本發明關於一種抗體及包含該抗體之醫藥組成物，尤其是用以處置癌症的醫藥組成物及該等的利用等，該抗體能特異性地辨識衍生自癌症抗原PVT1的胜肽與HLA分子的複合體。The present invention relates to an antibody and a pharmaceutical composition comprising the antibody, particularly a pharmaceutical composition for treating cancer and the use thereof. The antibody can specifically recognize a complex of a peptide derived from the cancer antigen PVT1 and an HLA molecule.
腫瘤抗原胜肽是腫瘤細胞中所表現的蛋白質經片段化所呈現出的胜肽。從而，在癌症免疫療法的開發中，重要的是尋找腫瘤細胞中所表現的蛋白質，特別是在正常細胞中不會觀察到表現，但在腫瘤細胞中會觀察到表現的腫瘤抗原蛋白質。Tumor antigen peptides are peptides that are fragmented and displayed by proteins expressed in tumor cells. Therefore, in the development of cancer immunotherapy, it is important to identify proteins expressed in tumor cells, particularly tumor antigen proteins that are not expressed in normal cells but are observed in tumor cells.
PVT1(Plasmacytoma variant translocation 1，漿細胞瘤變異轉位1)是與已知為原癌基因的MYC存在於相同染色體上的非編碼RNA，並且認為會作為MYC活化因子來發揮作用。此外，已報導有在觀察到MYC的複製數增加的多種癌症中，其複製數亦同樣地增加的情況，而顯示出PVT1基因的轉錄量的增加與細胞增殖及癌化有所關連。PVT1 (Plasmacytoma variant translocation 1) is a noncoding RNA expressed on the same chromosome as the proto-oncogene MYC and is thought to function as a MYC activator. Furthermore, increases in MYC copy number have been reported in various cancers where MYC copy number is increased, suggesting that increased PVT1 transcription is associated with cell proliferation and cancer progression.
在如此情況下，發現癌細胞中被預測為編碼有PVT1的開讀框(ORF，Open Reading Frame)之多肽的部分胜肽會進行抗原呈現，並且還發現衍生自PVT1的ORF的HF10在癌症疫苗等的免疫療法上是有用的(例如，專利文獻1、非專利文獻1等)。In this context, it was discovered that a partial peptide predicted to encode a polypeptide containing the open reading frame (ORF) of PVT1 is antigenically presented in cancer cells, and that HF10, derived from the PVT1 ORF, is useful in immunotherapy such as cancer vaccines (e.g., Patent Document 1, Non-Patent Document 1, etc.).
另一方面，截至目前的抗體醫藥品僅能對於過剩地表現在細胞表面的癌症抗原蛋白質進行目標標定，所以仍有目標抗原和目標疾病受限的問題。此外，雖然癌症抗原PVT1的癌症特異性高但是其表現位置在細胞內，所以仍有一般的抗體無法進行目標標定這樣的問題。 [先前技術文獻] (專利文獻)On the other hand, current antibody pharmaceuticals can only target cancer antigen proteins that are overexpressed on the cell surface, resulting in a limited range of target antigens and diseases. Furthermore, while the cancer antigen PVT1 is highly specific for cancer, it is expressed intracellularly, making it difficult for standard antibodies to target it.[Prior Art Literature](Patent Literature)
專利文獻1：國際公開第2017/115798號 (非專利文獻)Patent Document 1: International Publication No. 2017/115798(Non-patent Document)
非專利文獻1：Cancer Immunology Research 2021 Nov;9(11):1342-1353. doi:10.1158/2326-6066.CIR-20-0964.Non-patent reference 1: Cancer Immunology Research 2021 Nov;9(11):1342-1353. doi:10.1158/2326-6066.CIR-20-0964.
[發明所欲解決的問題] 本發明所欲解決的問題在於提供一種抗體及包含該抗體之醫藥組成物，尤其是用以處置癌症的醫藥組成物及該等的利用、癌症的處置方法等，該抗體辨識癌細胞表面的HLA-A24/HF10胜肽複合體。 [解決問題的技術手段][Problem to be Solved by the Invention]The problem to be solved by the present invention is to provide an antibody and a pharmaceutical composition containing the antibody, particularly a pharmaceutical composition for treating cancer, its use, and methods for treating cancer. The antibody recognizes the HLA-A24/HF10 peptide complex on the surface of cancer cells.[Technical Means for Solving the Problem]
發明人著眼於PVT1的ORF衍生胜肽HF10會呈現於細胞表面的MHC分子這點，開發可辨識癌細胞表面的MHC分子/HF10胜肽複合體的抗體(有時會簡稱為本案的抗體)。在持續進行該抗體的開發時，使用基於源自健康人體的周邊血液產生的噬菌體呈現抗體庫，成功地分離出可辨識PVT1的ORF衍生胜肽HF10與HLA-A24複合體的抗體片段(scFv)。並且，基於流式細胞計數(FACS)分析，選出對於呈現於HLA-A24之HF10胜肽的特異性反應性高的scFv選殖株(clone)，並進一步作成scFv-hIgG1型(2價)抗體而使結合力增加來選出特異性高者。當針對特異性高的HF10 scFv選殖株製作與CD3 scFv的雙特異性抗體時，具有經由T細胞的細胞毒殺活性及能誘導細胞激素釋放以及在體內能抑制腫瘤體積，並且還發現以下全新的見解：HF10 CAR-T細胞可辨識細胞表面的HLA-A24/HF10胜肽複合體並產生細胞激素IFNg及CF107a陽性細胞毒性顆粒。The inventors, focusing on the fact that the PVT1 ORF-derived peptide HF10 is presented on MHC molecules on the cell surface, developed an antibody (sometimes referred to as the antibody in this case) that recognizes the MHC molecule/HF10 peptide complex on the surface of cancer cells. During ongoing development of this antibody, using a phage-displayed antibody library generated from peripheral blood of healthy individuals, they successfully isolated an antibody fragment (scFv) that recognizes the complex of the PVT1 ORF-derived peptide HF10 and HLA-A24. Furthermore, based on flow cytometry (FACS) analysis, scFv clones with high specific reactivity to the HF10 peptide presented on HLA-A24 were selected. ScFv-hIgG1 (bivalent) antibodies were then generated to enhance binding and select those with high specificity. When bispecific antibodies were generated against the highly specific HF10 scFv clone and CD3 scFv, they exhibited cytotoxic activity via T cells, induced cytokine release, and inhibited tumor growth in vivo. Furthermore, the researchers revealed the following novel insight: HF10 CAR-T cells can recognize the HLA-A24/HF10 peptide complex on the cell surface and produce the cytokine IFNg and CF107a-positive cytotoxic particles.
亦即，本發明關於下述揭示的技術。 [1] 一種多特異性抗體，其具有辨識由序列編號3表示的多肽與MHC分子之複合體的抗原結合部位。 [2] 如[1]所述之多特異性抗體，其中，辨識由序列編號3表示的多肽與MHC分子之複合體的抗原結合部位包含：在序列編號4～8的任一序列中所記載的胺基酸序列或GRD的胺基酸序列、或者胺基酸序列編號4～8的任一序列中所記載的胺基酸序列中的1個或2個胺基酸經取代而成之胺基酸序列。 [3] 如[1]所述之多特異性抗體，其中，具有表示於序列編號19的可變區域的胺基酸序列，並且1個或2個的胺基酸可受到取代。 [4] 如[1]所述之多特異性抗體，其中，進一步具有辨識CD3的抗原結合部位。 [5] 一種醫藥組成物，其包含抗體，該抗體辨識由序列編號3表示的多肽與MHC分子之複合體。 [6] 如[5]所述之醫藥組成物，其用以預防及/或處置癌症。 [7] 如[5]所述之醫藥組成物，其中，MHC分子為HLA分子。 [8] 如[5]所述之醫藥組成物，其中，抗體的抗原結合部位包含：序列編號4～18中的任一序列所記載的胺基酸序列；或，GRD、GKN或NDN的胺基酸序列。 [9] 如[5]所述之醫藥組成物，其中，抗體為[1]～[4]中任一項所述之多特異性抗體。 [10] 一種二價人工抗體，其包含 (1)辨識由序列編號3表示的多肽與MHC分子之複合體的2個抗體片段(scFv)及 (2)人類IgG的Fc區域。 [11] 一種嵌合抗原受體，其包含：辨識由序列編號3表示的多肽與MHC分子之複合體的抗原結合部位以及CD3ζ鏈。 [12] 如[11]所述之嵌合抗原受體，其中，進一步包含協同刺激分子。 [13] 一種基因改造T細胞、基因改造NK細胞或基因改造巨噬細胞(MΦ)(較佳是基因改造T細胞)，其特徵在於導入有[11]或[12]所述之嵌合抗原受體。 [14] 一種人工細胞毒性T細胞，其包含T細胞受體，該T細胞受體辨識由序列編號3表示的多肽與MHC分子之複合體。That is, the present invention relates to the following disclosed technologies. [1] A multispecific antibody having an antigen binding site that recognizes a complex of a polypeptide represented by sequence number 3 and an MHC molecule. [2] The multispecific antibody as described in [1], wherein the antigen binding site that recognizes a complex of a polypeptide represented by sequence number 3 and an MHC molecule comprises: an amino acid sequence described in any one of sequence numbers 4 to 8 or an amino acid sequence of GRD, or an amino acid sequence in which one or two amino acids in the amino acid sequence described in any one of sequence numbers 4 to 8 are substituted. [3] The multispecific antibody as described in [1], wherein the multispecific antibody has an amino acid sequence represented in a variable region of sequence number 19, and one or two amino acids are substituted. [4] The multispecific antibody as described in [1], further comprising an antigen binding site that recognizes CD3.[5] A pharmaceutical composition comprising an antibody that recognizes a complex of the polypeptide represented by sequence number 3 and an MHC molecule.[6] The pharmaceutical composition as described in [5], for preventing and/or treating cancer.[7] The pharmaceutical composition as described in [5], wherein the MHC molecule is an HLA molecule.[8] The pharmaceutical composition as described in [5], wherein the antigen binding site of the antibody comprises: an amino acid sequence described in any one of sequence numbers 4 to 18; or an amino acid sequence of GRD, GKN, or NDN.[9] The pharmaceutical composition as described in [5], wherein the antibody is the multispecific antibody as described in any one of [1] to [4]. [10] A bivalent artificial antibody comprising (1) two antibody fragments (scFv) that recognize a complex of a polypeptide represented by sequence number 3 and an MHC molecule and (2) the Fc region of human IgG. [11] A chimeric antigen receptor comprising: an antigen binding site that recognizes a complex of a polypeptide represented by sequence number 3 and an MHC molecule and a CD3ζ chain. [12] The chimeric antigen receptor as described in [11], further comprising a co-stimulatory molecule. [13] A genetically modified T cell, genetically modified NK cell or genetically modified macrophage (MΦ) (preferably a genetically modified T cell), characterized in that the chimeric antigen receptor as described in [11] or [12] is introduced. [14] An artificial cytotoxic T cell comprising a T cell receptor that recognizes a complex of a polypeptide represented by sequence number 3 and an MHC molecule.
此外，本發明可關於下述揭示的技術。 [1A] 一種抗體，其具有辨識由序列編號3表示的多肽與HLA-A24分子之複合體的抗原結合部位， 該抗原結合部位為由VH以及VL所構成之抗原結合部位，該VH具有 (a)由序列編號4的胺基酸序列所構成之VH-CDR1、 (b)由序列編號5的胺基酸序列所構成之VH-CDR2及 (c)由序列編號6的胺基酸序列所構成之VH-CDR3， 該VL具有 (d)由序列編號7的胺基酸序列所構成之VL-CDR1、 (e)由GRD的胺基酸序列所構成之VL-CDR2及 (f)由序列編號8的胺基酸序列所構成之VL-CDR3；並且， 在(a)～(f)中的任一或多個CDR中，各自且任意的1個或2個胺基酸殘基可被取代為其他胺基酸。 [2A] 如[1A]所述之抗體，其中，辨識由序列編號3表示的多肽與HLA-A24分子之複合體的抗原結合部位具有VH與VL，該VH由與表示於序列編號22(選殖株1的VH)的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列所構成，該VL由與表示於序列編號23(選殖株1的VL)的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列所構成。 [3A] 如[1A]或[2A]所述之抗體，其為人工抗體，該人工抗體包含 (1)辨識由序列編號3表示的多肽與HLA-A24分子之複合體的抗原結合部位為scFv、 (2)人類IgG的Fc區域。 [4A] 如[3A]所述之抗體，其中，scFv包含記載於序列編號19的胺基酸序列。 [5A] 如[1A]～[4A]中任一項所述之抗體，其中，進一步具有辨識CD3的抗原結合部位。 [6A] 如[5A]所述之抗體，其中，辨識CD3的抗原結合部位為包含記載於序列編號32的胺基酸序列之scFv。 [7A] 如[5A]或[6A]所述之抗體，其為(1)雙特異性T細胞衍生抗體。 [8A] 一種嵌合抗原受體，其包含辨識由序列編號3表示的多肽與HLA-A24分子之複合體的抗原結合部位以及CD3ζ鏈， 該抗原結合部位為由VH以及VL所構成之抗原結合部位，該VH具有 (a)由序列編號4的胺基酸序列所構成之VH-CDR1、 (b)由序列編號5的胺基酸序列所構成之VH-CDR2及 (c)由序列編號6的胺基酸序列所構成之VH-CDR3， 該VL具有 (d)由序列編號7的胺基酸序列所構成之VL-CDR1、 (e)由GRD的胺基酸序列所構成之VL-CDR2及 (f)由序列編號8的胺基酸序列所構成之VL-CDR3；並且， 在(a)～(f)中的任一或多個CDR中，各自且任意的1個或2個胺基酸殘基可被取代為其他胺基酸。 [9A] 如[8A]所述之嵌合抗原受體，其中，辨識由序列編號3表示的多肽與HLA-A24分子之複合體的抗原結合部位具有VH與VL，該VH由與表示於序列編號22(選殖株1的VH)的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列所構成，該VL由與表示於序列編號23(選殖株1的VL)的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列所構成。 [10A] 如[8A]或[9A]所述之嵌合抗原受體，其中，進一步包含協同刺激分子(較佳是CD28)。 [11A] 一種基因改造免疫細胞(較佳是基因改造T細胞(CAR-T)、基因改造NK細胞(CAR-NK)或基因改造巨噬細胞(MΦ)，更佳是基因改造T細胞(CAR-T))，其特徵在於導入有[8A]～[10A]中任一項所述之嵌合抗原受體。 [12A] 一種醫藥組成物，其包含[1A]～[7A]中任一項所述之抗體或[11A]所述之基因改造免疫細胞。 [13A] 如[12A]所述之醫藥組成物，其中，進一步包含至少一種藥學上可接受的載體。 [14A] 如[12A]所述之醫藥組成物，其用以預防及/或處置癌症。 [15A] 一種[1A]～[7A]中任一項所述之抗體、[11A]所述之基因改造免疫細胞或[12A]～[14A]中任一項所述之醫藥組成物的用途，其用於用以製造預防或處置癌症的醫藥。 [16A] 一種預防或處置癌症的方法，其包含如下步驟：對有需求的對象投予有效量的[1A]～[7A]中任一項所述之抗體、[11A]所述之基因改造免疫細胞或[12A]～[14A]中任一項所述之醫藥組成物。 [17A] 如[1A]～[7A]中任一項所述之抗體、[11A]所述之基因改造免疫細胞或[12A]～[14A]中任一項所述之醫藥組成物，其使用於癌症的預防或處置。 [18] 一種癌症的預防劑或處置劑，其含有[1A]～[7A]中任一項所述之抗體、[11A]所述之基因改造免疫細胞或[12A]～[14A]中任一項所述之醫藥組成物。 [發明的效果]In addition, the present invention may relate to the technology disclosed below. [1A] An antibody having an antigen-binding site that recognizes a complex of a polypeptide represented by Sequence Number 3 and an HLA-A24 molecule, The antigen-binding site is an antigen-binding site composed of VH and VL, the VH having (a) VH-CDR1 composed of the amino acid sequence of Sequence Number 4, (b) VH-CDR2 composed of the amino acid sequence of Sequence Number 5, and (c) VH-CDR3 composed of the amino acid sequence of Sequence Number 6, The VL having (d) VL-CDR1 composed of the amino acid sequence of Sequence Number 7, (e) VL-CDR2 composed of the amino acid sequence of GRD, and (f) VL-CDR3 composed of the amino acid sequence of Sequence Number 8; and, In any one or more CDRs in (a) to (f), any one or two amino acid residues may be substituted with other amino acids.[2A] The antibody according to [1A], wherein the antigen-binding site that recognizes the complex of the polypeptide represented by SEQ ID NO: 3 and the HLA-A24 molecule comprises VH and VL, wherein the VH comprises an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 22 (VH of clone 1), and the VL comprises an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 23 (VL of clone 1). [3A] The antibody described in [1A] or [2A], which is an artificial antibody comprising:(1) an scFv that recognizes a complex of the polypeptide represented by Sequence Number 3 and an HLA-A24 molecule;(2) an Fc region of human IgG.[4A] The antibody described in [3A], wherein the scFv comprises the amino acid sequence set forth in Sequence Number 19.[5A] The antibody described in any one of [1A] to [4A], which further comprises an antigen binding site that recognizes CD3.[6A] The antibody described in [5A], wherein the antigen binding site that recognizes CD3 is an scFv that comprises the amino acid sequence set forth in Sequence Number 32. [7A] The antibody as described in [5A] or [6A], which is (1) a bispecific T cell-derived antibody. [8A] A chimeric antigen receptor comprising an antigen binding site that recognizes a complex of a polypeptide represented by sequence number 3 and an HLA-A24 molecule and a CD3ζ chain, wherein the antigen binding site is an antigen binding site composed of VH and VL, wherein the VH has (a) a VH-CDR1 composed of the amino acid sequence of sequence number 4, (b) a VH-CDR2 composed of the amino acid sequence of sequence number 5, and (c) a VH-CDR3 composed of the amino acid sequence of sequence number 6, and wherein the VL has (d) a VL-CDR1 composed of the amino acid sequence of sequence number 7, (e) a VL-CDR2 composed of the amino acid sequence of GRD, and (f) a VL-CDR3 composed of the amino acid sequence of sequence number 8; and In any one or more CDRs in (a) to (f), any one or two amino acid residues may be substituted with other amino acids.[9A] The chimeric antigen receptor according to [8A], wherein the antigen binding site that recognizes the complex of the polypeptide represented by SEQ ID NO: 3 and the HLA-A24 molecule comprises VH and VL, wherein the VH comprises an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence represented by SEQ ID NO: 22 (VH of clone 1), and the VL comprises an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to the amino acid sequence represented by SEQ ID NO: 23 (VL of clone 1). [10A] The chimeric antigen receptor as described in [8A] or [9A], further comprising a co-stimulatory molecule (preferably CD28).[11A] A genetically modified immune cell (preferably a genetically modified T cell (CAR-T), a genetically modified NK cell (CAR-NK) or a genetically modified macrophage (MΦ), more preferably a genetically modified T cell (CAR-T)), characterized by the introduction of the chimeric antigen receptor as described in any one of [8A] to [10A].[12A] A pharmaceutical composition comprising the antibody as described in any one of [1A] to [7A] or the genetically modified immune cell as described in [11A].[13A] The pharmaceutical composition as described in [12A], further comprising at least one pharmaceutically acceptable carrier. [14A] The pharmaceutical composition as described in [12A], which is used for preventing and/or treating cancer.[15A] Use of the antibody described in any one of [1A] to [7A], the genetically modified immune cell described in [11A], or the pharmaceutical composition described in any one of [12A] to [14A], for manufacturing a medicament for preventing or treating cancer.[16A] A method for preventing or treating cancer, comprising the steps of administering an effective amount of the antibody described in any one of [1A] to [7A], the genetically modified immune cell described in [11A], or the pharmaceutical composition described in any one of [12A] to [14A] to a subject in need thereof. [17A] The antibody described in any one of [1A] to [7A], the genetically modified immune cell described in [11A], or the pharmaceutical composition described in any one of [12A] to [14A], for use in the prevention or treatment of cancer.[18] A preventive or therapeutic agent for cancer, comprising the antibody described in any one of [1A] to [7A], the genetically modified immune cell described in [11A], or the pharmaceutical composition described in any one of [12A] to [14A].[Effects of the Invention]
根據本案的抗體，能夠特異性地辨識HLA-A24/HF10胜肽複合體。此外，例如根據本發明的雙特異性抗體，能夠誘導經由T細胞的細胞激素生成及能夠在體內抑制腫瘤體積，並且能夠對定位於細胞內的癌症抗原PVT1進行特異性的目標標定。 此外，例如，本案的抗體會對癌症特異性高的PVT1進行目標標定，所以能進行對於健康器官的副作用低的治療。此外，本案的抗體的特異性高，所以可能能夠改善對於既存的治療沒有反應的癌症末期患者的預後。The antibodies described in this application are capable of specifically recognizing HLA-A24/HF10 peptide complexes. Furthermore, for example, the bispecific antibodies described in this application can induce cytokine production by T cells and inhibit tumor growth in vivo, specifically targeting the intracellular cancer antigen PVT1.Furthermore, for example, because the antibodies described in this application target PVT1, which has high cancer specificity, they enable treatment with minimal side effects on healthy organs. Furthermore, the high specificity of these antibodies has the potential to improve the prognosis of terminally ill cancer patients who have not responded to existing treatments.
以下，詳細地說明本發明。 本說明書中，只要沒有進行另外的定義，用於本說明書中的全部的技術用語及科學用語具有與所屬技術領域中具有通常知識者所一般性理解的定義具有相同的意義。在本說明書中參照的全部的專利、申請案及其他刊物和資訊，藉由參照將其整體援引至本說明書中。此外，當本說明書中所參照的刊物與本說明書的記載存在矛盾時，以本說明書的記載為優先。The present invention is described below in detail.In this specification, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. All patents, applications, and other publications and materials referenced in this specification are incorporated herein by reference in their entirety. In the event of a conflict between a referenced publication and this specification, the disclosure in this specification shall prevail.
本說明書中，所謂「抗原決定胜肽(epitope peptide)」或「抗原胜肽」，意指一種胜肽，其會與主要組織相容性複合體(MHC(在人體內為人類白血球抗原(HLA)))分子結合並且會在細胞表面進行抗原呈現且具有抗原性(可被T細胞辨識)。抗原決定胜肽中，包含CTL(細胞毒性T細胞)抗原決定胜肽及輔助抗原決定胜肽，該CTL(細胞毒性T細胞)抗原決定胜肽為會與MHC I類分子(MHC class I)結合而抗原呈現並受到CD8陽性T細胞辨識的抗原決定胜肽，該輔助抗原決定胜肽為會與MHC II類分子(MHC class II)結合而抗原呈現並受到CD4陽性T細胞辨識的抗原決定胜肽。As used herein, the term "epitope peptide" or "antigenic peptide" refers to a peptide that binds to a major histocompatibility complex (MHC) (human leukocyte antigen (HLA) in humans) molecule, presents itself on the cell surface, and possesses antigenicity (can be recognized by T cells). Antigenic peptides include CTL (cytotoxic T cell) antigenic peptides and helper antigenic peptides. CTL (cytotoxic T cell) antigenic peptides are antigenic peptides that bind to MHC class I molecules (MHC class I), present antigens, and are recognized by CD8-positive T cells. Helper antigenic peptides are antigenic peptides that bind to MHC class II molecules (MHC class II), present antigens, and are recognized by CD4-positive T cells.
抗原決定胜肽中，將在腫瘤細胞中會特異性或過剩地表現的衍生自蛋白質的胜肽特別稱為腫瘤抗原胜肽。所謂抗原呈現，意指存在於細胞內的胜肽與MHC結合並且該MHC/抗原胜肽複合體定位於細胞表面的現象。已知呈現於細胞表面的抗原在受到T細胞等的辨識後，會活化細胞免疫及體液免疫，呈現於MHC I類分子的抗原會活化細胞免疫，並且受到初始T細胞的T細胞受體辨識，將初始T細胞誘導為具有細胞毒殺活性之CTL，所以在免疫療法中，作為腫瘤抗原胜肽，一般會使用可與MHC I類分子結合而抗原呈現的胜肽。Among antigenic peptides, peptides derived from proteins that are specifically or excessively expressed in tumor cells are specifically called tumor antigen peptides. Antigen presentation refers to the phenomenon in which a peptide present within a cell binds to MHC, and the MHC/antigen peptide complex is localized on the cell surface. Antigens presented on the cell surface are known to activate both cellular and humoral immunity after being recognized by T cells and other molecules. Antigens presented on MHC class I molecules activate cellular immunity and are recognized by T cell receptors on naive T cells, inducing them to become CTLs with cytotoxic activity. Therefore, in immunotherapy, peptides that can bind to MHC class I molecules for antigen presentation are generally used as tumor antigen peptides.
已知多數會與MHC結合的胜肽具有一定的特徵。本案中，將該特徵稱為「結合模體(motif)」。對於所屬技術領域中具有通常知識者而言，已知何種MHC會與具有何種結合模體之胜肽結合。例如，人類MHC的其中之一的HLA-A24的結合模體自N端起的第2個胺基酸為酪胺酸、苯丙胺酸、甲硫胺酸或色胺酸，且C端的胺基酸為離胺酸、異離胺酸或苯丙胺酸。此外，HLA-A02的結合模體自N端起的第2個胺基酸為離胺酸、異離胺酸或甲硫胺酸及/或C端的胺基酸為纈胺酸、離胺酸或異離胺酸。It is known that most peptides that bind to MHC have certain characteristics. In this case, these characteristics are referred to as "binding motifs." Those skilled in the art know which MHC types bind to peptides with which binding motifs. For example, the binding motif of HLA-A24, one of the human MHCs, has a second amino acid from the N-terminus that is tyrosine, phenylalanine, methionine, or tryptophan, and a C-terminal amino acid that is lysine, isolysine, or phenylalanine. Furthermore, the binding motif of HLA-A02 has a second amino acid from the N-terminus that is lysine, isolysine, or methionine, and/or a C-terminal amino acid that is valine, lysine, or isolysine.
本案中，所謂「天然胜肽」，意指實際上抗原呈現於細胞表面的胜肽。此外，所謂「天然抗原胜肽」，意指天然胜肽中能夠確認到抗原性的胜肽。將天然抗原胜肽與MHC分子之複合體自癌細胞分離出來，決定天然抗原胜肽的序列及其來源，藉此即能夠選出本案的抗體可辨識的候選細胞表面抗原。 本案中，「腫瘤(tumor)」包含良性腫瘤及惡性腫瘤(癌症、惡性贅生物)。癌症(cancer)包含造血系統的腫瘤、上皮性的惡性腫瘤(癌，carcinoma)與非上皮性的惡性腫瘤(肉瘤，sarcoma)。癌症(cancer)包含：大腸癌(例如直腸癌等)、肺癌(例如非小細胞肺癌等)、乳癌、骨髓瘤、頭頸癌(例如口腔癌、口腔鱗狀細胞癌、頭頸鱗狀細胞癌、咽喉癌、喉癌、舌癌、甲狀腺癌、聽神經鞘瘤等)、骨肉瘤、胰癌(胰臟癌)、皮膚癌(例如惡性黑色素瘤等)、前列腺癌、卵巢癌、淋巴瘤(例如B細胞淋巴瘤、T細胞淋巴瘤等)、葡萄膜惡性黑色素瘤、胸腺瘤、間皮瘤、食道癌、胃癌、十二指腸癌、肝細胞癌、膽管癌、膽囊癌、腎細胞癌、腎盂/輸尿管癌、膀胱癌、陰莖癌、睪丸癌、子宮癌、陰道癌、外陰癌、惡性骨腫瘤、軟組織肉瘤、軟骨肉瘤、白血病(例如急性骨髓性白血病、急性淋巴性白血病、慢性骨髓性白血病、慢性淋巴性白血病等)、骨髓化生不良症候群或多發性骨髓瘤等。In this case, "natural peptides" refer to peptides that are actually antigens presented on the cell surface. Furthermore, "natural antigenic peptides" refer to peptides within natural peptides that can be confirmed as antigenic. By isolating the natural antigenic peptide complex with an MHC molecule from cancer cells, the sequence and origin of the natural antigenic peptide can be determined, thereby enabling the selection of candidate cell surface antigens recognizable by the antibodies in this case.In this case, "tumor" includes both benign and malignant tumors (cancer, malignant tumors). Cancer includes hematopoietic tumors, epithelial malignancies (carcinomas), and non-epithelial malignancies (sarcomas). Cancer includes: colorectal cancer (such as rectal cancer, etc.), lung cancer (such as non-small cell lung cancer, etc.), breast cancer, myeloma, head and neck cancer (such as oral cancer, oral squamous cell carcinoma, head and neck squamous cell carcinoma, pharyngeal cancer, laryngeal cancer, tongue cancer, thyroid cancer, acoustic neurothelial carcinoma, etc.), osteosarcoma, pancreatic cancer (pancreatic cancer), skin cancer (such as malignant melanoma, etc.), prostate cancer, ovarian cancer, lymphoma (such as B cell lymphoma, T cell lymphoma, etc.), uveal tract cancer, ureteral carcinoma, thyroid ... Malignant melanoma, thymoma, mesothelioma, esophageal cancer, gastric cancer, duodenal cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, renal cell carcinoma, renal pelvis/ureteral cancer, bladder cancer, penile cancer, testicular cancer, uterine cancer, vaginal cancer, vulvar cancer, malignant bone tumor, soft tissue sarcoma, chondrosarcoma, leukemia (such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, etc.), myelodysplastic syndrome or multiple myeloma, etc.
PVT1(Plasmacytoma variant translocation 1)是與已知為原癌基因的MYC存在於相同染色體上的非編碼RNA，並且認為會作為MYC活化因子來發揮作用。此外，已報導有在觀察到MYC的複製數增加的多種癌症中，其複製數亦同樣地增加的情況，而顯示出PVT1基因的轉錄量的增加與細胞增殖及癌化有所關連。此外，如同上述，當考量到PVT1為非編碼RNA時，發現在癌細胞中被預測為編碼有PVT1的HF10胜肽受到抗原呈現，並且顯現出在癌細胞中胜肽可能自PVT1的ORF被轉譯而進行表現的情況。PVT1 (Plasmacytoma variant translocation 1) is a noncoding RNA that colocalizes with MYC, a known proto-oncogene, and is thought to function as a MYC activator. Furthermore, increases in MYC copy number have been reported in various cancers where MYC copy number is increased, suggesting that increased transcription of the PVT1 gene is associated with cell proliferation and cancer progression. Furthermore, considering that PVT1 is a noncoding RNA, the HF10 peptide, predicted to encode PVT1, was found to be antigenically presented in cancer cells, suggesting that the peptide may be expressed in cancer cells through translation from the PVT1 ORF.
在多數的大腸癌中會檢測到PVT1的mRNA的表現，並且即使在正常組織中雖可在卵巢中發現少量的表現，其表現量仍低(非專利文獻1)。從衍生自PVT1的HF10胜肽(序列編號3)顯示與HLA-A24的高結合性這點來看，認為其作為免疫治療的標的是有用的(專利文獻1)。此外，從該基因在癌細胞中會特異性地表現這點來看，可能與細胞癌化和細胞的生存性有所關連，所以也可期待其作為藉由抑制表現等來進行核酸治療等的標的產生的效果。PVT1 mRNA expression is detected in most colorectal cancers, and even in normal tissues, although a small amount can be found in the ovary, its expression level is still low (Non-Patent Document 1). The HF10 peptide (SEQ ID NO: 3) derived from PVT1 exhibits high binding to HLA-A24, suggesting its usefulness as a target for immunotherapy (Patent Document 1). Furthermore, since this gene is specifically expressed in cancer cells, it may be involved in cell carcinogenesis and cell survival, and thus its expression inhibition could be expected to yield benefits as a target for nucleic acid therapy.
本案中，例如當如「PVT1」這樣僅以基因名標示時，只要沒有另外記載，意指由該基因名所表示且具有習知的核酸序列之基因，通常表示cDNA或mRNA序列，並且只要所屬技術領域中具有通常知識者可理解該基因的序列時，則不限於此。例如，作為本案中較佳的基因及其核酸序列的示例，可列舉由序列編號1表示的鹼基序列，但是該等基因的基因多態性等(例如SNPs等)、可理解為與該等基因相同的該基因的序列，也包含在本案的基因中。 從而，作為本案的基因表現產物的mRNA，有時僅藉由基因名的記載來表示。In this case, when a gene name is used alone, such as "PVT1," unless otherwise specified, it refers to the gene represented by that name and having a known nucleic acid sequence, typically a cDNA or mRNA sequence. This is not limited to this, as long as the gene sequence is understandable to those skilled in the art. For example, the base sequence represented by SEQ ID NO: 1 is an example of a preferred gene and its nucleic acid sequence in this case. However, genetic polymorphisms (e.g., SNPs) of such genes, as well as sequences of such genes that are understood to be identical to such genes, are also included in the genes in this case.Thus, the mRNA, the expression product of a gene in this case, is sometimes indicated simply by the gene name.
本案中，「衍生自PVT1的ORF的抗原胜肽」意指在構成PVT1的ORF的胺基酸序列中由連續的一部分的序列所構成之部分胜肽，並且為具有作為上述抗原胜肽的性質者。本案的較佳態樣中，「衍生自PVT1的ORF的抗原胜肽」具體而言意指由序列編號3所表示的多肽也就是HF10。In this application, an "antigenic peptide derived from the ORF of PVT1" refers to a partial peptide consisting of a continuous portion of the amino acid sequence constituting the ORF of PVT1, and possessing the properties of such an antigenic peptide. In a preferred embodiment of this application, the "antigenic peptide derived from the ORF of PVT1" specifically refers to the polypeptide represented by SEQ ID NO: 3, namely, HF10.
＜1＞本案的抗體 本案的一實施形態，關於一種抗體，其具有可特異性地辨識癌細胞中特異性表現的基因、例如衍生自PVT1的ORF的抗原胜肽HF10與MHC分子之複合體。原則上，該抗體具有辨識前述抗原胜肽與MHC分子之複合體的抗原結合部位。<1> Antibodies of the Present ApplicationOne embodiment of the present application relates to an antibody that specifically recognizes a complex of an antigenic peptide HF10 derived from the ORF of a gene specifically expressed in cancer cells, such as PVT1, and an MHC molecule. In principle, the antibody has an antigen-binding site that recognizes the complex of the antigenic peptide and MHC molecule.
本案中所謂的「抗體」，意指一種蛋白質，其具有抗原結合部位，並且該抗原結合部位具有與所辨識的分子結合的性質，典型上為免疫球蛋白但是不限於此。從而，不僅是免疫球蛋白分子，例如能由免疫球蛋白的抗原結合部位所生成的抗體的功能性片段(抗原結合性片段)等也包含在本案的抗體中。作為該抗原結合性片段，典型上可列舉Ｆ(ab’)2片段、Fab’片段、Fab片段、Fv片段、rIgG片段等，除此之外也包含scFv、dsFv、雙抗體(diabody)及sc(Fv)2等。例如，該等片段可藉由恆定區域和抗體鏈區域中的雙硫鍵進行連結，也可以是利用連接子(linker)將各區域結合而單鏈化而成者(scFv)。在較佳的一態樣中，本案的抗體為單鏈抗體(scFv)。在較佳的一態樣中，本案的抗體為scFv-IgG型人工抗體。作為scFv-IgG型人工抗體，可列舉一種人工抗體，其包含具有辨識由序列編號3表示的多肽與HLA分子之複合體之抗原結合部位之抗體片段(scFv)及(2)人類IgG的Fc區域。作為其他的一較佳態樣，本案的抗體為IgG型，較佳是IgG1型。此外，本案的抗體中，免疫球蛋白和其功能性片段的多聚物(例如二聚物、三聚物、四聚物、聚合物)也包含於本案的抗體中。The term "antibody" as used herein refers to a protein having an antigen-binding site capable of binding to a target molecule, typically an immunoglobulin, but not limited thereto. Therefore, the term "antibody" encompasses not only immunoglobulin molecules but also functional fragments of antibodies (antigen-binding fragments) that can be generated from the antigen-binding site of an immunoglobulin. Typical examples of such antigen-binding fragments include F(ab')2 fragments, Fab' fragments, Fab fragments, Fv fragments, and rIgG fragments. These fragments also include scFv, dsFv, diabodies, and sc(Fv)2 . For example, these fragments can be linked via disulfide bonds in the constant region and the antibody chain region, or they can be formed by linking the regions together using a linker to form a single chain (scFv). In a preferred embodiment, the antibody of the present invention is a single-chain antibody (scFv). In a preferred embodiment, the antibody of the present invention is an scFv-IgG type artificial antibody. As an scFv-IgG type artificial antibody, an artificial antibody can be cited, which comprises an antibody fragment (scFv) having an antigen binding site that recognizes a complex of a polypeptide represented by sequence number 3 and an HLA molecule and (2) the Fc region of human IgG. As another preferred embodiment, the antibody of the present invention is an IgG type, preferably an IgG1 type. In addition, in the antibody of the present invention, polymers (such as dimers, trimers, tetramers, and polymers) of immunoglobulins and their functional fragments are also included in the antibody of the present invention.
本案的抗體，只要是可辨識存在於細胞表面的癌細胞抗原胜肽與MHC分子之複合體者即可，並無特別限定，可以是多株抗體，也可以是單株抗體。此外，只要具有可辨識癌細胞抗原胜肽與MHC分子之複合體的抗原結合部位即可，並無特別限定，例如可以具有其他的抗原結合部位和用以與其他蛋白質結合的結合區域等。從而，某一態樣中，本案的抗體可以是多特異性抗體。在一較佳態樣中，本案的抗體為雙特異性抗體。The antibodies in this application are not particularly limited, as long as they can recognize the complex of a cancer cell antigen peptide and an MHC molecule present on the cell surface. They can be either polyclonal or monoclonal. Furthermore, as long as they possess an antigen-binding site capable of recognizing the complex of a cancer cell antigen peptide and an MHC molecule, they are not particularly limited. For example, they may possess other antigen-binding sites or binding regions for binding to other proteins. Thus, in one embodiment, the antibodies in this application can be multispecific antibodies. In a preferred embodiment, the antibodies in this application are bispecific antibodies.
本案的抗體，能夠辨識存在於癌細胞表面的癌細胞抗原胜肽與MHC分子之複合體並進行結合，所以能夠用於各式各樣的用途。在一態樣中，本案的抗體能用於檢測癌細胞。在另一態樣中，本案的抗體能用於處置癌細胞(亦即，處置癌症)。當本案的抗體用於處置癌細胞時，可以是利用細胞免疫的方法，也可以是利用體液免疫的方法。作為利用細胞免疫的態樣，可列舉例如過繼免疫細胞療法等，其使用了導入有嵌合抗原受體(CAR)之免疫細胞(例如：T細胞(CAR-T細胞)、NK細胞(CAR-NK細胞)、巨噬細胞(MΦ)(CAR-MΦ)等)，該嵌合抗原受體組合有本案的抗體所具有的抗原結合部位。作為利用體液免疫的態樣，可列舉例如利用抗體的抗體依賴性細胞媒介之細胞毒性(ADCC)活性、補體依賴性細胞媒介之細胞毒性(CDCC)活性的方法等。此外，在另一態樣中，本案的抗體包括一種嵌合抗原受體(CAR)，其組合有辨識癌細胞抗原胜肽與MHC分子之複合體的抗原結合部位。The antibodies described herein can recognize and bind to complexes of cancer cell antigen peptides and MHC molecules present on the surface of cancer cells, and therefore can be used in a variety of applications. In one embodiment, the antibodies described herein can be used to detect cancer cells. In another embodiment, the antibodies described herein can be used to treat cancer cells (i.e., to treat cancer). When the antibodies described herein are used to treat cancer cells, either cellular or humoral immunity can be used. Examples of methods utilizing cellular immunity include secondary immune cell therapy, which utilizes immune cells (e.g., T cells (CAR-T cells), NK cells (CAR-NK cells), macrophages (MΦ) (CAR-MΦ), etc.) that have been introduced with chimeric antigen receptors (CARs) that incorporate the antigen-binding site of the antibodies described herein. Examples of methods utilizing humoral immunity include methods that utilize the antibody-dependent cell-mediated cytotoxicity (ADCC) activity and complement-dependent cell-mediated cytotoxicity (CDCC) activity of antibodies. In another embodiment, the antibody of the present invention comprises a chimeric antigen receptor (CAR) that is combined with an antigen binding site that recognizes a complex of a cancer cell antigen peptide and an MHC molecule.
如同上述，本案的抗體具有抗原結合部位，其可特異性地辨識衍生自癌細胞中特異性表現的基因的表現產物即PVT1的ORF的抗原胜肽HF10、與MHC分子之複合體。選擇辨識特定的抗原的抗原結合部位或具有該抗原結合部位之抗體的方法，只要使用該技術領域中的習知方法即可。作為如此的方法，可列舉例如噬菌體呈現方法等。As described above, the antibodies in this case possess an antigen-binding site that specifically recognizes a complex of an antigenic peptide, HF10, derived from the ORF of PVT1, a gene expressed specifically in cancer cells, and an MHC molecule. Methods for selecting an antigen-binding site that recognizes a specific antigen, or an antibody possessing such an antigen-binding site, can be employed using methods known in the art. Examples of such methods include phage display.
抗原胜肽只要可與MHC結合即可，並無特別限定。與MHC結合的胜肽，具有因其MHC的型態而異的特徵。例如，當為人類MHC即HLA時，結合於HLA I類分子(HLA class I)的胜肽，長度約為8～14個胺基酸左右，較佳是長度約為8～10個胺基酸左右，相對於此，HLA II類分子會與長度10個胺基酸以上、例如長度約10～30個胺基酸之胜肽結合。此外，在HLA I類分子之中，例如與HLA-A02結合的胜肽，具有自N端起的第2個胺基酸為離胺酸、異離胺酸或甲硫胺酸及/或C端的胺基酸為纈胺酸、離胺酸或異離胺酸之結合模體，並且與HLA-A24結合的胜肽，具有自N端起的第2個胺基酸為酪胺酸、苯丙胺酸、甲硫胺酸或色胺酸及/或C端的胺基酸為離胺酸、異離胺酸或苯丙胺酸之結合模體。There are no particular limitations on the type of antigenic peptide, as long as it can bind to MHC. Peptides that bind to MHC have characteristics that vary depending on the type of MHC. For example, in the case of human MHC (HLA), peptides that bind to HLA class I molecules are approximately 8 to 14 amino acids in length, preferably 8 to 10 amino acids in length. In contrast, HLA class II molecules bind to peptides longer than 10 amino acids, for example, approximately 10 to 30 amino acids in length. Furthermore, among HLA class I molecules, for example, peptides that bind to HLA-A02 have a binding motif in which the second amino acid from the N-terminus is lysine, iso-lysine, or methionine and/or the C-terminal amino acid is valine, lysine, or iso-lysine. Furthermore, peptides that bind to HLA-A24 have a binding motif in which the second amino acid from the N-terminus is tyrosine, phenylalanine, methionine, or tryptophan and/or the C-terminal amino acid is lysine, iso-lysine, or phenylalanine.
抗原胜肽可以以藉由由蛋白質的全長序列並基於結合模體來預測MHC限制性胜肽的方式來決定，實際上可藉由鑑定抗原呈現出的天然胜肽來決定。作為天然胜肽的鑑定方法，可以是在該技術領域中習知的方法或該等方法之組合，並且可列舉例如國際公開第2015/050259號、國際公開第2017/115798號所記載的方法等。本案的抗體，由於是可辨識抗原呈現於細胞表面的抗原胜肽者，所以較佳是抗原胜肽為天然胜肽。根據發明人的操作，HF10(HWNDTRPAHF(序列編號3))被鑑定為人類癌症細胞中的PVT1的天然胜肽。從而，在一更佳態樣中，抗原胜肽為由序列編號3表示的HF10。Antigenic peptides can be determined by predicting MHC-restricted peptides based on binding motifs from the full-length protein sequence. In practice, they can be determined by identifying naturally occurring peptides presented by the antigen. Methods for identifying naturally occurring peptides can be methods known in the art, or a combination of such methods, including those described in International Publication Nos. 2015/050259 and 2017/115798. Because the antibodies in this application are designed to recognize antigenic peptides presented on the cell surface, naturally occurring peptides are preferred. According to the inventors' work, HF10 (HWNDTRPAHF (SEQ ID NO: 3)) was identified as a naturally occurring peptide of PVT1 in human cancer cells. Therefore, in a more preferred embodiment, the antigenic peptide is HF10 represented by SEQ ID NO: 3.
在本案的一較佳態樣中，MHC為HLA。在一更佳態樣中，MHC為HLA I類分子。在一進一步較佳態樣中，MHC為HLA-A02。在另一進一步更佳態樣中，MHC為HLA-A24。In a preferred embodiment of the present invention, the MHC is HLA. In an even more preferred embodiment, the MHC is an HLA class I molecule. In an even more preferred embodiment, the MHC is HLA-A02. In another even more preferred embodiment, the MHC is HLA-A24.
如同上述，本案的抗體只要能特異性地辨識抗原胜肽HF10與MHC分子之複合體即可，並無特別限定，也可以為可進一步辨識其他分子並結合的抗體。從而，本案的抗體，在一較佳態樣中，除了辨識癌細胞抗原胜肽HF10與MHC分子之複合體的抗原結合部位以外，還可以具有對於其他分子的結合區域。該結合區域，典型上是抗原結合部位，但是不限於此，例如也可以是相對於細胞表面受體的配體。在一較佳態樣中，本案的抗體是可對於2個以上的分子特異性地結合的抗體、即多特異性抗體。在此處，本案中所謂的「多特異性抗體」，意指具有至少1個抗原結合部位並可對於2個以上的分子特異性地結合的抗體。從而，例如當諸如本案的雙特異性抗體時，意指如下抗體，其為具有至少1個抗原結合部位並可對於2個以上的分子特異性地結合的抗體，並且至少1個抗原結合部位為辨識癌細胞抗原胜肽HF10與MHC分子之複合體的抗原結合部位，還進一步能夠與另一個其他分子特異性地結合。對於其他分子的特異性結合，可藉由其他抗原結合部位來達成，也可以藉由其他方法例如對於其他分子的配體區域等來達成。As mentioned above, the antibodies in this case are not particularly limited as long as they can specifically recognize the complex of the antigen peptide HF10 and the MHC molecule. They can also be antibodies that can further recognize and bind to other molecules. Therefore, in a preferred embodiment, the antibodies in this case, in addition to the antigen-binding site that recognizes the complex of the cancer cell antigen peptide HF10 and the MHC molecule, can also have binding regions for other molecules. This binding region is typically an antigen-binding site, but is not limited to this. For example, it can also be a ligand for a cell surface receptor. In a preferred embodiment, the antibodies in this case are antibodies that can specifically bind to two or more molecules, that is, multispecific antibodies. As used herein, "multispecific antibodies" refer to antibodies that possess at least one antigen-binding site and can specifically bind to two or more molecules. Thus, for example, bispecific antibodies, as used herein, refer to antibodies that possess at least one antigen-binding site and can specifically bind to two or more molecules, with at least one antigen-binding site specifically binding to a complex of the cancer cell antigen peptide HF10 and an MHC molecule, and further specifically binding to another molecule. Specific binding to another molecule can be achieved through other antigen-binding sites or by other methods, such as binding to a ligand region of another molecule.
本案的抗體當為多特異性抗體時，本案的抗體所辨識的其他抗原並無特別限定，較佳是有助於癌細胞的處置者。作為該抗原，可列舉例如表現於CD3、CD28、CD40、PD1、CTLA4、TIGIT、OX40、CD137等的免疫細胞的細胞表面的蛋白質等。在本案的一較佳態樣中，作為其他抗原，可列舉CD3和CD28等的T細胞表面蛋白質。從而，本案的多特異性抗體，在一較佳態樣中，在辨識癌細胞抗原胜肽與MHC分子之複合體的抗原結合部位以外，具有對於CD3及/或CD28的結合區域。該結合區域可以是抗原結合部位，也可以例如是CD80作為對於CD28的配體等。When the antibody in this case is a multispecific antibody, the additional antigens recognized by the antibody are not particularly limited, but preferably contribute to the treatment of cancer cells. Examples of such antigens include proteins expressed on the cell surface of immune cells such as CD3, CD28, CD40, PD1, CTLA4, TIGIT, OX40, and CD137. In a preferred embodiment of this case, additional antigens include T cell surface proteins such as CD3 and CD28. Therefore, in a preferred embodiment, the multispecific antibody in this case possesses binding regions for CD3 and/or CD28 in addition to the antigen-binding site that recognizes the complex of a cancer cell antigen peptide and an MHC molecule. The binding region may be an antigen binding site, or may be, for example, CD80 as a ligand for CD28.
根據發明人的操作，針對上述天然抗原胜肽HF10，具有辨識該等抗原胜肽與HLA-A24之複合體的抗原結合部位之抗體會被篩選出來。從而，本案的一較佳態樣中，辨識癌細胞抗原胜肽HF10與MHC分子之複合體的抗原結合部位，為包含在序列編號4～8的任一序列中所記載的胺基酸序列或GRD、GKN或者NDN的胺基酸序列者。在本案的另一較佳態樣中，辨識癌細胞抗原胜肽HF10與MHC分子之複合體的抗原結合部位，可以是包含在序列編號4～18的任一序列中所記載的胺基酸序列中的1個或2個胺基酸經取代而成之胺基酸序列者、或者為GRD、GKN或者NDN的胺基酸序列。該胺基酸取代，較佳是保守取代，其是酸性胺基酸彼此的取代、鹼性胺基酸彼此的取代或者中性胺基酸彼此的取代等。According to the inventors' work, antibodies with antigen-binding sites that recognize the complex of the aforementioned natural antigen peptide HF10 and HLA-A24 are screened. Therefore, in a preferred embodiment of the present invention, the antigen-binding site that recognizes the complex of the cancer cell antigen peptide HF10 and the MHC molecule comprises an amino acid sequence described in any of SEQ ID NOs. 4-8, or an amino acid sequence of GRD, GKN, or NDN. In another preferred embodiment of the present invention, the antigen-binding site that recognizes the complex of the cancer cell antigen peptide HF10 and the MHC molecule may comprise an amino acid sequence in which one or two amino acids are substituted in the amino acid sequence described in any of SEQ ID NOs. 4-18, or an amino acid sequence of GRD, GKN, or NDN. The amino acid substitution is preferably a conservative substitution, such as substitution of acidic amino acids, substitution of basic amino acids, or substitution of neutral amino acids.
作為本案的抗體的典型示例，可列舉：具有辨識由序列編號3記載的胺基酸序列所構成之癌細胞抗原胜肽(HF10)與HLA-A24之複合體的抗原結合部位之抗體；具有辨識由序列編號3記載的胺基酸序列所構成之癌細胞抗原胜肽(HF10)與HLA-A24之複合體的抗原結合部位及辨識CD3的抗原結合部位之雙特異性抗體(作為一較佳態樣可列舉BiTE(雙特異性T細胞衍生抗體))；具有辨識由序列編號3記載的胺基酸序列所構成之癌細胞抗原胜肽(HF10)與HLA-A24之複合體的抗原結合部位、辨識CD3的抗原結合部位及辨識CD28的抗原結合部位之三特異性抗體；具有辨識由序列編號3記載的胺基酸序列所構成之癌細胞抗原胜肽(HF10)與HLA-A24之複合體的抗原結合部位、辨識CD3的抗原結合部位及CD80區域之三特異性抗體等。Typical examples of antibodies in this case include: antibodies having an antigen binding site that recognizes a complex of a cancer cell antigen peptide (HF10) composed of the amino acid sequence set forth in SEQ ID NO: 3 and HLA-A24; bispecific antibodies having an antigen binding site that recognizes a complex of a cancer cell antigen peptide (HF10) composed of the amino acid sequence set forth in SEQ ID NO: 3 and HLA-A24 and an antigen binding site that recognizes CD3 (a preferred embodiment includes BiTE (bispecific T cell-derived antibody)). a trispecific antibody having an antigen-binding site that recognizes a complex of a cancer cell antigen peptide (HF10) composed of the amino acid sequence set forth in SEQ ID NO: 3 and HLA-A24, an antigen-binding site that recognizes CD3, and an antigen-binding site that recognizes CD28; and a trispecific antibody having an antigen-binding site that recognizes a complex of a cancer cell antigen peptide (HF10) composed of the amino acid sequence set forth in SEQ ID NO: 3 and HLA-A24, an antigen-binding site that recognizes CD3, and a CD80 region.
本案的抗體較佳是：在抗原結合部位包含互補決定區域(CDR，complementarity-determining regions)1、互補決定區域(CDR)2及互補決定區域(CDR)3中的至少1種，並且在重鏈及輕鏈這兩處包含互補決定區域(CDR)1、互補決定區域(CDR)2及互補決定區域(CDR)3中的至少1種。 進一步，在一較佳態樣中，本案的抗體的抗原結合部位可以是包含在胺基酸序列編號4～18的任一序列中所記載的胺基酸序列中的1個或2個胺基酸經取代而成之胺基酸序列者。 此外，在一較佳態樣中，本案的抗體在抗原結合部位，特別是包含序列編號4及/或序列編號7所記載的胺基酸序列作為互補決定區域(CDR)1、序列編號5及/或GRD的胺基酸序列作為互補決定區域(CDR)2以及序列編號6及/或序列編號8所記載的胺基酸序列作為互補決定區域(CDR)3。 此外，在另一較佳態樣中，本案的抗體在抗原結合部位，特別是包含序列編號9及/或序列編號12所記載的胺基酸序列作為互補決定區域(CDR)1、序列編號10及/或GNK的胺基酸序列作為互補決定區域(CDR)2以及序列編號11及/或序列編號13所記載的胺基酸序列作為互補決定區域(CDR)3。 此外，在另一進一步較佳態樣中，本案的抗體在抗原結合部位，特別是包含序列編號14及/或序列編號17所記載的胺基酸序列作為互補決定區域(CDR)1、序列編號15及/或NDN的胺基酸序列作為互補決定區域(CDR)2以及序列編號16及/或序列編號18所記載的胺基酸序列作為互補決定區域(CDR)3。 進一步，在一較佳態樣中，本案的抗體的抗原結合部位可以是包含在序列編號4～18的任一序列中所記載的胺基酸序列中的1個或2個胺基酸經取代而成之胺基酸序列者。The antibody of the present invention preferably comprises at least one of complementarity-determining regions (CDRs) 1, 2, and 3 in its antigen-binding site, and at least one of CDRs 1, 2, and 3 in both the heavy and light chains.Furthermore, in a preferred embodiment, the antigen-binding site of the antibody of the present invention may comprise an amino acid sequence in which one or two amino acids in any of the amino acid sequences set forth in amino acid sequences 4 to 18 are substituted.Furthermore, in a preferred embodiment, the antibody of the present invention comprises, in its antigen-binding site, the amino acid sequence set forth in SEQ ID NO: 4 and/or SEQ ID NO: 7 as complementary determining region (CDR) 1, the amino acid sequence set forth in SEQ ID NO: 5 and/or GRD as complementary determining region (CDR) 2, and the amino acid sequence set forth in SEQ ID NO: 6 and/or SEQ ID NO: 8 as complementary determining region (CDR) 3. Furthermore, in another preferred embodiment, the antibody of the present invention comprises, in its antigen-binding site, the amino acid sequence set forth in SEQ ID NO: 9 and/or SEQ ID NO: 12 as complementary determining region (CDR) 1, the amino acid sequence set forth in SEQ ID NO: 10 and/or GNK as complementary determining region (CDR) 2, and the amino acid sequence set forth in SEQ ID NO: 11 and/or SEQ ID NO: 13 as complementary determining region (CDR) 3. In another more preferred embodiment, the antibody of the present invention comprises, in its antigen-binding site, the amino acid sequence set forth in SEQ ID NO: 14 and/or SEQ ID NO: 17 as complementary determining region (CDR) 1, the amino acid sequence set forth in SEQ ID NO: 15 and/or NDN as complementary determining region (CDR) 2, and the amino acid sequence set forth in SEQ ID NO: 16 and/or SEQ ID NO: 18 as complementary determining region (CDR) 3.Furthermore, in a more preferred embodiment, the antigen-binding site of the antibody of the present invention may comprise an amino acid sequence in which one or two amino acids in the amino acid sequence set forth in any of SEQ ID NOs: 4 to 18 are substituted.
進一步，在一較佳態樣中，本案的抗體可以是具有VH與VL者，該VH由與表示於序列編號22(選殖株1的VH)的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列所構成，該VL由與表示於序列編號23(選殖株1的VL)的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列所構成。 進一步，在一較佳態樣中，本案的抗體可以是具有VH與VL者，該VH由與表示於序列編號24(選殖株2的VH)的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列所構成，該VL由與表示於序列編號25(選殖株2的VL)的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列所構成。 進一步，在一較佳態樣中，本案的抗體可以是具有VH與VL者，該VH由與表示於序列編號26(選殖株5的VH)的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列所構成，該VL由與表示於序列編號27(選殖株5的VL)的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列所構成。 進一步，在一較佳態樣中，本案的抗體可以是包含VH及VL之scFv，VH及VL的較佳態樣如同上述。作為scFv，較佳是具有與由序列編號19～21中任一序列表示的胺基酸序列至少80%、90%、95%、98%或99%相同的胺基酸序列者。 此外，本案的抗體可以是衍生自嵌合體抗體、人源抗體及人類抗體中的任一抗體者。 再者，本案的抗體中，3個CDR之中，CDR3尤其在抗原結合方面是重要的。Furthermore, in a preferred embodiment, the antibody of the present invention may comprise a VH and a VL, wherein the VH is composed of an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 22 (VH of clone 1), and the VL is composed of an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 23 (VL of clone 1). Furthermore, in a preferred embodiment, the antibody of the present invention may comprise a VH and a VL, wherein the VH is composed of an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 24 (VH of clone 2), and the VL is composed of an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 25 (VL of clone 2). Furthermore, in a preferred embodiment, the antibody of the present invention may comprise a VH and a VL, wherein the VH comprises an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 26 (VH from clone 5), and the VL comprises an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 27 (VL from clone 5).Further, in a preferred embodiment, the antibody of the present invention may be an scFv comprising VH and VL, and preferred embodiments of VH and VL are as described above. The scFv preferably comprises an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by any of SEQ ID NOs: 19-21. Furthermore, the antibodies in this application may be derived from chimeric, human, or human antibodies.Furthermore, among the three CDRs in the antibodies in this application, CDR3 is particularly important for antigen binding.
＜2＞本案的醫藥組成物 本案的一實施形態，關於一種醫藥組成物，其包含本案的抗體。如同上述，本案的抗體能夠特異性地辨識抗原呈現於癌細胞的表面的癌細胞抗原胜肽，因此能作為各種用途的醫藥組成物的有效成分來使用。從而，作為在本案的醫藥組成物中可包含來作為有效成分的本案的抗體，能夠使用上述＜1＞已詳述的任意抗體。 本案的醫藥組成物，包含本案的抗體作為有效成分。本案的醫藥組成物可進一步包含至少一種藥學上可接受的載體。在此處，作為藥學上可接受的載體，可列舉例如：穩定劑、增溶劑、懸浮劑、乳化劑、止痛劑、緩衝劑、保鮮劑、防腐劑、pH調整劑及抗氧化劑等。<2> Pharmaceutical Compositions of the Present ApplicationOne embodiment of the present application relates to a pharmaceutical composition comprising the antibody of the present application. As described above, the antibody of the present application is capable of specifically recognizing cancer cell antigen peptides displayed on the surface of cancer cells and can therefore be used as an active ingredient in pharmaceutical compositions for various applications. Therefore, any of the antibodies described in detail in <1> above can be used as the active ingredient of the antibody of the present application that can be included in the pharmaceutical composition of the present application.The pharmaceutical composition of the present application comprises the antibody of the present application as an active ingredient. The pharmaceutical composition of the present application may further comprise at least one pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include, for example, stabilizers, solubilizers, suspending agents, emulsifiers, analgesics, buffers, preservatives, preservatives, pH adjusters, and antioxidants.
本案的醫藥組成物不僅能作為用以處置癌細胞的處置劑(亦即，癌治療劑)來利用，例如也能夠用於癌細胞的檢測劑、用以診斷作為處置對象的患者對於疫苗免疫療法的有效性的伴隨式診斷劑等的用途。 亦即，本案的抗體，本案的抗體藉由辨識呈現於癌細胞的表面的癌細胞抗原胜肽而結合於癌細胞表面，藉此能發揮對於該癌細胞的毒殺活性。從而，在一較佳態樣中，本案的醫藥組成物是用以預防及/或處置癌症的醫藥組成物。The pharmaceutical composition of this invention can be used not only as a therapeutic agent for treating cancer cells (i.e., a cancer treatment agent), but can also be used, for example, as a cancer cell detection agent or as a companion diagnostic agent for diagnosing the effectiveness of vaccine immunotherapy in patients being treated.That is, the antibodies of this invention bind to the surface of cancer cells by recognizing cancer cell antigen peptides presented on the surface of cancer cells, thereby exerting cytotoxic activity against the cancer cells. Therefore, in a preferred embodiment, the pharmaceutical composition of this invention is a pharmaceutical composition for preventing and/or treating cancer.
在此處的癌症的「預防」中，不僅是對於患者的癌症罹患的預防，還包括：對於藉由手術切除原發性腫瘤的患者的復發預防；無法藉由手術、放射性療法或者藥物療法等的癌症治療完全去除的腫瘤的轉移預防等。此外，癌症的「處置」中，不僅是使癌症縮小的癌症的治療及症狀改善，還包括進程防止等，該進程防止為抑制癌症細胞的增殖、腫瘤的擴大或來自原發性腫瘤的癌細胞的轉移。The term "cancer prevention" here encompasses not only the prevention of cancer in patients but also the prevention of recurrence in patients whose primary tumors have been surgically removed, and the prevention of metastasis of tumors that cannot be completely removed by surgery, radiation therapy, or medication. Furthermore, the term "cancer treatment" encompasses not only cancer shrinkage and symptom improvement but also the prevention of cancer progression by inhibiting cancer cell proliferation, tumor expansion, or metastasis from the primary tumor.
本案的醫藥組成物能夠對於可能會有腫瘤的任何生物個體進行投予，但是較佳是人類及非人哺乳動物(例如，小鼠、大鼠、天竺鼠、倉鼠等的囓齒類；黑猩猩等的靈長類；牛、山羊、綿羊等的偶蹄目；馬等的奇蹄目；兔、狗、貓等)的個體，更佳是將人類個體設為投予對象者。The pharmaceutical composition of this invention can be administered to any biological subject that may have a tumor, but preferably to humans and non-human mammals (e.g., rodents such as mice, rats, guinea pigs, and hamsters; primates such as chimpanzees; artiodactyls such as cattle, goats, and sheep; perissodactyls such as horses; rabbits, dogs, and cats), and more preferably, to humans.
此外，當將本案的醫藥組成物用作腫瘤檢測劑時，檢測對象的細胞集團，能夠對於源自由上述生物個體所獲得的任意活體樣品的細胞集團使用，但是較佳是源自由人類所獲得的活體樣品的細胞集團，更佳是如下細胞集團，該細胞集團包含源自已確認在組織的細胞中大致不會表現PVT1的卵巢以外的組織，例如選自由心臟、大腦、胎盤、肺、肝臟、骨骼肌、腎臟、胰臟、脾臟、胸腺、攝護腺、小腸、大腸及血液所組成之群組中的1種或2種以上的活體樣品的細胞。Furthermore, when the pharmaceutical composition of the present invention is used as a tumor detection agent, the cell population to be detected can be a cell population derived from any living sample obtained from the aforementioned biological individuals, but preferably a cell population derived from a living sample obtained from a human. More preferably, the cell population comprises cells derived from one or more living samples of tissues other than the ovary, which have been confirmed to substantially not express PVT1 in the cells of the tissue, such as heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, small intestine, large intestine, and blood.
如同上述，本案的抗體的一較佳態樣，是具有辨識衍生自PVT1的ORF的癌細胞抗原胜肽HF10與HLA-A24之複合體的抗原結合部位者。從而，本案的醫藥組成物，尤其能夠適合用於罹患有會表現PVT1的癌症的對象。此外，尤其能夠適合用於具有HLA-A24作為HLA之對象。具體而言，能夠使用來用以預防或處置大腸癌、肺癌、乳癌、骨髓瘤、口腔癌、口腔鱗狀細胞癌、骨肉瘤、胰臟癌、皮膚癌、攝護腺癌等的癌症(腫瘤)。As mentioned above, a preferred embodiment of the antibody of this invention possesses an antigen-binding site that recognizes a complex of the cancer cell antigen peptide HF10, derived from the ORF of PVT1, and HLA-A24. Therefore, the pharmaceutical composition of this invention is particularly suitable for use in subjects suffering from cancers expressing PVT1. Furthermore, it is particularly suitable for use in subjects possessing HLA-A24 as their HLA. Specifically, it can be used to prevent or treat cancers (tumors) such as colorectal cancer, lung cancer, breast cancer, myeloma, oral cancer, oral squamous cell carcinoma, osteosarcoma, pancreatic cancer, skin cancer, and prostate cancer.
此外，近年來已知由於癌細胞會藉由遮蔽來自免疫細胞的攻擊來避免受到免疫系統排除，而該遮蔽是利用被稱為「免疫檢查點」的機制等，免疫檢查點原本是用以抑制對自體過度的免疫反應和對於正常組織的傷害而具備者。從而，藉由在癌細胞中抑制免疫檢查點的功能，即能夠使來自免疫細胞的攻擊變得有效。本案的醫藥組成物，在一態樣中，是利用能傷害會與作為有效成分的抗體結合之癌細胞的免疫細胞，藉此發揮抗腫瘤效果，所以認為藉由一併抑制免疫檢查點的功能，能發揮更高的治療效果。從而，在一較佳態樣中，本案的醫藥組成物也能與免疫檢查點抑制劑一起使用。Furthermore, it has been recently discovered that cancer cells avoid immune elimination by shielding themselves from attacks by immune cells. This shielding utilizes mechanisms known as "immune checkpoints," which are designed to suppress excessive immune responses to the body and damage to normal tissues. Therefore, by inhibiting the function of immune checkpoints in cancer cells, the attack by immune cells can be made more effective. In one aspect, the pharmaceutical composition of this case utilizes immune cells that can damage cancer cells that bind to the antibody as an active ingredient, thereby exerting its anti-tumor effect. Therefore, by also inhibiting the function of immune checkpoints, it is believed that a higher therapeutic effect can be achieved. Therefore, in a preferred embodiment, the pharmaceutical composition of the present invention can also be used together with an immune checkpoint inhibitor.
本案中，當將某劑A與其他劑B「一起使用」或「併用」時，意指在劑A發揮效果的期間使得劑B成為可發揮效果的狀態。從而，可以在與投予劑A的同時將劑B進行投予，也可以在投予劑A後隔出固定的間隔再將劑B進行投予。此外，劑A與劑B可以是相同的投予形態，也可以是不同的投予形態。進一步，只要劑A或劑B不會喪失其效果，即可混合劑A與劑B而作成一組成物。In this case, when agent A is used "together" or "in combination" with another agent B, it means that agent B is in a state where it can exert its effect while agent A is exerting its effect. Therefore, agent B can be administered simultaneously with agent A or a fixed interval after agent A. Furthermore, agent A and agent B can be administered in the same or different forms. Furthermore, agent A and agent B can be mixed to form a single composition, as long as the effectiveness of either agent A or agent B is not compromised.
作為本態樣中的免疫檢查點抑制劑，只要不會妨礙本案的醫藥組成物的抗原辨識能力，即能夠使用習知且任意的劑來作為免疫檢查點抑制劑。作為習知為免疫檢查點抑制劑者，雖然不限於此，但可列舉例如：抗PD-1抗體、抗PD-L1抗體、抗CTLA-4抗體、抗TIM-3抗體、抗LAG-3抗體、抗B7-H3抗體、抗B7-H4抗體、抗B7-H5抗體、抗TIGIT抗體等。As immune checkpoint inhibitors in this embodiment, any known and arbitrary agent can be used as an immune checkpoint inhibitor, as long as it does not interfere with the antigen recognition ability of the pharmaceutical composition of this invention. Examples of known immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-B7-H5 antibodies, and anti-TIGIT antibodies.
此外，作為本案的醫藥組成物的劑型，並無特別限定，可列舉：油乳液(乳液製劑)、高分子奈米粒子、脂質體製劑、結合於直徑數μm的珠子的顆粒狀製劑、結合有脂質之製劑、微球製劑、微膠囊製劑等。 作為投予方法，可列舉皮內注射投予、皮下注射投予、肌肉注射投予、靜脈注射投予等習知且任意的投予方法。製劑中的本案的醫藥組成物的投予量，能基於治療的目標疾病、患者的年齡、體重等適當地調整，但是一般是0.0001 mg～1000 mg，較佳是0.001 mg～1000 mg，更佳是0.1 mg～10 mg，並且較佳是在數天或數個月內投予一次。Furthermore, the dosage form of the pharmaceutical composition in this case is not particularly limited, and examples include: oil emulsions (emulsion preparations), polymer nanoparticles, liposome preparations, particulate preparations bound to beads with a diameter of several microns, lipid-bound preparations, microsphere preparations, microcapsule preparations, etc.Administration methods include any known and arbitrary methods, such as intradermal injection, subcutaneous injection, intramuscular injection, and intravenous injection. The dosage of the pharmaceutical composition of this invention in the formulation can be appropriately adjusted based on the target disease, patient age, weight, etc., but is generally 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, and more preferably 0.1 mg to 10 mg. It is preferably administered once every few days or months.
此外，如同上述，也能夠藉由過繼免疫細胞療法來處置癌症，其使用了導入有嵌合抗原受體(CAR)之T細胞(CAR-T細胞)，該嵌合抗原受體組合有本案的抗體所具有的抗原結合部位。本案中，「嵌合抗原受體」是以如下方式設計而成之嵌合體蛋白質分子：使N末端側具有單鏈抗體(scFv)並使C末端側具有構成T細胞受體(TCR)/CD3複合體的分子中的CD3ζ鏈，該單鏈抗體(scFv)是可辨識存在於癌細胞的細胞表面的分子之抗體的抗體可變區域的輕鏈與重鏈直線地排列而成者。該嵌合抗原受體，若以scFv區域辨識特定的抗原，即可經由CD3ζ鏈產生T細胞的活化。為了增加T細胞的活化，在scFv與ζ鏈之間可組合1個或2個以上的協同刺激分子(例如CD28、4-1BB、ICOS等)。從而，作為scFv能夠使用本案的抗體來製作CAR。辨識癌細胞抗原胜肽與MHC之複合體的CAR，能夠辨識呈現有會受到CTL標記的癌細胞抗原胜肽之癌細胞、吞噬癌細胞而在MHC I類分子上呈現有腫瘤抗原胜肽的樹狀細胞等，所以包含導入前述CAR之基因改造T細胞(CAR-T細胞)之醫藥組成物也包括在本案的醫藥組成物中。此外，本案的醫藥組成物中，也包括：包含導入前述CAR之基因改造NK細胞(CAR-T細胞)之醫藥組成物、包含導入前述CAR之基因改造巨噬細胞(MΦ)(CAR-MΦ)之醫藥組成物。Furthermore, as mentioned above, cancer can also be treated through relay immune cell therapy, which uses T cells (CAR-T cells) that have been introduced with a chimeric antigen receptor (CAR) that combines the antigen-binding site of the antibody described herein. In this case, the "CAR" is a chimeric protein molecule designed as follows: a single-chain antibody (scFv) is attached to the N-terminus, and the CD3ζ chain of a molecule that constitutes the T cell receptor (TCR)/CD3 complex is attached to the C-terminus. The single-chain antibody (scFv) is composed of a linear arrangement of the light and heavy chains of the variable region of an antibody that recognizes molecules present on the cell surface of cancer cells. If the chimeric antigen receptor (CAR) recognizes a specific antigen using the scFv region, it can activate T cells via the CD3ζ chain. To increase T cell activation, one or more co-stimulatory molecules (such as CD28, 4-1BB, ICOS, etc.) can be combined between the scFv and the ζ chain. Thus, the antibody in this case can be used as an scFv to create a CAR. CARs that recognize complexes of cancer cell antigen peptides and MHC can recognize cancer cells that present cancer cell antigen peptides that are labeled by CTLs, dendritic cells that phagocytize cancer cells and present tumor antigen peptides on MHC class I molecules, and so on. Therefore, pharmaceutical compositions containing genetically modified T cells (CAR-T cells) that have been introduced with the aforementioned CAR are also included in the pharmaceutical compositions of this application. Furthermore, pharmaceutical compositions of this application also include pharmaceutical compositions containing genetically modified NK cells (CAR-T cells) that have been introduced with the aforementioned CAR, and pharmaceutical compositions containing genetically modified macrophages (MΦ) that have been introduced with the aforementioned CAR (CAR-MΦ).
＜3＞腫瘤的檢測方法(檢查方法、診斷方法) 本案的一實施態樣，關於一種腫瘤的檢測方法(檢查方法、診斷方法)，其利用本案的抗體。 使用本案的抗體之本案的檢測方法(診斷方法)，典型上如下：採取受試者的血液或是利用活體組織切片採取懷疑為腫瘤的受試組織的一部分，藉由本案的抗體檢測並測定包含於其中的具有癌細胞抗原胜肽與MHC分子之複合體的細胞量，藉此檢測、檢查或診斷是否罹患大腸癌、肺癌、乳癌、骨髓瘤、口腔癌、胰臟癌、皮膚癌、攝護腺癌等的癌症(腫瘤)或者其罹患程度。<3> Tumor Detection Method (Inspection Method, Diagnostic Method)One embodiment of the present invention relates to a tumor detection method (inspection method, diagnostic method) utilizing the present antibody.The present detection method (diagnostic method) utilizing the present antibody typically involves sampling blood from a subject or obtaining a portion of a tissue suspected of being a tumor using a biopsy. The antibody is then used to detect and measure the amount of cells containing a complex of a cancer cell antigen peptide and a MHC molecule, thereby detecting, examining, or diagnosing the presence or severity of a cancer (tumor) such as colorectal cancer, lung cancer, breast cancer, myeloma, oral cancer, pancreatic cancer, skin cancer, or prostate cancer.
使用本案的抗體之本案的檢測(檢查)方法的特定態樣是包含如下步驟(a)及(b)及任意的(c)者。 (a)使自受試者所獲得的活體樣品與本案的腫瘤檢測劑接觸的步驟； (b)以與上述腫瘤檢測劑結合的細胞量作為指標，來測定該活體樣品中的呈現出癌細胞抗原胜肽與HLA抗原之複合體的細胞量的步驟； (c)根據步驟(b)的結果來判斷是否罹患癌症的步驟。 使用本案的抗體之本案的診斷方法的特定態樣包含上述步驟(a)、(b)及(c)。 作為在此處使用的活體樣品，能夠列舉由受試者的活體組織(懷疑存在癌細胞的組織及其周邊組織或者血液等)所調製成的樣品。具體而言，可列舉包含自該組織所採取到的組織細胞之樣品等。A specific embodiment of the detection (examination) method of the present invention using the antibody of the present invention comprises the following steps (a) and (b), and optionally (c).(a) contacting a biopsy sample obtained from a subject with the tumor detection agent of the present invention;(b) measuring the amount of cells in the biopsy sample that express a complex of a cancer cell antigen peptide and an HLA antigen, using the amount of cells bound to the tumor detection agent as an indicator;(c) determining whether or not a subject has cancer based on the result of step (b).A specific embodiment of the diagnostic method of the present invention using the antibody of the present invention comprises the above steps (a), (b), and (c). Examples of biological samples used herein include samples prepared from a subject's biological tissue (tissue suspected of containing cancer cells, surrounding tissue, or blood, etc.). Specifically, examples include samples containing tissue cells collected from the tissue.
有無腫瘤的預測、判定、判斷或診斷，例如能夠藉由測定受試者的血液或懷疑為腫瘤的受試組織中的與本案的抗體結合的細胞量來實行。此時，能夠根據情況藉由如下方式來實行：以正常的相對應組織中的與本案的抗體結合的細胞程度等為基準值，比較該基準值與由受試者所獲得的樣品中的前述程度來判定兩者的差異。 在此處，受試者的受試組織與正常的相對應組織之前述程度的比較，能夠與藉由以受試者的活體樣品與正常人的活體樣品作為對象的測定一併實行。在沒有一併實行的情況下，能夠使用複數(至少2個，較佳是3個以上，更佳是5個以上)的正常組織，利用統一的測定條件進行測定，將測定所獲得的與本案的抗體結合的細胞量的平均值或統計上的中間值視為正常人的數值也就是基準值，來進行比較。The prediction, determination, judgment, or diagnosis of the presence or absence of a tumor can be performed, for example, by measuring the amount of cells binding to the antibody in a subject's blood or a test tissue suspected of harboring a tumor. Depending on the circumstances, this can be achieved by comparing the level of cells binding to the antibody in a sample obtained from the subject with the baseline level, using the baseline level as a benchmark, and determining the difference between the two.Here, the comparison of the levels in the test tissue of the subject and the corresponding normal tissue can be performed in conjunction with measurements of the subject's biopsy sample and those of a healthy individual. If this is not possible, multiple (at least 2, preferably 3 or more, and more preferably 5 or more) normal tissues can be used for measurement using standardized measurement conditions. The average or statistical median value of the number of cells binding to the antibody in question obtained from the measurement can be used as the normal value, or benchmark, for comparison.
受試者是否罹患癌症的判斷，例如能夠以如下情況為指標來實行：該受試者的組織中的與本案的抗體結合的細胞比起正常者的抗體結合的細胞的程度，例如多於2倍以上，較佳是3倍以上。Whether a subject has cancer can be determined, for example, by using as an indicator that the level of cells in the subject's tissues that bind to the antibody of this invention is, for example, more than twice, preferably more than three times, that of cells in a normal subject that bind to the antibody.
＜4＞ 癌症的預防及/或處置方法 本案的一實施態樣也關於一種預防及/或處置對象的癌症的方法，該方法包含如下步驟：將本案的抗體或CAR-T細胞的有效量對需要進行投予的對象進行投予。 本案中的「對象」，只要是會罹患癌症的生物個體即可，可以是任何生物個體，較佳是人類及非人哺乳類(例如，小鼠、大鼠、天竺鼠、倉鼠等的囓齒類；黑猩猩等的靈長類；牛、山羊、綿羊等的偶蹄目；馬等的奇蹄目；兔、狗、貓等)的個體，更佳是人類個體。本案中，對象可以設為健康個體，也可以設為罹患某種疾病的個體，但是意圖進行癌症的預防及/或處置的情況下，典型上意指罹患癌症或者具有罹患癌症的風險之對象。本案的一態樣中，對象為HLA-A24或HLA-A02陽性。本案的一較佳態樣中，對象罹患PVT1陽性的癌症或具有罹患的風險。本案的一態樣中，對象為HLA-A24陽性並且罹患PVT1陽性的癌症或具有罹患的風險。<4> Methods for Preventing and/or Treating CancerOne embodiment of the present invention also relates to a method for preventing and/or treating cancer in a subject, comprising administering an effective amount of an antibody or CAR-T cell described herein to a subject in need of administration.The term "subject" herein refers to any biological subject susceptible to cancer, preferably humans and non-human mammals (e.g., rodents such as mice, rats, guinea pigs, and hamsters; primates such as chimpanzees; even-toed ungulates such as cattle, goats, and sheep; and horses; rabbits, dogs, and cats), and more preferably humans. In this case, the subject can be a healthy individual or an individual suffering from a certain disease. However, when the purpose is to prevent and/or treat cancer, it typically refers to a subject suffering from or at risk of developing cancer. In one aspect of this case, the subject is HLA-A24 or HLA-A02 positive. In a preferred aspect of this case, the subject suffers from or is at risk of developing PVT1-positive cancer. In another aspect of this case, the subject is both HLA-A24-positive and PVT1-positive and suffers from or is at risk of developing cancer.
作為用於本案的預防/處置方法的本案的抗體及CAR-T細胞，可列舉本說明書中記載的任意者。所謂本案中的有效量，例如是可降低癌症的症狀或延遲或停止癌症的進程的量，較佳是可抑制或治癒癌症的量。此外，較佳是不會產生超過由投予產生的利益的不良影響的量。該量能夠藉由使用培養細胞等的體外(in vitro)試驗或針對小鼠、大鼠等的模式生物的試驗來適當決定，並且如此的試驗法為發明所屬技術領域中具有通常知識者所習知。有效成分的具體性用量，可考慮有關設為需要的對象的各種條件來決定，該條件例如為症狀的嚴重度、對象的一般健康狀態、年齡、體重、對象的性別、飲食、投予的時期及頻率、併用的醫藥、對於處置的反應性、劑型及對於處置的依從性等。The antibodies and CAR-T cells of the present invention used in the prevention/treatment methods of the present invention include any of those described in this specification. The so-called effective amount in the present invention is, for example, an amount that can reduce cancer symptoms or delay or halt cancer progression, and preferably an amount that can inhibit or cure cancer. Furthermore, it is preferably an amount that does not produce adverse effects that outweigh the benefits of administration. This amount can be appropriately determined by in vitro assays using cultured cells or other model organisms such as mice and rats, and such assays are known to those skilled in the art. The specific dosage of the active ingredient can be determined by considering various conditions related to the subject in need thereof, such as the severity of symptoms, the subject's general health condition, age, weight, sex, diet, time and frequency of administration, concomitant medications, responsiveness to treatment, dosage form, and compliance with treatment.
作為具體性的用量，例如當為本案的抗體的情況下，通常是0.0001 mg～2000 mg，較佳是0.001 mg～2000 mg，並且較佳是將其在1週～4週內進行一次投予。當為本案的CAR-T細胞時，通常為1×104～1×108個，較佳是1×105～1×107個，並且較佳是將其在1天～4週內進行一次投予。此外，作為投予方法，能夠使用皮內注射投予、皮下注射投予、肌肉注射投予、靜脈注射投予等的已知且任意的投予方法。Specific dosages, for example, for the antibodies of the present invention, are typically 0.0001 mg to 2000 mg, preferably 0.001 mg to 2000 mg, and are preferably administered once every 1 to 4 weeks. For CAR-T cells of the present invention, the dosage is typically 1×104 to 1×108 cells, preferably 1×105 to 1×107 cells, and are preferably administered once every 1 to 4 weeks. Any known administration method, such as intradermal, subcutaneous, intramuscular, or intravenous injection, can be used.
本案的預防/處置方法的一態樣，在進行投予的步驟之前，進一步包含選擇HLA-A24或HLA-A02陽性的對象作為預防/處置對象的步驟。本案的該態樣，在上述進行選擇的步驟之前，可進一步包含決定對象的HLA型的步驟。對象的HLA型的決定，能藉由已知的任意的手法來實行。此外，本案的預防/處置方法的一態樣，在進行投予的步驟之前，進一步包含選擇具有PVT1陽性的癌症之對象作為預防/處置對象的步驟。本案的該態樣，在上述進行選擇的步驟之前，可進一步包含檢測對象的PVT1陽性的癌症的步驟。對象的PVT1陽性的癌症的檢測，能夠使用上述＜3＞所述之腫瘤的檢測方法。本案的預防/處置方法的一態樣，在進行投予的步驟之前，進一步包含選擇HLA-A24陽性且具有PVT1陽性的癌症之對象作為預防/處置對象的步驟。本案的該態樣，在上述進行選擇的步驟之前，可進一步包含決定對象的HLA型的步驟及檢測對象的PVT1陽性的癌症的步驟。One aspect of the prevention/treatment method of this invention further includes, before the administration step, the step of selecting a subject who is HLA-A24 or HLA-A02 positive as the prevention/treatment subject. This aspect of the invention may further include, before the selection step, the step of determining the subject's HLA type. The subject's HLA type can be determined using any known method. Furthermore, one aspect of the prevention/treatment method of this invention further includes, before the administration step, the step of selecting a subject who has PVT1-positive cancer as the prevention/treatment subject. This aspect of the invention may further include, before the selection step, the step of detecting PVT1-positive cancer in the subject. Detection of PVT1-positive cancer in a subject can be performed using the tumor detection method described in <3> above. One aspect of the prevention/treatment method of this invention further includes, before the administration step, the step of selecting a subject with HLA-A24-positive and PVT1-positive cancer as a candidate for prevention/treatment. This aspect of the invention may further include, before the selection step, the steps of determining the subject's HLA type and detecting the subject's PVT1-positive cancer.
以下，藉由實施例具體地說明本發明，但是本發明不限於該等實施例。對於發明所屬技術領域中具有通常知識者而言能夠基於本發明的記載進行各種變更、修飾，並且該等變更、修飾也包括在本發明中。 [實施例]The present invention is described in detail below using examples. However, the present invention is not limited to these examples. Those skilled in the art would be able to make various changes and modifications based on the present invention, and such changes and modifications are also included in the present invention.[Examples]
例1.PVT1基因的表現CD8+T細胞可辨識HLA I類分子所呈現的胜肽。基於HLA配體組學的蛋白質體學分析(proteogenomic analysis of HLA ligandomes)顯示存在有源自基因體的非編碼區域的胜肽的子集(subset)。作為其中之一，顯示了如下情況：在多數的大腸癌組織中作為長鏈非編碼RNA(long noncoding RNA(lncRNA))的PVT1受到片段性地翻譯，成為PVT1胜肽(HF10)並且與細胞表面的HLA I類分子結合而形成複合體，而成為CD8+T細胞的目標(專利文獻1、非專利文獻1)。 因此，針對各種癌細胞中的PVT1 mRNA的表現進行研究。發現PVT1在以大腸癌為首的各種的癌症腫瘤中會過剩地表現(第1圖B)。尤其是，在大腸癌細胞株Colo320、肺癌細胞株LHK2、口腔鱗狀細胞癌細胞株MO-1000、骨肉瘤細胞株OS-2000等中發現較高的表現。除了在正常組織中可在卵巢中發現輕度的表現之外，表現程度較低(非專利文獻1)。Example1. PVT1Gene Expression: CD8+ T cells can recognize peptides presented by HLA class I molecules. Proteogenomic analysis of HLA ligandomes based on HLA ligandomics has revealed the presence of a subset of peptides derived from noncoding regions of the genome. One such finding has been that in most colorectal cancer tissues, PVT1, expressed as a long noncoding RNA (lncRNA), is fragmentarily translated into a PVT1 peptide (HF10). This peptide binds to HLA class I molecules on the cell surface, forming a complex that serves as a target for CD8+ T cells (Patent Document 1, Non-Patent Document 1). Therefore, research is underway to investigate the expression of PVT1 mRNA in various cancer cells. PVT1 has been found to be overexpressed in various cancer tumors, including colorectal cancer (Figure 1B). In particular, high expression is observed in the colorectal cancer cell line Colo320, the lung cancer cell line LHK2, the oral squamous cell carcinoma cell line MO-1000, and the osteosarcoma cell line OS-2000. While mild expression may be observed in normal tissues, expression is relatively low in the ovary (Non-Patent Reference 1).
例2.對於HLA-A24/天然抗原胜肽複合體具特異性的抗體的篩選(1)抗體噬菌體的作成 使用衍生自PVT1的ORF的天然抗原胜肽HF10(序列編號3)作為癌細胞抗原胜肽，並使用與Tsukahara et al., J Biol Chem., 2014, Aug 8;289(32):22035-47記載的噬菌體呈現法相同的方法來鑑定HLA-A24/HF10複合體。再者，以下的實施例3～6中，「癌細胞抗原胜肽」意指HF10。 具體而言，首先，基於scFv資料庫製作組合有DNA之嗜菌粒載體來感染大腸桿菌，該DNA編碼有以連接子將抗體的重鏈可變區域(VH區域)及輕鏈可變區域(VL區域)結合而成之胜肽。進一步使大腸桿菌感染輔助嗜菌體(Helper Phage)，來製作M13嗜菌體(抗體嗜菌體)，其呈現以連接子結合VH區域及VL區域之scFv。Example2.Screening of Antibodies SpecifictoHLA-A24/NativeAntigen Peptide Complexes (1) Preparation of Antibody Phages The natural antigen peptide HF10 (SEQ ID NO: 3) derived from the ORF of PVT1 was used as a cancer cell antigen peptide, and the HLA-A24/HF10 complex was identified using the same phage display method as described in Tsukahara et al., J Biol Chem., 2014, Aug 8;289(32):22035-47. In Examples 3 to 6 below, "cancer cell antigen peptide" refers to HF10. Specifically, a phagemid vector containing DNA encoding a peptide consisting of the antibody's heavy chain variable region (VH region) and light chain variable region (VL region) bound by a linker was first generated based on a scFv library and used to infect E. coli. The vector was then infected with a helper phage to produce M13 phage (antibody phage), which displays the scFv containing the linker-bound VH and VL regions.
(2)生物淘洗(biopanning) 在生物淘洗之前，使用以生物素修飾HLA-A24與HIV胜肽(RYLRDQQLLGI(序列編號28))之複合體而成之胜肽與卵白素結合磁珠，自嗜菌體資料庫去除非特異性結合嗜菌體。使用生物素修飾HLA-A24/HF10複合體與卵白素結合磁珠，重複進行3次自剩下的抗體嗜菌體篩選出(正向淘洗)會與各個複合體結合的抗體嗜菌體的步驟，獲得會對HLA-A24/HF10複合體具特異性結合的候選抗體嗜菌體。(2) BiopanningPrior to biopanning, a peptide consisting of a complex of HLA-A24 and HIV peptide (RYLRDQQLLGI (SEQ ID NO. 28)) modified with biotin and avidin-conjugated magnetic beads was used to remove non-specifically binding phages from the phage database. The steps of screening (forward panning) the antibody phages that bind to each complex from the remaining antibody phages using the biotin-modified HLA-A24/HF10 complex and avidin-conjugated magnetic beads were repeated three times to obtain candidate antibody phages that specifically bind to the HLA-A24/HF10 complex.
(3)scFv抗體向IgG1型的轉換與FACS分析 自所獲得的候選抗體嗜菌體分離出scFv部分，在VL區域的後方標示FLAG標記，使用ELISA分析對於HLA-A24/HF10複合體的反應性。分析的結果，選擇出反應性高的12個選殖株。對於HF10胜肽的親和性，依序為scFv選殖株1＜scFv選殖株2＜scFv選殖株5，並且選殖株5最高。 此外，使用FACS及SPR進行分析，分析的結果，選擇對於HF10胜肽的特異性反應性較高的scFv選殖株(選殖株1、2、5)(第3圖、第5圖)並以IgG1的重鏈Fc區域(CH2及CH3區域，序列編號30(將其編碼的鹼基序列顯示於序列編號29))取代FLAG標記進行連結，來將scFv抗體轉換為IgG1型。 使用已轉換為IgG1的抗體(scFv-hIgG1)，進一步實行FACS分析，藉由SPR產生的親和性較低，但是選擇出反應性特別高的選殖株1(第6圖)，使用於以下的實施例。(3) Conversion of scFv antibodies to IgG1 type and FACS analysisThe scFv portion was isolated from the obtained candidate antibody phages, and a FLAG tag was added after the VL region. ELISA was used to analyze the reactivity to the HLA-A24/HF10 complex. The results of the analysis selected 12 clones with high reactivity. The affinity for the HF10 peptide was in the order of scFv clone 1 < scFv clone 2 < scFv clone 5, with clone 5 having the highest affinity. Furthermore, FACS and SPR analysis revealed that scFv clones with high specific reactivity to the HF10 peptide (Clones 1, 2, and 5) were selected (Figures 3 and 5). The FLAG tag was replaced by the heavy chain Fc region of IgG1 (CH2 and CH3 regions, SEQ ID NO: 30 (the base sequence encoded by this is shown in SEQ ID NO: 29)), thereby converting the scFv antibody to an IgG1 type.Further FACS analysis was performed using the IgG1-converted antibody (scFv-hIgG1). Although the affinity detected by SPR was low, Clone 1 (Figure 6) was selected for its particularly high reactivity and used in the following examples.
該等的選殖株(選殖株1、2、5)的CDR1～3區域的序列如同以下。 選殖株1 VH區域 CDR1區域的序列：GYSFTTYG(序列編號4) CDR2區域的序列：ISAYTGDT(序列編號5) CDR3區域的序列：CARGSSNFYGMDVW(序列編號6) 選殖株1 VL區域 CDR1區域的序列：SLRNYY(序列編號7) CDR2區域的序列：GRD CDR3區域的序列：CQSRDISGNPQNVVF(序列編號8) 選殖株2 VH領域 CDR1區域的序列：GYSFTSYW(序列編號9) CDR2區域的序列：IYPGDSDT(序列編號10) CDR3區域的序列：CARQSGSYGGTFDIW(序列編號11) 選殖株2 VL領域 CDR1區域的序列：SLRNYY(序列編號12) CDR2區域的序列：GKN CDR3區域的序列：CSSRDSAGKHLVF(序列編號13) 選殖株5 VH領域 CDR1區域的序列：GFTFSSYA(序列編號14) CDR2區域的序列：SSYGGGTT(序列編號15) CDR3區域的序列：CASSITVRGSLRNW(序列編號16) 選殖株5 VL領域 CDR1區域的序列：SLRSYY(序列編號17) CDR2區域的序列：NDN CDR3區域的序列：CSSRDSSGDPVIF(序列編號18)The sequences of the CDR1-3 regions of these selected strains (strains 1, 2, and 5) are as follows.Clone 1 VH regionCDR1 region sequence: GYSFTTYG (SEQ ID NO. 4)CDR2 region sequence: ISAYTGDT (SEQ ID NO. 5)CDR3 region sequence: CARGSSNFYGMDVW (SEQ ID NO. 6)Clone 1 VL regionCDR1 region sequence: SLRNYY (SEQ ID NO. 7)CDR2 region sequence: GRDCDR3 region sequence: CQSRDISGNPQNVVF (SEQ ID NO. 8)Clone 2 VH regionCDR1 region sequence: GYSFTSYW (SEQ ID NO. 9)CDR2 region sequence: IYPGDSDT (SEQ ID NO. 10)CDR3 region sequence: CARQSGSYGGTFDIW (SEQ ID NO. 11)Clone 2 VL regionCDR1 region sequence: SLRNYY (SEQ ID NO. 12) CDR2 region sequence: GKNCDR3 region sequence: CSSRDSAGKHLVF (SEQ ID NO. 13)VH domain of clone 5CDR1 region sequence: GFTFSSYA (SEQ ID NO. 14)CDR2 region sequence: SSYGGGTT (SEQ ID NO. 15)CDR3 region sequence: CASSITVRGSLRNW (SEQ ID NO. 16)VL domain of clone 5CDR1 region sequence: SLRSYY (SEQ ID NO. 17)CDR2 region sequence: NDNCDR3 region sequence: CSSRDSSGDPVIF (SEQ ID NO. 18)
此外，包含選殖株1、選殖株2及選殖株5的可變區域(VH、VL)之scFv的胺基酸序列如同以下。 選殖株1 QVQLVQSGAEVKKPGASVKVSCKASGYSFTTYGISWVRQAPGQGLEWMGWISAYTGDTNYAQKFQGRVTVTTDTSTGTVYMELRTLRSDDSAVYYCARGSSNFYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQRAGQAPVLVFYGRDNRPSGIPDRFSGSTSGSTASLTITGAQAEDEADYYCQSRDISGNPQNVVFGGGTKVTVL(序列編號19) 選殖株2 QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQSGSYGGTFDIWGQGTMVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQTPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADFYCSSRDSAGKHLVFGTGTKVPVL(序列編號20) 選殖株5 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGAGVGLNSSYGGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSITVRGSLRNWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPTLVLYNDNNRPSGIPDRFSGSTSGTTASLTITGAQAEDDADYYCSSRDSSGDPVIFGGGTKLTVL(序列編號21)In addition, the amino acid sequences of the scFvs comprising the variable regions (VH, VL) of clones 1, 2, and 5 are as follows.Clone 1QVQLVQSGAEVKKPGASVKVSCKASGYSFTTYGISWVRQAPGQGLEWMGWISAYTGDTNYAQKFQGRVTVTTDTSTGTVYMELRTLRSDDSAVYYCARGSSNFYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQRAGQAPVLVFYGRDNRPSGIPDRFSGSTSGSTASLTITGAQAEDEADYYCQSRDISGNPQNVVFGGGTKVTVL (SEQ ID NO: 19)Clone 2 QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQSGSYGGTFDIWGQGTMVTVSSGGGGS GGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQTPVLVIYGKNNRPSGIPDRFSGSSSSGNTASLTITGAQAEDEADFYCSSRDSAGKHLVFGTGTKVPVL (serial number 20) Selected strain 5 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGAGVGLNSSYGGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSITVRGSLRNWGQGTLVTVSSGGGGSG GGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPTLVLYNDNNRPSGIPDRFSGSTSGTTASLTITGAQAEDDADYYCSSRDSSGDPVIFGGGTKLTVL (serial number 21)
此外，選殖株1、選殖株2及選殖株5的可變區域(VH、VL)的胺基酸序列如同以下。 選殖株1的VH QVQLVQSGAEVKKPGASVKVSCKASGYSFTTYGISWVRQAPGQGLEWMGWISAYTGDTNYAQKFQGRVTVTTDTSTGTVYMELRTLRSDDSAVYYCARGSSNFYGMDVWGQGTTVTVSS(序列編號22) 選殖株1的VL SSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQRAGQAPVLVFYGRDNRPSGIPDRFSGSTSGSTASLTITGAQAEDEADYYCQSRDISGNPQNVVFGGGTKVTVL(序列編號23) 選殖株2的VH QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQSGSYGGTFDIWGQGTMVTVSS(序列編號24) 選殖株2的VL SSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQTPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADFYCSSRDSAGKHLVFGTGTKVPVL(序列編號25) 選殖株5的VH QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGAGVGLNSSYGGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSITVRGSLRNWGQGTLVTVSS(序列編號26) 選殖株5的VL SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPTLVLYNDNNRPSGIPDRFSGSTSGTTASLTITGAQAEDDADYYCSSRDSSGDPVIFGGGTKLTVL(序列編號27)In addition, the amino acid sequences of the variable regions (VH, VL) of strains 1, 2, and 5 are as follows.VH of strain 1QVQLVQSGAEVKKPGASVKVSCKASGYSFTTYGISWVRQAPGQGLEWMGWISAYTGDTNYAQKFQGRVTVTTDTSTGTVYMELRTLRSDDSAVYYCARGSSNFYGMDVWGQGTTVTVSS (SEQ ID NO: 22)VL of strain 1SSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQRAGQAPVLVFYGRDNRPSGIPDRFSGSTSGSTASLTITGAQAEDEADYYCQSRDISGNPQNVVFGGGTKVTVL (SEQ ID NO: 23)VH of strain 2QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQSGSYGGTFDIWGQGTMVTVSS (SEQ ID NO: 24)VL from clone 2SSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQTPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADFYCSSRDSAGKHLVFGTGTKVPVL (SEQ ID NO: 25)VH from clone 5 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGAGVGLNSSYGGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSITVRGSLRNWGQGTLVTVSS (serial number 26) VL of selected clone 5 SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPTLVLYNDNNRPSGIPDRFSGSTSGTTASLTITGAQAEDDADYYCSSRDSSGDPVIFGGGTKLTVL (serial number 27)
例3.(1)胜肽脈衝293T-A24細胞中的細胞表面的HLA-A24/HF10胜肽複合體的檢測對由例2所獲得的scFv-hIgG1抗體實際試驗是否會與在細胞表面呈現抗原的細胞進行反應。對於以調製為最終濃度50 μg/mL的HF10對於293T-A24細胞進行脈衝後的細胞，以最終濃度為10 μg/mL的方式使由例2所獲得的scFv-hIgG1抗體進行反應，以PE接合抗人IgG1抗體作為螢光標示並於細胞核染色使用DAPI來進行觀察。在細胞表面存在HLA-A24/HF10胜肽複合體而scFv-hIgG1抗體結合的情況下，細胞周邊會被染色為紅色。癌細胞的細胞核會由於DAPI被染色為藍色。 將結果顯示於第7圖。在利用癌細胞抗原胜肽HF10進行脈衝而成的293T-A24中，檢測出細胞表面的HLA-A24/HF10胜肽複合體。Example3. (1) Detection ofHLA-A24/HF10peptide complexeson the cell surface ofpeptide-pulsed293T-A24 cells The scFv-hIgG1 antibody obtained in Example 2 was tested to see if it would react with cells presenting antigens on their cell surfaces. 293T-A24 cells pulsed with HF10 at a final concentration of 50 μg/mL were reacted with the scFv-hIgG1 antibody obtained in Example 2 at a final concentration of 10 μg/mL. PE-conjugated anti-human IgG1 antibody was used as a fluorescent marker and DAPI was used to stain the cell nuclei for observation. When the HLA-A24/HF10 peptide complex is present on the cell surface and bound by the scFv-hIgG1 antibody, the cell periphery is stained red. The nuclei of cancer cells are stained blue with DAPI. The results are shown in Figure 7. In 293T-A24 cells pulsed with the cancer cell antigen peptide HF10, the HLA-A24/HF10 peptide complex was detected on the cell surface.
(2)表現HF10的癌細胞中的細胞表面的HLA-A24/HF10胜肽複合體的檢測 繼而試驗對於實際的癌細胞的反應性。對人類大腸癌細胞株Colo320(HLA-A24(+)及HF10(+))及人類口腔鱗狀細胞癌細胞株MO-1000(HLA-A24(+)及HF10(+))進行與(1)相同的試驗。 將結果顯示於第8圖。實際的癌細胞也與293T-A24細胞同樣地可利用流式細胞計數(第8圖左)及螢光顯微鏡(第8圖右)檢測出細胞表面的HLA-A24/HF10胜肽複合體。從而，在內源性地呈現有癌細胞抗原之癌細胞中也檢測到細胞表面的HLA-A24/HF10胜肽複合體。從而顯示出本案的抗體能夠檢測出內源性地呈現天然抗原胜肽的細胞的細胞表面的HLA-A24/HF10胜肽複合體。(2) Detection of HLA-A24/HF10 peptide complex on the cell surface of HF10-expressing cancer cellsNext, the reactivity to actual cancer cells was tested. The same test as in (1) was performed on the human colorectal cancer cell line Colo320 (HLA-A24(+) and HF10(+)) and the human oral squamous cell carcinoma cell line MO-1000 (HLA-A24(+) and HF10(+)).The results are shown in Figure 8. Similar to 293T-A24 cells, actual cancer cells also exhibited HLA-A24/HF10 peptide complexes on their cell surfaces using flow cytometry (Figure 8, left) and fluorescence microscopy (Figure 8, right). Thus, HLA-A24/HF10 peptide complexes were also detected on the cell surfaces of cancer cells that endogenously display cancer cell antigens. This demonstrates that the antibodies in this case can detect HLA-A24/HF10 peptide complexes on the cell surfaces of cells that endogenously display natural antigen peptides.
例4.雙特異性抗體BiTE的分析(1)BiTE(雙特異性T細胞衍生抗體)的製作及與各細胞的結合性 使用與Stadler et al., Nature Medicine volume 23, pages 815-817 (2017)記載的方法相同的方法，於選殖株1的抗體的scFv連結抗CD3的scFv(序列編號32)(將其編碼的鹼基序列顯示於序列編號31)，製成雙特異性抗體BiTE。使用該BiTE，試驗對於脈衝有癌細胞抗原胜肽而成之各種癌細胞、PBMC的結合性。BiTE會與脈衝有癌細胞抗原胜肽而成之大腸癌細胞株colo320及肺癌細胞株LHK2反應而產生IFN-γ(第9圖A)。Example4.Analysis ofBispecific AntibodyBiTE (1) Preparation of BiTE (Bispecific T cell-derived Antibody) and Binding to Various Cells Using the same method as described in Stadler et al., Nature Medicine volume 23, pages 815-817 (2017), the scFv of the antibody from clone 1 was linked to an anti-CD3 scFv (SEQ ID NO. 32) (the base sequence encoded by it is shown in SEQ ID NO. 31) to prepare a bispecific antibody BiTE. Using this BiTE, the binding to various cancer cells and PBMC pulsed with cancer cell antigen peptides was tested. BiTEs reacted with the colorectal cancer cell line colo320 and the lung cancer cell line LHK2, which were pulsed with cancer cell antigen peptides, and produced IFN-γ (Figure 9A).
(2)細胞毒殺活性 將由(1)製成的BiTE與PBMC混合，然後與經癌細胞抗原胜肽進行脈衝而成之293T-A24細胞進行共培養。CTL的誘導的有無，例如能夠藉由V-Plex Cytokine Panel 1 Human Kit測定CTL對抗原胜肽呈現細胞有反應而產生的各種細胞激素(例如IFN-γ、IL-2、TNFα)的量，來藉此確認。 如同第9圖B所示，藉由雙特異性抗體BiTE，在293T-A24細胞及大腸癌細胞株colo320中，各種的細胞激素的量呈現增加。 然而，如同第9圖C所示，只有CD3 scFv及HF10 scFv無法誘導出經由T細胞的細胞激素產生。(2) Cytotoxic activityThe BiTE prepared in (1) was mixed with PBMC and then co-cultured with 293T-A24 cells pulsed with cancer cell antigen peptides. The presence or absence of CTL induction can be confirmed by measuring the amount of various cytokines (e.g., IFN-γ, IL-2, TNFα) produced by CTL in response to antigen peptide-presenting cells using the V-Plex Cytokine Panel 1 Human Kit.As shown in Figure 9B, the amount of various cytokines increased in 293T-A24 cells and the colorectal cancer cell line colo320 by the bispecific antibody BiTE. However, as shown in Figure 9C, only CD3 scFv and HF10 scFv failed to induce cytokine production via T cells.
例5.細胞毒殺活性測定經由由例4所獲得的雙特異性抗體BiTE，使用Maestro Z, Axion Biosystems來試驗T細胞對於癌細胞的細胞毒殺活性。詳言之，使用在底面附有電極之96孔盤。若播種目標細胞，伴隨對底面的黏著及增殖而電阻會上升，若引發細胞死亡則目標細胞會自底面剝離，因而造成電阻降低。以即時方式測定該電阻。 將結果顯示於第10圖A～第10圖C。雙特異性抗體BiTE對於骨肉瘤OS-2000細胞株顯示出細胞毒殺活性。(A)顯示伴隨時間經過的阻抗變化。(B)、(C)顯示自阻抗變化算出的細胞毒殺活性結果。(B)左側為20小時後，(C)右側為40小時後的細胞毒殺活性。Example5.Cytotoxic Activity Assay : The bispecific antibody BiTE obtained in Example 4 was used to assay the cytotoxic activity of T cells against cancer cells using the Maestro Z (Axion Biosystems). Specifically, a 96-well plate with an electrode attached to the bottom was used. When target cells were seeded, the electrical resistance increased as they adhered to the bottom surface and proliferated. When cell death was induced, the target cells detached from the bottom surface, resulting in a decrease in electrical resistance. This electrical resistance was measured in real time. The results are shown in Figures 10A to 10C. The bispecific antibody BiTE demonstrated cytotoxic activity against the osteosarcoma OS-2000 cell line. (A) Shows impedance changes over time. (B) and (C) Show cytotoxic activity calculated from impedance changes. (B) Left: 20 hours, (C) Right: 40 hours.
例6.HF10 CAR-T細胞的細胞毒殺活性測定如同第11圖A所示，建構了使用了HF10 scFv之HF10 CAR結構物。作成編碼有HF10 CAR結構之反轉錄病毒載體(pMX)，使其感染並侵入健康人體的周邊血液單核細胞(PBMC)。之後，使用HLA-A24/HF10胜肽四聚物檢測CAR(第11圖B)。 HF10 CAR-T細胞可辨識細胞表面的HLA-A24/HF10胜肽複合體，產生細胞激素IFN g及CD107a陽性細胞毒性顆粒(第12圖)。 此外，HF10 CAR-T細胞顯示出對於經HF10胜肽脈衝之T2-A24細胞的細胞毒殺活性，並觀察到源自目標細胞的LDH(乳酸脫氫酶)釋放(第13圖)。Example 6.Cytotoxic Activity Assay ofHF10 CAR-T Cells: As shown in Figure 11A, an HF10 CAR construct using the HF10 scFv was constructed. A retroviral vector (pMX) encoding the HF10 CAR construct was generated and used to infect and colonize peripheral blood mononuclear cells (PBMCs) from healthy individuals. The CAR was then detected using an HLA-A24/HF10 peptide tetramer (Figure 11B). HF10 CAR-T cells recognized the HLA-A24/HF10 peptide complex on the cell surface and produced the cytokine IFN-g and CD107a-positive cytotoxic granules (Figure 12). Furthermore, HF10 CAR-T cells demonstrated cytotoxic activity against T2-A24 cells pulsed with HF10 peptide, and LDH (lactate dehydrogenase) release from target cells was observed (Figure 13).
例7.雙特異性抗體BiTE的在體內的抗腫瘤效果以對於每隻NSG小鼠2×10e6個人類口腔鱗狀細胞癌細胞株MO-1000細胞的條件進行皮下移植於右背部(第-12天)。在11天後確認腫瘤植入(第-1天)，隔天以每隻小鼠1×10e7個PBMC的條件進行腹腔內投予(第0天)。隔天以每隻小鼠抗體3 mg/kg/天的條件進行投予10天(第1天～第10天)。 抗體使用由例4所獲得的雙特異性抗體BiTE，小鼠區分為以下的組別。 No treatment(未治療組)：小鼠6隻 Control(控制組)(PBMC+PBS投予組)：小鼠7隻 Treatment(治療組)(PBMC+抗體投予組)：小鼠7隻 在第1天～第10天(抗體投予期間)中，比起No treatment組及PBS控制組(PBMC+PBS)，在Treatment組中發現抑制腫瘤體積的效果(第14圖B～第14圖E)。 抗體投予結束後，在Treatment組中也發現腫瘤的增大傾向(第14圖B)。 [產業上的可利用性]Example7.In vivo antitumor efficacy ofa bispecific antibodyBiTE. NSG mice were subcutaneously implanted with 2×10e6 human oral squamous cell carcinoma MO-1000 cells per mouse on the right dorsum (day -12). Tumor implantation was confirmed 11 days later (day -1), and the next day, 1×10e7 PBMCs were intraperitoneally administered per mouse (day 0). The antibody was then administered at 3 mg/kg/day per mouse for 10 days (days 1 to 10). The bispecific antibody BiTE obtained in Example 4 was used, and mice were divided into the following groups. No treatment group: 6 mice; Control group (PBMC + PBS administration group): 7 mice; Treatment group (PBMC + antibody administration group): 7 mice. From day 1 to day 10 (during antibody administration), the treatment group showed an inhibitory effect on tumor growth compared to the no treatment group and the PBS control group (PBMC + PBS) (Figures 14B to 14E). After the completion of antibody administration, tumors also showed a tendency to grow in the treatment group (Figure 14B). [Industrial Applicability]
本案的抗體能夠對於內源性地呈現的天然抗原胜肽產生反應，進一步藉由體液免疫反應顯示細胞毒殺活性。因此，能對於表現PVT1的患者進行以癌細胞為標的的免疫療法。此外，細胞毒殺機制是體液免疫，所以能實施不會依賴投予對象的免疫力的疫苗免疫療法，並且能期待作為抗癌劑和作為免疫療法的較高效果。The antibodies in this case react to endogenously expressed natural antigenic peptides and further exhibit cytotoxic activity through humoral immune responses. Therefore, immunotherapy targeting cancer cells is possible for patients expressing PVT1. Furthermore, since the cytotoxic mechanism is humoral immunity, vaccine immunotherapy can be implemented that is independent of the recipient's immunity, and is expected to be highly effective as both an anticancer agent and an immunotherapy.
無without
[第1圖]第1圖A是顯示PVT1基因在染色體上的位置的示意圖，並且擴大地表示PVT1基因的外顯子1～外顯子2的鹼基序列(序列編號1)及ORF的胺基酸序列(序列編號2)。HF10胜肽(序列編號3)是由10個胺基酸殘基所構成之胜肽，由447個鹼基(149個胺基酸)的ORF所編碼。第1圖B是顯示大腸癌、肺癌、口腔鱗狀細胞癌、骨肉瘤等的各種癌細胞株中的PVT1 mRNA的相對表現量的圖。 [第2圖]第2圖A是顯示藉由噬菌體呈現法進行的scFv抗體的篩選的概要的示意圖。首先以是否會對HLA與HIV之複合體進行反應的條件對scFv噬菌體呈現抗體庫進行篩選，將會進行反應者視為非特異性抗體噬菌體而去除。之後，以會與HLA-A24/天然抗原胜肽複合體進行反應者視為特異性抗體噬菌體，將不會進行反應者視為非特異性抗體噬菌體的方式進行選擇，並重複3次此流程。由特異性抗體噬菌體獲得可溶性scFv。第2圖B顯示對於94個scFv選殖株的抗原的反應性的篩選結果。A1/H12是顯示對照組(blank)的圖。選擇出反應性高的前12個選殖株(第2圖B左)。[Figure 1] Figure 1A is a schematic diagram showing the location of the PVT1 gene on chromosomes, and shows an enlarged view of the base sequence of exons 1 and 2 of the PVT1 gene (SEQ ID NO: 1) and the amino acid sequence of the ORF (SEQ ID NO: 2). The HF10 peptide (SEQ ID NO: 3) is a peptide composed of 10 amino acid residues and encoded by an ORF of 447 bases (149 amino acids). Figure 1B is a graph showing the relative expression levels of PVT1 mRNA in various cancer cell lines, including colorectal cancer, lung cancer, oral squamous cell carcinoma, and osteosarcoma.[Figure 2] Figure 2A is a schematic diagram showing the overview of scFv antibody screening by phage display. First, the scFv phage displaying antibody library was screened for reactivity to HLA and HIV complexes. Phage that reacted were considered non-specific and removed. Subsequently, phage that reacted to HLA-A24/natural antigen peptide complexes were considered specific, while those that did not react were considered non-specific. This process was repeated three times. Soluble scFv was obtained from the specific antibody phage. Figure 2B shows the results of screening for antigen reactivity of 94 scFv clones. A1/H12 shows the control group (blank). The top 12 clones with the highest reactivity were selected (Figure 2B, left).
[第3圖]第3圖是顯示基於流式細胞計數(FACS)分析的PVT1 HF10 scFv的反應性的研究結果的圖。對於抗原呈現細胞T2-A24細胞以50 μM的條件將HF10或病毒衍生胜肽(EBV、CMV)進行脈衝後使其與scFv選殖株進行反應，然後以二次抗體進行染色。 [第4圖]第4圖是顯示本案的人工抗體辨識出呈現於癌細胞表面的PVT1胜肽並與癌細胞結合的情況的圖。左圖是當為細胞毒殺性T細胞的T細胞受體的情況的結合示意圖，右圖是當為本案的抗體的情況的結合示意圖。 [第5圖]第5圖是顯示藉由表面電漿共振(Surface Plasmon Resonance：SPR)進行的PVT1 HF10 scFv的親和性結果的圖。[Figure 3] Figure 3 shows the results of a flow cytometry (FACS) analysis of the reactivity of the PVT1 HF10 scFv. Antigen-presenting T2-A24 cells were pulsed with HF10 or a viral peptide (EBV, CMV) at 50 μM, reacted with the scFv clone, and then stained with a secondary antibody.[Figure 4] Figure 4 shows the artificial antibody in this case recognizing and binding to the PVT1 peptide presented on the surface of cancer cells. The left figure shows the binding behavior of the T cell receptor of a cytotoxic T cell, and the right figure shows the binding behavior of the antibody in this case.[Figure 5] Figure 5 shows the affinity analysis results of PVT1 HF10 scFv using surface plasmon resonance (SPR).
[第6圖]第6圖是顯示利用FACS分析由scFv(一價)轉換為hIgG1型(二價)的本案的scFv-IgG1型人工抗體的對於HLA-A24/PVT1 HF10胜肽複合體的反應特異性的結果的圖。 [第7圖]第7圖是使本案的scFv-IgG1型人工抗體反應於胜肽脈衝293T-A24細胞(HLA-A24陽性293T細胞)並利用螢光顯微鏡進行觀察時的照片。為將scFv-IgG1設為抗體並使用HF10作為天然抗原胜肽時的照片。可知：在經脈衝HF10抗原胜肽後的細胞的細胞膜上，呈現於HLA-A24的HF10抗原胜肽與scFv-IgG1型人工抗體(HF10scFv-IgG1型人工抗體)已結合並受到螢光標誌PE染色。細胞核受到DAPI染色。 [第8圖]第8圖左分別與第8圖右對應，並且為顯示針對大腸癌細胞株及口腔鱗狀細胞癌細胞株進行流式細胞計數(FACS)分析及螢光顯微鏡觀察的scFv-IgG1的反應性的研究結果的圖。[Figure 6] Figure 6 shows the results of FACS analysis of the reactivity specificity of the present scFv-IgG1 artificial antibody, converted from scFv (monovalent) to hIgG1 (bivalent), toward the HLA-A24/PVT1 HF10 peptide complex.[Figure 7] Figure 7 shows a photograph of the present scFv-IgG1 artificial antibody reacting with peptide-pulsed 293T-A24 cells (HLA-A24-positive 293T cells) and observed under a fluorescence microscope. This photograph shows the case where scFv-IgG1 was used as the antibody and HF10 was used as the natural antigen peptide. On the cell membranes of cells pulsed with the HF10 antigen peptide, the HF10 antigen peptide, which is expressed on HLA-A24, binds to the scFv-IgG1 artificial antibody (HF10scFv-IgG1), which is stained with the fluorescent marker PE. Cell nuclei are stained with DAPI.[Figure 8] Figure 8 (left) corresponds to Figure 8 (right), and shows the results of a study of the reactivity of scFv-IgG1 with colorectal cancer cell lines and oral squamous cell carcinoma cell lines, as determined by flow cytometry (FACS) analysis and fluorescence microscopy observation.
[第9圖]第9圖是顯示周邊血液單核細胞(Peripheral Blood Mononuclear Cell：PBMC)經由本案的雙特異性抗體對於癌細胞產生反應的圖。第9圖A顯示藉由PBMC對於癌細胞的反應而產生細胞激素(IFNg)的情況。第9圖B顯示藉由PBMC對於癌細胞的反應而產生細胞激素(IFNg、IL-2、TNFα)的情況。第9圖C顯示僅有CD3 scFv及僅有HF10 scFv無法誘導經由T細胞的細胞激素生成的情況。 [第10圖]第10圖是顯示使用Maestro Z, Axion Biosystems之阻抗檢驗進行的細胞毒殺活性測定的圖。第10圖A顯示T細胞經由本案的雙特異性抗體對於OS-2000表現出細胞毒殺活性的情況。第10圖B表示在20小時的細胞毒殺活性，第10圖C表示在40小時的細胞毒殺活性。[Figure 9] Figure 9 shows peripheral blood mononuclear cells (PBMCs) responding to cancer cells via the bispecific antibody described herein. Figure 9A shows the production of cytokines (IFNg) by PBMCs in response to cancer cells. Figure 9B shows the production of cytokines (IFNg, IL-2, and TNFα) by PBMCs in response to cancer cells. Figure 9C shows that CD3 scFv alone and HF10 scFv alone fail to induce cytokine production by T cells. [Figure 10] Figure 10 shows cytotoxic activity assays performed using the Maestro Z, Axion Biosystems impedance assay. Figure 10A shows the cytotoxic activity of T cells against OS-2000 via the bispecific antibody described herein. Figure 10B shows the cytotoxic activity at 20 hours, and Figure 10C shows the cytotoxic activity at 40 hours.
[第11圖]第11圖是顯示CAR-T細胞的生成的圖。第11圖A表示使用了HF10 scFv之HF10 CAR結構的示意圖。第11圖B是顯示作成編碼有HF10 CAR結構之反轉錄病毒載體(pMX)並使其感染而導入健康人體的周邊血液單核細胞(PBMC)時的使用了HLA-A24/HF10胜肽四聚物之CAR的檢測。 [第12圖]第12圖是顯示HF10 CAR-T細胞辨識細胞表面的HLA-A24/HF10胜肽複合體並產生細胞激素IFNg及CD107a陽性細胞毒性顆粒的情況的圖。 [第13圖]第13圖是顯示HF10 CAR-T細胞對於經HF10胜肽脈衝之T2-A24細胞表現細胞毒殺活性並觀察到源自目標細胞的LDH(乳酸脫氫酶)釋放的情況的圖。 [第14圖]第14圖是顯示使用了本案的雙特異性抗體之小鼠中的抗腫瘤效果的圖。第14圖A顯示本試驗的投予時間表的示意圖。第14圖B顯示處理(Treatment)組在PBMC投予後20天的腫瘤體積的變化。第14圖C顯示控制(Control)組在PBMC投予後20天的腫瘤體積的變化。第14圖D顯示未處理(No treatment)組20天的腫瘤體積的變化。第14圖E顯示將各組中的腫瘤體積的變化進行平均後的數值。第14圖F顯示在第10天(Day 10)時的各組(由左起為處理組、控制組、未處理組)的比較。[Figure 11] Figure 11 shows the generation of CAR-T cells. Figure 11A is a schematic diagram of the HF10 CAR structure using the HF10 scFv. Figure 11B shows the detection of CAR using the HLA-A24/HF10 peptide tetramer when a retroviral vector (pMX) encoding the HF10 CAR structure was created and introduced into peripheral blood mononuclear cells (PBMCs) from a healthy individual.[Figure 12] Figure 12 shows that HF10 CAR-T cells recognize the HLA-A24/HF10 peptide complex on the cell surface and produce the cytokine IFNg and CD107a-positive cytotoxic granules.[Figure 13] Figure 13 shows that HF10 CAR-T cells exhibit cytotoxic activity against T2-A24 cells pulsed with HF10 peptide, and LDH (lactate dehydrogenase) release from target cells is observed.[Figure 14] Figure 14 shows the antitumor effect in mice treated with the bispecific antibody described in this study. Figure 14A shows a schematic diagram of the administration schedule for this study. Figure 14B shows changes in tumor volume in the treatment group 20 days after PBMC administration. Figure 14C shows changes in tumor volume in the control group 20 days after PBMC administration. Figure 14D shows the change in tumor volume in the no-treatment group over 20 days. Figure 14E shows the average change in tumor volume across all groups. Figure 14F shows a comparison of the groups (from left: treatment group, control group, and no-treatment group) on Day 10.
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