred embodiment, the dendritic cells are hAFP 137-145 , residues 137-145 of SEQ ID NO: 2; hAFP 158-166 , residues 158-166 of SEQ ID NO: 2; hAFP 325-334 , residues 325-334 of SEQ ID NO: 2; And hAFP 542-550 , a mixture of residues 542-550 of SEQ ID NO: 2. In another preferred embodiment, the dendritic cells are administered 2 to 5 times. In a particularly preferred embodiment, the dendritic cells are administered three times. In another preferred embodiment, the dendritic cells are administered three times at two week intervals.
바람직한 구체예에서, 수상 세포는 피내적으로 투여되지만, 본 명세서를 참고로 당업자에 의해 이해되는 바와 같이 그 밖의 투여경로도 적합하다. 하나의 구체예에서, 수상 세포는 약 1 x 105 내지 1 x 108 의 용량으로 투여된다. 또 다른 바람직한 구체예에서, 수상 세포는 약 1 x 106 내지 1 x 107 의 용량으로 투여된다. 특히 바람직한 구체예에서, 수상 세포는 약 5 x 106 의 용량으로 투여된다.In a preferred embodiment, dendritic cells are administered intradermally, although other routes of administration are also suitable, as will be understood by those skilled in the art by reference to the specification. In one embodiment, the dendritic cells are administered at a dose of about 1 × 10 5 to 1 × 10 8 . In another preferred embodiment, the dendritic cells are administered at a dose of about 1 × 10 6 to 1 × 10 7 . In a particularly preferred embodiment, the dendritic cells are administered at a dose of about 5 x 10 6 .
수상 세포를 당분야에 공지된 기술에 따라 과립구-마크로파지 콜로니 자극 인자(GM-CSF) 및 인터루킨-4(IL-4)에 조직 배양액중에서 1주 동안노출된 부착성 자가 말초혈 단핵 세포로부터 제조하였다. 단핵 세포를 Ficoll-Hypaque 원심분리에 의한 단일 백혈구분리반출법으로부터 수득된 세포로부터 단리하였다. 해동된 단핵 세포를 염수중에 1회 세척하고 2.5 내지 5 x 106 생세포/ml의 농도로 2 내지 4 x 107 세포/25cm2 코스타 플라스크(Costar flask)로 플레이팅하였다(RPMI 1640 + 5% 열-불활성화된 자가 혈청 + 젠타마이신). 37℃에서 2시간 동안 부착시킨 후에, 비부착 세포를 염수로 세척함으로써 제거하였다. 부착 세포를 rhGM-CSF(800 U/ml) 및 rhIL-4(500 U/ml)의 존재하에 7일 동안 완전 배지에서 배양하였다. 임상 등급 GM-CSF는 이뮤넥스(Immunex), IL-4는 쉐링-플로프(Schering-Plough)로부터 입수하였다.Dendritic cells were prepared from adherent autologous peripheral blood mononuclear cells exposed to granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 1 week in tissue culture according to techniques known in the art. . Monocytes were isolated from cells obtained from single leukocyte export by Ficoll-Hypaque centrifugation. Thawed mononuclear cells were washed once in saline and plated into 2-4 x 10 7 cells / 25 cm 2 Costa flask at a concentration of 2.5-5 x 10 6 viable cells / ml (RPMI 1640 + 5% heat). -Inactivated autologous serum + gentamycin). After attaching at 37 ° C. for 2 hours, non-adherent cells were removed by washing with saline. Adherent cells were cultured in complete medium for 7 days in the presence of rhGM-CSF (800 U / ml) and rhIL-4 (500 U / ml). Clinical grade GM-CSF was obtained from Imunex and IL-4 from Schering-Plough.
환자에게 백혈구분리반출법을 수행하여 2 x 109 이상의 PBL을 수득하고, 이를 70% RPMI 1640, 20% 자가 혈청 및 10% DMSO중에 냉동보존하였다. 분취액을 연구하는 날-7, 7 및 21일에 해동시켰다. 자가 혈청을 위한 혈액(60 ml)을 백혈구분리반출법 수행시 및 제 1 면역화 시행일에 해동시키고, 이는 모든 세포 배양물에 대해 충분하였다. 본 발명의 AFP-유래된 면역도미넌트 펩티드를 당분야에 공지된 기술에 따라 제조하고 정제하였다.Patients were subjected to leukocyte separation to obtain at least 2 × 10 9 PBLs, which were cryopreserved in 70% RPMI 1640, 20% autologous serum and 10% DMSO. Aliquots were thawed on days 7, 7, and 21 of the study. Blood for autologous serum (60 ml) was thawed at the time of leukopening and on the first immunization run, which was sufficient for all cell cultures. AFP-derived immunodominant peptides of the invention were prepared and purified according to techniques known in the art.
환자를 다음과 같이 면역화시켰다. 면역화 제 1일에, 수상 세포를 수집하고, 멸균 염수로 1회 세척하고 1 mL의 무혈청 RPMI 1640 및 각각 별도의 50 mg/ml의 4가지 면역도미넌트 펩티드중에 적절한 농도, 예컨대 106으로 재현탁시켰다. 최소 1시간 인큐베이션 후에, AFP 펩티드/DC를 펠렛화시키고 멸균 염수중에 3회 세척하였다. 세포를 트립판 블루에서 카운팅하고 내피 주입을 위해 0.1 ml 멸균 염수중에 재현탁시켰다.Patients were immunized as follows. On day 1 of immunization, dendritic cells were collected, washed once with sterile saline and resuspended at an appropriate concentration, such as 10 6 , in 1 mL of serum-free RPMI 1640 and 4 separate immunodominant peptides of 50 mg / ml each. I was. After a minimum of 1 hour incubation, AFP peptides / DCs were pelleted and washed three times in sterile saline. Cells were counted on trypan blue and resuspended in 0.1 ml sterile saline for endothelial injection.
전체 용량을 투여하기 전에, 피검자는 염수 0.1 ml중의 이들 용량의 1/100을 사용하는 피부 시험을 받을 것이다. 30분 관찰한 후에, 피검자의 겨드랑이 아래 측면부, 또는 서혜부의 아래 또는 위에 염수 0.1 ml중에 피내적으로 투여된 AFP 펩티드-펄싱된 수상 세포의 전체 용량을 투여할 것이다. 환자를 면역화후 2시간 동안 모니터링하였다. 바람직하게는, 환자를 50 mg의 디펜하이드라민 및 650 mg의 타이레놀을 사용하여 경구적으로 전처리하였다.Prior to administering the full dose, the subject will have a skin test using 1/100 of these doses in 0.1 ml saline. After 30 minutes of observation, the entire dose of AFP peptide-pulsed dendritic cells administered intradermally in 0.1 ml of saline will be administered either under the subject's underarm side, or below or above the inguinal tract. Patients were monitored for 2 hours after immunization. Preferably, the patient is pretreated orally with 50 mg diphenhydramine and 650 mg tylenol.
실시예 IIIExample III
사람 AFP 아데노바이러스-형질도입된 수상 세포를 투여함으로써 간세포암을 예방하거나 치료하는 방법How to prevent or treat hepatocellular carcinoma by administering human AFP adenovirus-transduced dendritic cells
본 발명의 하나의 구체예에 따라, 간세포암에 걸린 환자를 예방하거나 치료하는 방법이 제공된다. 상기 방법은 AFP 포지티브 간세포암에 걸린 HLA-A*0201+ 환자와 같은 적합한 환자를 선택하는 것을 포함한다. 다음으로, 상기 환자에게 사람 AFP 아데노바이러스-형질도입된 수상 세포가 투여된다. 수상 세포는 충분한 용량으로 투여되며, 바람직하게는, 투여는 AFP에 대한 면역 반응을 생성시키기 위해 여러번 반복되며, 이로써 간세포암에 대한 면역 반응이 생성된다.According to one embodiment of the present invention, a method of preventing or treating a patient with hepatocellular carcinoma is provided. The method includes selecting a suitable patient, such as a HLA-A * 0201 + patient with AFP positive hepatocellular carcinoma. Next, the patient is administered human AFP adenovirus-transduced dendritic cells. The dendritic cells are administered in sufficient doses, preferably the administration is repeated several times to produce an immune response against AFP, thereby producing an immune response against hepatocellular carcinoma.
또 다른 바람직한 구체예에서, 수상 세포는 2회 내지 5회 투여된다. 특히 바람직한 구체예에서, 수상 세포는 3회 투여된다. 또 다른 바람직한 구체예에서, 수상 세포는 2주 간격으로 3회 투여된다.In another preferred embodiment, the dendritic cells are administered 2 to 5 times. In a particularly preferred embodiment, the dendritic cells are administered three times. In another preferred embodiment, the dendritic cells are administered three times at two week intervals.
바람직한 구체예에서, 수상 세포는 겨드랑이 또는 서혜부 아래 측면 부위에 피내적으로 투여되지만, 본 명세서를 참조로 당업자에 의해 이해되는 바와 같이 그 밖의 투여경로도 적합하다. 하나의 구체예에서, 수상 세포는 약 1 x 105 내지 1 x 108의 용량으로 투여된다. 또 다른 바람직한 구체예에서, 수상 세포는 약 1 x 106 내지 1 x 107의 용량으로 투여된다. 특히 바람직한 구체예에서, 수상 세포는 약 5 x 106의 용량으로 투여된다.In a preferred embodiment, dendritic cells are administered intradermally to the flanks under the armpits or inguinal tract, but other routes of administration are also suitable, as will be understood by one of ordinary skill in the art with reference to this specification. In one embodiment, the dendritic cells are administered at a dose of about 1 × 10 5 to 1 × 10 8 . In another preferred embodiment, the dendritic cells are administered at a dose of about 1 × 10 6 to 1 × 10 7 . In a particularly preferred embodiment, the dendritic cells are administered at a dose of about 5 x 10 6 .
단핵 세포를 Ficoll-Hypaque 원심분리에 의한 백혈구분리반출 생성물로부터 단리하여 10% DMSO/20% 자가 혈청중에 보관하였다. DC 백신접종 1주일 전에, 세포를 해동시키고, PBS중에 1회 세척하고 2.5 내지 5 x 106 생세포/ml(RPMI 1640 + 5% 열-불활성화된 자가 혈청)의 농도로 2 내지 4 x 107 세포/25cm2 코스타 플라스크로 플레이팅하였다. 37℃에서 2시간 동안 부착시킨 후에, 비부착 세포를 PBS로 세척함으로써 온화하게 제거하였다. 부착 세포를 rhGM-CSF (800 U/ml) 및 rhIL-4 (500 U/ml)의 존재하에 7일 동안 완전 배지에서 배양시켰다. 임상 등급 GM-CSF 및 IL-4는 쉐링-플로프로부터 입수하였다.Monocytes were isolated from leukocyte export products by Ficoll-Hypaque centrifugation and stored in 10% DMSO / 20% autologous serum. One week prior to DC vaccination, cells were thawed, washed once in PBS and 2 to 4 x 10 7 at a concentration of 2.5 to 5 x 10 6 viable cells / ml (RPMI 1640 + 5% heat-inactivated autologous serum). Plated into cells / 25 cm 2 Costa flask. After attaching at 37 ° C. for 2 hours, non-adherent cells were gently removed by washing with PBS. Adherent cells were cultured in complete medium for 7 days in the presence of rhGM-CSF (800 U / ml) and rhIL-4 (500 U / ml). Clinical grade GM-CSF and IL-4 were obtained from Schering-Flop.
AdVhAFP는 사람 AFP cDNA가 CMV 인핸서/프로모터에 의해 유래된 E1-결실된 복제-결함 타입 5 아데노바이러스 벡터이다. 각각의 최종 바이러스 생성 로트에 대한 바이러스 역가를 지놈 DNA 정량화 및 감염 역가 둘 모두에 기초하여 제공하였다. 100:1 미만의 바이러스 입자 대 생물학적으로 활성인 바이러스의 생성 비가 허용가능한 비라고 생각되었다.AdVhAFP is an E1-deleted replication-defect type 5 adenovirus vector in which human AFP cDNA is derived by a CMV enhancer / promoter. Virus titers for each final virus producing lot were provided based on both genome DNA quantification and infection titers. It was thought that the production ratio of virus particles less than 100: 1 to biologically active virus was an acceptable ratio.
환자를 다음과 같이 면역화시켰다. 면역화 당일에, 수상 세포를 수집하고 멸균 염수중에 1회 세척하고 2% 자가 혈청-RPMI 1640 1 mL 및 AdVhAFP 109 내지 1010 pfu/ml중에 106 내지 107의 농도로 재현탁시켰다(다중감염도 = 1000:1). 37℃에서 2시간 인큐베이션시킨 후에, AdVhAFP/DC를 RPMI-1640 + 5% 자가 혈청중에 재현탁시켜 비-흡수된 아데노바이러스 벡터를 불활성화시킨 다음, 펠렛화시키고 염수중에 3회 세척하였다. 세포를 트립판 블루에서 카운팅하고 적당한 수(환자 그룹에 따라 1 x 105 내지 1 x 108)를 내피적 주입을 위해 멸균 염수중에 재현탁시켰다.Patients were immunized as follows. On the day of immunization, dendritic cells were collected and washed once in sterile saline and resuspended at a concentration of 10 6 to 10 7 in 1 mL of 2% autologous serum-RPMI 1640 and AdVhAFP 10 9 to 10 10 pfu / ml (multi-infection). Degrees = 1000: 1). After 2 hours incubation at 37 ° C., AdVhAFP / DC was resuspended in RPMI-1640 + 5% autologous serum to inactivate the non-absorbed adenovirus vector, then pelletized and washed three times in saline. Cells were counted in trypan blue and appropriate numbers (1 × 10 5 to 1 × 10 8 depending on patient group) were resuspended in sterile saline for endothelial injection.
환자에게 겨드랑이 또는 서혜부 아래 측면 부위에 0.1 ml 정상 염수중에 피내적으로 주입된 AdVhAFP 형질도입된 DC를 투여하였다. 환자를 면역화후 2시간 동안 모니터링하였다. 바람직하게는, 환자를 50 mg의 디펜하이드라민 및 650 mg의 타이레놀을 사용하여 경구적으로 전처리하였다.Patients received AdVhAFP transduced DC intradermally injected in 0.1 ml normal saline to the side of the armpit or inguinal area. Patients were monitored for 2 hours after immunization. Preferably, the patient is pretreated orally with 50 mg diphenhydramine and 650 mg tylenol.
본 발명이 특정한 바람직한 구체예에 대해 참조로 매우 상세하게 논의되었지만, 그 밖의 구체예들이 가능하다. 그러므로, 첨부된 청구항의 범위는 본 명세서에 포함된 바람직한 구체예들의 설명에 제한되지 않아야 한다.Although the present invention has been discussed in great detail with reference to certain preferred embodiments, other embodiments are possible. Therefore, the scope of the appended claims should not be limited to the description of the preferred embodiments contained herein.
SEQUENCE LISTING <110> The Regents of the University of California Economou, James S. Butterfield, Lisa H. Ribas Bruguera, Antoni <120> Method and Compositions for Treating Hepatocellular Cancer <130> 11969-5PCT <140> to be assigned <141> 2001-02-12 <150> US 60/181,966 <151> 2000-02-10 <150> US 09/660,252 <151> 2000-09-12 <150> US 09/662,505 <151> 2000-09-14 <160> 2 <170> PatentIn version 3.0 <210> 1 <211> 2032 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (48)..(1874) <223> <400> 1 tccatattgt gcttccacca ctgccaataa caaaataact agcaacc atg aag tgg 56 Met Lys Trp 1 gtg gaa tca att ttt tta att ttc cta cta aat ttt act gaa tcc aga 104 Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr Glu Ser Arg 5 10 15 aca ctg cat aga aat gaa tat gga ata gct tcc ata ttg gat tct tac 152 Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr 20 25 30 35 caa tgt act gca gag ata agt tta gct gac ctg gct acc ata ttt ttt 200 Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe 40 45 50 gcc cag ttt gtt caa gaa gcc act tac aag gaa gta agc aaa atg gtg 248 Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val 55 60 65 aaa gat gca ttg act gca att gag aaa ccc act gga gat gaa cag tct 296 Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser 70 75 80 tca ggg tgt tta gaa aac cag cta cct gcc ttt ctg gaa gaa ctt tgc 344 Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys 85 90 95 cat gag aaa gaa att ttg gag aag tac gga cat tca gac tgc tgc agc 392 His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser 100 105 110 115 caa agt gaa gag gga aga cat aac tgt ttt ctt gca cac aaa aag ccc 440 Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro 120 125 130 act cca gca tcg atc cca ctt ttc caa gtt cca gaa cct gtc aca agc 488 Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser 135 140 145 tgt gaa gca tat gaa gaa gac agg gag aca ttc atg aac aaa ttc att 536 Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile 150 155 160 tat gag ata gca aga agg cat ccc ttc ctg tat gca cct aca att ctt 584 Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu 165 170 175 ctt tgg gct gct cgc tat gac aaa ata att cca tct tgc tgc aaa gct 632 Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys Cys Lys Ala 180 185 190 195 gaa aat gca gtt gaa tgc ttc caa aca aag gca gca aca gtt aca aaa 680 Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys 200 205 210 gaa tta aga gaa agc agc ttg tta aat caa cat gca tgt gca gta atg 728 Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Ala Val Met 215 220 225 aaa aat ttt ggg acc cga act ttc caa gcc ata act gtt act aaa ctg 776 Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu 230 235 240 agt cag aag ttt acc aaa gtt aat ttt act gaa atc cag aaa cta gtc 824 Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Val 245 250 255 ctg gat gtg gcc cat gta cat gag cac tgt tgc aga gga gat gtg ctg 872 Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly Asp Val Leu 260 265 270 275 gat tgt ctg cag gat ggg gaa aaa atc atg tcc tac ata tgt tct caa 920 Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln 280 285 290 caa gac act ctg tca aac aaa ata aca gaa tgc tgc aaa ctg acc acg 968 Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr 295 300 305 ctg gaa cgt ggt caa tgt ata att cat gca gaa aat gat gaa aaa cct 1016 Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro 310 315 320 gaa ggt cta tct cca aat cta aac agg ttt tta gga gat aga gat ttt 1064 Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe 325 330 335 aac caa ttt tct tca ggg gaa aaa aat atc ttc ttg gca agt ttt gtt 1112 Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val 340 345 350 355 cat gaa tat tca aga aga cat cct cag ctt gct gtc tca gta att cta 1160 His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser Val Ile Leu 360 365 370 aga gtt gct aaa gga tac cag gag tta ttg gag aag tgt ttc cag act 1208 Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr 375 380 385 gaa aac cct ctt gaa tgc caa gat aaa gga gaa gaa gaa tta cag aaa 1256 Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys 390 395 400 tac atc cag gag agc caa gca ttg gca aag cga agc tgc ggc ctc ttc 1304 Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe 405 410 415 cag aaa cta gga gaa tat tac tta caa aat gcg ttt ctc gtt gct tac 1352 Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu Val Ala Tyr 420 425 430 435 aca aag aaa gcc ccc cag ctg acc tcg tcg gag ctg atg gcc atc acc 1400 Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr 440 445 450 aga aaa atg gca gcc aca gca gcc act tgt tgc caa ctc agt gag gac 1448 Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp 455 460 465 aaa cta ttg gcc tgt ggc gag gga gcg gct gac att att atc gga cac 1496 Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His 470 475 480 tta tgt atc aga cat gaa atg act cca gta aac cct ggt gtt ggc cag 1544 Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly Val Gly Gln 485 490 495 tgc tgc act tct tca tat gcc aac agg agg cca tgc ttc agc agc ttg 1592 Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu 500 505 510 515 gtg gtg gat gaa aca tat gtc cct cct gca ttc tct gat gac aag ttc 1640 Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Phe 520 525 530 att ttc cat aag gat ctg tgc caa gct cag ggt gta gcg ctg caa acg 1688 Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala Leu Gln Thr 535 540 545 atg aag caa gag ttt ctc att aac ctt gtg aag caa aag cca caa ata 1736 Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys Pro Gln Ile 550 555 560 aca gag gaa caa ctt gag gct gtc att gca gat ttc tca ggc ctg ttg 1784 Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser Gly Leu Leu 565 570 575 gag aaa tgc tgc caa ggc cag gaa cag gaa gtc tgc ttt gct gaa gag 1832 Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Glu 580 585 590 595 gga caa aaa ctg att tca aaa act cgt gct gct ttg gga gtt 1874 Gly Gln Lys Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly Val 600 605 taaattactt caggggaaga gaagacaaaa cgagtctttc attcggtgtg aacttttctc 1934 tttaatttta actgatttaa cactttttgt gaattaatga aatgataaag acttttatgt 1994 gagatttcct tatcacagaa ataaaatatc tccaaatg 2032 <210> 2 <211> 609 <212> PRT <213> Homo sapiens <400> 2 Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr 1 5 10 15 Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu 20 25 30 Asp Ser Tyr Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr 35 40 45 Ile Phe Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser 50 55 60 Lys Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp 65 70 75 80 Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu 85 90 95 Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp 100 105 110 Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His 115 120 125 Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro 130 135 140 Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn 145 150 155 160 Lys Phe Ile Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro 165 170 175 Thr Ile Leu Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys 180 185 190 Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr 195 200 205 Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys 210 215 220 Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val 225 230 235 240 Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln 245 250 255 Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly 260 265 270 Asp Val Leu Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile 275 280 285 Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys 290 295 300 Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp 305 310 315 320 Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp 325 330 335 Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala 340 345 350 Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser 355 360 365 Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys 370 375 380 Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu 385 390 395 400 Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys 405 410 415 Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu 420 425 430 Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met 435 440 445 Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu 450 455 460 Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile 465 470 475 480 Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly 485 490 495 Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp 515 520 525 Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala 530 535 540 Leu Gln Thr Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys 545 550 555 560 Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser 565 570 575 Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe 580 585 590 Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly 595 600 605 ValSEQUENCE LISTING <110> The Regents of the University of California Economou, James S. Butterfield, Lisa H. Ribas Bruguera, Antoni <120> Method and Compositions for Treating Hepatocellular Cancer <130> 11969-5PCT <140> to be assigned <141> 2001-02-12 <150> US 60 / 181,966 <151> 2000-02-10 <150> US 09 / 660,252 <151> 2000-09-12 <150> US 09 / 662,505 <151> 2000-09-14 <160> 2 <170> PatentIn version 3.0 <210> 1 <211> 2032 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (48) .. (1874) <223> <400> 1 tccatattgt gcttccacca ctgccaataa caaaataact agcaacc atg aag tgg 56 Met lys trp One gtg gaa tca att ttt tta att ttc cta cta aat ttt act gaa tcc aga 104 Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr Glu Ser Arg 5 10 15 aca ctg cat aga aat gaa tat gga ata gct tcc ata ttg gat tct tac 152 Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr 20 25 30 35 caa tgt act gca gag ata agt tta gct gac ctg gct acc ata ttt ttt 200 Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe 40 45 50 gcc cag ttt gtt caa gaa gcc act tac aag gaa gta agc aaa atg gtg 248 Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val 55 60 65 aaa gat gca ttg act gca att gag aaa ccc act gga gat gaa cag tct 296 Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser 70 75 80 tca ggg tgt tta gaa aac cag cta cct gcc ttt ctg gaa gaa ctt tgc 344 Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys 85 90 95 cat gag aaa gaa att ttg gag aag tac gga cat tca gac tgc tgc agc 392 His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser 100 105 110 115 caa agt gaa gag gga aga cat aac tgt ttt ctt gca cac aaa aag ccc 440 Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro 120 125 130 act cca gca tcg atc cca ctt ttc caa gtt cca gaa cct gtc aca agc 488 Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser 135 140 145 tgt gaa gca tat gaa gaa gac agg gag aca ttc atg aac aaa ttc att 536 Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile 150 155 160 tat gag ata gca aga agg cat ccc ttc ctg tat gca cct aca att ctt 584 Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu 165 170 175 ctt tgg gct gct cgc tat gac aaa ata att cca tct tgc tgc aaa gct 632 Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys Cys Lys Ala 180 185 190 195 gaa aat gca gtt gaa tgc ttc caa aca aag gca gca aca gtt aca aaa 680 Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys 200 205 210 gaa tta aga gaa agc agc ttg tta aat caa cat gca tgt gca gta atg 728 Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Ala Val Met 215 220 225 aaa aat ttt ggg acc cga act ttc caa gcc ata act gtt act aaa ctg 776 Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu 230 235 240 agt cag aag ttt acc aaa gtt aat ttt act gaa atc cag aaa cta gtc 824 Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Val 245 250 255 ctg gat gtg gcc cat gta cat gag cac tgt tgc aga gga gat gtg ctg 872 Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly Asp Val Leu 260 265 270 275 gat tgt ctg cag gat ggg gaa aaa atc atg tcc tac ata tgt tct caa 920 Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln 280 285 290 caa gac act ctg tca aac aaa ata aca gaa tgc tgc aaa ctg acc acg 968 Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr 295 300 305 ctg gaa cgt ggt caa tgt ata att cat gca gaa aat gat gaa aaa cct 1016 Leu Glu Arg Gly Gln Cys Ile His Ala Glu Asn Asp Glu Lys Pro 310 315 320 gaa ggt cta tct cca aat cta aac agg ttt tta gga gat aga gat ttt 1064 Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe 325 330 335 aac caa ttt tct tca ggg gaa aaa aat atc ttc ttg gca agt ttt gtt 1112 Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val 340 345 350 355 cat gaa tat tca aga aga cat cct cag ctt gct gtc tca gta att cta 1160 His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser Val Ile Leu 360 365 370 aga gtt gct aaa gga tac cag gag tta ttg gag aag tgt ttc cag act 1208 Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr 375 380 385 gaa aac cct ctt gaa tgc caa gat aaa gga gaa gaa gaa tta cag aaa 1256 Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys 390 395 400 tac atc cag gag agc caa gca ttg gca aag cga agc tgc ggc ctc ttc 1304 Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe 405 410 415 cag aaa cta gga gaa tat tac tta caa aat gcg ttt ctc gtt gct tac 1352 Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu Val Ala Tyr 420 425 430 435 aca aag aaa gcc ccc cag ctg acc tcg tcg gag ctg atg gcc atc acc 1400 Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr 440 445 450 aga aaa atg gca gcc aca gca gcc act tgt tgc caa ctc agt gag gac 1448 Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp 455 460 465 aaa cta ttg gcc tgt ggc gag gga gcg gct gac att att atc gga cac 1496 Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His 470 475 480 tta tgt atc aga cat gaa atg act cca gta aac cct ggt gtt ggc cag 1544 Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly Val Gly Gln 485 490 495 tgc tgc act tct tca tat gcc aac agg agg cca tgc ttc agc agc ttg 1592 Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu 500 505 510 515 gtg gtg gat gaa aca tat gtc cct cct gca ttc tct gat gac aag ttc 1640 Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Phe 520 525 530 att ttc cat aag gat ctg tgc caa gct cag ggt gta gcg ctg caa acg 1688 Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala Leu Gln Thr 535 540 545 atg aag caa gag ttt ctc att aac ctt gtg aag caa aag cca caa ata 1736 Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys Pro Gln Ile 550 555 560 aca gag gaa caa ctt gag gct gtc att gca gat ttc tca ggc ctg ttg 1784 Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser Gly Leu Leu 565 570 575 gag aaa tgc tgc caa ggc cag gaa cag gaa gtc tgc ttt gct gaa gag 1832 Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Glu 580 585 590 595 gga caa aaa ctg att tca aaa act cgt gct gct ttg gga gtt 1874 Gly Gln Lys Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly Val 600 605 taaattactt caggggaaga gaagacaaaa cgagtctttc attcggtgtg aacttttctc 1934 tttaatttta actgatttaa cactttttgt gaattaatga aatgataaag acttttatgt 1994 gagatttcct tatcacagaa ataaaatatc tccaaatg 2032 <210> 2 <211> 609 <212> PRT <213> Homo sapiens <400> 2 Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr 1 5 10 15 Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu 20 25 30 Asp Ser Tyr Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr 35 40 45 Ile Phe Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser 50 55 60 Lys Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp 65 70 75 80 Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu 85 90 95 Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp 100 105 110 Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His 115 120 125 Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro 130 135 140 Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn 145 150 155 160 Lys Phe Ile Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro 165 170 175 Thr Ile Leu Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys 180 185 190 Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr 195 200 205 Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys 210 215 220 Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val 225 230 235 240 Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln 245 250 255 Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly 260 265 270 Asp Val Leu Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile 275 280 285 Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys 290 295 300 Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp 305 310 315 320 Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp 325 330 335 Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala 340 345 350 Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser 355 360 365 Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys 370 375 380 Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu 385 390 395 400 Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys 405 410 415 Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu 420 425 430 Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met 435 440 445 Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu 450 455 460 Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile 465 470 475 480 Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly 485 490 495 Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp 515 520 525 Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala 530 535 540 Leu Gln Thr Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys 545 550 555 560 Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser 565 570 575 Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe 580 585 590 Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly 595 600 605 Val
Claims (38)
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
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US18196600P | 2000-02-10 | 2000-02-10 | |
US60/181,966 | 2000-02-10 | ||
US66025200A | 2000-09-12 | 2000-09-12 | |
US09/660,252 | 2000-09-12 | ||
US66250500A | 2000-09-14 | 2000-09-14 | |
US09/662,505 | 2000-09-14 |
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KR20020073203A KR20020073203A (en) | 2002-09-19 |
KR100482920B1 true KR100482920B1 (en) | 2005-04-14 |
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KR10-2002-7010174A KR100482920B1 (en) | 2000-02-10 | 2001-02-12 | Compositions for treating hepatocellular cancer |
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EP (1) | EP1253930A4 (en) |
JP (1) | JP3876162B2 (en) |
KR (1) | KR100482920B1 (en) |
CN (1) | CN1255427C (en) |
AU (1) | AU2001238179A1 (en) |
HK (1) | HK1046853A1 (en) |
WO (1) | WO2001058922A2 (en) |
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KR100985914B1 (en) * | 2006-12-13 | 2010-10-08 | 주식회사 바이오리더스 | Cell Surface Expression Vector for liver Cancer Specific Antigen Alpha-fetoprotein and Microorganism Transformed by Thereof |
WO2008113970A2 (en) * | 2007-03-16 | 2008-09-25 | Ucl Business Plc | Peptides |
KR100900742B1 (en) * | 2007-05-17 | 2009-06-08 | 크레아젠 주식회사 | Animal Models Carrying Tumors Expressing Human Liver Cancer-Specific Antigen and Method for Analyzing Prevention and Treatment Efficacy of Dendritic Cells-Derived Immunotherapeutics Using the Above |
US8541543B2 (en) * | 2007-11-20 | 2013-09-24 | Academia Sinica | Peptides specific for hepatocellular carcinoma cells and applications thereof |
JP6903587B2 (en) * | 2015-04-03 | 2021-07-14 | ユーリカ セラピューティックス, インコーポレイテッド | Constructs targeting AFP peptide / MHC complexes and their use |
CN113072636B (en) * | 2020-01-06 | 2024-05-28 | 香雪生命科学技术(广东)有限公司 | T cell receptor for identifying AFP and coding sequence thereof |
CN113321725B (en) * | 2020-02-28 | 2024-06-11 | 香雪生命科学技术(广东)有限公司 | T cell receptor for identifying AFP |
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WO1996022787A1 (en) * | 1995-01-24 | 1996-08-01 | Murgita Robert A | Recombinant human alpha-fetoprotein and uses thereof |
WO1998035981A1 (en) * | 1997-02-13 | 1998-08-20 | The Regents Of The University Of California | Prevention and treatment of hepatocellular cancer |
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2001
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- 2001-02-12 EP EP01910587A patent/EP1253930A4/en not_active Ceased
- 2001-02-12 WO PCT/US2001/004539 patent/WO2001058922A2/en not_active Application Discontinuation
- 2001-02-12 AU AU2001238179A patent/AU2001238179A1/en not_active Abandoned
- 2001-02-12 KR KR10-2002-7010174A patent/KR100482920B1/en not_active IP Right Cessation
- 2001-02-12 CN CNB018047580A patent/CN1255427C/en not_active Expired - Fee Related
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JP3876162B2 (en) | 2007-01-31 |
WO2001058922A9 (en) | 2002-10-17 |
EP1253930A4 (en) | 2003-05-21 |
JP2003522195A (en) | 2003-07-22 |
WO2001058922A3 (en) | 2002-02-14 |
AU2001238179A1 (en) | 2001-08-20 |
KR20020073203A (en) | 2002-09-19 |
CN1255427C (en) | 2006-05-10 |
HK1046853A1 (en) | 2003-01-30 |
CN1398187A (en) | 2003-02-19 |
WO2001058922A2 (en) | 2001-08-16 |
EP1253930A2 (en) | 2002-11-06 |
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