TW202144404A - High-affinity t cell receptor capable of recognizing AFP antigen - Google Patents
High-affinity t cell receptor capable of recognizing AFP antigen Download PDFInfo
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Abstract
Description
本發明涉及生物技術領域,更具體地涉及能夠辨識衍生自AFP蛋白多肽的T細胞受體(T cell receptor, TCR)。本發明還涉及所述受體的製備和用途。The present invention relates to the field of biotechnology, and more particularly, to a T cell receptor (TCR) capable of recognizing polypeptides derived from AFP proteins. The present invention also relates to the preparation and use of said receptors.
僅僅有兩種類型的分子能夠以特異性的方式辨識抗原。其中一種是免疫球蛋白或抗體;另一種是T細胞受體(TCR),它是由α鏈/β鏈或者γ鏈/δ鏈以異二聚體形式存在的細胞膜表面的糖蛋白。免疫系統的TCR組庫的組成是在胸腺中通過V(D)J重組,然後進行陽性和陰性選擇而產生的。在周邊環境中,TCR媒介了T細胞對主組織相容性複合體-胜肽複合物(pMHC)的特異性辨識,因此其對免疫系統的細胞免疫功能是至關重要的。Only two types of molecules are capable of recognizing antigens in a specific manner. One of them is immunoglobulin or antibody; the other is T cell receptor (TCR), which is a glycoprotein on the surface of the cell membrane in the form of heterodimers of α chain/β chain or γ chain/δ chain. The composition of the immune system's TCR repertoire is generated in the thymus by V(D)J recombination followed by positive and negative selection. In the surrounding environment, TCR mediates the specific recognition of the major histocompatibility complex-peptide complex (pMHC) by T cells and is therefore crucial for the cellular immune function of the immune system.
TCR是呈現在主組織相容性複合體(MHC)上的特異性抗原胜肽的唯一受體,這種外源胜肽或內源胜肽可能會是細胞出現異常的唯一跡象。在免疫系統中,通過抗原特異性的TCR與pMHC複合物的結合引發T細胞與抗原呈現細胞(APC)直接的物理接觸,然後T細胞及APC兩者的其他細胞膜表面分子就發生相互作用,這就引起了一系列後續的細胞信號傳遞和其他生理反應,從而使得不同抗原特異性的T細胞對其標的細胞發揮免疫效應。TCR is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC), and this exogenous or endogenous peptide may be the only sign of abnormal cells. In the immune system, direct physical contact between T cells and antigen-presenting cells (APCs) is triggered by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact. It causes a series of subsequent cell signaling and other physiological responses, so that T cells with different antigen specificities exert immune effects on their target cells.
與TCR相對應的MHC I類和II類分子配體也是免疫球蛋白超家族的蛋白質但對於抗原的呈現具有特異性,不同的個體有不同的MHC,從而能呈現一種蛋白抗原中不同的短胜肽到各自的APC細胞表面。人類的MHC通常稱為HLA基因或HLA複合體。The MHC class I and class II molecular ligands corresponding to TCR are also proteins of the immunoglobulin superfamily, but they are specific for the presentation of antigens. Different individuals have different MHCs, so that they can present different short-term proteins in a protein antigen. Peptides to the respective APC cell surfaces. The human MHC is often referred to as the HLA gene or HLA complex.
AFP (αFetoprotein)也稱甲胎蛋白,是胚胎發育過程中表現的一種蛋白,是胚胎血清的主要成分。在發育過程中,AFP在卵黃囊及肝臟中有比較高的表現位準,隨後被抑制。在肝癌中,AFP的表現被活化(Butterfield et al. J Immunol., 2001, Apr 15; 166(8): 5300-8)。AFP在細胞內生成後被加工處理為抗原胜肽,並與MHC(主要組織相容性複合體)分子結合形成複合物,被呈現到細胞表面。TSSELMAITR是衍生自AFP抗原的短胜肽,是AFP相關疾病治療的一種標的。AFP (αFetoprotein), also known as alpha-fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP is expressed at relatively high levels in the yolk sac and liver and is subsequently inhibited. In liver cancer, the expression of AFP is activated (Butterfield et al. J Immunol., 2001, Apr 15; 166(8): 5300-8). After AFP is generated in cells, it is processed into antigenic peptides, and combined with MHC (major histocompatibility complex) molecules to form complexes, which are presented on the cell surface. TSSELMAITR is a short peptide derived from the AFP antigen and is a target for the treatment of AFP-related diseases.
因此,TSSELMAITR-HLA A1101複合物提供了一種TCR可標靶腫瘤細胞的標記。能夠結合TSSELMAITR -HLA A1101複合物的TCR對腫瘤的治療具有很高的應用價值。例如,能夠標靶該腫瘤細胞標記的TCR可用於將細胞毒性劑或免疫刺激劑遞送到標的細胞,或被轉形入T細胞,使表現該TCR的T細胞能夠破壞腫瘤細胞,以便在被稱為過繼免疫治療的治療過程中給予患者。對於前一目的,理想的TCR是具有較高的親和力的,從而使該TCR能夠長期駐留在所標靶的細胞上面。對於後一目的,則優選使用中等親和力的TCR。因此,本領域技術人員致力於開發可用於滿足不同目的的標靶腫瘤細胞標記的TCR。Thus, the TSSELMAITR-HLA A1101 complex provides a marker for TCR-targeted tumor cells. The TCR that can bind to the TSSELMAITR-HLA A1101 complex has a high application value in the treatment of tumors. For example, a TCR capable of targeting the tumor cell marker can be used to deliver cytotoxic or immunostimulatory agents to target cells, or be transformed into T cells so that T cells expressing the TCR can destroy tumor cells for Administer to patients during the course of treatment for adoptive immunotherapy. For the former purpose, the ideal TCR has a high affinity, so that the TCR can reside on the target cell for a long time. For the latter purpose, it is preferred to use a medium affinity TCR. Therefore, those skilled in the art are devoted to developing TCRs that can be used to target tumor cell markers for different purposes.
本發明的目的在於提供一種對TSSELMAITR-HLA A1101複合物具有較高親和力的TCR。The purpose of the present invention is to provide a TCR with higher affinity for the TSSELMAITR-HLA A1101 complex.
本發明的再一目的是提供一種上述類型TCR的製備方法及上述類型TCR的用途。Another object of the present invention is to provide a preparation method of the above-mentioned type of TCR and the use of the above-mentioned type of TCR.
本發明的第一方面,提供了一種包含了α鏈可變域和β鏈可變域的T細胞受體(TCR),其具有結合TSSELMAITR-HLA A1101複合物的活性,並且所述TCRα鏈可變域的胺基酸序列與SEQ ID NO: 1所示的胺基酸序列有至少90%的序列同源性和所述TCRβ鏈可變域的胺基酸序列與SEQ ID NO: 2所示的胺基酸序列有至少90%的序列同源性。A first aspect of the present invention provides a T cell receptor (TCR) comprising an α-chain variable domain and a β-chain variable domain, which has the activity of binding the TSSELMAITR-HLA A1101 complex, and the TCRα chain can The amino acid sequence of the variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence of the TCR beta chain variable domain is shown in SEQ ID NO: 2 have at least 90% sequence homology.
在一優選例中,所述TCRα鏈可變域的胺基酸序列和所述TCRβ鏈可變域的胺基酸序列不同時為野生型TCRα鏈可變域的胺基酸序列和野生型TCRβ鏈可變域的胺基酸序列。In a preferred embodiment, the amino acid sequence of the variable domain of the TCRα chain and the amino acid sequence of the variable domain of the TCRβ chain are not both the amino acid sequence of the variable domain of the wild-type TCRα chain and the amino acid sequence of the variable domain of the wild-type TCRβ The amino acid sequence of the chain variable domain.
在進一步的優選例中,所述TCRα鏈可變域的胺基酸序列不是SEQ ID NO: 1所示的胺基酸序列,和/或所述TCRβ鏈可變域的胺基酸序列不是SEQ ID NO: 2所示的胺基酸序列。In a further preferred embodiment, the amino acid sequence of the variable domain of the TCRα chain is not the amino acid sequence shown in SEQ ID NO: 1, and/or the amino acid sequence of the variable domain of the TCRβ chain is not SEQ ID NO: 1 The amino acid sequence shown in ID NO: 2.
在另一優選例中,所述TCR的α鏈可變域包含與SEQ ID NO: 1所示的序列有至少91%、92%、93%、94%、95%、96%、97%、98%或99%的序列同源性的胺基酸序列。In another preferred embodiment, the α chain variable domain of the TCR comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% sequence homology.
在另一優選例中,所述TCR的β鏈可變域為與SEQ ID NO: 2所示的序列有至少91%、92%、93%、94%、95%、96%、97%、98%、99%或100%的序列同源性的胺基酸序列。In another preferred embodiment, the β chain variable domain of the TCR is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98%, 99% or 100% sequence homology.
在另一優選例中,所述TCRα鏈可變域的胺基酸序列與SEQ ID NO: 2所示的胺基酸序列有至少95%的序列同源性。In another preferred embodiment, the amino acid sequence of the variable domain of the TCRα chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 2.
在另一優選例中,所述TCRβ鏈可變域的胺基酸序列與SEQ ID NO: 2所示的胺基酸序列有至少95%的序列同源性。In another preferred example, the amino acid sequence of the variable domain of the TCR beta chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 2.
在另一優選例中,所述TCRα鏈可變域的3個CDR區(互補決定區)的基準序列如下,
CDR1α:YGATPY
CDR2α:YFSGDTLV
CDR3α:AVVATDSWGKLQ,並且CDR3α含有至少一個下列突變:
在另一優選例中,所述CDR3α中胺基酸突變包含:
在另一優選例中,所述CDR3α中胺基酸突變包含:
在另一優選例中,所述CDR3α中胺基酸突變包含:
在另一優選例中,所述CDR3α中胺基酸突變個數為2-5個,具體地,突變個數為2個或3個或4個或5個,優選地,突變個數為3個或4個。In another preferred example, the number of amino acid mutations in the CDR3α is 2-5, specifically, the number of mutations is 2 or 3 or 4 or 5, preferably, the number of mutations is 3 or 4.
在另一優選例中,所述TCR與TSSELMAITR-HLA A1101複合物的親和力是野生型TCR的至少2倍。In another preferred embodiment, the affinity of the TCR to the TSSELMAITR-HLA A1101 complex is at least 2 times that of the wild-type TCR.
在另一優選例中,所述TCRα鏈的CDR3α選自:AVASLKSWGKLQ、AVASMDSWGKLQ、AVASMQSWGKLQ、AVASYQSWGKLQ和AVASLSSWGKLQ。In another preferred embodiment, the CDR3α of the TCRα chain is selected from: AVASLKSWGKLQ, AVASMDSWGKLQ, AVASMQSWGKLQ, AVASYQSWGKLQ and AVASLSWGKLQ.
在另一優選例中,所述TCRβ鏈可變域的3個CDR為: CDR1β:SGHVS; CDR2β:FQNEAQ;和 CDR3β:ASSLVAGARTDTQY。In another preferred embodiment, the three CDRs of the variable domain of the TCRβ chain are: CDR1β:SGHVS; CDR2β:FQNEAQ; and CDR3β:ASSLVAGARTDTQY.
在另一優選例中,所述TCRβ鏈可變域的胺基酸序列為SEQ ID NO: 2。In another preferred embodiment, the amino acid sequence of the variable domain of the TCRβ chain is SEQ ID NO: 2.
在另一優選例中,所述TCRβ鏈可變域的3個CDR的基準序列如下,
CDR1β:SGHVS
CDR2β:FQNEAQ
CDR3β:ASSLVAGARTDTQY,並且CDR3β含有至少一個下列突變:
在另一優選例中,所述TCRα鏈可變域包含CDR1α、CDR2α和CDR3α,其中CDR1α的胺基酸序列為YGATPY,CDR2α的胺基酸序列為YFSGDTLV,並且CDR3α的胺基酸序列為:AV[3αX1][3αX2][3αX3][3αX4]SWGKLQ,其中[3αX1]為V或A或P,和/或[3αX2]為A或S或D或G,和/或[3αX3]為T或L或M或Y或I,和/或[3αX4]為D或K或Q或S或E或A或H或N或P或R。In another preferred embodiment, the TCRα chain variable domain comprises CDR1α, CDR2α and CDR3α, wherein the amino acid sequence of CDR1α is YGATPY, the amino acid sequence of CDR2α is YFSGDTLV, and the amino acid sequence of CDR3α is: AV [3αX1][3αX2][3αX3][3αX4]SWGKLQ, where [3αX1] is V or A or P, and/or [3αX2] is A or S or D or G, and/or [3αX3] is T or L or M or Y or I, and/or [3αX4] is D or K or Q or S or E or A or H or N or P or R.
在另一優選例中,所述TCR在SEQ ID NO: 1所示的α鏈可變域中發生突變,所述突變選自V94A/P、A95S/D/G、T96L/M/Y/I、D97K/Q/S/E/A/H/N/P/R中的一組或幾組,其中,胺基酸殘基編號採用SEQ ID NO: 1所示的編號。In another preferred embodiment, the TCR is mutated in the α chain variable domain shown in SEQ ID NO: 1, and the mutation is selected from V94A/P, A95S/D/G, T96L/M/Y/I , one or several groups of D97K/Q/S/E/A/H/N/P/R, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1.
在另一優選例中,所述TCRβ鏈可變域包含CDR1β、CDR2β和CDR3β,其中CDR1β的胺基酸序列為SGHVS,CDR2β的胺基酸序列為FQNEAQ,並且CDR3β的胺基酸序列為:AS[3βX1][3βX2][3βX3][3βX4]GARTDTQY,其中[3βX1]為S或T,和/或[3βX2]為L或M或W,和/或[3βX3]為V或L或I,和/或[3βX4]為A或G。In another preferred embodiment, the TCRβ chain variable domain comprises CDR1β, CDR2β and CDR3β, wherein the amino acid sequence of CDR1β is SGHVS, the amino acid sequence of CDR2β is FQNEAQ, and the amino acid sequence of CDR3β is: AS [3βX1][3βX2][3βX3][3βX4]GARTDTQY, where [3βX1] is S or T, and/or [3βX2] is L or M or W, and/or [3βX3] is V or L or I, and /or [3βX4] is A or G.
在另一優選例中,所述TCR在SEQ ID NO: 2所示的β鏈可變域中發生突變,所述突變選自S95T、L96M/W、V97L/I、A98G中的一組或幾組,其中,胺基酸殘基編號採用SEQ ID NO: 2所示的編號。In another preferred embodiment, the TCR is mutated in the β chain variable domain shown in SEQ ID NO: 2, and the mutation is selected from one or more of S95T, L96M/W, V97L/I, and A98G. group, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 2.
在另一優選例中,所述TCR具有選自下組的CDR:
在另一優選例中,所述TCR是可溶的。In another preferred embodiment, the TCR is soluble.
在另一優選例中,所述TCR為αβ異質二聚TCR,包含α鏈TRAC恆定區序列和β鏈TRBC1或TRBC2恆定區序列。In another preferred embodiment, the TCR is an αβ heterodimeric TCR, comprising the α chain TRAC constant region sequence and the β chain TRBC1 or TRBC2 constant region sequence.
在另一優選例中,所述TCR包含(i)除其跨膜結構域以外的全部或部分TCRα鏈,和(ii)除其跨膜結構域以外的全部或部分TCRβ鏈,其中(i)和(ii) 均包含TCR鏈的可變域和至少一部分恆定域。In another preferred embodiment, the TCR comprises (i) all or part of the TCRα chain excluding its transmembrane domain, and (ii) all or part of the TCRβ chain excluding its transmembrane domain, wherein (i) and (ii) each comprise the variable domain and at least a portion of the constant domain of the TCR chain.
在另一優選例中,所述TCR的α鏈恆定區與β鏈恆定區之間含有人工鏈間二硫鍵。In another preferred embodiment, an artificial interchain disulfide bond is contained between the constant region of the α chain and the constant region of the β chain of the TCR.
在另一優選例中,在所述TCRα與β鏈的恆定區之間形成人工鏈間二硫鍵的半胱胺酸殘基取代了選自下列的一組或多組位址:
TRAC*01外顯子1的Thr48和TRBC1*01或TRBC2*01外顯子1的Ser57;
TRAC*01外顯子1的Thr45和TRBC1*01或TRBC2*01外顯子1的Ser77;
TRAC*01外顯子1的Tyr10和TRBC1*01或TRBC2*01外顯子1的Ser17;
TRAC*01外顯子1的Thr45和TRBC1*01或TRBC2*01外顯子1的Asp59;
TRAC*01外顯子1的Ser15和TRBC1*01或TRBC2*01外顯子1的Glu15;
TRAC*01外顯子1的Arg53和TRBC1*01或TRBC2*01外顯子1的Ser54;
TRAC*01外顯子1的Pro89和TRBC1*01或TRBC2*01外顯子1的Ala19;
和TRAC*01外顯子1的Tyr10和TRBC1*01或TRBC2*01外顯子1的Glu20。In another preferred embodiment, the cysteine residue that forms an artificial interchain disulfide bond between the constant region of the TCRα and the β chain is substituted for one or more sets of addresses selected from the following:
Thr48 in
在另一優選例中,所述TCR的α鏈可變域胺基酸序列為SEQ ID NO: 1、13-32之一;和/或所述TCR的β鏈可變域胺基酸序列為SEQ ID NO: 2、33-36之一。In another preferred embodiment, the amino acid sequence of the α chain variable domain of the TCR is one of SEQ ID NOs: 1, 13-32; and/or the amino acid sequence of the β chain variable domain of the TCR is One of SEQ ID NO: 2, 33-36.
在另一優選例中,所述TCR選自下組:
在另一優選例中,所述TCR為單鏈TCR。In another preferred embodiment, the TCR is a single-chain TCR.
在另一優選例中,所述TCR是由α鏈可變域和β鏈可變域組成的單鏈TCR,所述α鏈可變域和β鏈可變域由一柔性短胜肽序列(linker)連接。In another preferred embodiment, the TCR is a single-chain TCR composed of an α-chain variable domain and a β-chain variable domain, and the α-chain variable domain and the β-chain variable domain are composed of a flexible short peptide sequence ( linker) connection.
在另一優選例中,所述TCR的α鏈和/或β鏈的C-或N-末端結合有偶聯物,優選地,所述偶聯物為可檢測標記物、治療劑、PK修飾部分或任何這些物質的組合。In another preferred example, a conjugate is bound to the C- or N-terminus of the α chain and/or β chain of the TCR, preferably, the conjugate is a detectable label, a therapeutic agent, or a PK modification some or a combination of any of these substances.
在另一優選例中,與所述TCR結合的治療劑為連接於所述TCR的α或β鏈的C-或N-末端的抗-CD3抗體。In another preferred embodiment, the therapeutic agent bound to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the α or β chain of the TCR.
本發明的第二方面,提供了一種多價TCR複合物,其包含至少兩個TCR分子,並且其中的至少一個TCR分子為上述請求項中任一項所述的TCR。A second aspect of the present invention provides a multivalent TCR complex comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR described in any one of the above claims.
本發明的第三方面,提供了一種核酸分子,所述核酸分子包含編碼本發明第一方面所述的TCR分子或者本發明第二方面所述的多價TCR複合物的核酸序列或其互補序列。The third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or the multivalent TCR complex described in the second aspect of the present invention or a complementary sequence thereof .
本發明的第四方面,提供了一種載體,所述的載體含有本發明第三方面所述的所述的核酸分子。The fourth aspect of the present invention provides a vector, the vector contains the nucleic acid molecule described in the third aspect of the present invention.
本發明的第五方面,提供了一種宿主細胞,所述的宿主細胞中含有本發明第四方面所述的載體或染色體中併入了外源的本發明第三方面所述的核酸分子。The fifth aspect of the present invention provides a host cell containing the vector of the fourth aspect of the present invention or incorporating the exogenous nucleic acid molecule of the third aspect of the present invention into a chromosome.
本發明的第六方面,提供了一種分離的細胞,所述細胞表現本發明第一方面所述的TCR。The sixth aspect of the present invention provides an isolated cell expressing the TCR of the first aspect of the present invention.
本發明的第七方面,提供了一種藥物組成物,所述組成物含有藥學上可接受的載劑以及本發明第一方面所述的TCR、或本發明第二方面所述的TCR複合物、或本發明第六方面所述的細胞。The seventh aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell according to the sixth aspect of the present invention.
本發明的第八方面,提供了一種治療疾病的方法,包括給需要治療的對象施用適量的本發明第一方面所述的TCR、或本發明第二方面所述的TCR複合物、或本發明第六方面所述的細胞、或本發明第七方面所述的藥物組成物,優選地,所述疾病為AFP陽性腫瘤,更優選地,所述腫瘤為肝癌,最優選地,所述腫瘤為肝細胞癌。The eighth aspect of the present invention provides a method for treating a disease, comprising administering an appropriate amount of the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the present invention to a subject in need of treatment. The cell according to the sixth aspect or the pharmaceutical composition according to the seventh aspect of the present invention, preferably, the disease is an AFP positive tumor, more preferably, the tumor is liver cancer, most preferably, the tumor is hepatocellular carcinoma.
本發明的第九方面,提供了本發明第一方面所述的TCR、或本發明第二方面所述的TCR複合物、或本發明第六方面所述的細胞的用途,用於製備治療腫瘤的藥物,優選地,所述腫瘤為AFP陽性腫瘤,更優選地,所述腫瘤為肝癌,最優選地,所述腫瘤為肝細胞癌。The ninth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell described in the sixth aspect of the present invention, for preparing and treating tumors Preferably, the tumor is AFP positive tumor, more preferably, the tumor is liver cancer, most preferably, the tumor is hepatocellular carcinoma.
本發明的第十方面,提供了一種製備本發明第一方面所述的T細胞受體的方法,包括步驟: (i) 培養本發明第五方面所述的宿主細胞,從而表現本發明第一方面所述的T細胞受體; (ii) 分離或純化出所述的T細胞受體。A tenth aspect of the present invention provides a method for preparing the T cell receptor described in the first aspect of the present invention, comprising the steps of: (i) culturing the host cell of the fifth aspect of the present invention to express the T cell receptor of the first aspect of the present invention; (ii) isolating or purifying said T cell receptor.
應理解,在本發明範圍內中,本發明的上述各技術特徵和在下文(如實施例)中具體描述的各技術特徵之間都可以互相組合,從而構成新的或優選的技術方案。限於篇幅,在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.
具體實施方式detailed description
本發明通過廣泛而深入的研究,獲得一種辨識TSSELMAITR短胜肽(衍生自AFP蛋白)的高親和性T細胞受體(TCR),所述TSSELMAITR短胜肽以胜肽-HLA A1101複合物的形式被呈現。所述高親和性TCR在其α鏈可變域的3個CDR區: CDR1α:YGATPY CDR2α:YFSGDTLV CDR3α:AVVATDSWGKLQ中發生突變;和/或在其β鏈可變域的3個CDR區: CDR1β:SGHVS CDR2β:FQNEAQ CDR3β:ASSLVAGARTDTQY中發生突變;並且,突變後本發明TCR對上述TSSELMAITR-HLA A1101複合物的親和力和/或結合半衰期是野生型TCR的至少2倍。The present invention has obtained a high-affinity T cell receptor (TCR) that recognizes a short peptide of TSSELMAITR (derived from AFP protein) through extensive and in-depth research. The short peptide of TSSELMAITR is in the form of a peptide-HLA A1101 complex. is presented. The high-affinity TCR has 3 CDR regions in its alpha chain variable domain: CDR1α: YGATPY CDR2α: YFSGDTLV CDR3α: mutated in AVVATDSWGKLQ; and/or in the 3 CDR regions of its β chain variable domain: CDR1β: SGHVS CDR2β: FQNEAQ CDR3β: A mutation occurs in ASSLVAGARTDTQY; and the affinity and/or binding half-life of the TCR of the present invention to the above-mentioned TSSELMAITR-HLA A1101 complex after the mutation is at least 2 times that of the wild-type TCR.
在描述本發明之前,應當理解本發明不限於所述的具體方法和實驗條件,因為這類方法和條件可以變動。還應當理解本文所用的術語其目的僅在於描述具體實施方案,並且其意圖不是限制性的,本發明的範圍將僅由所附的申請專利範圍限制。Before the present invention is described, it is to be understood that this invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the scope of the appended claims.
除非另外定義,否則本文中所用的全部技術與科學術語均具有如本發明所屬領域的普通技術人員通常理解的相同含義。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
雖然在本發明的實施或測試中可以使用與本發明中所述相似或等價的任何方法和材料,本文在此處例舉優選的方法和材料。術語 T 細胞受體 (T cell receptor,TCR) Although any methods and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present invention, the preferred methods and materials are exemplified herein. Term T cell receptor (TCR)
可以採用國際免疫遺傳學資訊系統(IMGT)來描述TCR。天然αβ異源二聚TCR具有α鏈和β鏈。廣義上講,各鏈包含可變區、連接區和恆定區,β鏈通常還在可變區和連接區之間含有短的多變區,但該多變區常視作連接區的一部分。通過獨特的IMGT的TRAJ 和TRBJ確定TCR的連接區,通過IMGT的TRAC 和TRBC確定TCR的恆定區。TCRs can be described using the International Information System on Immunogenetics (IMGT). Native αβ heterodimeric TCRs have α and β chains. Broadly speaking, each chain contains a variable region, a linker region and a constant region, and the beta chain typically also contains a short variable region between the variable region and the linker region, but the variable region is often considered part of the linker region. The junctional region of the TCR is determined by TRAJ and TRBJ of the unique IMGT, and the constant region of the TCR is determined by the TRAC and TRBC of the IMGT.
各可變區包含嵌合在框架序列中的3個CDR (互補決定區),CDR1、CDR2和CDR3。在IMGT命名法中,TRAV和TRBV的不同編號分別指代不同Vα類型和Vβ的類型。在IMGT系統中,α鏈恆定結構域具有以下的符號:TRAC*01,其中“TR”表示T細胞受體基因;“A”表示α鏈基因;C表示恆定區;“*01”表示等位基因1。β鏈恆定結構域具有以下的符號:TRBC1*01或TRBC2*01,其中“TR”表示T細胞受體基因;“B”表示β鏈基因;C表示恆定區;“*01”表示等位基因1。α鏈的恆定區是唯一確定的,在β鏈的形式中,存在兩個可能的恆定區基因“C1”和“C2”。本領域技術人員通過公開的IMGT資料庫可以獲得TCRα與β鏈的恆定區基因序列。Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimerically incorporated in a framework sequence. In the IMGT nomenclature, the different numbers of TRAV and TRBV refer to different Vα types and Vβ types, respectively. In the IMGT system, the alpha chain constant domain has the following symbols: TRAC*01, where "TR" represents the T cell receptor gene; "A" represents the alpha chain gene; C represents the constant region; "*01" represents the
TCR的α和β鏈一般看作各有兩個“結構域”即可變域和恆定結構域。可變域由連接的可變區和連接區構成。因此,在本申請的說明書和申請專利範圍中,“TCRα鏈可變域”指連接的TRAV和TRAJ區,同樣地,“TCRβ鏈可變域”指連接的TRBV和TRBD/TRBJ區。TCRα鏈可變域的3個CDR分別為CDR1α、CDR2α和CDR3α;TCRβ鏈可變域的3個CDR分別為CDR1β、CDR2β和CDR3β。本發明TCR可變域的框架序列可以為鼠源的或人源的,優選為人源的。TCR的恆定結構域包含胞內部分、跨膜區和胞外部分。The alpha and beta chains of a TCR are generally viewed as having two "domains" each, a variable domain and a constant domain. A variable domain consists of linked variable and linker regions. Thus, in the specification and scope of this application, "TCR alpha chain variable domain" refers to linked TRAV and TRAJ regions, and similarly, "TCR beta chain variable domain" refers to linked TRBV and TRBD/TRBJ regions. The three CDRs of the variable domain of the TCRα chain are CDR1α, CDR2α and CDR3α, respectively; the three CDRs of the variable domain of the TCRβ chain are CDR1β, CDR2β and CDR3β, respectively. The framework sequences of the TCR variable domains of the present invention may be of murine or human origin, preferably human. The constant domains of TCRs comprise an intracellular portion, a transmembrane region and an extracellular portion.
本發明中,能夠結合TSSELMAITR-HLA A1101複合物的野生型TCR的α與β鏈可變域胺基酸序列分別為SEQ ID NO: 1和SEQ ID NO: 2,如圖1a和圖1b所示。本發明中所述可溶性“參考TCR”的α鏈胺基酸序列及β鏈胺基酸序列分別為SEQ ID NO: 11和SEQ ID NO: 12,如圖6a和圖6b所示。本發明中所述“野生型TCR”的α鏈胞外胺基酸序列及β鏈胞外胺基酸序列分別為SEQ ID NO: 37和SEQ ID NO: 38,如圖9a和圖9b所示。本發明中所用的TCR序列為人源的。本發明中所述“野生型TCR”的α鏈胺基酸序列及β鏈胺基酸序列分別為SEQ ID NO: 39和SEQ ID NO: 40,如圖10a和10b所示。在本發明中,術語“本發明多肽”、“本發明的TCR”、“本發明的T細胞受體”可互換使用。天然鏈間二硫鍵與人工鏈間二硫鍵 In the present invention, the amino acid sequences of the α and β chain variable domains of the wild-type TCR capable of binding to the TSSELMAITR-HLA A1101 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Figure 1a and Figure 1b . The amino acid sequence of α chain and the amino acid sequence of β chain of the soluble "reference TCR" in the present invention are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b. The α-chain extracellular amino acid sequence and the β-chain extracellular amino acid sequence of the "wild-type TCR" described in the present invention are SEQ ID NO: 37 and SEQ ID NO: 38, respectively, as shown in Figure 9a and Figure 9b . The TCR sequences used in the present invention are of human origin. The amino acid sequence of the α chain and the amino acid sequence of the β chain of the "wild-type TCR" in the present invention are SEQ ID NO: 39 and SEQ ID NO: 40, respectively, as shown in Figures 10a and 10b. In the present invention, the terms "polypeptide of the present invention", "TCR of the present invention", "T cell receptor of the present invention" are used interchangeably. Natural interchain disulfide bonds and artificial interchain disulfide bonds
在天然TCR的近膜區Cα與Cβ鏈間存在一組二硫鍵,本發明中稱為“天然鏈間二硫鍵”。在本發明中,將人工引入的,位置與天然鏈間二硫鍵的位置不同的鏈間共價二硫鍵稱為“人工鏈間二硫鍵”。There is a set of disulfide bonds between the Cα and Cβ chains in the near-membrane region of the native TCR, which are referred to as "native interchain disulfide bonds" in the present invention. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as "artificial interchain disulfide bond".
為方便描述,本發明中TRAC*01與TRBC1*01或TRBC2*01胺基酸序列的位置編號按從N端到C端依次的順序進行位置編號,如TRBC1*01或TRBC2*01中,按從N端到C端依次的順序第60個胺基酸為P (脯胺酸),則本發明中可將其描述為TRBC1*01或TRBC2*01外顯子1的Pro60,也可將其表述為TRBC1*01或TRBC2*01外顯子1的第60位胺基酸,又如TRBC1*01或TRBC2*01中,按從N端到C端依次的順序第61個胺基酸為Q (麩醯胺酸),則本發明中可將其描述為TRBC1*01或TRBC2*01外顯子1的Gln61,也可將其表述為TRBC1*01或TRBC2*01外顯子1的第61位胺基酸,其他以此類推。本發明中,可變區TRAV與TRBV的胺基酸序列的位置編號,按照IMGT中列出的位置編號。如TRAV中的某個胺基酸,IMGT中列出的位置編號為46,則本發明中將其描述為TRAV第46位胺基酸,其他以此類推。本發明中,其他胺基酸的序列位置編號有特殊說明的,則按特殊說明。腫瘤 For the convenience of description, the position numbering of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention is numbered in sequence from the N-terminus to the C-terminus. For example, in TRBC1*01 or TRBC2*01, press The 60th amino acid in the sequence from the N-terminal to the C-terminal is P (proline), then in the present invention, it can be described as Pro60 of
術語“腫瘤”指包括所有類型的癌細胞生長或致癌過程,轉移性組織或惡性轉化細胞、組織或器官,不管病理類型或侵染的階段。腫瘤的實施例非限制性地包括:固態腫瘤,軟組織瘤,和轉移性病灶。固態腫瘤的實施例包括:不同器官系統的惡性腫瘤,例如肉瘤,肺鱗狀細胞癌和癌症。例如:感染的前列腺,肺,乳房,淋巴,腸胃(例如:結腸),和生殖泌尿道(例如:腎臟,上皮細胞),咽喉。肺鱗狀細胞癌包括惡性腫瘤,例如,多數的結腸癌,直腸癌,腎細胞癌,肝癌,肺部的非小細胞癌,小腸癌和食道癌。上述癌症的轉移性病變可同樣用本發明的方法和組成物來治療和預防。發明詳述 The term "tumor" is meant to encompass all types of cancer cell growth or oncogenic processes, metastatic or malignantly transformed cells, tissues or organs, regardless of the type of pathology or stage of infection. Examples of tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignancies of various organ systems such as sarcomas, lung squamous cell carcinomas, and cancers. For example: infected prostate, lung, breast, lymph, gastrointestinal (eg: colon), and genitourinary tract (eg: kidney, epithelial cells), throat. Squamous cell carcinoma of the lung includes malignancies such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell carcinomas of the lungs, small bowel cancers and esophageal cancers. Metastatic lesions of the aforementioned cancers can likewise be treated and prevented with the methods and compositions of the present invention. Detailed description of the invention
眾所周知,TCR的α鏈可變域與β鏈可變域各含有3個CDR,類似於抗體的互補決定區。CDR3與抗原短胜肽相互作用,CDR1和CDR2與HLA相互作用。因此,TCR分子的CDR決定了其與抗原短胜肽-HLA複合物的相互作用。能夠結合抗原短胜肽TSSELMAITR與HLA A1101複合物(即,TSSELMAITR-HLA A1101複合物)的野生型TCR的α鏈可變域胺基酸序列與β鏈可變域胺基酸序列分別為SEQ ID NO: 1和SEQ ID NO: 2, 該序列為本發明人首次發現。其具有下列CDR區: α鏈可變域CDR CDR1α:YGATPY CDR2α:YFSGDTLV CDR3α:AVVATDSWGKLQ 和β鏈可變域CDR CDR1β:SGHVS CDR2β:FQNEAQ和 CDR3β:ASSLVAGARTDTQY。It is well known that the α chain variable domain and the β chain variable domain of TCR each contain three CDRs, similar to the complementarity determining regions of antibodies. CDR3 interacts with antigenic short peptides, and CDR1 and CDR2 interact with HLA. Therefore, the CDRs of the TCR molecule determine its interaction with the antigenic short peptide-HLA complex. SEQ ID NO: 1 and SEQ ID NO: 2, the sequence was discovered by the inventor for the first time. It has the following CDR regions: Alpha chain variable domain CDRs CDR1α: YGATPY CDR2α: YFSGDTLV CDR3α:AVVATDSWGKLQ and β-chain variable domain CDRs CDR1β: SGHVS CDR2β: FQNEAQ and CDR3β:ASSLVAGARTDTQY.
本發明通過對上述CDR區進行突變篩選,獲得了與TSSELMAITR-HLA A1101複合物的親和力是野生型TCR與TSSELMAITR-HLA A1101複合物親和力至少2倍的高親和力TCR。The present invention obtains a high-affinity TCR whose affinity with the TSSELMAITR-HLA A1101 complex is at least 2 times that of the wild-type TCR and the TSSELMAITR-HLA A1101 complex through mutation screening of the above-mentioned CDR region.
進一步,本發明所述TCR是αβ異質二聚TCR,所述TCR的α鏈可變域包含與SEQ ID NO: 1所示的胺基酸序列有至少85%;優選地,至少90%;更優選地,至少92%;更優選地,至少94% (如,可以是至少88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的序列同源性)的序列同源性的胺基酸序列;和/或所述TCR的β鏈可變域包含與SEQ ID NO: 2所示的胺基酸序列有至少90%,優選地,至少92%;更優選地,至少94%(如,可以是至少91%、92%、93%、94%、95%、96%、97%、98%、99%或100%的序列同源性)的序列同源性的胺基酸序列。Further, the TCR of the present invention is an αβ heterodimeric TCR, and the α chain variable domain of the TCR comprises at least 85% of the amino acid sequence shown in SEQ ID NO: 1; preferably, at least 90%; more Preferably, at least 92%; more preferably, at least 94% (eg, can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) the amino acid sequence of sequence homology; And/or the β chain variable domain of described TCR comprises the amino acid sequence shown in SEQ ID NO: 2 with at least 90%, preferably, at least 92%; more preferably, at least 94% (eg, can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology) amino acid sequence of sequence homology.
進一步,本發明所述TCR是單鏈TCR,所述TCR的α鏈可變域包含與SEQ ID NO: 3所示的胺基酸序列有至少85%,優選地,至少90%;更優選地,至少92%;最優選地,至少94% (如,可以是至少88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的序列同源性)的序列同源性的胺基酸序列;和/或所述TCR的β鏈可變域包含與SEQ ID NO: 4所示的胺基酸序列有至少85%,優選地,至少90%;更優選地,至少92%;最優選地,至少94%;(如,可以是至少91%、92%、93%、94%、95%、96%、97%、98%、99%的序列同源性)的序列同源性的胺基酸序列。Further, the TCR of the present invention is a single-chain TCR, and the α-chain variable domain of the TCR comprises at least 85%, preferably, at least 90% of the amino acid sequence shown in SEQ ID NO: 3; more preferably , at least 92%; most preferably, at least 94% (eg, can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% sequence homology) amino acid sequence of sequence homology; and/or the β chain variable domain of the TCR comprises at least 85% amino acid sequence with the amino acid sequence shown in SEQ ID NO: 4 , preferably, at least 90%; more preferably, at least 92%; most preferably, at least 94%; (eg, can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% sequence homology) amino acid sequence of sequence homology.
本發明中野生型TCRα鏈可變域SEQ ID NO: 1的3個CDR即CDR1、CDR2和CDR3分別位於SEQ ID NO: 1的第27-32位、第50-57位和第92-103位。據此,胺基酸殘基編號採用SEQ ID NO: 1所示的編號,94V即為CDR3α的第3位V、95A即為CDR3α的第4位A、96T即為CDR3α的第5位T、97D即為CDR3α的第6位D。In the present invention, the three CDRs of the wild-type TCRα chain variable domain SEQ ID NO: 1, namely CDR1, CDR2 and CDR3, are respectively located at positions 27-32, 50-57 and 92-103 of SEQ ID NO: 1 . Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1, 94V is the 3rd position V of CDR3α, 95A is the 4th position A of CDR3α, 96T is the 5th position T of CDR3α, 97D is the 6th D of CDR3α.
本發明提供具有結合TSSELMAITR-HLA A1101複合物的特性的TCR,並包含α鏈可變域和β鏈可變域,其中,所述TCR在SEQ ID NO: 1所示的α鏈可變域中發生突變,所述突變的胺基酸殘基位址包括94V、95A、96T和97D中的一個或多個,其中,胺基酸殘基編號採用SEQ ID NO: 1所示的編號。The present invention provides a TCR having the property of binding to the TSSELMAITR-HLA A1101 complex, and comprising an α-chain variable domain and a β-chain variable domain, wherein the TCR is in the α-chain variable domain shown in SEQ ID NO: 1 A mutation occurs, and the mutated amino acid residue addresses include one or more of 94V, 95A, 96T and 97D, wherein the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 1.
優選地,突變後的所述TCRα鏈可變域包括選自下組的一個或多個胺基酸殘基:94A或94P;95S或95D或95G;96L或96M或96Y或96I;97K或97Q或97S或97E或97A或97H或97N或97P或97R,其中,胺基酸殘基編號採用SEQ ID NO: 1所示的編號。Preferably, the mutated TCRα chain variable domain comprises one or more amino acid residues selected from the group consisting of: 94A or 94P; 95S or 95D or 95G; 96L or 96M or 96Y or 96I; 97K or 97Q or 97S or 97E or 97A or 97H or 97N or 97P or 97R, wherein the numbering of the amino acid residues adopts the numbering shown in SEQ ID NO: 1.
更具體地,α鏈可變域中所述突變的具體形式包括V94A/P、A95S/D/G、T96L/M/Y/I、D97K/Q/S/E/A/H/N/P/R中的一組或幾組。More specifically, specific forms of the mutation in the alpha chain variable domain include V94A/P, A95S/D/G, T96L/M/Y/I, D97K/Q/S/E/A/H/N/P A group or groups in /R.
本發明中野生型TCRβ鏈可變域SEQ ID NO: 2的3個CDR即CDR1、CDR2和CDR3分別位於SEQ ID NO: 2的第27-31位、第49-54位和第93-106位。據此,胺基酸殘基編號採用SEQ ID NO: 2所示的編號,95S即為CDR3β的第3位S、96L即為CDR3β的第4位L、97V即為CDR3β的第5位V、98A即為CDR3β的第6位A。In the present invention, the three CDRs of the wild-type TCRβ chain variable domain SEQ ID NO: 2, namely CDR1, CDR2 and CDR3, are respectively located at positions 27-31, 49-54 and 93-106 of SEQ ID NO: 2 . Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 2, 95S is the 3rd S of CDR3β, 96L is the 4th L of CDR3β, 97V is the 5th V of CDR3β, 98A is the 6th A of CDR3β.
本發明提供具有結合TSSELMAITR-HLA A1101複合物的特性的TCR,並包含β鏈可變域和β鏈可變域,其中,所述TCR在SEQ ID NO: 2所示的β鏈可變域中發生突變,所述突變的胺基酸殘基位址包括95S、96L、97V、98A中的一個或多個,其中,胺基酸殘基編號採用SEQ ID NO: 2所示的編號。The present invention provides a TCR having the property of binding to the TSSELMAITR-HLA A1101 complex, and comprising a beta chain variable domain and a beta chain variable domain, wherein the TCR is in the beta chain variable domain shown in SEQ ID NO: 2 Mutation occurs, and the mutated amino acid residue addresses include one or more of 95S, 96L, 97V, and 98A, wherein the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 2.
優選地,突變後的所述TCRβ鏈可變域包括選自下組的一個或多個胺基酸殘基:95T;96M或96W;97L或97I;98G,其中,胺基酸殘基編號採用SEQ ID NO: 2所示的編號。Preferably, the mutated TCRβ chain variable domain comprises one or more amino acid residues selected from the group consisting of: 95T; 96M or 96W; 97L or 97I; 98G, wherein the amino acid residues are numbered using The numbering shown in SEQ ID NO: 2.
更具體地,β鏈可變域中所述突變的具體形式包括S95T、L96M/W、V97L/I、A98G中的一組或幾組。More specifically, the specific form of the mutation in the beta chain variable domain includes one or several groups of S95T, L96M/W, V97L/I, A98G.
應理解,本文中胺基酸名稱採用國際通用的單英文字母標識,與其相對應的胺基酸名稱三英文字母簡寫分別是:Ala (A)、Arg (R)、Asn (N)、Asp (D)、Cys (C)、Gln (Q)、Glu (E)、Gly (G)、His (H)、Ile (I)、Leu (L)、Lys (K)、Met (M)、Phe (F)、Pro (P)、Ser (S)、Thr (T)、Trp (W)、Tyr (Y)、Val (V);It should be understood that the name of amino acid in this paper adopts the international single English letter identification, and the three English letter abbreviations of the corresponding amino acid name are respectively: Ala (A), Arg (R), Asn (N), Asp ( D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe ( F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V);
本發明中,Pro60或者60P均表示第60位脯胺酸。另外,本發明中所述突變的具體形式的表述方式如“V94A/P”代表第94位的V被A取代或被G取代,其他以此類推。In the present invention, both Pro60 or 60P represent proline at the 60th position. In addition, the expression of the specific form of the mutation in the present invention, such as "V94A/P", represents that V at position 94 is substituted by A or by G, and so on.
根據本領域技術人員熟知的定點突變的方法,將野生型TCRα鏈恆定區TRAC*01外顯子1的Thr48突變為半胱胺酸,β鏈恆定區TRBC1*01或TRBC2*01外顯子1的Ser57突變為半胱胺酸,即得到參考TCR,其胺基酸序列分別為SEQ ID NO: 11和SEQ ID NO: 12,如圖6a和圖6b所示,突變後的半胱胺酸殘基以加粗字母表示。上述半胱胺酸取代能使參考TCR的α與β鏈的恆定區之間形成人工鏈間二硫鍵,以形成更加穩定的可溶性TCR,從而能夠更加方便地評估TCR與TSSELMAITR-HLA A1101複合物之間的結合親和力和/或結合半衰期。應理解,TCR可變區的CDR區決定了其與pMHC複合物之間的親和力,因此,上述TCR恆定區的半胱胺酸取代並不會對TCR的結合親和力和/或結合半衰期產生影響。所以,在本發明中,測得的參考TCR與TSSELMAITR-HLA A1101複合物之間的結合親和力即認為是野生型TCR與TSSELMAITR-HLA A1101複合物之間的結合親和力。同樣地,如果測得本發明TCR與TSSELMAITR-HLA A1101複合物之間的結合親和力是參考TCR與TSSELMAITR-HLA A1101複合物之間的結合親和力的至少10倍,即等同於本發明TCR與TSSELMAITR-HLA A1101複合物之間的結合親和力是野生型TCR與TSSELMAITR-HLA A1101複合物之間的結合親和力的至少10倍。According to the method of site-directed mutagenesis well known to those skilled in the art, Thr48 of TRAC*01
可通過任何合適的方法測定結合親和力(與解離平衡常數KD 成反比)和結合半衰期(表示為T1/2 ),如採用表面電漿共振技術進行檢測。應瞭解,TCR的親和力翻倍將導致KD 減半。T1/2 計算為In2除以解離速率(Koff )。因此,T1/2 翻倍會導致Koff 減半。優選採用相同的試驗方案檢測給定TCR的結合親和力或結合半衰期數次,例如3次或更多,取結果的平均值。在優選的實施方式中,採用本文實施例中的表面電漿共振(BIAcore)方法檢測可溶性TCR的親和力,條件為:溫度25℃,pH值為7.1-7.5。該方法檢測到參考TCR對TSSELMAITR-HLA A1101複合物的解離平衡常數KD 為2.98E-05M,即29.8 μM,本發明中即認為野生型TCR對TSSELMAITR-HLA A1101複合物的解離平衡常數KD 也為29.8 μM。由於TCR的親和力翻倍將導致KD 減半,所以若檢測到高親和力TCR對TSSELMAITR-HLA A1101複合物的解離平衡常數KD 為2.98E-06M,即2.98 μM,則說明該高親和力TCR對TSSELMAITR-HLA A1101複合物的親和力是野生型TCR對TSSELMAITR-HLA A1101複合物的親和力的10倍。本領域技術人員熟知KD 值單位間的換算關係,即1 M = 106 μM,1 μM = 1000 nM,1 nM = 1000 pM。在本發明中,所述TCR與TSSELMAITR-HLA A1101複合物的親和力是野生型TCR的至少2倍。The binding affinity may be measured by any suitable method (the equilibrium dissociation constant K D is inversely proportional to) the half-life and binding (expressed as T 1/2), such as using surface plasmon resonance technology for detection. It should be understood, TCR affinity K D doubled the lead in half. T 1/2 was calculated as In2 divided by the dissociation rate (K off ). Therefore, doubling T 1/2 results in a halving of K off. Preferably, the same assay protocol is used to measure the binding affinity or binding half-life of a given TCR several times, eg, 3 times or more, and the results are averaged. In a preferred embodiment, the surface plasmon resonance (BIAcore) method in the examples herein is used to detect the affinity of soluble TCR, and the conditions are: the temperature is 25° C., and the pH value is 7.1-7.5. This method detects that the dissociation equilibrium constant K D of the reference TCR to the TSSELMAITR-HLA A1101 complex is 2.98E-05M, that is, 29.8 μM. In the present invention, it is considered that the dissociation equilibrium constant K D of the wild-type TCR to the TSSELMAITR-HLA A1101 complex Also 29.8 μM. Since the doubling of the affinity of TCR will lead to the halving of the K D , if the dissociation equilibrium constant K D of the high-affinity TCR for the TSSELMAITR-HLA A1101 complex is detected to be 2.98E-06M, that is, 2.98 μM, it means that the high-affinity TCR pair The affinity of the TSSELMAITR-HLA A1101 complex was 10 times higher than that of the wild-type TCR for the TSSELMAITR-HLA A1101 complex. Those skilled in the art are familiar with the conversion relationship between K D value units, namely 1 M = 10 6 μM, 1 μM = 1000 nM, 1 nM = 1000 pM. In the present invention, the affinity of the TCR to the TSSELMAITR-HLA A1101 complex is at least 2 times that of the wild-type TCR.
可採用任何合適的方法進行突變,包括但不限於依據聚合酶鏈式反應(PCR)的那些、依據限制酶的選殖或不依賴連接的選殖(LIC)方法。許多標準分子生物學教材詳述了這些方法。聚合酶鏈式反應(PCR)誘變和依據限制酶的選殖的更多細節可參見Sambrook和Russell, (2001)分子選殖-實驗室手冊(Molecular Cloning-A Laboratory Manual)(第三版) CSHL出版社。LIC方法的更多資訊可見(Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6)。Mutagenesis can be performed using any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme based cloning or ligation independent cloning (LIC) methods. These methods are detailed in many standard molecular biology textbooks. More details on polymerase chain reaction (PCR) mutagenesis and cloning by restriction enzymes can be found in Sambrook and Russell, (2001) Molecular Cloning - A Laboratory Manual (Third Edition) CSHL Press. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6).
產生本發明的TCR的方法可以是但不限於從展示此類TCR的噬菌體顆粒的多樣性序列庫中篩選出對TSSELMAITR-HLA-A1101複合物具有高親和性的TCR,如文獻(Li,et al (2005) Nature Biotech 23(3): 349-354)中所述。The method for generating the TCRs of the present invention can be, but is not limited to, screening TCRs with high affinity for the TSSELMAITR-HLA-A1101 complex from a diverse sequence library of phage particles displaying such TCRs, as described in the literature (Li, et al. (2005) Nature Biotech 23(3): 349-354).
應理解,表現野生型TCRα和β鏈可變域胺基酸的基因或者表現略作修飾的野生型TCR的α和β鏈可變域胺基酸的基因都可用來製備模板TCR。然後在編碼該模板TCR的可變域的DNA中引入產生本發明的高親和力TCR所需的改變。It will be appreciated that either genes expressing wild-type TCR alpha and beta chain variable domain amino acids or genes expressing slightly modified wild-type TCR alpha and beta chain variable domain amino acids can be used to prepare template TCRs. The changes required to generate the high affinity TCRs of the invention are then introduced into the DNA encoding the variable domains of the template TCR.
本發明的高親和性TCR包含α鏈可變域胺基酸序列為SEQ ID NO: 1、13-32之一;和/或所述TCR的β鏈可變域胺基酸序列為SEQ ID NO: 2、33-36之一。本發明中,形成異質二聚TCR分子的α鏈可變域與β鏈可變域的胺基酸序列優選自下表1:
表1
基於本發明的目的,本發明TCR是具有至少一個TCRα和/或TCRβ鏈可變域的部分。它們通常同時包含TCRα鏈可變域和TCRβ鏈可變域。它們可以是αβ異源二聚體或是單鏈形式或是其他任何能夠穩定存在的形式。在過繼性免疫治療中,可將αβ異源二聚TCR的全長鏈(包含細胞質和跨膜結構域)進行轉染。本發明TCR可用作將治療劑遞送至抗原呈現細胞的標靶劑或與其他分子結合製備雙功能多肽來定向效應細胞,此時TCR優選為可溶形式。For the purposes of the present invention, a TCR of the present invention is a portion having at least one variable domain of the TCRα and/or TCRβ chain. They usually contain both the TCRα chain variable domain and the TCRβ chain variable domain. They can be alpha beta heterodimers or single-chain forms or any other form that can be stably present. In adoptive immunotherapy, the full-length chain of alpha beta heterodimeric TCR (comprising cytoplasmic and transmembrane domains) can be transfected. The TCR of the present invention can be used as a targeting agent for delivery of therapeutic agents to antigen-presenting cells or combined with other molecules to produce bifunctional polypeptides to target effector cells, in which case the TCR is preferably in a soluble form.
對於穩定性而言,現有技術中公開了在TCR的α與β鏈恆定域之間引入人工鏈間二硫鍵能夠獲得可溶且穩定的TCR分子,如專利文獻PCT/CN2015/093806中所述。因此,本發明TCR可以是在其α和β鏈恆定域的殘基之間引入人工鏈間二硫鍵的TCR。半胱胺酸殘基在所述TCR的α和β鏈恆定域間形成人工鏈間二硫鍵。半胱胺酸殘基可以取代在天然TCR中合適位址的其他胺基酸殘基以形成人工鏈間二硫鍵。例如,取代TRAC*01外顯子1的Thr48和取代TRBC1*01或TRBC2*01外顯子1的Ser57來形成二硫鍵。引入半胱胺酸殘基以形成二硫鍵的其他位址還可以是:TRAC*01外顯子1的Thr45和TRBC1*01或TRBC2*01外顯子1的Ser77;TRAC*01外顯子1的Tyr10和TRBC1*01或TRBC2*01外顯子1的Ser17;TRAC*01外顯子1的Thr45和TRBC1*01或TRBC2*01外顯子1的Asp59;TRAC*01外顯子1的Ser15和TRBC1*01或TRBC2*01外顯子1的Glu15;TRAC*01外顯子1的Arg53和TRBC1*01或TRBC2*01外顯子1的Ser54;TRAC*01外顯子1的Pro89和TRBC1*01或TRBC2*01外顯子1的Ala19;或TRAC*01外顯子1的Tyr10和TRBC1*01或TRBC2*01外顯子1的Glu20。即半胱胺酸殘基取代了上述α與β鏈恆定域中任一組位址。可在本發明TCR恆定域的一個或多個C末端截短最多15個、或最多10個、或最多8個或更少的胺基酸,以使其不包括半胱胺酸殘基來達到缺失天然鏈間二硫鍵的目的,也可通過將形成天然鏈間二硫鍵的半胱胺酸殘基突變為另一胺基酸來達到上述目的。For stability, it is disclosed in the prior art that soluble and stable TCR molecules can be obtained by introducing artificial interchain disulfide bonds between the constant domains of the α and β chains of TCR, as described in the patent document PCT/CN2015/093806 . Thus, the TCR of the present invention may be one in which artificial interchain disulfide bonds are introduced between residues of its alpha and beta chain constant domains. Cysteine residues form artificial interchain disulfide bonds between the constant domains of the alpha and beta chains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate addresses in native TCRs to form artificial interchain disulfide bonds. For example, substitution of Thr48 in
如上所述,本發明的TCR可以包含在其α和β鏈恆定域的殘基間引入的人工鏈間二硫鍵。應注意,恆定域間含或不含上文所述的引入的人工二硫鍵,本發明的TCR均可含有TRAC恆定域序列和TRBC1或TRBC2恆定域序列。TCR的TRAC恆定域序列和TRBC1或TRBC2恆定域序列可通過存在於TCR中的天然鏈間二硫鍵連接。As described above, the TCRs of the present invention may comprise artificial interchain disulfide bonds introduced between residues of their alpha and beta chain constant domains. It should be noted that the TCRs of the invention may contain both a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bonds introduced above between the constant domains. The TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may be linked by natural interchain disulfide bonds present in the TCR.
另外,對於穩定性而言,專利文獻PCT/CN2016/077680還公開了在TCR的α鏈可變區與β鏈恆定區之間引入人工鏈間二硫鍵能夠使TCR的穩定性顯著提高。因此,本發明的高親和力TCR的α鏈可變區與β鏈恆定區之間還可以含有人工鏈間二硫鍵。具體地,在所述TCR的α鏈可變區與β鏈恆定區之間形成人工鏈間二硫鍵的半胱胺酸殘基取代了:TRAV的第46位胺基酸和TRBC1*01或TRBC2*01外顯子1的第60位胺基酸;TRAV的第47位胺基酸和TRBC1*01或TRBC2*01外顯子1的61位胺基酸;TRAV的第46位胺基酸和TRBC1*01或TRBC2*01外顯子1的第61位胺基酸;或TRAV的第47位胺基酸和TRBC1*01或TRBC2*01外顯子1的第60位胺基酸。優選地,這樣的TCR可以包含(i)除其跨膜結構域以外的全部或部分TCRα鏈,和(ii)除其跨膜結構域以外的全部或部分TCRβ鏈,其中(i)和(ii) 均包含TCR鏈的可變域和至少一部分恆定域,α鏈與β鏈形成異質二聚體。更優選地,這樣的TCR可以包含α鏈可變域和β鏈可變域以及除跨膜結構域以外的全部或部分β鏈恆定域,但其不包含α鏈恆定域,所述TCR的α鏈可變域與β鏈形成異質二聚體。In addition, in terms of stability, patent document PCT/CN2016/077680 also discloses that the introduction of artificial interchain disulfide bonds between the α chain variable region and the β chain constant region of TCR can significantly improve the stability of TCR. Therefore, the high-affinity TCR of the present invention may also contain artificial interchain disulfide bonds between the variable region of the α chain and the constant region of the β chain. Specifically, the cysteine residue that forms an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR is substituted for: the 46th amino acid of TRAV and TRBC1*01 or
對於穩定性而言,另一方面,本發明TCR還包括在其疏水核心區域發生突變的TCR,這些疏水核心區域的突變優選為能夠使本發明TCR的穩定性提高的突變,如在公開號為WO2014/206304的專利文獻中所述。這樣的TCR可在其下列可變域疏水核心位置發生突變:(α和/或β鏈)可變區胺基酸第11、13、19、21、53、76、89、91、94位,和/或α鏈J基因(TRAJ)短胜肽胺基酸位置倒數第3、5、7位,和/或β鏈J基因(TRBJ)短胜肽胺基酸位置倒數第2、4、6位,其中胺基酸序列的位置編號按國際免疫遺傳學資訊系統(IMGT)中列出的位置編號。本領域技術人員知曉上述國際免疫遺傳學資訊系統,並可根據該資料庫得到不同TCR的胺基酸殘基在IMGT中的位置編號。In terms of stability, on the other hand, the TCRs of the present invention also include TCRs with mutations in their hydrophobic core regions, and the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the TCRs of the present invention, such as those published in Publication No. Described in the patent document of WO2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable
更具體地,本發明中疏水核心區域發生突變的TCR可以是由一柔性胜肽鏈連接TCR的α鏈與β鏈的可變域而構成的高穩定性單鏈TCR。TCR可變區的CDR區決定了其與短胜肽-HLA複合物之間的親和力,疏水核心的突變能夠使TCR更加穩定,但並不會影響其與短胜肽-HLA複合物之間的親和力。應注意,本發明中柔性胜肽鏈可以是任何適合連接TCRα與β鏈可變域的胜肽鏈。本發明實施例1中建構的用於篩選高親和性TCR的模板鏈即為上述含有疏水核心突變的高穩定性單鏈TCR。採用穩定性較高的TCR,能夠更方便的評估TCR與TSSELMAITR-HLA-A1101複合物之間的親和力。More specifically, in the present invention, the TCR in which the hydrophobic core region is mutated can be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the α chain and the β chain of the TCR. The CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex, and the mutation of the hydrophobic core can make the TCR more stable, but does not affect its affinity with the short peptide-HLA complex. Affinity. It should be noted that the flexible peptide chain in the present invention can be any peptide chain suitable for linking the variable domains of the TCRα and β chains. The template chain constructed in Example 1 of the present invention for screening high-affinity TCR is the above-mentioned high-stability single-chain TCR containing a hydrophobic core mutation. Using TCR with higher stability can more conveniently evaluate the affinity between TCR and TSSELMAITR-HLA-A1101 complex.
該單鏈模板TCR的α鏈可變域及β鏈可變域的CDR區與野生型TCR的CDR區完全相同。即α鏈可變域的3個CDR分別為CDR1α:YGATPY;CDR2α:YFSGDTLV;CDR3α:AVVATDSWGKLQ和β鏈可變域的3個CDR分別為CDR1β:SGHVS;CDR2β:FQNEAQ;CDR3β:ASSLVAGARTDTQY。該單鏈模板TCR的胺基酸序列(SEQ ID NO: 9)及核苷酸序列(SEQ ID NO: 10)分別如圖5a和5b所示。以此篩選出對TSSELMAITR-HLA A1101複合物具有高親和性的由α鏈可變域和β鏈可變域構成的單鏈TCR。The CDR regions of the α-chain variable domain and the β-chain variable domain of the single-chain template TCR are identical to those of the wild-type TCR. That is, the three CDRs of the α chain variable domain are CDR1α: YGATPY; CDR2α: YFSGDTLV; CDR3α: AVVATDSWGKLQ and the three CDRs of the β chain variable domain are CDR1β: SGHVS; CDR2β: FQNEAQ; CDR3β: ASSLVAGARTDTQY. The amino acid sequence (SEQ ID NO: 9) and nucleotide sequence (SEQ ID NO: 10) of the single-stranded template TCR are shown in Figures 5a and 5b, respectively. In this way, a single-chain TCR composed of α-chain variable domain and β-chain variable domain with high affinity for TSSELMAITR-HLA A1101 complex was screened out.
本發明的對TSSELMAITR-HLA-A1101複合物具有高親和性的αβ異質二聚體的獲得是通過將篩選出的高親和性單鏈TCR的α與β鏈可變域的CDR區轉移到野生型TCRα鏈可變域(SEQ ID NO: 1)與β鏈可變域(SEQ ID NO: 2)的相應位置而得到。The αβ heterodimer with high affinity for the TSSELMAITR-HLA-A1101 complex of the present invention is obtained by transferring the CDR regions of the α and β chain variable domains of the selected high-affinity single-chain TCR to wild type The corresponding positions of the TCR alpha chain variable domain (SEQ ID NO: 1) and beta chain variable domain (SEQ ID NO: 2) were obtained.
本發明的TCR也可以多價複合體的形式提供。本發明的多價TCR複合體包含兩個、三個、四個或更多個本發明TCR相結合而形成的多聚物,如可以用p53的四聚結構域來產生四聚體,或多個本發明TCR與另一分子結合而形成的複合物。本發明的TCR複合物可用於體外或體內追蹤或標靶呈現特定抗原的細胞,也可用於產生具有此類應用的其他多價TCR複合物的中間物。The TCRs of the present invention may also be provided in the form of multivalent complexes. The multivalent TCR complexes of the present invention comprise two, three, four or more multimers formed by combining the TCRs of the present invention, for example, the tetramerization domain of p53 can be used to generate tetramers, or multiple A complex formed by combining a TCR of the present invention with another molecule. The TCR complexes of the present invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes with such applications.
本發明的TCR可以單獨使用,也可與偶聯物以共價或其他方式結合,優選以共價方式結合。所述偶聯物包括可檢測標記物(為診斷目的,其中所述TCR用於檢測呈現TSSELMAITR-HLA- A1101複合物的細胞的存在)、治療劑、PK (蛋白激酶)修飾部分或任何以上這些物質的組合結合或偶聯。The TCR of the present invention can be used alone, or can be combined with the conjugate in a covalent or other manner, preferably in a covalent manner. The conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the TSSELMAITR-HLA-A1101 complex), a therapeutic agent, a PK (protein kinase) modification moiety, or any of the above Combination binding or coupling of substances.
用於診斷目的的可檢測標記物包括但不限於:螢光或發光標記物、放射性標記物、MRI (核磁共振成像)或CT (電腦X射線斷層掃描技術)造影劑、或能夠產生可檢測產物的酶。Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.
可與本發明TCR結合或偶聯的治療劑包括但不限於: 1. 放射性核種(Koppe等,2005,癌轉移評論(Cancer metastasis reviews)24,539);2. 生物毒(Chaudhary等,1989,自然(Nature)339,394;Epel等,2002,癌症免疫學和免疫治療(Cancer Immunology and Immunotherapy)51,565);3. 細胞激素如IL-2等(Gillies等,1992,美國國家科學院院刊(PNAS)89,1428;Card等,2004,癌症免疫學和免疫治療(Cancer Immunology and Immunotherapy) 53,345;Halin等,2003,癌症研究(Cancer Research)63,3202);4. 抗體Fc片段(Mosquera等,2005,免疫學雜誌(The Journal Of Immunology) 174,4381);5. 抗體scFv片段 (Zhu等,1995,癌症國際期刊(International Journal of Cancer)62, 319) ;6. 金奈米顆粒/奈米棒(Lapotko等,2005,癌症通信(Cancer letters )239,36;Huang等,2006,美國化學學會雜誌(Journal of the American Chemical Society)128,2115);7. 病毒顆粒(Peng等,2004,基因治療(Gene therapy) 11,1234);8. 脂質體(Mamot等,2005,癌症研究(Cancer research)65,11631);9. 奈米磁粒;10.前驅藥活化酶(例如,DT-心肌黃酶(DTD)或聯苯基水解酶-樣蛋白質(BPHL));11.化療劑(例如,順鉑)或任何形式的奈米顆粒等。Therapeutic agents that can be bound or conjugated to the TCRs of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biological toxicants (Chaudhary et al., 1989, Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2, etc. (Gillies et al., 1992, Proceedings of the National Academy of Sciences of the United States of America) (PNAS) 89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. Antibody Fc fragments ( Mosquera et al, 2005, The Journal Of Immunology 174, 4381); 5. Antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62, 319); 6. Gold nanoparticles / Nanorods (Lapotko et al., 2005,
與本發明TCR結合的抗體或其片段包括抗-T細胞或NK-細胞決定抗體,如抗-CD3或抗-CD28或抗-CD16抗體,上述抗體或其片段與TCR的結合能夠對效應細胞進行定向來更好地標靶標的細胞。一個優選的實施方式是本發明TCR與抗-CD3抗體或所述抗-CD3抗體的功能片段或變體結合。具體地,本發明的TCR與抗CD3單鏈抗體的融合分子包括選自TCRα鏈可變域胺基酸序列為SEQ ID NO: 1、13-32之一;和/或所述TCR的β鏈可變域胺基酸序列為SEQ ID NO: 2、33-36之一。Antibodies or fragments thereof that bind to the TCR of the present invention include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies, the binding of such antibodies or fragments thereof to TCR is capable of effecting effector cells. Oriented to better target cells. A preferred embodiment is that the TCR of the present invention binds to an anti-CD3 antibody or a functional fragment or variant of said anti-CD3 antibody. Specifically, the fusion molecule of the TCR and anti-CD3 single-chain antibody of the present invention comprises one selected from the amino acid sequence of the variable domain of the TCR α chain variable domain of SEQ ID NO: 1, 13-32; and/or the β chain of the TCR The variable domain amino acid sequence is one of SEQ ID NOs: 2, 33-36.
本發明還涉及編碼本發明TCR的核酸分子。本發明的核酸分子可以是DNA形式或RNA形式。DNA可以是編碼鏈或非編碼鏈。例如,編碼本發明TCR的核酸序列可以與本發明附圖中所示的核酸序列相同或是簡併的變異體。舉例說明“簡併的變異體”的含義,如本文所用,“簡併的變異體”在本發明中是指編碼具有SEQ ID NO: 3的蛋白序列,但與SEQ ID NO: 5的序列有差別的核酸序列。The present invention also relates to nucleic acid molecules encoding the TCRs of the present invention. The nucleic acid molecules of the present invention may be in the form of DNA or RNA. DNA can be the coding or non-coding strand. For example, the nucleic acid sequences encoding the TCRs of the present invention may be identical or degenerate variants of the nucleic acid sequences shown in the figures of the present invention. To illustrate the meaning of "degenerate variant", as used herein, "degenerate variant" in the present invention refers to encoding a protein sequence having SEQ ID NO: 3, but having the same sequence as SEQ ID NO: 5. Different nucleic acid sequences.
本發明的核酸分子全長序列或其片段通常可以用但不限於PCR擴增法、重組法或人工合成的方法獲得。目前,已經可以完全通過化學合成來得到編碼本發明TCR(或其片段,或其衍生物)的DNA序列。然後可將該DNA序列引入本領域中已知的各種現有的DNA分子(或如載體)和細胞中。The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification method, recombinant method or artificial synthesis method. At present, the DNA sequences encoding the TCRs of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
本發明也涉及包含本發明的核酸分子的載體,以及用本發明的載體或編碼序列經基因工程產生的宿主細胞。The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, as well as host cells genetically engineered with the vectors or coding sequences of the present invention.
本發明還包括表現本發明TCR的分離細胞,特別是T細胞。有許多方法適合於用編碼本發明的高親和力TCR的DNA或RNA進行T細胞轉染(如,Robbins等.,(2008) J. Immunol. 180: 6116-6131)。表現本發明高親和性TCR的T細胞可以用於過繼免疫治療。本領域技術人員能夠知曉進行過繼性治療的許多合適方法(如,Rosenberg等.,(2008) Nat Rev Cancer 8(4): 299-308)。The present invention also includes isolated cells, particularly T cells, expressing the TCRs of the present invention. There are a number of methods suitable for transfection of T cells with DNA or RNA encoding the high affinity TCRs of the invention (eg, Robbins et al., (2008) J. Immunol. 180: 6116-6131). T cells expressing the high-affinity TCR of the present invention can be used for adoptive immunotherapy. Those skilled in the art are aware of many suitable methods for adoptive therapy (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).
本發明還提供一種藥物組成物,所述藥物組成物含有藥學上可接受的載劑以及本發明TCR、或本發明TCR複合物、或呈現本發明TCR的細胞。The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR of the present invention, or the TCR complex of the present invention, or a cell that exhibits the TCR of the present invention.
本發明還提供了一種治療疾病的方法,包括給需要治療的對象施用適量的本發明TCR、或本發明TCR複合物、或呈現本發明TCR的細胞、或本發明的藥物組成物。The present invention also provides a method of treating a disease, comprising administering to a subject in need of treatment an appropriate amount of a TCR of the present invention, or a TCR complex of the present invention, or a cell exhibiting the TCR of the present invention, or a pharmaceutical composition of the present invention.
在本領域中,用性能相近或相似的胺基酸進行取代時,通常不會改變蛋白質的功能。在C末端和/或N末端添加一個或數個胺基酸通常也不會改變蛋白質的結構和功能。因此,本發明TCR還包括本發明TCR的至多5個,較佳地至多3個,更佳地至多2個,最佳地1個胺基酸(尤其是位於CDR區之外的胺基酸),被性質相似或相近的胺基酸所替換,並仍能夠保持其功能性的TCR。In the art, substitution with amino acids with similar or similar properties usually does not change the function of the protein. The addition of one or several amino acids to the C-terminus and/or N-terminus also generally does not alter protein structure and function. Therefore, the TCRs of the present invention also include up to 5, preferably up to 3, more preferably up to 2, and optimally 1 amino acid (especially those located outside the CDR region) of the TCR of the present invention , is replaced by amino acids with similar or similar properties, and still retains its functional TCR.
本發明還包括對本發明TCR略作修飾後的TCR。修飾(通常不改變一級結構)形式包括:本發明TCR的化學衍生形式如乙醯化或羧基化。修飾還包括糖基化,如那些在本發明TCR的合成和加工中或進一步加工步驟中進行糖基化修飾而產生的TCR。這種修飾可以通過將TCR暴露於進行糖基化的酶(如哺乳動物的糖基化酶或去糖基化酶)而完成。修飾形式還包括具有磷酸化胺基酸殘基(如磷酸酪胺酸,磷酸絲胺酸,磷酸蘇胺酸)的序列。還包括被修飾從而提高了其抗蛋白水解性能或最適化了溶解性能的TCR。The present invention also includes slightly modified TCRs of the present invention. Modified (generally without altering the primary structure) forms include: chemically derivatized forms of the TCRs of the present invention such as acetylated or carboxylated. Modifications also include glycosylation, such as those TCRs resulting from glycosylation modifications in the synthesis and processing of the TCRs of the invention or in further processing steps. This modification can be accomplished by exposing the TCR to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that have been modified to increase their resistance to proteolysis or to optimize their solubility properties.
本發明的TCR、TCR複合物或本發明TCR轉染的T細胞可與藥學上可接受的載劑一起在藥物組成物中提供。本發明的TCR、多價TCR複合物或細胞通常作為無菌藥物組成物的一部分提供,所述組成物通常包括藥學上可接受的載劑。該藥物組成物可以是任何合適的形式(取決於給予患者的所需方法)。其可採用單位劑型提供,通常在密封的容器中提供,可作為套組的一部分提供。此類套組(但非必需)包括使用說明書。其可包括多個所述單位劑型。The TCRs of the present invention, TCR complexes or TCR-transfected T cells of the present invention can be provided in pharmaceutical compositions together with a pharmaceutically acceptable carrier. The TCRs, multivalent TCR complexes or cells of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It is presented in unit dosage form, usually in a sealed container, as part of a kit. Such kits (but not required) include instructions for use. It may comprise a plurality of such unit dosage forms.
此外,本發明的TCR可以單用,也可與其他治療劑結合或偶聯在一起使用(如配製在同一藥物組成物中)。In addition, the TCRs of the present invention can be used alone, or in combination or conjugation with other therapeutic agents (eg, formulated in the same pharmaceutical composition).
藥物組成物還可含有藥學上可接受的載劑。術語“藥學上可接受的載劑”指用於治療劑給藥的載劑。該術語指這樣一些藥劑載劑:它們本身不誘導產生對接受該組成物的個體有害的抗體,且給藥後沒有過分的毒性。這些載劑是本領域普通技術人員所熟知的。在雷明頓藥物科學(Remington's Pharmaceutical Sciences (Mack Pub. Co.,N.J. 1991))中可找到關於藥學上可接受的賦形劑的充分討論。這類載劑包括(但並不限於):鹽水、緩衝液、葡萄糖、水、甘油、乙醇、佐劑、及其組合。The pharmaceutical compositions may also contain pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies that are detrimental to the individual receiving the composition, and are not undue toxicity upon administration. These carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
治療性組成物中藥學上可接受的載劑可含有液體,如水、鹽水、甘油和乙醇。另外,這些載劑中還可能存在輔助性的物質,如潤濕劑或乳化劑、pH緩衝物質等。Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
通常,可將治療性組成物製成可注射劑,例如液體溶液或懸浮液;還可製成在注射前適合配入溶液或懸浮液中、液體載劑的固體形式。Generally, therapeutic compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared.
一旦配成本發明的組成物,可將其通過常規途徑進行給藥,其中包括(但並不限於):眼內、肌內、靜脈內、皮下、皮內、或局部給藥,優選為胃腸外包括皮下、肌肉內或靜脈內。待預防或治療的對象可以是動物;尤其是人。Once formulated, the compositions of the present invention can be administered by conventional routes including, but not limited to, intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenterally Including subcutaneous, intramuscular or intravenous. The subject to be prevented or treated can be an animal; especially a human.
當本發明的藥物組成物被用於實際治療時,可根據使用情況而採用各種不同劑型的藥物組成物。較佳地,可以例舉的有針劑、口服劑等。When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be adopted according to the usage. Preferably, injections, oral preparations and the like can be exemplified.
這些藥物組成物可根據常規方法通過混合、稀釋或溶解而進行配製,並且偶爾添加合適的藥物添加劑,如賦形劑、崩解劑、粘合劑、潤滑劑、稀釋劑、緩衝劑、等滲劑(isotonicities)、防腐劑、潤濕劑、乳化劑、分散劑、穩定劑和助溶劑,而且該配製過程可根據劑型用慣常方式進行。These pharmaceutical compositions can be formulated according to conventional methods by mixing, diluting or dissolving, with occasional addition of suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.
本發明的藥物組成物還可以緩釋劑形式給藥。例如,本發明TCR可被摻入以緩釋聚合物為載劑的藥丸或微膠囊中,然後將該藥丸或微膠囊通過手術植入待治療的組織。作為緩釋聚合物的例子,可例舉的有乙烯-乙烯基乙酸酯共聚物、聚羥基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯醯胺、聚乙烯吡咯烷酮、甲基纖維素、乳酸聚合物、乳酸-乙醇酸共聚物等,較佳地可例舉的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。The pharmaceutical compositions of the present invention can also be administered in the form of sustained release formulations. For example, the TCRs of the present invention can be incorporated into pellets or microcapsules with a slow release polymer as a carrier, and the pellets or microcapsules are then surgically implanted into the tissue to be treated. As examples of sustained-release polymers, ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer can be exemplified , lactic acid-glycolic acid copolymers, etc., preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
當本發明的藥物組成物被用於實際治療時,作為活性成分的本發明TCR或TCR複合物或呈現本發明TCR的細胞,可根據待治療的每個病人的體重、年齡、性別、症狀程度而合理地加以確定,最終由醫師決定合理的用量。本發明的主要優點在於: When the pharmaceutical composition of the present invention is used for actual treatment, the TCR or the TCR complex of the present invention or the cells exhibiting the TCR of the present invention as an active ingredient can be used according to the weight, age, sex, symptom level of each patient to be treated And be reasonably determined, and ultimately by the physician to decide a reasonable dosage. The main advantages of the present invention are:
(1)本發明的高親和力TCR對所述TSSELMAITR-HLA-A1101複合物的親和力和/或結合半衰期是野生型TCR的至少2倍。(1) The affinity and/or binding half-life of the high-affinity TCR of the present invention to the TSSELMAITR-HLA-A1101 complex is at least 2 times that of the wild-type TCR.
(2)本發明的高親和力TCR能夠與所述TSSELMAITR-HLA A1101特異性結合,同時轉染了本發明高親和力TCR的細胞能夠被特異性活化與增殖。(2) The high-affinity TCR of the present invention can specifically bind to the TSSELMAITR-HLA A1101, and the cells transfected with the high-affinity TCR of the present invention can be specifically activated and proliferated.
(3)轉染本發明的高親和力TCR的效應細胞具有強的特異性毒殺作用。(3) The effector cells transfected with the high-affinity TCR of the present invention have a strong specific poisoning effect.
下面的具體實施例,進一步闡述本發明。應理解,這些實施例僅用於說明本發明而不用於限制本發明的範圍。下列實施例中未註明具體條件的實驗方法,通常按照常規條件,例如(Sambrook和Russell等人,分子選殖:實驗室手冊(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的條件,或按照製造廠商所建議的條件。除非另外說明,否則百分比和份數按重量計算。材料和方法 The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions, such as (Sambrook and Russell et al., Molecular Cloning: Laboratory Manual (Molecular Cloning-A Laboratory Manual) (Third Edition) (2001) CSHL Publishing company), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Materials and methods
本發明實施例中所用的實驗材料如無特殊說明均可從市售管道獲得,其中,E.coli DH5α購自Tiangen、E.coli BL21(DE3)購自Tiangen、E. coli Tuner (DE3)購自Novagen、質體pET28a購自Novagen。實施例 1 疏水核心突變的穩定性單鏈 TCR 模板鏈的產生 The experimental materials used in the examples of the present invention can be obtained from commercially available pipelines unless otherwise specified, wherein, E. coli DH5α was purchased from Tiangen, E. coli BL21 (DE3) was purchased from Tiangen, and E. coli Tuner (DE3) was purchased from Tiangen From Novagen, plastid pET28a was purchased from Novagen. Example 1 Generation of stable single-stranded TCR template strands with hydrophobic core mutations
本發明利用定點突變的方法,根據專利文獻WO2014/206304中所述,建構了以一個柔性短胜肽(linker)連接TCRα與β鏈可變域而構成的穩定性單鏈TCR分子,其胺基酸及DNA序列分別為SEQ ID NO: 9和SEQ ID NO: 10,如圖5a和圖5b所示。並以該單鏈TCR分子為模板進行高親和性TCR分子的篩選。該模板鏈的α鏈可變域(SEQ ID NO: 3)及β鏈可變域(SEQ ID NO: 4)的胺基酸序列如圖2a和2b所示;其對應的DNA序列分別為SEQ ID NO: 5和SEQ ID NO: 6,如圖3a和3b所示;柔性短胜肽(linker)的胺基酸序列及DNA序列分別為SEQ ID NO: 7和8,如圖4a和4b所示。The present invention utilizes the method of site-directed mutagenesis, according to the patent document WO2014/206304, to construct a stable single-chain TCR molecule composed of a flexible short peptide (linker) connecting the TCRα and β chain variable domains, and its amine group The acid and DNA sequences are SEQ ID NO: 9 and SEQ ID NO: 10, respectively, as shown in Figures 5a and 5b. And use the single-chain TCR molecule as a template to screen high-affinity TCR molecules. The amino acid sequences of the α chain variable domain (SEQ ID NO: 3) and the β chain variable domain (SEQ ID NO: 4) of the template chain are shown in Figures 2a and 2b; their corresponding DNA sequences are SEQ ID NO: 4 respectively. ID NO: 5 and SEQ ID NO: 6, as shown in Figure 3a and 3b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are respectively SEQ ID NO: 7 and 8, as shown in Figure 4a and 4b Show.
將攜帶模板鏈的目標基因經NcoⅠ和NotⅠ雙限制酶切割,與經過NcoⅠ和NotⅠ雙限制酶切割的pET28a載體連接。連接產物轉形至E.coli DH5α,塗布含卡那黴素的LB培養盤,37℃倒置培養過夜,挑取陽性選殖株進行PCR篩選,對陽性重組體進行定序,確定序列正確後萃取重組質體轉形至E.coli BL21 (DE3)用於表現。實施例 2 實施例 1 中建構的穩定性單鏈 TCR 的表現、復性和純化 The target gene carrying the template chain was cut with NcoI and NotI double restriction enzymes, and ligated with the pET28a vector cut with NcoI and NotI double restriction enzymes. The ligation product was transformed into E.coli DH5α, coated with kanamycin-containing LB culture plate, and cultured overnight at 37°C by inversion. The positive clones were selected for PCR screening, and the positive recombinants were sequenced to confirm that the sequence was correct and then extracted. Recombinant plastids were transformed into E. coli BL21 (DE3) for expression. Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1
將實施例1中製備的含有重組質體pET28a-模板鏈的BL21 (DE 3)菌落全部接種於含有卡那黴素的LB培養基中,37℃培養至OD600 為0.6-0.8,加入IPTG至終濃度為0.5 mM,37℃繼續培養4 h。5000 rpm 離心15 min收取細胞沉澱物,用Bugbuster Master Mix (Merck)裂解細胞沉澱物,6000 rpm離心15 min回收包涵體,再用Bugbuster (Merck)進行洗滌以除去細胞碎片和膜組分,6000 rpm 離心15 min,收集包涵體。將包涵體溶解在緩衝液(20 mM Tris-HCl pH 8.0, 8 M尿素)中,高速離心去除不溶物,上清液用BCA法定量後進行分裝,於-80℃保存備用。All the BL21 (DE 3) colonies containing the recombinant plastid pET28a-template chain prepared in Example 1 were inoculated into LB medium containing kanamycin, and cultured at 37°C until the OD 600 was 0.6-0.8, and IPTG was added to the end. The concentration was 0.5 mM, and the cells were incubated at 37 °C for 4 h. Cell pellets were harvested by centrifugation at 5000 rpm for 15 min, lysed with Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000 rpm for 15 min, and washed with Bugbuster (Merck) to remove cell debris and membrane components, 6000 rpm Inclusion bodies were collected by centrifugation for 15 min. The inclusion bodies were dissolved in buffer (20 mM Tris-HCl pH 8.0, 8 M urea), and the insolubles were removed by high-speed centrifugation. The supernatant was quantified by BCA method, and then aliquoted and stored at -80 °C for future use.
向5 mg溶解的單鏈TCR包涵體蛋白中,加入2.5 mL緩衝液(6 M Gua-HCl,50 mM Tris-HCl pH 8.1,100 mM NaCl,10 mM EDTA),再加入DTT至終濃度為10 mM,37℃處理30 min。用注射器向125 mL復性緩衝液(100 mM Tris-HCl pH 8.1,0.4 M L-精胺酸,5 M 尿素,2 mM EDTA,6.5 mM β-mercapthoethylamine,1.87 mM Cystamine)中滴加上述處理後的單鏈TCR,4℃攪拌10 min,然後將復性液裝入截留量為4 kDa的纖維素膜透析袋,透析袋置於1 L 預冷的水中,4℃緩慢攪拌過夜。17小時後,將透析液換成1L 預冷的緩衝液(20 mM Tris-HCl pH 8.0),4℃繼續透析8 h,然後將透析液換成相同的新鮮緩衝液繼續透析過夜。17小時後,樣品經0.45 µm濾膜過濾,真空脫氣後通過陰離子交換管柱(HiTrap Q HP, GE Healthcare), 用20 mM Tris-HCl pH 8.0配製的0-1M NaCl線性梯度洗提液純化蛋白,收集的洗提組分進行SDS-PAGE分析,包含單鏈TCR的組分濃縮後進一步用凝膠過濾管柱(Superdex 75 10/300, GE Healthcare)進行純化,目標組分也進行SDS-PAGE分析。To 5 mg of solubilized single-chain TCR inclusion body protein, add 2.5 mL buffer (6 M Gua-HCl, 50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM EDTA) followed by DTT to a final concentration of 10 mM, treated at 37°C for 30 min. 125 mL of renaturation buffer (100 mM Tris-HCl pH 8.1, 0.4 M L-arginine, 5 M urea, 2 mM EDTA, 6.5 mM β-mercapthoethylamine, 1.87 mM Cystamine) was added dropwise with a syringe after the above treatment. The single-chain TCR was stirred at 4 °C for 10 min, and then the renaturation solution was put into a cellulose membrane dialysis bag with a cut-off of 4 kDa, and the dialysis bag was placed in 1 L of pre-cooled water, and slowly stirred at 4 °C overnight. After 17 hours, the dialysate was replaced with 1 L of pre-cooled buffer (20 mM Tris-HCl pH 8.0), and the dialysis was continued for 8 h at 4°C, and then the dialysate was replaced with the same fresh buffer and continued dialysis overnight. After 17 hours, the samples were filtered through a 0.45 µm filter, degassed in vacuo and passed through an anion exchange column (HiTrap Q HP, GE Healthcare) and purified with a linear gradient of 0-1 M NaCl in 20 mM Tris-HCl pH 8.0 Protein, the collected elution fractions were analyzed by SDS-PAGE, the fractions containing single-chain TCR were concentrated and further purified by gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also subjected to SDS-PAGE. PAGE analysis.
用於BIAcore分析的洗提組分進一步採用凝膠過濾法測試其純度。條件為:層析管柱Agilent Bio SEC-3(300 A,φ7.8×300 mm),流動相為150 mM磷酸鹽緩衝液,流速0.5 mL/min,柱溫25℃,紫外檢測波長214 nm。實施例 3 結合表徵 BIAcore分析The eluted fractions for BIAcore analysis were further tested for purity by gel filtration. The conditions are: chromatographic column Agilent Bio SEC-3 (300 A, φ7.8×300 mm), the mobile phase is 150 mM phosphate buffer, the flow rate is 0.5 mL/min, the column temperature is 25 °C, and the UV detection wavelength is 214 nm. . Example 3 Binding Characterization BIAcore Analysis
使用BIAcore T200即時分析系統檢測TCR分子與 TSSELMAITR-HLA-A1101複合物的結合活性。將抗鏈黴親和素的抗體(GenScript)加入偶聯緩衝液(10 mM醋酸鈉緩衝液,pH 4.77),然後將抗體流過預先用EDC和NHS活化過的CM5晶片,使抗體固定在晶片表面,最後用乙醇胺的鹽酸溶液封阻未反應的活化表面,完成偶聯過程,偶聯位準約為15,000 RU。條件為:溫度25℃,PH值為7.1-7.5。The binding activity of TCR molecules to the TSSELMAITR-HLA-A1101 complex was detected using the BIAcore T200 point-of-care analysis system. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), and the antibody was immobilized on the surface of the wafer by flowing the antibody over a CM5 wafer previously activated with EDC and NHS , and finally blocked the unreacted activated surface with ethanolamine hydrochloric acid solution to complete the coupling process with a coupling level of about 15,000 RU. The conditions are: the temperature is 25°C, and the pH value is 7.1-7.5.
使低濃度的鏈黴親和素流過已塗佈抗體的晶片表面,然後將TSSELMAITR-HLA-A1101複合物流過檢測通道,另一通道作為參考通道,再將0.05 mM的生物素以10 µL/min的流速流過晶片2 min,封阻鏈黴親和素剩餘的結合位址。採用單迴圈動力學分析方法測定其親和力,將TCR用HEPES-EP緩衝液(10 mM HEPES,150 mM NaCl,3 mM EDTA,0.005% P20,pH 7.4)稀釋成幾個不同的濃度,以30 µL/min的流速,依次流過晶片表面,每次進樣的結合時間為120 s,最後一次進樣結束後讓其解離600 s。每一輪測定結束後用pH 1.75 的10 mM Gly-HCl再生晶片。利用BIAcore Evaluation軟體計算動力學參數。Flow a low concentration of streptavidin over the antibody-coated wafer surface, then flow the TSSELMAITR-HLA-A1101 complex through the detection channel, the other channel as the reference channel, and 0.05 mM biotin at 10 µL/min. flow through the wafer for 2 min, blocking the remaining binding sites of streptavidin. Affinity was determined using a single-loop kinetic assay, and the TCR was diluted to several different concentrations in HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) at 30 The flow rate of µL/min was successively flowed over the wafer surface, and the bonding time of each injection was 120 s, and the dissociation was 600 s after the last injection. The wafers were regenerated with 10 mM Gly-HCl pH 1.75 after each round of assays. Kinetic parameters were calculated using BIAcore Evaluation software.
上述TSSELMAITR-HLA-A1101複合物的製備過程如下:
a. 純化:收集100 ml誘導表現重鏈或輕鏈的E. coli
菌液,於4℃ 8000 g離心10 min後用10 ml PBS洗滌菌體一次,之後用5 ml BugBuster Master Mix Extraction Reagents(Merck) 劇烈震盪重新懸浮菌體,並於室溫旋轉培育20 min,之後於4℃,6000 g 離心15 min,棄去上清,收集包涵體。
將上述包涵體重新懸浮於5 ml BugBuster Master Mix中,室溫旋轉培育5 min;加30 ml稀釋10倍的BugBuster,混勻,4℃ 6000 g離心15 min;棄去上清,加30 ml稀釋10倍的BugBuster重新懸浮包涵體,混勻,4℃ 6000 g離心15 min,重複兩次,加30 ml 20 mM Tris-HCl pH 8.0重新懸浮包涵體,混勻,4℃ 6000 g離心15 min,最後用20 mM Tris-HCl 8M尿素溶解包涵體,SDS-PAGE檢測包涵體純度,BCA套組測濃度。
b. 復性:將合成的短胜肽TSSELMAITR (北京賽百盛基因技術有限公司)溶解於DMSO至20 mg/ml的濃度。輕鏈和重鏈的包涵體用8 M尿素、20 mM Tris pH 8.0、10 mM DTT來溶解,復性前加入3 M鹽酸胍、10 mM醋酸鈉、10 mM EDTA進一步變性。將TSSELMAITR胜肽以25 mg/L (終濃度)加入復性緩衝液(0.4 M L-精胺酸、100 mM Tris pH 8.3、2 mM EDTA、0.5 mM 氧化性麩胱苷肽、5 mM 還原型麩胱苷肽、0.2 mM PMSF,冷卻至4℃),然後依次加入20 mg/L的輕鏈和90 mg/L的重鏈(終濃度,重鏈分三次加入,8 h/次),復性在4℃進行至少3天至完成,SDS-PAGE檢測能否復性成功。
c. 復性後純化:用10體積的20 mM Tris pH 8.0作透析來更換復性緩衝液,至少更換緩衝液兩次來充分降低溶液的離子強度。透析後用0.45 μm 醋酸纖維素濾膜過濾蛋白質溶液,然後載入到HiTrap Q HP (GE通用電氣公司)陰離子交換管柱上(5 ml床體積)。利用Akta純化儀(GE通用電氣公司),20 mM Tris pH 8.0配製的0-400 mM NaCl線性梯度液洗提蛋白,pMHC約在250 mM NaCl處洗提,收集諸峰組分,SDS-PAGE檢測純度。
d. 生物素化:用Millipore超濾管將純化的pMHC分子濃縮,同時將緩衝液置換為20 mM Tris pH 8.0,然後加入生物素化試劑0.05 M Bicine pH 8.3、10 mM ATP、10 mM MgOAc、50 μM D-Biotin、100 μg/ml BirA酶(GST-BirA),室溫培育混合物過夜,SDS-PAGE檢測生物素化是否完全。
e. 純化生物素化後的複合物:用Millipore超濾管將生物素化標記後的pMHC分子濃縮至1 ml,採用凝膠過濾層析純化生物素化的pMHC,利用Akta純化儀(GE通用電氣公司),用過濾過的PBS預平衡HiPrepTM
16/60 S200 HR管柱(GE通用電氣公司),載入1 ml濃縮過的生物素化pMHC分子,然後用PBS以1 ml/min流速洗提。生物素化的pMHC分子在約55 ml時作為單峰洗提出現。合併含有蛋白質的組分,用Millipore超濾管濃縮,BCA法(Thermo)測定蛋白質濃度,加入蛋白酶抑制劑cocktail (Roche)將生物素化的pMHC分子分裝保存在-80℃。實施例 4 高親和力 TCR 的產生 The preparation process of the above TSSELMAITR-HLA-A1101 complex is as follows: a. Purification: collect 100 ml of E. coli bacteria that induce heavy or light chain expression, centrifuge at 8000 g for 10 min at 4°C, and wash the cells with 10 ml PBS Once, the cells were resuspended with 5 ml of BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, and incubated at room temperature for 20 min by rotation, and then centrifuged at 6000 g for 15 min at 4°C. The supernatant was discarded and the inclusion bodies were collected. Resuspend the above inclusion bodies in 5 ml of BugBuster Master Mix and incubate at room temperature for 5 min; add 30 ml of 10-fold diluted BugBuster, mix well, and centrifuge at 6000 g at 4°C for 15 min; discard the supernatant and add 30 ml to dilute Resuspend the inclusion bodies with 10x BugBuster, mix well, centrifuge at 6000 g for 15 min at 4°C, repeat twice, add 30 ml of 20 mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mix well, centrifuge at 6000 g at 4°C for 15 min, Finally, the inclusion bodies were dissolved with 20 mM Tris-HCl 8M urea, the purity of inclusion bodies was detected by SDS-PAGE, and the concentration was determined by BCA kit. b. Refolding: Dissolve the synthetic short peptide TSSELMAITR (Beijing Saibaisheng Gene Technology Co., Ltd.) in DMSO to a concentration of 20 mg/ml. Inclusion bodies of light and heavy chains were solubilized with 8 M urea, 20 mM Tris pH 8.0, 10 mM DTT, and further denatured by the addition of 3 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA prior to renaturation. TSSELMAITR peptide was added at 25 mg/L (final concentration) to refolding buffer (0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced form Glutathione, 0.2 mM PMSF, cooled to 4°C), then added 20 mg/L light chain and 90 mg/L heavy chain in sequence (final concentration, heavy chain was added in three times, 8 h/time), repeated The renaturation was carried out at 4°C for at least 3 days to complete, and SDS-PAGE was used to detect whether the renaturation was successful. c. Post-renaturation purification: Dialyze against 10 volumes of 20 mM Tris pH 8.0 to replace the renaturation buffer, at least twice to reduce the ionic strength of the solution. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and loaded onto a HiTrap Q HP (GE) anion exchange column (5 ml bed volume). Proteins were eluted with a linear gradient of 0-400 mM NaCl prepared in 20 mM Tris pH 8.0 using an Akta purifier (GE), and pMHC was eluted at approximately 250 mM NaCl, and peak fractions were collected and detected by SDS-PAGE. purity. d. Biotinylation: Purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while buffer exchanged to 20 mM Tris pH 8.0, followed by addition of biotinylation reagents 0.05 M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 μM D-Biotin, 100 μg/ml BirA enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and the complete biotinylation was detected by SDS-PAGE. e. Purify the biotinylated complex: Concentrate the biotinylated pMHC molecules to 1 ml with a Millipore ultrafiltration tube, purify the biotinylated pMHC by gel filtration chromatography, and use an Akta purifier (GE Universal Electric Corporation),
噬菌體展示技術是產生TCR高親和力變體序列庫以篩選高親和力變體的一種手段。將Li等 ((2005) Nature Biotech 23(3): 349-354)描述的TCR噬菌體展示和篩選方法應用於實施例1中的單鏈TCR模板。通過突變該模板鏈的CDR區來建立高親和性TCR的序列庫並進行淘選。經過幾輪淘選後的噬菌體序列庫均和相應抗原有特異性結合,從中挑取單選殖株,並進行分析。Phage display technology is a means of generating TCR high-affinity variant sequence libraries for screening high-affinity variants. The TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3): 349-354) was applied to the single-chain TCR template in Example 1. A sequence library of high-affinity TCRs was created and panned by mutating the CDR regions of the template strand. After several rounds of panning, the phage sequence library has specific binding to the corresponding antigen, and the single selection clones are selected and analyzed.
將篩選到的高親和力的單鏈TCR的CDR區突變引入到αβ異質二聚TCR的可變域的相應位址中,並通過BIAcore來檢測其與TSSELMAITR-HLA-A1101複合物的親和力。上述CDR區高親和力突變點的引入採用本領域技術人員熟知的定點突變的方法。上述野生型TCR的α鏈與β鏈可變域胺基酸序列分別如圖1a (SEQ ID NO: 1)和1b(SEQ ID NO: 2)所示。The CDR region of the screened high-affinity single-chain TCR was mutated into the corresponding address of the variable domain of the αβ heterodimeric TCR, and its affinity with the TSSELMAITR-HLA-A1101 complex was detected by BIAcore. The introduction of the high-affinity mutation points in the above-mentioned CDR regions adopts the method of site-directed mutagenesis well known to those skilled in the art. The amino acid sequences of the α chain and β chain variable domains of the wild-type TCR are shown in Figure 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), respectively.
應注意,為獲得更加穩定的可溶性TCR,以便更方便地評估TCR與TSSELMAITR-HLA A1101複合物之間的結合親和力和/或結合半衰期,αβ異質二聚TCR可以是在α和β鏈的恆定區中分別引入了一個半胱胺酸殘基以形成人工鏈間二硫鍵的TCR,本實施例中引入半胱胺酸殘基後TCRα與β鏈的胺基酸序列分別如圖6a (SEQ ID NO: 11)和6b所示 (SEQ ID NO: 12),引入的半胱胺酸殘基以加粗字母表示。It should be noted that in order to obtain a more stable soluble TCR for easier assessment of the binding affinity and/or binding half-life between the TCR and the TSSELMAITR-HLA A1101 complex, the αβ heterodimeric TCR can be in the constant region of the α and β chains. A cysteine residue was introduced in the TCR to form an artificial interchain disulfide bond, and the amino acid sequences of the TCRα and β chains after the introduction of the cysteine residue in the present example were shown in Figure 6a (SEQ ID NO: 11) and 6b (SEQ ID NO: 12), the introduced cysteine residues are shown in bold letters.
通過«分子選殖實驗室手冊»(Molecular Cloning a Laboratory Manual)(第三版,Sambrook和Russell)中描述的標準方法將待表現的TCRα和β鏈的胞外序列基因經合成後分別插入到表現載體pET28a+(Novagene),上下游的選殖位址分別是NcoI和NotI。CDR區的突變通過本領域技術人員熟知的重疊PCR(overlap PCR)引入。插入片段經過定序確認無誤。實施例 5 高親和力 TCR 的表現、復性和純化 The extracellular sequence genes for the TCR alpha and beta chains to be expressed were synthesized and inserted into the expressed TCR alpha and beta chains, respectively, by standard methods as described in Molecular Cloning a Laboratory Manual (Third Edition, Sambrook and Russell) In the vector pET28a+ (Novagene), the upstream and downstream breeding sites are NcoI and NotI, respectively. Mutations in the CDR regions are introduced by overlapping PCR (overlap PCR) well known to those skilled in the art. The insert was sequenced and confirmed to be correct. Example 5 Expression, renaturation and purification of high-affinity TCR
將TCR α和β鏈的表現載體分別通過化學轉形法轉形進入表現細菌BL21(DE3), 細菌用LB培養液生長,於OD600 = 0.6時用終濃度0.5 mM IPTG誘導,TCR的α和β鏈表現後形成的包涵體通過BugBuster Mix (Novagene)進行萃取,並且經BugBuster 溶液反復多次洗滌,包涵體最後溶解於6 M 鹽酸胍,10 mM 二硫蘇糖醇(DTT),10 mM乙二胺四乙酸(EDTA),20 mM Tris(pH 8.1)中。The expression vectors of TCR α and β chains were transformed into the expression bacteria BL21 (DE3) by chemical transformation, respectively. The bacteria were grown in LB medium and induced with a final concentration of 0.5 mM IPTG at OD 600 = 0.6. The inclusion bodies formed after the expression of β chains were extracted with BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution. The inclusion bodies were finally dissolved in 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethyl acetate. Diaminetetraacetic acid (EDTA), 20 mM Tris (pH 8.1).
溶解後的TCR α和β鏈以1: 1的品質比快速混合於5 M 尿素,0.4 M 精胺酸,20 mM Tris (pH 8.1),3.7 mM cystamine,6.6 mM β-mercapoethylamine (4℃)中,終濃度為60 mg/mL。混合後將溶液置於10倍體積的去離子水中透析(4℃),12小時後將去離子水換成緩衝液(20 mM Tris, pH 8.0)繼續於4℃透析12小時。透析完成後的溶液經0.45 μM的濾膜過濾後,通過陰離子交換管柱(HiTrap Q HP, 5ml, GE Healthcare)純化。洗提峰含有復性成功的α和β二聚體的TCR通過SDS-PAGE膠確認。TCR隨後通過凝膠過濾層析(HiPrep 16/60,Sephacryl S-100 HR,GE Healthcare)進一步純化。純化後的TCR純度經過SDS-PAGE測定大於90%,濃度由BCA法確定。實施例 6 BIAcore 分析結果 Solubilized TCR α and β chains were rapidly mixed in 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, 6.6 mM β-mercapoethylamine (4°C) at a mass ratio of 1:1 , the final concentration was 60 mg/mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4°C). After 12 hours, the deionized water was exchanged for buffer (20 mM Tris, pH 8.0) and dialyzed at 4°C for 12 hours. The dialyzed solution was filtered through a 0.45 μM filter and purified by an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare). The eluted peaks of TCR containing successfully renatured α and β dimers were confirmed by SDS-PAGE gel. The TCR was then further purified by gel filtration chromatography (
採用實施例3中所述方法檢測引入高親和力CDR區的αβ異質二聚TCR與TSSELMAITR-HLA-A1101複合物的親和力。The method described in Example 3 was used to test the affinity of the αβ heterodimeric TCR introduced with the high-affinity CDR region to the TSSELMAITR-HLA-A1101 complex.
本發明得到新的TCRα鏈和β鏈可變域胺基酸序列,分別如圖7(1)-(20)和圖8(1)-(4)所示。由於TCR分子的CDR區決定了其與相應的pMHC複合物的親和力,所以本領域技術人員能夠預料引入高親和力突變點的αβ異質二聚TCR也具有對TSSELMAITR-HLA-A1101複合物的高親和力。利用實施例4中所述方法建構表現載體,利用實施例5中所述方法對上述引入高親和力突變的αβ異質二聚TCR進行表現、復性和純化,然後利用BIAcore T200測定其與TSSELMAITR-HLA-A1101複合物的親和力,如下表2所示。
表2
由上表2可知,所述異質二聚TCR的親和力是野生型TCR對TSSELMAITR-HLA-A1101複合物的親和力的至少2倍。實施例 7 抗 -CD3 抗體與高親和性 αβ 異質二聚 TCR 的融合體的表現、復性和純化 It can be seen from Table 2 above that the affinity of the heterodimeric TCR is at least 2 times that of the wild-type TCR for the TSSELMAITR-HLA-A1101 complex. Example 7 Expression, renaturation and purification of fusions of anti- CD3 antibodies and high-affinity αβ heterodimeric TCRs
將抗-CD3的單鏈抗體(scFv)與αβ異質二聚TCR融合,製備融合分子。抗-CD3的scFv與TCR的β鏈融合,該TCRβ鏈可以包含任一上述高親和性αβ異質二聚TCR的β鏈可變域,融合分子的TCRα鏈可以包含任一上述高親和性αβ異質二聚TCR的α鏈可變域。 融合分子表現載體的建構Fusion molecules were prepared by fusing anti-CD3 single chain antibody (scFv) to αβ heterodimeric TCR. The anti-CD3 scFv is fused to the β chain of TCR, the TCR β chain may comprise any of the above-mentioned high-affinity αβ heterodimeric β-chain variable domains, and the TCRα chain of the fusion molecule may comprise any of the above-mentioned high-affinity αβ heterodimers Alpha chain variable domains of dimeric TCRs. Construction of Fusion Molecular Expression Vectors
1. α鏈表現載體的建構:將攜帶αβ異質二聚TCR的α鏈的目標基因經NcoⅠ和NotⅠ雙限制酶切割,與經過NcoⅠ和NotⅠ雙限制酶切割的pET28a載體連接。連接產物轉形至E.coli DH5α,塗布於含卡那黴素的LB培養盤,37℃倒置培養過夜,挑取陽性選殖株進行PCR篩選,對陽性重組體進行定序,確定序列正確後萃取重組質體轉形至E.coli Tuner (DE3),用於表現。1. Construction of α chain expression vector: The target gene of α chain carrying αβ heterodimeric TCR was cut with NcoI and NotI double restriction enzymes, and then ligated with the pET28a vector cut with NcoI and NotI double restriction enzymes. The ligation product was transformed into E.coli DH5α, spread on LB culture plate containing kanamycin, and incubated overnight at 37°C upside down. The positive clones were selected for PCR screening, and the positive recombinants were sequenced to confirm that the sequence was correct. Extracted recombinant plastids were transformed into E. coli Tuner (DE3) for expression.
2. 抗-CD3(scFv)-β鏈表現載體的建構:通過重疊(overlap)PCR的方法,設計引子將抗-CD3 scFv和高親和性異質二聚TCRβ鏈基因連接起來,中間的連接短胜肽(linker)為GGGGS (SEQ ID NO: 31),並且使抗-CD3的scFv與高親和性異質二聚TCRβ鏈的融合蛋白的基因片段帶上限制性內切酶位址NcoⅠ (CCATGG(SEQ ID NO: 32))和NotⅠ (GCGGCCGC(SEQ ID NO: 33))。將PCR擴增產物經NcoⅠ和NotⅠ雙限制酶切割,與經過NcoⅠ和NotⅠ雙限制酶切割的pET28a載體連接。連接產物轉形至E.coli DH5α勝任細胞,塗布含卡那黴素的LB培養盤,37℃倒置培養過夜,挑取陽性選殖株進行PCR篩選,對陽性重組體進行定序,確定序列正確後萃取重組質體轉形至E.coli Tuner (DE3)勝任細胞,用於表現。 融合蛋白的表現、復性及純化2. Construction of anti-CD3 (scFv)-β chain expression vector: by overlapping PCR method, design primers to connect anti-CD3 scFv and high-affinity heterodimeric TCRβ chain gene, and the connection in the middle is short The peptide (linker) is GGGGS (SEQ ID NO: 31), and the gene fragment of the fusion protein of the anti-CD3 scFv and the high-affinity heterodimeric TCRβ chain is given the restriction endonuclease address NcoI (CCATGG (SEQ ID NO: 31). ID NO: 32)) and NotI (GCGGCCGC (SEQ ID NO: 33)). The PCR amplification product was cut with NcoI and NotI double restriction enzymes, and ligated with pET28a vector cut with NcoI and NotI double restriction enzymes. The ligation products were transformed into E.coli DH5α competent cells, coated with LB culture plates containing kanamycin, and cultured overnight at 37°C upside down. The positive clones were selected for PCR screening, and the positive recombinants were sequenced to confirm that the sequence was correct. Recombinant plastids were then extracted and transformed into E. coli Tuner (DE3) competent cells for expression. Expression, renaturation and purification of fusion proteins
將表現質體分別轉形進入E. coli
Tuner (DE3)勝任細胞,塗布LB培養盤(卡那黴素50 μg/mL)置於37℃培養過夜。次日,挑選殖株接種至10 mL LB液體培養基(卡那黴素50 μg/mL)培養2-3 h,按體積比1:100接種至1L LB培養基中,繼續培養至OD600為0.5-0.8,加入終濃度為1 mM IPTG誘導目標蛋白的表現。誘導4小時以後,以6000 rpm離心10 min收取細胞。PBS緩衝液洗滌菌體一次,並且分裝菌體,取相當於200 mL的細菌培養物的菌體用5 mL BugBuster Master Mix (Merck)裂解細菌,以6000 g離心15 min收集包涵體。然後進行4次洗滌劑洗滌以去除細胞碎片和膜組分。然後,用緩衝液如PBS洗滌包涵體以除去洗滌劑和鹽。最終,將包涵體用含6M鹽酸胍,10 mM 二硫蘇糖醇(DTT),10 mM 乙二胺四乙酸(EDTA),20 mM Tris,pH 8.1緩衝溶液溶解,並測定包涵體濃度,將其分裝後置於-80℃冷凍保存。The expressing plastids were transformed into E. coli Tuner (DE3) competent cells, coated with LB plates (
溶解後的TCRα鏈和抗-CD3 (scFv)-β鏈以2:5的品質比快速混合於5 M 尿素(urea),0.4 M L-精胺酸(L-arginine),20 mM Tris pH 8.1,3.7 mM cystamine,6.6 mM β-mercapoethylamine (4℃),終濃度α鏈和抗-CD3 (scFv)-β鏈分別為0.1 mg/mL,0.25 mg/mL。Solubilized TCRα chain and anti-CD3 (scFv)-β chain were rapidly mixed at a mass ratio of 2:5 in 5 M urea (urea), 0.4 M L-arginine, 20 mM Tris pH 8.1 , 3.7 mM cystamine, 6.6 mM β-mercapoethylamine (4°C), the final concentrations of α chain and anti-CD3 (scFv)-β chain were 0.1 mg/mL and 0.25 mg/mL, respectively.
混合後將溶液置於10倍體積的去離子水中透析(4℃),12小時後將去離子水換成緩衝液(10 mM Tris, pH 8.0)繼續於4℃透析12小時。 透析完成後的溶液經0.45 μM的濾膜過濾後, 通過陰離子交換管柱(HiTrap Q HP 5ml, GE healthcare)純化。洗提峰含有復性成功的TCRα鏈與抗-CD3 (scFv)-β鏈二聚體的TCR通過SDS-PAGE膠確認。TCR融合分子隨後通過粒徑篩析層析法(S-100 16/60,GE healthcare)進一步純化,以及陰離子交換管柱(HiTrap Q HP 5ml, GE healthcare)再次純化。純化後的TCR融合分子純度經過SDS-PAGE測定大於90%,濃度由BCA法測定。實施例 8 針對攜載短胜肽的 T2 細胞,轉染本發明高親和力 TCR 的效應細胞的活化功能實驗 After mixing, the solution was dialyzed against 10 volumes of deionized water (4°C). After 12 hours, the deionized water was exchanged for buffer (10 mM Tris, pH 8.0) and dialyzed at 4°C for 12 hours. After the dialysis was completed, the solution was filtered through a 0.45 μM filter membrane and purified by an anion exchange column (
IFN-γ是活化的T淋巴細胞產生的一種強有力的免疫調節因數,由此本實施例通過本領域技術人員熟知的ELISPOT實驗檢測IFN-γ數以驗證轉染本發明高親和力TCR的細胞的活化功能及抗原特異性。將本發明高親和力TCR轉染至從健康志願者的血液中分離到的CD3+T細胞作為效應細胞,並以同一志願者轉染了野生型TCR(WT-TCR)或轉染其他TCR(A6)的CD3+T細胞作為對照。所用的標的細胞為攜載了AFP抗原短胜肽TSSELMAITR的T2-A11 (即轉染了HLA-A1101的T2細胞,下同)、攜載了其他短胜肽的T2-A11和空載的T2-A11。IFN-γ is a powerful immunoregulatory factor produced by activated T lymphocytes, so in this example, the number of IFN-γ was detected by the ELISPOT assay well-known to those skilled in the art to verify that the cells transfected with the high-affinity TCR of the present invention were efficacious. Activation function and antigen specificity. The high-affinity TCR of the present invention was transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and the same volunteer was transfected with wild-type TCR (WT-TCR) or other TCR (A6 ) of CD3+ T cells as a control. The target cells used are T2-A11 carrying the AFP antigen short peptide TSSELMAITR (that is, T2 cells transfected with HLA-A1101, the same below), T2-A11 carrying other short peptides and empty T2 -A11.
以下分兩個批次(I)、(II)先後進行實驗:
(I)所述高親和力TCR可從表2獲悉,分別為
以上兩個批次均進行以下實驗步驟:首先準備ELISPOT培養盤。ELISPOT培養盤乙醇活化塗佈,4℃過夜。實驗第1天,去掉塗佈液,洗滌封阻,室溫下培育兩個小時,去除封阻液,將試驗的各個組分加入ELISPOT培養盤:標的細胞為1*104 個/孔,效應細胞為2*103 個/孔(按抗體的陽性率計算),並設置二個重複孔。加入對應短胜肽,使短胜肽在ELISPOT孔盤中的終濃度為1×10-6 M。恆溫培育過夜(37℃,5% CO2 )。實驗第2天,洗滌培養盤並進行二級檢測和顯色,乾燥培養盤,再利用免疫斑點培養盤讀數計(ELISPOT READER system; AID20 公司)計數膜上形成的斑點。The following experimental steps were carried out for the above two batches: First, prepare the ELISPOT culture plate. The ELISPOT plates were ethanol-activated and coated overnight at 4°C. On the first day of the experiment, remove the coating solution, wash the blocking solution, incubate at room temperature for two hours, remove the blocking solution, and add the components of the test to the ELISPOT culture plate: the target cells are 1*10 4 cells/well, and the effect The cells were 2*10 3 cells/well (calculated according to the positive rate of the antibody), and two duplicate wells were set. The corresponding short peptides were added so that the final concentration of the short peptides in the ELISPOT plate was 1×10 -6 M. Incubate overnight at constant temperature (37°C, 5% CO 2 ). On the second day of the experiment, the plates were washed and subjected to secondary detection and color development. The plates were dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 Company).
實驗結果如圖12a和圖12b所示,針對攜載了AFP抗原短胜肽TSSELMAITR的標的細胞,轉染本發明高親和力TCR的效應細胞相比於轉染野生型的效應細胞起更明顯的活化效應,而轉染其他TCR的效應細胞無活化狀態;同時,轉染本發明高親和力TCR的效應細胞未被攜載其他短胜肽或空載的標的細胞活化。實施例 9 針對腫瘤細胞株,轉染本發明高親和力 TCR 的效應細胞的活化功能實驗 The experimental results are shown in Figure 12a and Figure 12b. For the target cells carrying the AFP antigen short peptide TSSELMAITR, the effector cells transfected with the high-affinity TCR of the present invention are more significantly activated than the effector cells transfected with the wild type. The effector cells transfected with other TCRs have no activation state; meanwhile, the effector cells transfected with the high-affinity TCR of the present invention are not activated by other short peptides or empty target cells. Example 9 Activation function experiment of effector cells transfected with high-affinity TCR of the present invention for tumor cell lines
本實施例利用腫瘤細胞株再次驗證轉染本發明高親和力TCR的效應細胞的活化功能及特異性。同樣是通過本領域技術人員熟知的ELISPOT實驗進行檢測。將本發明高親和力TCR轉染至從健康志願者的血液中分離到的CD3+T細胞作為效應細胞,並以同一志願者轉染其他TCR(A6)的或空轉染(NC)的CD3+T細胞作為陰性對照。以下分兩個批次(I)、(II)先後進行實驗:
(I)所述高親和力TCR可從表2獲悉,分別為
實驗結果如圖13a和圖13b所示,轉染本發明高親和力TCR的效應細胞能夠很好地被AFP陽性腫瘤細胞株活化,活化效果明顯,同時無非特異性產生。轉染其他TCR的或空轉染的T細胞未被AFP陽性腫瘤細胞株活化。實施例 10 針對梯度攜載短胜肽的 T2 細胞,轉染本發明高親和力 TCR 的效應細胞的毒殺功能實驗 The experimental results are shown in Figure 13a and Figure 13b, the effector cells transfected with the high-affinity TCR of the present invention can be well activated by the AFP-positive tumor cell line, and the activation effect is obvious, and there is no non-specific production. T cells transfected with other TCRs or empty-transfected were not activated by AFP-positive tumor cell lines. Example 10 Toxicity test of effector cells transfected with the high-affinity TCR of the present invention for T2 cells carrying a gradient of short peptides
乳酸脫氫酶(LDH)在胞漿內含量豐富,正常時不能通過細胞膜,當細胞受損傷或死亡時可釋放到細胞外,此時細胞培養液中LDH活性與細胞死亡數目成正比。本實施例通過本領域技術人員熟知的非放射性細胞毒性實驗,測定LDH的釋放,從而驗證轉染本發明TCR的細胞的毒殺功能。該試驗是51Cr釋放細胞毒性試驗的比色替代試驗,定量測定細胞裂解後釋放的LDH。採用30分鐘偶聯的酶反應來檢測釋放在培養基中的LDH,在酶反應中LDH可使一種四唑鹽(INT)轉化為紅色的甲臢(formazan)。生成的紅色產物的量與裂解的細胞數成正比。可以用標準的96孔盤讀取儀收集490nm可見光吸光值資料。計算公式: %細胞毒性=100%×(實驗-效應細胞自發-標的細胞自發)/(標的細胞最大-標的細胞自發)。Lactate dehydrogenase (LDH) is abundant in the cytoplasm and cannot pass through the cell membrane under normal conditions, but can be released to the outside of the cell when the cell is damaged or died. At this time, the activity of LDH in the cell culture medium is proportional to the number of cells dying. In this example, non-radioactive cytotoxicity experiments well known to those skilled in the art are used to measure the release of LDH, thereby verifying the cytotoxicity of cells transfected with the TCR of the present invention. This assay is a colorimetric alternative to the 51Cr release cytotoxicity assay and quantifies the LDH released after cell lysis. LDH released in the medium was detected using a 30 min coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is proportional to the number of cells lysed. Visible absorbance data at 490 nm can be collected using a standard 96-well plate reader. Calculation formula: % cytotoxicity=100%×(experimental-effector cell spontaneous-target cell spontaneous)/(target cell maximum-target cell spontaneous).
本實施例將轉染本發明高親和力TCR至從健康志願者的血液中分離到的CD3+T細胞作為效應細胞,並以同一志願者轉染其他TCR(A6)或空轉染(NC)的CD3+ T細胞作為陰性對照。其中所述高親和力TCR以及其編號從表2獲悉,分別為TCR1 (α鏈可變域SEQ ID NO: 13,β鏈可變域SEQ ID NO: 2)、TCR2 (α鏈可變域SEQ ID NO: 14,β鏈可變域SEQ ID NO: 2)和TCR3 (α鏈可變域SEQ ID NO: 15,β鏈可變域SEQ ID NO: 2)。標的細胞為攜載TSSELMAITR胜肽的T2-A11 (轉染HLA-A1101的T2細胞,下同)、攜載其他短胜肽的或空載的T2-A11。In this example, the high-affinity TCR of the present invention will be transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and the same volunteer will be transfected with other TCR (A6) or empty transfected (NC) cells. CD3+ T cells served as a negative control. Wherein the high-affinity TCR and its numbering are learned from Table 2, respectively TCR1 (alpha chain variable domain SEQ ID NO: 13, beta chain variable domain SEQ ID NO: 2), TCR2 (alpha chain variable domain SEQ ID NO: 2) NO: 14, beta chain variable domain SEQ ID NO: 2) and TCR3 (alpha chain variable domain SEQ ID NO: 15, beta chain variable domain SEQ ID NO: 2). The target cells are T2-A11 carrying TSSELMAITR peptide (T2 cells transfected with HLA-A1101, the same below), T2-A11 carrying other short peptides or empty.
首先準備LDH培養盤,先按標的細胞3×104 個細胞/孔、效應細胞3×104 個細胞/孔(按抗體陽性率計算)加入對應孔中,然後在實驗組加入AFP抗原短胜肽TSSELMAITR,且使其短胜肽在ELISPOT孔盤中的終濃度依次為1×10-13 M到1×10-6 M,共8個梯度;在對照組加入其他短胜肽,且使其短胜肽終濃度為依次為1×10-8 M到1×10-6 M,共3個梯度,並各設置三個重複孔。同時設置效應細胞自發孔,標的細胞自發孔,標的細胞最大孔,體積校正對照孔及培養基背景對照孔。恆溫培育過夜(37℃,5% CO2 )。實驗第2天,檢測顯色,終止反應後用酵素標示讀取儀(Bioteck)在490nm記錄吸光值。LDH is first prepared culture plate, to press the target cells 3 × 10 4 cells / well, effector cells 3 × 10 4 cells / well (in terms of antibody positive rate) added to the corresponding wells, followed by addition of AFP antigen short wins in the experiment Peptide TSSELMAITR, and the final concentration of the short peptide in the ELISPOT well plate was 1 × 10 -13 M to 1 × 10 -6 M, a total of 8 gradients; in the control group, other short peptides were added and made The final concentration of the short peptides was 1×10 -8 M to 1×10 -6 M in sequence, with a total of 3 gradients, and each set of three replicate wells. Simultaneously set effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells and medium background control wells. Incubate overnight at constant temperature (37°C, 5% CO 2 ). On the second day of the experiment, color development was detected, and the absorbance value was recorded at 490 nm with an enzyme label reader (Bioteck) after the reaction was terminated.
實驗結果如圖14所示,針對梯度攜載AFP抗原短胜肽TSSELMAITR的標的細胞,轉染本發明高親和力TCR的效應細胞表現出很強的毒殺作用,且在上述特定短胜肽濃度較低時即起反應,而轉染其他TCR的或空轉染的效應細胞自始無毒殺作用;同時,轉染本發明高親和力TCR的效應細胞對攜載其他短胜肽或空載的標的細胞均無毒殺效應,說明其具有很好的特異性。實施例 11 針對腫瘤細胞株,轉染本發明高親和力 TCR 的效應細胞的毒殺功能實驗 The experimental results are shown in Figure 14. For the target cells carrying the AFP antigen short peptide TSSELMAITR in a gradient, the effector cells transfected with the high-affinity TCR of the present invention showed a strong killing effect, and the concentration of the above-mentioned specific short peptide was low. It reacts immediately, and the effector cells transfected with other TCRs or empty-transfected have no cytotoxic effect from the beginning; at the same time, the effector cells transfected with the high-affinity TCR of the present invention have no effect on the target cells carrying other short peptides or empty-loading. No poisoning effect, indicating that it has good specificity. Example 11 Experiment on the killing function of effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines
本實施例同樣通過本領域技術人員熟知的非放射性細胞毒性實驗,測定LDH的釋放,從而驗證轉染本發明TCR的細胞的毒殺功能。本實施例LDH實驗用從健康志願者的血液中分離到的CD3+T細胞轉染本發明高親和力TCR作為效應細胞,並以同一志願者轉染其他TCR(A6)的或空轉染(NC)的CD3+T細胞作為陰性對照。In this example, the release of LDH is also measured by non-radioactive cytotoxicity experiments well known to those skilled in the art, so as to verify the killing function of cells transfected with the TCR of the present invention. In the LDH experiment in this example, CD3+ T cells isolated from the blood of healthy volunteers were used to transfect the high-affinity TCR of the present invention as effector cells, and the same volunteer was used to transfect other TCRs (A6) or empty transfection (NC). ) CD3+ T cells as a negative control.
以下分三個批次(I)、(II)、(III)先後進行實驗: (I)所述高親和力TCR以及其編號從表2獲悉,分別為TCR6 (α鏈可變域SEQ ID NO: 18,β鏈可變域SEQ ID NO: 2)、TCR1 (α鏈可變域SEQ ID NO: 13,β鏈可變域SEQ ID NO: 2)、TCR5 (α鏈可變域SEQ ID NO: 17,β鏈可變域SEQ ID NO: 2)和TCR3 (α鏈可變域SEQ ID NO: 15,β鏈可變域SEQ ID NO: 2)。該批次使用的AFP陽性腫瘤細胞株為:HepG2-A11(HLA-A1101過表現)、SK-MEL-28-AFP(AFP過表現)和SNU423-AFP(AFP過表現),陰性腫瘤細胞株為HepG2、SK-MEL-28和SNU423。實驗步驟如下:首先準備LDH培養盤,按以下順序將試驗的各個組分加入培養盤:標的細胞3×104 個細胞/孔、效應細胞3×104 個細胞/孔(按抗體陽性率計算)加入對應孔中,並設置三個重複孔。同時設置效應細胞自發孔,標的細胞自發孔,標的細胞最大孔,體積校正對照孔及培養基背景對照孔。恆溫培育過夜(37℃,5% CO2 )。實驗第2天,檢測顯色,終止反應後用酵素標示讀取儀(Bioteck)在490nm記錄吸光值。 (II)所述高親和力TCR以及其編號從表2獲悉,分別為TCR2 (α鏈可變域SEQ ID NO: 14,β鏈可變域SEQ ID NO: 2)和TCR13 (α鏈可變域SEQ ID NO: 21,β鏈可變域SEQ ID NO: 2)。該批次使用的AFP陽性腫瘤細胞株為SK-MEL-28-AFP(AFP過表現),陰性腫瘤細胞株為HepG2、HUCCT1、SK-MEL-28和SNU423。實驗步驟與批次(I)相同。 (III)所述高親和力TCR以及其編號從表2獲悉,其為TCR4 (α鏈可變域SEQ ID NO: 16,β鏈可變域SEQ ID NO: 2)。該批次使用的陽性腫瘤細胞株為SK-MEL-28-AFP(AFP過表現),陰性腫瘤細胞株為LCLs-150909A、SK-MEL-28。首先準備LDH培養盤,按以下順序將試驗的各個組分加入培養盤:標的細胞2×104 個細胞/孔、效應細胞2×104 個細胞/孔(按抗體陽性率計算)加入對應孔中,並設置三個重複孔。其餘實驗步驟與批次(I)相同。The experiments are carried out successively in three batches (I), (II) and (III) as follows: (1) The high-affinity TCR and its number are learned from Table 2, which are respectively TCR6 (α chain variable domain SEQ ID NO: 18, β chain variable domain SEQ ID NO: 2), TCR1 (α chain variable domain SEQ ID NO: 13, β chain variable domain SEQ ID NO: 2), TCR5 (α chain variable domain SEQ ID NO: 2) 17, beta chain variable domain SEQ ID NO: 2) and TCR3 (alpha chain variable domain SEQ ID NO: 15, beta chain variable domain SEQ ID NO: 2). The AFP positive tumor cell lines used in this batch are: HepG2-A11 (HLA-A1101 overexpression), SK-MEL-28-AFP (AFP overexpression) and SNU423-AFP (AFP overexpression), and the negative tumor cell lines are HepG2, SK-MEL-28 and SNU423. The experimental steps are as follows: first prepare the LDH culture plate, and add the components of the test to the culture plate in the following order: target cells 3×10 4 cells/well, effector cells 3×10 4 cells/well (calculated according to the antibody positive rate) ) into the corresponding wells and set up three replicate wells. Simultaneously set effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells and medium background control wells. Incubate overnight at constant temperature (37°C, 5% CO 2 ). On the second day of the experiment, color development was detected, and the absorbance value was recorded at 490 nm with an enzyme label reader (Bioteck) after the reaction was terminated. (II) The high-affinity TCR and its number are learned from Table 2, which are respectively TCR2 (alpha chain variable domain SEQ ID NO: 14, beta chain variable domain SEQ ID NO: 2) and TCR13 (alpha chain variable domain SEQ ID NO: 21, beta chain variable domain SEQ ID NO: 2). The AFP-positive tumor cell lines used in this batch were SK-MEL-28-AFP (AFP overexpression), and the negative tumor cell lines were HepG2, HUCCT1, SK-MEL-28 and SNU423. The experimental procedure was the same as batch (I). (III) The high-affinity TCR and its numbering are obtained from Table 2, which is TCR4 (alpha chain variable domain SEQ ID NO: 16, beta chain variable domain SEQ ID NO: 2). The positive tumor cell lines used in this batch were SK-MEL-28-AFP (AFP overexpression), and the negative tumor cell lines were LCLs-150909A and SK-MEL-28. First prepare the LDH culture plate, and add the components of the test to the culture plate in the following order: target cells 2×10 4 cells/well, effector cells 2×10 4 cells/well (calculated according to the antibody positive rate) into the corresponding wells , and set up three repeating wells. The rest of the experimental procedures were the same as batch (I).
實驗結果如圖15a、圖15b和圖15c所示,針對AFP陽性腫瘤細胞株,轉染本發明高親和力TCR的效應細胞仍表現出強毒殺效力,而轉染其他TCR或空轉染的效應細胞基本不起反應;同時,轉染本發明高親和力TCR的T細胞對陰性腫瘤細胞株基本無毒殺。本實驗進一步體現了轉染本發明高親和力TCR的細胞的很好的特異性毒殺功能。The experimental results are shown in Figure 15a, Figure 15b and Figure 15c, for AFP-positive tumor cell lines, the effector cells transfected with the high-affinity TCR of the present invention still showed potent killing effect, while the effector cells transfected with other TCRs or empty transfected Basically no reaction; at the same time, the T cells transfected with the high-affinity TCR of the present invention are basically non-toxic to negative tumor cell lines. This experiment further demonstrated the good specific killing function of cells transfected with the high-affinity TCR of the present invention.
在本發明提及的所有文獻都在本申請中引用作為參考,就如同每一篇文獻被單獨引用作為參考那樣。此外應理解,在閱讀了本發明的上述講授內容之後,本領域技術人員可以對本發明作各種改動或修改,這些等價形式同樣落於本申請所附申請專利範圍所限定的範圍。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended patent scope of the present application.
圖1a和圖1b分別顯示了對TSSELMAITR-HLA A1101複合物能夠特異性結合的野生型TCRα與β鏈可變域胺基酸序列。Figure 1a and Figure 1b show the amino acid sequences of wild-type TCRα and β chain variable domains that can specifically bind to the TSSELMAITR-HLA A1101 complex, respectively.
圖2a和圖2b分別為本發明建構的單鏈模板TCR的α鏈可變域的胺基酸序列和β鏈可變域的胺基酸序列。Figure 2a and Figure 2b are respectively the amino acid sequence of the α chain variable domain and the amino acid sequence of the β chain variable domain of the single-chain template TCR constructed by the present invention.
圖3a和圖3b分別為本發明建構的單鏈模板TCR的α鏈可變域的DNA序列和β鏈可變域的DNA序列。Figure 3a and Figure 3b are respectively the DNA sequence of the variable domain of the α chain and the DNA sequence of the variable domain of the β chain of the single-chain template TCR constructed by the present invention.
圖4a和圖4b分別為本發明建構的單鏈模板TCR的連接短胜肽(linker)的胺基酸序列和DNA序列。Figure 4a and Figure 4b are respectively the amino acid sequence and DNA sequence of the linker of the single-chain template TCR constructed by the present invention.
圖5a、圖5b分別為本發明建構的單鏈模板TCR的胺基酸序列和DNA序列。Figure 5a and Figure 5b are respectively the amino acid sequence and DNA sequence of the single-stranded template TCR constructed by the present invention.
圖6a和圖6b分別為本發明中可溶性參考TCRα鏈與β鏈的胺基酸序列。Figure 6a and Figure 6b are respectively the amino acid sequences of the soluble reference TCR α chain and β chain in the present invention.
圖7(1)-(20)分別顯示了對TSSELMAITR-HLA A1101複合物具有高親和力的異質二聚TCR的α鏈可變域胺基酸序列,突變的殘基以加底線表示。Figures 7(1)-(20) show the alpha chain variable domain amino acid sequences of heterodimeric TCRs with high affinity for the TSSELMAITR-HLA A1101 complex, respectively, with mutated residues underlined.
圖8(1)-(4)分別顯示了對TSSELMAITR-HLA A1101複合物具有高親和力的異質二聚TCR的β鏈可變域胺基酸序列,突變的殘基以加底線表示。Figures 8(1)-(4) show the β-chain variable domain amino acid sequences of heterodimeric TCRs with high affinity for the TSSELMAITR-HLA A1101 complex, respectively, with mutated residues underlined.
圖9a和圖9b分別顯示了對TSSELMAITR-HLA A1101複合物能夠特異性結合的野生型TCRα鏈與β鏈的胞外胺基酸序列。Figures 9a and 9b show the extracellular amino acid sequences of wild-type TCR alpha and beta chains, respectively, that can specifically bind to the TSSELMAITR-HLA A1101 complex.
圖10a和圖10b分別顯示了對TSSELMAITR-HLA A1101複合物能夠特異性結合的野生型TCRα鏈與β鏈的胺基酸序列。Figures 10a and 10b show the amino acid sequences of wild-type TCR alpha chain and beta chain, respectively, that can specifically bind to the TSSELMAITR-HLA A1101 complex.
圖11為可溶性參考TCR即野生型TCR與TSSELMAITR-HLA A1101複合物的結合曲線。Figure 11 is a binding curve of a soluble reference TCR, wild-type TCR, to the TSSELMAITR-HLA A1101 complex.
圖12a和圖12b為針對攜載短胜肽的T2細胞,轉染本發明高親和力TCR的效應細胞的活化功能實驗結果。Figure 12a and Figure 12b are the results of the activation function experiment of effector cells transfected with the high-affinity TCR of the present invention for T2 cells carrying short peptides.
圖13a和圖13b為針對腫瘤細胞株,轉染本發明高親和力TCR的效應細胞的活化功能實驗結果。Fig. 13a and Fig. 13b are the results of the activation function experiment of effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines.
圖14為針對梯度攜載短胜肽的T2細胞,轉染本發明高親和力TCR的效應細胞的毒殺功能實驗結果。Fig. 14 shows the results of the toxicity test on the effector cells transfected with the high-affinity TCR of the present invention against T2 cells carrying the short peptides in a gradient.
圖15a、圖15b和圖15c為針對腫瘤細胞株,轉染本發明高親和力TCR的效應細胞的毒殺功能實驗結果。Fig. 15a, Fig. 15b and Fig. 15c are the results of the poisoning function experiment on the effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines.
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