技术领域Technical Field
本发明属于生物制药技术领域，具体地涉及PSMA抗体及其应用。更具体地，本发明涉及一种抗体或其抗原结合片段、双特异性抗体、核酸分子、表达载体、重组细胞、偶联物、药物组合物、试剂盒及它们的用途。The present invention belongs to the field of biopharmaceutical technology, and specifically relates to PSMA antibodies and their applications. More specifically, the present invention relates to an antibody or an antigen-binding fragment thereof, a bispecific antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a conjugate, a pharmaceutical composition, a kit and their uses.
背景技术Background Art
癌症是影响人类生存发展的重大疾病，根据最新数据，全球每年新发癌症病例1900万左右，每年死亡癌症病例1000万左右，并且发生率和死亡率呈增长趋势。Cancer is a major disease that affects human survival and development. According to the latest data, there are approximately 19 million new cases of cancer and 10 million cancer deaths each year worldwide, and the incidence and mortality rates are on the rise.
除手术切除外，化疗、放疗等传统癌症治疗手段副作用大、易复发。近年来，免疫治疗，包括肿瘤靶向抗体、免疫检查点抗体、双特异抗体等，成为了抗癌新热点和新希望。In addition to surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy have serious side effects and are prone to recurrence. In recent years, immunotherapy, including tumor-targeted antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hot spot and new hope in the fight against cancer.
近年来，以PD-1/L1为代表的免疫治疗展示了巨大的潜力，但是不能忽视的是，即便目前获批适应症最广的PD-1/L1疗法的总体响应率仍只有30％，仍有更多的患者无法从中受益。免疫检查点分子是表达于免疫细胞包括T、NK、单核巨噬细胞等表面的抑制性分子，与相应配体结合后，向免疫细胞内传递抑制性信号，抑制免疫细胞的抗癌功能。由于肿瘤、肿瘤浸润淋巴细胞的免疫检查点受配体表达具有非常大的异质性，导致单一种类的免疫检查点疗法无法适用于所有患者，多数患者无法从中获益；另一方面，部分接受过免疫检测点疗法的患者会复发肿瘤并产生对该免疫检查点疗法的耐受，继续给药也无法产生疗效；另外，T细胞通过表面的T细胞受体(TCR)识别新生抗原(neo-antigen)，也就是肿瘤基因突变的抗原，而部分肿瘤的基因突变频率低，新生抗原种类少，称为“冷肿瘤”。目前的免疫检查点疗法如PD-1/L1疗法，通过恢复T细胞自身的功能达到抗癌目的，而在“冷肿瘤”中，T细胞无法有效识别肿瘤，导致免疫检查点疗法对冷肿瘤无效。In recent years, immunotherapy represented by PD-1/L1 has shown great potential, but it cannot be ignored that even the overall response rate of PD-1/L1 therapy, which has the widest approved indications, is only 30%, and there are still more patients who cannot benefit from it. Immune checkpoint molecules are inhibitory molecules expressed on the surface of immune cells including T, NK, monocytes and macrophages. After binding to the corresponding ligand, they transmit inhibitory signals to the immune cells and inhibit the anti-cancer function of immune cells. Due to the very large heterogeneity of immune checkpoint ligand expression in tumors and tumor-infiltrating lymphocytes, a single type of immune checkpoint therapy cannot be applied to all patients, and most patients cannot benefit from it; on the other hand, some patients who have received immune checkpoint therapy will relapse and develop tolerance to the immune checkpoint therapy, and continued administration will not produce therapeutic effects; in addition, T cells recognize neo-antigens (neo-antigens), that is, antigens with tumor gene mutations, through the surface T cell receptors (TCR), and some tumors have low gene mutation frequencies and few types of neo-antigens, which are called "cold tumors." Current immune checkpoint therapies, such as PD-1/L1 therapy, achieve anti-cancer goals by restoring the function of T cells themselves. However, in "cold tumors", T cells cannot effectively recognize tumors, resulting in the ineffectiveness of immune checkpoint therapies for cold tumors.
。PSMA是种细胞膜蛋白，在大部分人正常组织中几乎不表达，在前列腺癌中呈现出特异性高表达，超过80％的前列腺癌患者高表达PSMA，且其高表达与不良预后相关。因此，PSMA是有潜力的治疗靶点，开发靶向PSMA的抗体或者基于PSMA单抗的双、多功能抗体或者CAR-T、CAR-NK疗法非常有价值。PSMA is a cell membrane protein that is barely expressed in most normal tissues, but is highly expressed in prostate cancer. More than 80% of prostate cancer patients highly express PSMA, and its high expression is associated with poor prognosis. Therefore, PSMA is a potential therapeutic target, and it is very valuable to develop antibodies targeting PSMA or bi- and multifunctional antibodies based on PSMA monoclonal antibodies or CAR-T and CAR-NK therapies.
发明内容Summary of the invention
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此，本发明提供了一种靶向PSMA的抗体或抗原结合片段，该抗体或抗原结合片段具有高PSMA结合亲和力和肿瘤杀伤力。The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. To this end, the present invention provides an antibody or antigen-binding fragment targeting PSMA, which has high PSMA binding affinity and tumor killing ability.
在本发明的第一方面，本发明提出了一种抗体或抗原结合片段。根据本发明的实施例，所述抗体或抗原结合片段包括选自下列至少之一的CDR：HCDR 1:GYSFTX1NW，其中，X1为S或H；HCDR 2:IYPGDSDT；HCDR 3:ARQTGFLWSSDLWGRGT；LCDR 1:X2QDISX3A，其中，X2为S或P，X3为S或Y；LCDR 2:DASX4，其中，X4为S或W；LCDR 3:QQFNSYPLX5，其中，X5为T或S；X1为S、X2为S、X3为S、X4为S、X5为T不同时存在。与野生型抗体相比，上述抗体或抗原结合片段具有高PSMA结合亲和力和肿瘤杀伤力，可检测PSMA。In the first aspect of the present invention, the present invention proposes an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises at least one of the following CDRs: HCDR 1: GYSFTX1 NW, wherein X1 is S or H; HCDR 2: IYPGDSDT; HCDR 3: ARQTGFLWSSDLWGRGT; LCDR 1: X2 QDISX3 A, wherein X2 is S or P, and X3 is S or Y; LCDR 2: DASX4 , wherein X4 is S or W; LCDR 3: QQFNSYPLX5 , wherein X5 is T or S; X1 is S, X2 is S, X 3 is S, X4 is S, andX 5is T, which do not exist at the same time. Compared with the wild-type antibody, the above-mentioned antibody or antigen-binding fragment has high PSMA binding affinity and tumor killing ability, and can detect PSMA.
在本发明的第二方面，本发明提出了一种多特异性抗体。根据本发明的实施例，所述多特异性抗体包括：第一结合区，所述第一结合区包括第一方面所述的抗体或抗原结合片段；第二结合区，所述第二结合区具有第一分子结合活性。本发明的多特异性抗体具有高PSMA结合亲和力和肿瘤杀伤力，可有效预防和/或治疗PSMA介导的相关疾病，尤其是预防和/或治疗前列腺癌。In the second aspect of the present invention, the present invention proposes a multispecific antibody. According to an embodiment of the present invention, the multispecific antibody comprises: a first binding region, the first binding region comprises the antibody or antigen-binding fragment described in the first aspect; a second binding region, the second binding region has a first molecule binding activity. The multispecific antibody of the present invention has high PSMA binding affinity and tumor killing ability, and can effectively prevent and/or treat PSMA-mediated related diseases, especially prevent and/or treat prostate cancer.
在本发明的第三方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体。本发明的核酸分子可编码第一方面的抗体或抗原结合片段、第二方面所述的多特异性抗体。In the third aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect, and the multispecific antibody described in the second aspect. The nucleic acid molecule of the present invention can encode the antibody or antigen-binding fragment of the first aspect, and the multispecific antibody described in the second aspect.
在本发明的第四方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体携带第三方面所述的核酸分子。由此，采用本发明的表达载体可有效实现第一方面的抗体或抗原结合片段或第二方面所述的多特异性抗体的表达，进而实现抗体或抗原结合片段或者多特异性抗体的体外大量获得。In a fourth aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect. Thus, the expression vector of the present invention can effectively achieve the expression of the antibody or antigen-binding fragment of the first aspect or the multispecific antibody of the second aspect, thereby achieving the in vitro acquisition of a large number of antibodies or antigen-binding fragments or multispecific antibodies.
在本发明的第五方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞包括携带第三方面所述的核酸分子或第四方面所述的表达载体；或表达第一方面所述的抗体或抗原结合片段、或者第二方面所述的多特异性抗体。利用该重组细胞在适合条件下，能够在细胞内有效地表达前述的抗体或抗原结合片段或者多特异性抗体。In the fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell includes carrying the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect; or expressing the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect. The recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment or multispecific antibody in the cell under suitable conditions.
在本发明的第六方面，本发明提出了一种偶联物。根据本发明的实施例，所述偶联物包含第一方面所述的抗体或抗原结合片段、或者第二方面所述的多特异性抗体；以及偶联部分，所述偶联部分与所述抗体或抗原结合片段或多特异性抗体相连。本发明的偶联物具有高PSMA结合亲和力，可用于检测PSMA、或者用于预防和/或治疗PSMA介导的相关疾病，尤其是用于检测前列腺癌、或预防和/或治疗前列腺癌。In the sixth aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect; and a coupling portion, wherein the coupling portion is connected to the antibody or antigen-binding fragment or multispecific antibody. The conjugate of the present invention has a high PSMA binding affinity and can be used to detect PSMA, or to prevent and/or treat PSMA-mediated related diseases, especially to detect prostate cancer, or to prevent and/or treat prostate cancer.
在本发明的第七方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体、第三方面所述的核酸分子、第四方面所述的表达载体、第五方面所述的重组细胞或第六方面所述的偶联物。本发明的药物组合物具有高PSMA结合亲和力和肿瘤杀伤力，可有效预防和/或治疗PSMA介导的相关疾病，尤其是用于预防和/或治疗前列腺癌。In the seventh aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the conjugate described in the sixth aspect. The pharmaceutical composition of the present invention has high PSMA binding affinity and tumor killing ability, and can effectively prevent and/or treat PSMA-mediated related diseases, especially for preventing and/or treating prostate cancer.
在本发明的第八方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体、第三方面所述的核酸分子、第四方面所述的表达载体、第五方面所述的重组细胞或第六方面所述的偶联物。本发明的试剂盒具有高PSMA结合亲和力，可有效检测PSMA、检测PSMA介导的相关疾病、诊断PSMA介导的相关疾病、对PSMA介导的相关疾病进行分期、或者评估PSMA介导的相关疾病预后。In the eighth aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit comprises the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the conjugate described in the sixth aspect. The kit of the present invention has high PSMA binding affinity and can effectively detect PSMA, detect PSMA-mediated related diseases, diagnose PSMA-mediated related diseases, stage PSMA-mediated related diseases, or evaluate the prognosis of PSMA-mediated related diseases.
在本发明的第九方面，本发明提出了一种第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体、第三方面所述的核酸分子、第四方面所述的表达载体、第五方面所述的重组细胞、第六方面所述的偶联物或第七方面所述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗PSMA介导的相关疾病，尤其是预防和/或治疗前列腺癌。In the ninth aspect of the present invention, the present invention proposes a use of the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect, the conjugate of the sixth aspect or the pharmaceutical composition of the seventh aspect in the preparation of a drug, wherein the drug is used for preventing and/or treating PSMA-mediated related diseases, especially preventing and/or treating prostate cancer.
在本发明的第十方面，本发明提出了一种第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体、第三方面所述的核酸分子、第四方面所述的表达载体、第五方面所述的重组细胞或第六方面所述的偶联物在制备试剂盒中的用途，所述试剂盒用于检测PSMA、检测PSMA介导的相关疾病、诊断PSMA介导的相关疾病、对PSMA介导的相关疾病进行分期、或者评估PSMA介导的相关疾病预后。In the tenth aspect of the present invention, the present invention proposes a use of the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect or the conjugate of the sixth aspect in preparing a kit, wherein the kit is used to detect PSMA, detect PSMA-mediated related diseases, diagnose PSMA-mediated related diseases, stage PSMA-mediated related diseases, or evaluate the prognosis of PSMA-mediated related diseases.
有益效果：Beneficial effects:
1)本发明获得的亲和力成熟PSMA抗体相较于母本抗体具有更慢的的解离速率(Kd)。1) The affinity matured PSMA antibody obtained in the present invention has a slower dissociation rate (Kd) than the parent antibody.
2)本发明获得的亲和力成熟PSMA抗体相较于母本抗体与肿瘤细胞结合的强度更高。2) The affinity-matured PSMA antibody obtained in the present invention binds to tumor cells more strongly than the parent antibody.
3)本发明获得的亲和力成熟抗体PSMA抗体相较于母本抗体具有更强的ADCC活性。3) The affinity matured PSMA antibody obtained in the present invention has stronger ADCC activity than the parent antibody.
4)本发明获得的CD3×PSMA双抗相较于母本抗体具有更强的结合活性和促杀伤活性。4) The CD3×PSMA bispecific antibody obtained in the present invention has stronger binding activity and promoting killing activity than the parent antibody.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:
图1为根据本发明实施例2的亲和力成熟PSMAv11抗体、母本抗体parental mAb的CDR序列对比图；FIG1 is a CDR sequence comparison diagram of the affinity matured PSMAv11 antibody and the parental mAb according to Example 2 of the present invention;
图2为根据本发明实施例3的亲和力成熟PSMAv11抗体、母本抗体parental mAb蛋白亲和力测定的SPR结果图；FIG2 is a graph showing the SPR results of affinity determination of affinity matured PSMAv11 antibody and parental mAb protein according to Example 3 of the present invention;
图3为根据本发明实施例5的亲和力成熟PSMAv11抗体、母本抗体parental mAb与22Rv1人前列腺癌细胞结合的流式细胞术结果图；FIG3 is a flow cytometry result diagram of the binding of affinity matured PSMAv11 antibody and parental mAb to 22Rv1 human prostate cancer cells according to Example 5 of the present invention;
图4为根据本发明实施例5的亲和力成熟PSMAv11抗体、母本抗体parental mAb与LNCaP人前列腺癌细胞结合的流式细胞术结果图；FIG4 is a flow cytometry result diagram of the binding of affinity matured PSMAv11 antibody and parental mAb to LNCaP human prostate cancer cells according to Example 5 of the present invention;
图5为根据本发明实施例6的亲和力成熟PSMAv11抗体、母本抗体parental mAb促进PBMC杀伤LNCaP人前列腺癌细胞的结果图；FIG5 is a graph showing the results of affinity-matured PSMAv11 antibody and parental mAb promoting PBMC to kill LNCaP human prostate cancer cells according to Example 6 of the present invention;
图6为根据本发明实施例6的亲和力成熟PSMAv11抗体、母本抗体parental mAb促进PBMC杀伤22Rv1人前列腺癌细胞的结果图；FIG6 is a graph showing the results of affinity-matured PSMAv11 antibody and parental mAb promoting PBMC to kill 22Rv1 human prostate cancer cells according to Example 6 of the present invention;
图7为根据本发明实施例7的CD3×PSMAv11抗体、CD3×parental PSMA双特异抗体与22Rv1人前列腺癌细胞结合的流式细胞术结果图；FIG7 is a flow cytometry result diagram of the binding of CD3×PSMAv11 antibody and CD3×parental PSMA bispecific antibody according to Example 7 of the present invention to 22Rv1 human prostate cancer cells;
图8为根据本发明实施例7的CD3×PSMAv11抗体、CD3×parental PSMA双特异抗体与LNCaP人前列腺癌细胞结合的流式细胞术结果图；FIG8 is a flow cytometry result diagram of the binding of CD3×PSMAv11 antibody and CD3×parental PSMA bispecific antibody according to Example 7 of the present invention to LNCaP human prostate cancer cells;
图9为根据本发明实施例8的CD3×PSMAv11抗体、CD3×parental PSMA双特异抗体与促进PBMC杀伤LNCaP人前列腺癌细胞的结果图；FIG9 is a graph showing the results of CD3×PSMAv11 antibody and CD3×parental PSMA bispecific antibody according to Example 8 of the present invention and promoting PBMC to kill LNCaP human prostate cancer cells;
图10为根据本发明实施例8的CD3×PSMAv11抗体、CD3×parental PSMA双特异抗体促进PBMC杀伤22Rv1人前列腺癌细胞的结果图。10 is a graph showing the results of CD3×PSMAv11 antibody and CD3×parental PSMA bispecific antibody according to Example 8 of the present invention promoting PBMC to kill 22Rv1 human prostate cancer cells.
具体实施方式DETAILED DESCRIPTION
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.
本发明详细说明Detailed description of the invention
定义及一般术语Definitions and general terms
在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.
在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
在本文中，术语“片段”是指目标蛋白或多肽，以及具有N-末端(N端)或C-末端(C端)截短、和/或内部删除的目标蛋白或多肽。Herein, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide having an N-terminal (N-terminus) or C-terminal (C-terminus) truncation, and/or internal deletion.
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文中使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
本发明所述的抗体或抗原结合片段通常由生物合成的方法制备。根据本发明所述的核苷酸序列，本领域技术人员可方便地用各种已知方法制得本发明的编码核酸。这些方法例如但不限于：PCR，DNA人工合成等，具体的方法可参见J.萨姆布鲁克，《分子克隆实验指南》。作为本发明的一种实施方式，可通过分段合成核苷酸序列再进行重叠延伸PCR的方法来构建本发明的编码核酸序列。其中，所述抗体或抗原片段是采用Kabat编号系统进行编号和定义的。The antibody or antigen-binding fragment of the present invention is usually prepared by a biosynthetic method. According to the nucleotide sequence of the present invention, a person skilled in the art can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods include but are not limited to: PCR, DNA artificial synthesis, etc. For specific methods, please refer to J. Sambrook, "Molecular Cloning Laboratory Guide". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing the nucleotide sequence in segments and then performing overlap extension PCR. Wherein, the antibody or antigen fragment is numbered and defined using the Kabat numbering system.
在本文中，术语“同一性”、“同源性”或“相似性”均用于描述相对于参考序列的氨基酸序列或核酸序列时，采用通过常规的方法进行确定两个氨基酸序列或核酸序列之间的相同氨基酸或核苷酸的百分比，例如参见，Ausubel等，编著(1995)，Current Protocols inMolecular Biology，第19章(Greene Publishing and Wiley-Interscience，New York)；和ALIGN程序(Dayhoff(1978)，Atlas of Protein Sequence and Structure5：Suppl.3(National Biomedical Research Foundation，Washington，D.C.)。关于比对序列和测定序列同一性有很多算法，包括，Needleman等(1970)J.Mol.Biol.48：443的同源性比对算法；Smith等(1981)Adv.Appl.Math.2：482的局部同源性算法；Pearson等(1988)Proc.Natl.Acad.Sci.85：2444的相似性搜索方法；Smith-Waterman算法(Meth.Mol.Biol.70：173-187(1997)；和BLASTP，BLASTN，和BLASTX算法(参见Altschul等(1990)J.Mol.Biol.215：403-410)。利用这些算法的计算机程序也是可获得的，并且包括但不限于：ALIGN或Megalign(DNASTAR)软件，或者WU-BLAST-2(Altschul等，Meth.Enzym.，266：460-480(1996))；或者GAP，BESTFIT，BLAST Altschul等，上文，FASTA，和TFASTA，在Genetics Computing Group(GCG)包，8版，Madison，Wisconsin，USA中可获得；和Intelligenetics，Mountain View，California提供的PC/Gene程序中的CLUSTAL。As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... l.70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC/Gene program provided by Intelligenetics, Mountain View, California.
在本文中，术语“至少80％同一性”指与各参考序列至少为80％，可为80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％的同一性。As used herein, the term "at least 80% identity" refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.
在本文中，术语“至少90％同一性”指与各参考序列至少为90％，可为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％的同一性。As used herein, the term "at least 90% identity" refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.
在本文中，术语“表达载体”通常是指能够插入在合适的宿主中自我复制的核酸分子，该核酸分子包含可表达目标蛋白的核苷酸序列，且可将该核酸分子转移到宿主细胞中和/或宿主细胞之间。所述表达载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述表达载体还包括具有多种上述功能的载体。所述表达载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述表达载体的合适的宿主细胞，所述表达载体可以产生期望的表达产物。In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, the nucleic acid molecule comprises a nucleotide sequence that can express a target protein, and the nucleic acid molecule can be transferred into a host cell and/or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and/or translation of DNA or RNA. The expression vector also includes vectors with a variety of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.
在本文中，术语“重组细胞”通常是指采用基因工程技术或细胞融合技术对宿主细胞的遗传物质进行修饰改造或重组，获得具有稳定遗传的独特性状的细胞。其中，术语“宿主细胞”是指可导入重组表达载体的原核细胞或真核细胞。本文所用术语“转化的”或“转染的”是指通过本领域已知的各种技术将核酸(例如表达载体)引入细胞。合适的宿主细胞可以用本发明的DNA序列转化或转染，并且可以用于靶蛋白的表达和/或分泌。可用于本发明的合适宿主细胞的例子包括永生化杂交瘤细胞、NS/0骨髓瘤细胞、293细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞、Cap细胞(人羊水来源的细胞)和CoS细胞。In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant expression vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as expression vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention, and can be used for the expression and/or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid) and CoS cells.
在本文中，术语“药物组合物”通常是指单位剂量形式，并且可以通过制药领域中熟知的方法的任何一种进行制备。所有的方法包括使活性成分与构成一种或多种附属成分的载体相结合的步骤。通常，通过均匀并充分地使活性抗体或抗原结合片段与液体载体、细碎固体载体或这两者相结合，制备组合物。As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active antibody or antigen-binding fragment with a liquid carrier, a finely divided solid carrier, or both.
在本文中，术语“药学上可接受的辅料”均可包括任何溶剂、固体赋形剂、稀释剂或其他液体赋形剂等等，适合于特有的目标剂型。除了任何常规的辅料与本发明的抗体或抗原结合片段不相容的范围，例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用，它们的用途也是本发明所考虑的范围。In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the antibody or antigen-binding fragment of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.
在本文中，术语“给药”指将预定量的物质通过某种适合的方式引入病人。本发明的抗体或抗原结合片段、多特异性抗体或药物组合物可以通过任何常见的途径被给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜、静脉注射、肌肉注射、皮下注射等等，但是本发明不限于这些已举例的给药方式。优选地，本发明的组合物采用静脉注射或皮下注射方式被给药。As used herein, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The antibody or antigen-binding fragment, multispecific antibody or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.
在本文中，术语“治疗”是指用于获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病或病症发生；(b)抑制疾病，例如阻滞疾病发展；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将药物或抗体或抗原结合片段给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药，包括但不限于将含本文所述抗体或抗原结合片段的药物给予有需要的个体。As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and/or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing a disease and/or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or antibody or antigen-binding fragment to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing the antibody or antigen-binding fragment described herein to an individual in need.
本发明抗新型冠状病毒抗体及其应用的详细说明Detailed description of the anti-novel coronavirus antibody and its application
本发明提出了一种抗体或抗原结合片段、多特异性抗体、核酸分子、表达载体、重组细胞、偶联物、药物组合物、试剂盒及它们的用途，下面将分别对其进行详细描述。The present invention provides an antibody or antigen-binding fragment, a multispecific antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a conjugate, a pharmaceutical composition, a kit and uses thereof, which will be described in detail below.
抗体或抗原结合片段Antibodies or antigen-binding fragments
在本发明的一个方面，本发明提出了一种抗体或抗原结合片段。根据本发明的实施例，所述抗体或抗原结合片段包括选自下列至少之一的CDR：HCDR 1:GYSFTX1NW，其中，X1为S或H；HCDR 2:IYPGDSDT；HCDR 3:ARQTGFLWSSDLWGRGT；LCDR 1:X2QDISX3A，其中，X2为S或P，X3为S或Y；LCDR 2:DASX4，其中，X4为S或W；LCDR 3:QQFNSYPLX5，其中，X5为T或S；X1为S、X2为S、X3为S、X4为S、X5为T不同时存在。与野生型抗体相比，上述抗体或抗原结合片段具有高PSMA结合亲和力和肿瘤杀伤力，可用于检测PSMA、或者用于预防和/或治疗PSMA介导的相关疾病。In one aspect of the present invention, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises at least one of the following CDRs: HCDR 1: GYSFTX1 NW, wherein X1 is S or H; HCDR 2: IYPGDSDT; HCDR 3: ARQTGFLWSSDLWGRGT; LCDR 1: X2 QDISX3 A, wherein X2 is S or P, and X3 is S or Y; LCDR 2: DASX4 , wherein X4 is S or W; LCDR 3: QQFNSYPLX5 , wherein X5 is T or S; X1 is S, X2 is S, X 3 is S, X4 is S, and X5 is T, which do not exist at the same time. Compared with the wild-type antibody, the above-mentioned antibody or antigen-binding fragmenthas high PSMA binding affinity and tumor killing ability, and can be used to detect PSMA, or to prevent and/or treat PSMA-mediated related diseases.
在本文中，术语“抗体”在最广义上使用，其可以包括全长单克隆抗体、多特异性抗体、以及嵌合抗体，具体结构不受限制，只要它们展示所需的生物学活性。其通常包括分子量较轻的轻链和分子量较重的重链，重链(H链)和轻链(L链)由二硫键连接形成的抗体分子。其中，肽链的氨基端(N端)氨基酸序列变化很大，称为可变区(V区)；羧基端(C端)相对稳定，变化很小，称为恒定区(C区)。L链和H链的V区分别称为VL和VH。In this article, the term "antibody" is used in the broadest sense, which can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited as long as they show the desired biological activity. It usually includes a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight, and the heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form an antibody molecule. Among them, the amino acid sequence of the amino terminal (N terminal) of the peptide chain varies greatly, which is called the variable region (V region); the carboxyl terminal (C terminal) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and the H chain are called VL and VH, respectively.
在本文中，重链互补决定区(重链可变区CDR)用“HCDRs”或“HCDR”表示，其包括HCDR1(又称CDR-H1)、HCDR2(又称CDR-H2)和HCDR3(又称CDR-H3)；轻链互补决定区(轻链可变区CDR)用“LCDRs”或“LCDR”表示，其包括LCDR1(又称CDR-L1)、LCDR2(又称CDR-L2)和LCDR3(又称CDR-L3)。本领域常用的CDR定义方案包括：Kabat定义、Chothia定义、IMGT定义、Contact定义和AbM定义。In this article, the heavy chain complementary determining region (heavy chain variable region CDR) is represented by "HCDRs" or "HCDR", which includes HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2) and HCDR3 (also known as CDR-H3); the light chain complementary determining region (light chain variable region CDR) is represented by "LCDRs" or "LCDR", which includes LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2) and LCDR3 (also known as CDR-L3). Commonly used CDR definition schemes in the art include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition.
如本文所述，“Kabat定义”是指Kabat等，U.S.Dept.of Health and HumanServices，“Sequence of Proteinsof Immunological Interest”(1983)所述的定义系统。“Chothia定义”参见Chothia等，J Mol Biol 196：901-917(1987)。示例性的定义的CDR列于下表A中，不同文献中的定义略有不同，在给定抗体的可变区氨基酸序列的情况下，本领域技术人员可以常规地确定哪些残基包含特定CDR。需要说明的是，本发明中的CDR包括不限于表A中的其他方法定义的CDR，基于本申请公开的重链可变区和轻链可变区采用本领域公开的其他规则确定的CDR也属于本公开的保护范围。As described herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the "Chothia definition", see Chothia et al., J Mol Biol 196:901-917 (1987). Exemplary defined CDRs are listed in Table A below. The definitions in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that the CDRs in the present invention include but are not limited to CDRs defined by other methods in Table A. CDRs determined based on the heavy chain variable region and light chain variable region disclosed in this application using other rules disclosed in the art also fall within the scope of protection of the present disclosure.
表A：CDR定义1Table A: CDR Definition1
1表A中所有CDR定义的编号是依据Kabat编号系统(参见下文)，重链上的氨基酸编号用“H+数字”表示，轻链上的氨基酸编号用“L+数字”表示。1 The numbering of all CDR definitions in Table A is based on the Kabat numbering system (see below), with the amino acid numbers on the heavy chain represented by "H+numbers" and the amino acid numbers on the light chain represented by "L+numbers".
2如表A中使用的“AbM”具有小写“b”，是指通过Oxford Molecular的“AbM”抗体建模软件定义的CDR。2 “AbM” as used in Table A with a lowercase “b” refers to CDRs defined by Oxford Molecular’s “AbM” antibody modeling software.
3如果H35A和H35B都不存在时，那么CDR-H1结束在35位；如果只有H35A存在时，那么CDR-H1结束在35A位；如果H35A和H35B同时存在，那么CDR-H1结束在35B位。3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.
4如果H35A和H35B都不存在时，那么CDR-H1结束在32位；如果只有H35A存在时，那么CDR-H1结束在33位；如果H35A和H35B同时存在，那么CDR-H1结束在34位。4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.
5如果H35A和H35B都不存在时，那么CDR-H1结束在33位；如果只有H35A存在时，那么CDR-H1结束在34位；如果H35A和H35B同时存在，那么CDR-H1结束在35位。5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.
Kabat等还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以明确地将该Kabat编号系统对应到任何可变区序列，而不依赖于序列本身之外的任何实验数据。如本文所述，“Kabat编号”是指采用《Kabat等，U.S.Dept.of Health andHumanServices，“Sequence of Proteins of ImmunologicalInterest”(1983)》所述的编号系统进行编号的，采用上述编号系统对本发明的抗体或抗原结合片段的HCDRs和LCDRs进行编号，具体编号结果参见表A。需要说明的是，本发明的序列表和表B中的多肽序列为根据Kabat编号系统进行编号。然而，本领域普通技术人员完全能够将序列表的序列Kabat编号转换为其他编号系统下的“HCDRs”和/或“LCDRs”，其均在本发明的保护范围内。Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. A person of ordinary skill in the art can clearly correspond the Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, "Kabat numbering" refers to the numbering system described in "Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983)", and the HCDRs and LCDRs of the antibody or antigen-binding fragment of the present invention are numbered using the above numbering system, and the specific numbering results are shown in Table A. It should be noted that the polypeptide sequences in the sequence table and Table B of the present invention are numbered according to the Kabat numbering system. However, a person of ordinary skill in the art is fully capable of converting the sequence Kabat numbering of the sequence table into "HCDRs" and/or "LCDRs" under other numbering systems, which are all within the scope of protection of the present invention.
在本文中，术语“抗原结合片段”是包含抗体的一部分或全部的片段，其缺乏至少一些存在于全长链中的氨基酸但仍能够特异性结合抗原的性能活性，例如，该片段可包含抗体CDR的一部分或全部。此类片段具生物活性，因为其结合至抗原，且可与其他抗原结合分子(包括完整抗体)竞争结合至给定表位。此类片段选自Fab、Fv、scFv或单域抗体。此类片段可通过重组核酸技术产生，或可通过抗原结合分子(包括完整抗体)的酶裂解或化学裂解产生。As used herein, the term "antigen-binding fragment" is a fragment comprising part or all of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen, for example, the fragment may comprise part or all of an antibody CDR. Such fragments are biologically active because they bind to an antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, Fv, scFv or single domain antibodies. Such fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic cleavage or chemical cleavage of antigen-binding molecules (including intact antibodies).
根据本发明的实施例，所述抗体或抗原结合片段还可以进一步包括如下技术特征的至少之一：According to an embodiment of the present invention, the antibody or antigen-binding fragment may further include at least one of the following technical features:
根据本发明的实施例，X1为S。According to an embodiment of the present invention,X1 is S.
根据本发明的实施例，X1为H。According to an embodiment of the present invention,X1 is H.
根据本发明的实施例，X2为S。According to an embodiment of the present invention,X2 is S.
根据本发明的实施例，X2为P。According to an embodiment of the present invention,X2 is P.
根据本发明的实施例，X3为S。According to an embodiment of the present invention,X3 is S.
根据本发明的实施例，X3为Y。According to an embodiment of the present invention,X3 is Y.
根据本发明的实施例，X4为S。According to an embodiment of the present invention,X4 is S.
根据本发明的实施例，X4为W。According to an embodiment of the present invention,X4 is W.
根据本发明的实施例，X5为T。According to an embodiment of the present invention,X5 is T.
根据本发明的实施例，X5为S。According to an embodiment of the present invention,X5 is S.
在本发明的一个实施例中，所述抗体或抗原结合片段包括如下其中一组的CDRs：In one embodiment of the present invention, the antibody or antigen-binding fragment comprises one of the following groups of CDRs:
根据本发明的实施例，所述抗体或抗原结合片段包括：分别如SEQ ID NO:1、2和3的氨基酸序列所示的HCDR1、HCDR2、HCDR3；和分别如SEQ ID NO:4、5和6的氨基酸序列所示的LCDR1、LCDR2、LCDR3。其中，该CDRs与上表中组别18相同。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: HCDR1, HCDR2, HCDR3 as shown in the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; and LCDR1, LCDR2, LCDR3 as shown in the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively. The CDRs are the same as group 18 in the above table.
根据本发明的实施例，所述抗体或其抗原结合片段特异性识别PSMA。According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof specifically recognizes PSMA.
根据本发明的实施例，所述抗体或抗原结合片段包括重链框架区和/或轻链框架区。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a heavy chain framework region and/or a light chain framework region.
根据本发明的实施例，所述重链框架区和/或轻链框架区的至少一部分来自于鼠源抗体、灵长目源抗体、牛源抗体、马源抗体、乳牛源抗体、猪源抗体、绵羊源抗体、山羊源抗体、狗源抗体、猫源抗体、兔源抗体、骆驼源抗体、驴源抗体、鹿源抗体、貂源抗体、鸡源抗体、鸭源抗体、鹅源抗体、火鸡源抗体、斗鸡源抗体或其突变体中的至少之一。According to an embodiment of the present invention, at least a portion of the heavy chain framework region and/or the light chain framework region is derived from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof.
根据本发明的实施例，所述重链框架区和/或轻链框架区的至少一部分来自于鼠源抗体和人源抗体中的至少之一。According to an embodiment of the present invention, at least a portion of the heavy chain framework region and/or the light chain framework region is derived from at least one of a murine antibody and a human antibody.
根据本发明的实施例，所述抗体或抗原结合片段包括：如SEQ ID NO:7所示的氨基酸序列或与其具有至少90％同源性的氨基酸序列的重链可变区；和如SEQ ID NO:8所示的氨基酸序列或与其具有至少90％同源性的氨基酸序列的轻链可变区。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 90% homology thereto; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 90% homology thereto.
根据本发明的实施例，所述抗体或抗原结合片段进一步包括重链恒定区和/或轻链恒定区。According to an embodiment of the present invention, the antibody or antigen-binding fragment further comprises a heavy chain constant region and/or a light chain constant region.
根据本发明的实施例，所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于鼠源抗体、灵长目源抗体、牛源抗体、马源抗体、乳牛源抗体、猪源抗体、绵羊源抗体、山羊源抗体、狗源抗体、猫源抗体、兔源抗体、骆驼源抗体、驴源抗体、鹿源抗体、貂源抗体、鸡源抗体、鸭源抗体、鹅源抗体、火鸡源抗体、斗鸡源抗体或其突变体中的至少之一。According to an embodiment of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof.
根据本发明的实施例，所述重链恒定区包括选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区。According to an embodiment of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.
根据本发明的实施例，所述轻链恒定区包括选自κ型或λ型轻链恒定区。According to an embodiment of the present invention, the light chain constant region comprises a light chain constant region selected from a κ type or a λ type.
根据本发明的实施例，所述轻链恒定区和重链恒定区均来自于鼠源抗体或其突变体、和/或人源抗体或其突变体。According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from a murine antibody or a mutant thereof, and/or a human antibody or a mutant thereof.
根据本发明的实施例，所述重链恒定区的N端与所述重链可变区的C端相连；和/或所述轻链恒定区的N端与所述轻链可变区的C端相连。According to an embodiment of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and/or the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.
根据本发明的实施例，所述重链恒定区包括如SEQ ID NO:9或与其具有至少80％同一性的氨基酸序列所示的重链恒定区；和/或所述轻链恒定区包括如SEQ ID NO:10或与其具有至少80％同一性的氨基酸序列所示的轻链恒定区。According to an embodiment of the present invention, the heavy chain constant region includes the heavy chain constant region shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto; and/or the light chain constant region includes the light chain constant region shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity thereto.
根据本发明的实施例，所述抗体包括多抗、全长单抗、Fab抗体、Fab’抗体、F(ab’)2抗体、Fv抗体、单链抗体、单域抗体以及最小识别单位中的至少之一；或者所述抗原结合片段包括F(ab’)2片段、Fab’片段、Fab片段、F(ab)2片段、Fv片段、scFv片段、scFv-Fc融合蛋白、scFv-Fv融合蛋白和最小识别单位中的至少之一。According to an embodiment of the present invention, the antibody includes at least one of a polyclonal antibody, a full-length monoclonal antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, a Fv antibody, a single-chain antibody, a single-domain antibody and a minimum recognition unit; or the antigen-binding fragment includes at least one of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit.
在本文中，术语“全长抗体”、“全长单抗”或“全长单克隆抗体”均是由至少两条相同的轻链和至少两条相同的重链通过链间二硫键连接而成，如免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白D(IgD)或免疫球蛋白E(IgE)。As used herein, the terms "full-length antibody", "full-length monoclonal antibody" or "full-length monoclonal antibody" are composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).
在本文中，术语“多抗”和“多特异性抗体”同义，均是指可识别多种抗原表位的抗体，例如可识别两种抗原表位的抗体(双特异性抗体，简称双抗)、三种抗原表位的抗体或者四种抗原表位的抗体，其为广义理解，具体结构不受限制，可识别多种抗原表位即可。在本发明中，多种抗原表位的其中至少之一来源于PSMA。In this article, the terms "polyantibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, referred to as bispecific antibodies), antibodies that recognize three antigenic epitopes, or antibodies that recognize four antigenic epitopes. They are understood in a broad sense, and the specific structure is not limited, as long as they can recognize multiple antigenic epitopes. In the present invention, at least one of the multiple antigenic epitopes is derived from PSMA.
在本文中，术语“单域抗体”、“纳米抗体”和“VHH抗体”可互换使用，其最初被描述为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白(可变)域(Hamers-CastermanC,AtarhouchT,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,HamersR.:“Naturallyoccurring antibodies devoid of light chains”；Nature 363,446-448(1993))，包含重链可变区(VH)和常规的CH2与CH3区，其通过重链可变区与抗原蛋白(例如PSMA)特异性结合。Herein, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably, which were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to an antigen protein (e.g., PSMA) through the heavy chain variable region.
在本文中，术语“Fab抗体”或“Fab片段”通常是指仅含Fab分子的抗体或片段，其由重链的VH和CH1以及完整的轻链构成，轻链和重链之间通过一个二硫键连接。As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and the heavy chain connected by a disulfide bond.
在本文中，术语“F(ab’)2抗体”或“F(ab’)2片段”具有通过二硫键连接在一起的两个抗原结合F(ab’)部分。As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by disulfide bonds.
在本文中，术语“Fv抗体”或“Fv片段”通常是指仅由轻链可变区(VL)和重链可变区(VH)通过非共价键连接而成的抗体或片段，是抗体分了保留完整抗原结合部位的最小功能片段。As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds, and is the smallest functional fragment of an antibody that retains a complete antigen binding site.
在本文中，术语“单链抗体”、“scFv片段”是由抗体重链可变区和轻链可变区通过短肽连接而成的抗体或片段。As used herein, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.
在本文中，术语“最小识别单位”和“MRU”均是指仅由一个CDR组成的抗体或片段，其分子量十分小仅占完全抗体的1％左右。In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, and their molecular weight is very small, accounting for only about 1% of the complete antibody.
根据本发明的实施例，所述抗体或抗原结合片段包括：如SEQ ID NO:11所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列的重链；和如SEQ ID NO:12所示的氨基酸序列或与其具有至少80％同源性的氨基酸序列的轻链。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto; and a light chain having an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto.
多特异性抗体Multispecific Antibodies
在本发明的第二方面，本发明提出了一种多特异性抗体。根据本发明的实施例，所述多特异性抗体包括：第一结合区，所述第一结合区包括第一方面所述的抗体或抗原结合片段；第二结合区，所述第二结合区具有第一分子结合活性。本发明的多特异性抗体具有高PSMA结合亲和力和肿瘤杀伤力，可有效预防和/或治疗PSMA介导的相关疾病，尤其是预防和/或治疗前列腺癌。In the second aspect of the present invention, the present invention proposes a multispecific antibody. According to an embodiment of the present invention, the multispecific antibody comprises: a first binding region, the first binding region comprises the antibody or antigen-binding fragment described in the first aspect; a second binding region, the second binding region has a first molecule binding activity. The multispecific antibody of the present invention has high PSMA binding affinity and tumor killing ability, and can effectively prevent and/or treat PSMA-mediated related diseases, especially prevent and/or treat prostate cancer.
根据本发明的实施例，所述第一分子选自免疫细胞表面抗原、肿瘤抗原、病毒、细菌、内毒素、细胞因子和细胞因子受体中的至少之一。According to an embodiment of the present invention, the first molecule is selected from at least one of immune cell surface antigens, tumor antigens, viruses, bacteria, endotoxins, cytokines and cytokine receptors.
在本文中，“免疫细胞表面抗原”应做广义理解，可以是指免疫细胞(例如T细胞、NK细胞、B细胞等)表面具有免疫原性的蛋白。包括但不限于CD3、BCMA、CTLA-4、LAG-3、TIGIT、CD38、SLAMF7、B7-H3、CD19、CD20、CD30、CD33、CD47、CD52、CD133、RANKL和CD16a等。In this article, "immune cell surface antigens" should be understood in a broad sense, and may refer to immunogenic proteins on the surface of immune cells (such as T cells, NK cells, B cells, etc.), including but not limited to CD3, BCMA, CTLA-4, LAG-3, TIGIT, CD38, SLAMF7, B7-H3, CD19, CD20, CD30, CD33, CD47, CD52, CD133, RANKL and CD16a, etc.
在本文中，“肿瘤抗原”应做广义理解，可以是指肿瘤细胞表面具有免疫原性的蛋白。包括但不限于PD-L1、PD-1、TGF-β、CEA、GD2和CD3等。In this article, "tumor antigen" should be understood in a broad sense, and may refer to proteins with immunogenicity on the surface of tumor cells, including but not limited to PD-L1, PD-1, TGF-β, CEA, GD2 and CD3.
在本文中，“细胞因子”应做广义理解，可以是指一类能在细胞间传递信息、具有免疫调节和效应功能的蛋白质或小分子多肽。包括但不限于IL-10、VEGF(包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E、VEGF-F和PIGF中的至少之一)、EpCAM、GM2和RANKL等。In this article, "cytokine" should be understood in a broad sense, and may refer to a class of proteins or small molecule polypeptides that can transmit information between cells and have immune regulation and effector functions, including but not limited to IL-10, VEGF (including at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF), EpCAM, GM2 and RANKL, etc.
在本文中，“细胞因子受体”应做广义理解，可以是指细胞表面上可与细胞因子结合的受体。包括但不限于Her2、EGFR和IL-10R等等。In this article, "cytokine receptor" should be understood in a broad sense, and may refer to receptors on the cell surface that can bind to cytokines, including but not limited to Her2, EGFR, and IL-10R, etc.
根据本发明的实施例，所述第一分子选自CD3、PD-L1、PD-1、IL-10、IL-10R、BCMA、VEGF、TGF-β、CTLA-4、LAG-3、TIGIT、CEA、CD38、SLAMF7、B7-H3、Her2、EpCAM、CD19、CD20、CD30、CD33、CD47、CD52、CD133、EGFR、GD2、CD3、GM2、RANKL和CD16a中的至少之一。According to an embodiment of the present invention, the first molecule is selected from at least one of CD3, PD-L1, PD-1, IL-10, IL-10R, BCMA, VEGF, TGF-β, CTLA-4, LAG-3, TIGIT, CEA, CD38, SLAMF7, B7-H3, Her2, EpCAM, CD19, CD20, CD30, CD33, CD47, CD52, CD133, EGFR, GD2, CD3, GM2, RANKL and CD16a.
根据本发明的实施例，所述第二结合区为所述第一分子的结合蛋白或其片段。According to an embodiment of the present invention, the second binding region is the binding protein of the first molecule or a fragment thereof.
根据本发明的实施例，所述第二结合区为所述第一分子的抗体或抗原结合片段、和所述第一分子的受体片段中的至少之一。According to an embodiment of the present invention, the second binding region is at least one of the antibody or antigen binding fragment of the first molecule, and the receptor fragment of the first molecule.
根据本发明的实施例，所述第二结合区为所述第一分子的单链抗体。According to an embodiment of the present invention, the second binding region is a single-chain antibody of the first molecule.
根据本发明的实施例，所述第二结合区进一步包括第一Fc片段。According to an embodiment of the present invention, the second binding region further includes a first Fc fragment.
根据本发明的实施例，所述第一结合区进一步包括第二Fc片段。According to an embodiment of the present invention, the first binding region further includes a second Fc fragment.
在本文中，无需特殊说明，Fc片段包括CH2、CH3区和任选地铰链区，例如第一Fc片段和/或第二Fc片段。在本发明的一个实施例中，所述CH2区的C端和所述CH3区的N端相连。在本发明的另一个实施例中，所述铰链区的C端和所述CH2区的N端，所述CH2区的C端和所述CH3区的N端相连。In this article, without special instructions, the Fc fragment includes CH2, CH3 region and optionally hinge region, such as the first Fc fragment and/or the second Fc fragment. In one embodiment of the present invention, the C-terminus of the CH2 region is connected to the N-terminus of the CH3 region. In another embodiment of the present invention, the C-terminus of the hinge region is connected to the N-terminus of the CH2 region, and the C-terminus of the CH2 region is connected to the N-terminus of the CH3 region.
根据本发明的实施例，所述第一Fc片段和第二Fc片段均为人Fc肽段。According to an embodiment of the present invention, both the first Fc fragment and the second Fc fragment are human Fc peptide segments.
根据本发明的实施例，所述人Fc肽段是人IgG1 Fc肽段。According to an embodiment of the present invention, the human Fc peptide segment is a human IgG1 Fc peptide segment.
根据本发明的实施例，所述第一Fc片段和所述第二Fc片段是通过knob-into-hole结构进行连接的。According to an embodiment of the present invention, the first Fc fragment and the second Fc fragment are connected via a knob-into-hole structure.
在本发明的一个实施例中，“knob into hole结构”为在抗体重链恒定区的CH3区形成钮(Knob)扣(hole)突变，便于重链咬合，形成异二聚体，例如，通过突变人IgG1重链恒定区CH3结构域中氨基酸(一条链中为T366S、L368A、Y407V、Y349C突变，即“hole”；另一链中为T366W、S354C突变，即“knob”)实现。In one embodiment of the present invention, the "knob into hole structure" is a knob (Knob) and a hole (hole) mutation formed in the CH3 region of the antibody heavy chain constant region to facilitate the heavy chain bite to form a heterodimer, for example, by mutating the amino acids in the CH3 domain of the human IgG1 heavy chain constant region (T366S, L368A, Y407V, Y349C mutations in one chain, i.e., the "hole"; T366W, S354C mutations in the other chain, i.e., the "knob").
根据本发明的实施例，所述第一分子的结合蛋白或其片段的C端与所述第一Fc片段的N端相连。According to an embodiment of the present invention, the C-terminus of the binding protein or fragment thereof of the first molecule is connected to the N-terminus of the first Fc fragment.
需要说明的是，当第一分子的结合蛋白或其片段为含有两条链的抗体，优选将含有重链的肽链与第一Fc片段相连。It should be noted that, when the first molecule of the binding protein or its fragment is an antibody containing two chains, it is preferred to connect the peptide chain containing the heavy chain to the first Fc fragment.
根据本发明的实施例，所述第二结合区进一步包括连接肽。According to an embodiment of the present invention, the second binding region further includes a connecting peptide.
根据本发明的实施例，所述第一分子的结合蛋白或其片段的C端与所述连接肽的N端相连，所述连接肽的C端与所述第一Fc片段的N端相连。According to an embodiment of the present invention, the C-terminus of the binding protein or fragment thereof of the first molecule is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the first Fc fragment.
根据本发明的实施例，所述连接肽具有如(GGGGS)n所示的氨基酸序列，其中n为大于或等于1的整数，优选为1、2、3、4、5、6、7、8、9或10。According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
根据本发明的实施例，所述双特异性抗体包括对称双特异性抗体或不对称双特异性抗体，优选为不对称双特异性抗体。According to an embodiment of the present invention, the bispecific antibody includes a symmetric bispecific antibody or an asymmetric bispecific antibody, preferably an asymmetric bispecific antibody.
根据本发明的实施例，所述第一分子为CD3，所述第二结合区包括抗CD3抗体。According to an embodiment of the present invention, the first molecule is CD3, and the second binding region includes an anti-CD3 antibody.
根据本发明的实施例，所述抗CD3抗体选自Fab抗体、Fab’抗体、F(ab’)2抗体、Fv抗体、单链抗体、单域抗体以及最小识别单位中的至少之一。According to an embodiment of the present invention, the anti-CD3 antibody is selected from at least one of a Fab antibody, a Fab' antibody, a F(ab')2 antibody, a Fv antibody, a single-chain antibody, a single-domain antibody and a minimum recognition unit.
根据本发明的实施例，所述抗CD3抗体为CD3单链抗体。According to an embodiment of the present invention, the anti-CD3 antibody is a CD3 single-chain antibody.
根据本发明的实施例，所述CD3单链抗体的C端与所述连接肽的N端相连，所述连接肽的C端与所述第一Fc片段的N端相连。According to an embodiment of the present invention, the C-terminus of the CD3 single-chain antibody is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the first Fc fragment.
根据本发明的实施例，所述CD3单链抗体具有如SEQ ID NO:13所示的氨基酸序列。According to an embodiment of the present invention, the CD3 single-chain antibody has an amino acid sequence as shown in SEQ ID NO:13.
根据本发明的实施例，所述连接肽具有如SEQ ID NO:14所示的氨基酸序列。According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:14.
根据本发明的实施例，所述第一Fc片段具有如SEQ ID NO:15所示的氨基酸序列。According to an embodiment of the present invention, the first Fc fragment has an amino acid sequence as shown in SEQ ID NO:15.
根据本发明的实施例，所述第一结合区具有如SEQ ID NO:17所示的氨基酸序列。According to an embodiment of the present invention, the first binding region has an amino acid sequence as shown in SEQ ID NO:17.
根据本发明的实施例，所述第一结合区中的抗体或抗原结合片段选自Fab抗体、Fab’抗体、F(ab’)2抗体、Fv抗体、单链抗体、单域抗体以及最小识别单位中的至少之一，优选为Fab抗体或单链抗体。According to an embodiment of the present invention, the antibody or antigen-binding fragment in the first binding region is selected from at least one of Fab antibody, Fab' antibody, F(ab')2 antibody, Fv antibody, single-chain antibody, single-domain antibody and minimum recognition unit, preferably Fab antibody or single-chain antibody.
根据本发明的实施例，所述第一结合区中的抗体或抗原结合片段为Fab抗体。According to an embodiment of the present invention, the antibody or antigen-binding fragment in the first binding region is a Fab antibody.
根据本发明的实施例，所述第一结合区中的Fab抗体包括第一方面所述的抗体或抗原结合片段中的CDRs。According to an embodiment of the present invention, the Fab antibody in the first binding region includes CDRs in the antibody or antigen-binding fragment described in the first aspect.
由前定义可知，Fab抗体包含两条链，即重链可变区+CH1、以及轻链可变区+CL，其中重链可变区和轻链可变区中CDRs与第一方面所述的抗体或抗原结合片段中所限定的CDR一致。As can be seen from the above definition, the Fab antibody comprises two chains, namely the heavy chain variable region+CH1, and the light chain variable region+CL, wherein the CDRs in the heavy chain variable region and the light chain variable region are consistent with the CDRs defined in the antibody or antigen-binding fragment described in the first aspect.
根据本发明的实施例，所述第一结合区中的Fab抗体包括第一方面所述的抗体或抗原结合片段中的重链可变区和轻链可变区。According to an embodiment of the present invention, the Fab antibody in the first binding region includes the heavy chain variable region and the light chain variable region in the antibody or antigen binding fragment described in the first aspect.
由前定义可知，Fab抗体包含两条链，即重链可变区+CH1、以及轻链可变区+CL(即为轻链恒定区)，其中重链可变区和轻链可变区与第一方面所述的抗体或抗原结合片段中所限定的重链可变区和轻链可变区一致。As can be seen from the previous definition, the Fab antibody comprises two chains, namely the heavy chain variable region + CH1, and the light chain variable region + CL (i.e. the light chain constant region), wherein the heavy chain variable region and the light chain variable region are consistent with the heavy chain variable region and the light chain variable region defined in the antibody or antigen-binding fragment described in the first aspect.
根据本发明的实施例，所述第一结合区中的CH1具有如SEQ ID NO:22所示的氨基酸序列。According to an embodiment of the present invention, CH1 in the first binding region has an amino acid sequence as shown in SEQ ID NO:22.
根据本发明的实施例，所述第一结合区中的Fab抗体中CH1的C端与所述第二Fc片段的N端相连。According to an embodiment of the present invention, the C-terminus of CH1 in the Fab antibody in the first binding region is connected to the N-terminus of the second Fc fragment.
根据本发明的实施例，所述第二Fc片段具有如SEQ ID NO:16所示的氨基酸序列。According to an embodiment of the present invention, the second Fc fragment has an amino acid sequence as shown in SEQ ID NO:16.
根据本发明的实施例，所述第一结合区包括：具有如SEQ ID NO:18所示氨基酸序列的第一肽链；具有如SEQ ID NO:12所示氨基酸序列的第二肽链。According to an embodiment of the present invention, the first binding region includes: a first peptide chain having an amino acid sequence as shown in SEQ ID NO:18; and a second peptide chain having an amino acid sequence as shown in SEQ ID NO:12.
根据本发明的实施例，所述双特异性抗体包括：具有如SEQ ID NO:18所示氨基酸序列的第一肽链；具有如SEQ ID NO:12所示氨基酸序列的第二肽链；以及具有如SEQ IDNO:17所示的氨基酸序列的第三肽链。According to an embodiment of the present invention, the bispecific antibody comprises: a first peptide chain having an amino acid sequence as shown in SEQ ID NO: 18; a second peptide chain having an amino acid sequence as shown in SEQ ID NO: 12; and a third peptide chain having an amino acid sequence as shown in SEQ ID NO: 17.
核酸分子、表达载体和重组细胞Nucleic acid molecules, expression vectors and recombinant cells
在本发明的第三方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体。本发明的核酸分子可编码第一方面的抗体或抗原结合片段、第二方面所述的多特异性抗体。In the third aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect, and the multispecific antibody described in the second aspect. The nucleic acid molecule of the present invention can encode the antibody or antigen-binding fragment of the first aspect, and the multispecific antibody described in the second aspect.
根据本发明的实施例，所述核酸分子为DNA。According to an embodiment of the present invention, the nucleic acid molecule is DNA.
需要说明的是，对于本文中所提及的核酸分子，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本发明中的核酸序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.
在本发明的第四方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体携带第三方面所述的核酸分子。在将上述核酸分子连接到表达载体上时，可以将所述核酸分子与表达载体上的控制元件直接或者间接相连，只要这些控制元件能够控制所述核酸分子的翻译和表达等即可。当然这些控制元件可以直接来自于表达载体本身，也可以是外源性的，即并非来自于表达载体本身。当然，所述核酸分子与控制元件进行可操作地连接即可。In the fourth aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect. When the above-mentioned nucleic acid molecule is connected to the expression vector, the nucleic acid molecule can be directly or indirectly connected to the control element on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the expression vector itself, or they can be exogenous, that is, not from the expression vector itself. Of course, the nucleic acid molecule can be operably connected to the control element.
本文中“可操作地连接”是指将外源基因连接到表达载体上，使得表达载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。常用的表达载体例如可以为质粒、噬菌体等等。Herein, "operably linked" means connecting the foreign gene to the expression vector so that the control elements in the expression vector, such as transcription control sequences and translation control sequences, can play their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors can be, for example, plasmids, bacteriophages, and the like.
根据本发明的一些具体实施例，所述表达载体导入合适的受体细胞后，可在调控系统的介导下，有效实现前述的抗体或抗原结合片段、前述的重组蛋白或者前述的多特异性抗体的表达，进而实现抗体或抗原结合片段、重组蛋白或者多特异性抗体的体外大量获得。According to some specific embodiments of the present invention, after the expression vector is introduced into a suitable recipient cell, it can effectively achieve the expression of the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein or the aforementioned multi-specific antibody under the mediation of the regulatory system, thereby achieving large-scale in vitro acquisition of the antibody or antigen-binding fragment, recombinant protein or multi-specific antibody.
根据本发明的实施例，所述表达载体包括选自真核表达载体或原核表达载体。According to an embodiment of the present invention, the expression vector comprises a vector selected from a eukaryotic expression vector or a prokaryotic expression vector.
在本发明的一个可选实施例中，所述表达载体为质粒表达载体、病毒表达载体，例如慢病毒表达载体。In an optional embodiment of the present invention, the expression vector is a plasmid expression vector, a viral expression vector, such as a lentiviral expression vector.
在本发明的第五方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞包括携带第三方面所述的核酸分子或第四方面所述的表达载体；或表达第一方面所述的抗体或抗原结合片段、或者第二方面所述的多特异性抗体。利用该重组细胞在适合条件下，能够在细胞内有效地表达前述的抗体或抗原结合片段或者多特异性抗体。In the fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell includes carrying the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect; or expressing the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect. The recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment or multispecific antibody in the cell under suitable conditions.
根据本发明的一些具体实施例，所述重组细胞在合适条件下可高效并大量表达抗体或其抗原结合片段，所述抗体或其抗原结合片段具有更强的特异性、更长的半衰期和更高的效力，能够在较少的用药剂量下，将抗体药物递送到靶细胞，实现PSMA介导的疾病的有效治疗或预防，其毒副作用低，具有更高的安全性。According to some specific embodiments of the present invention, the recombinant cells can efficiently and massively express antibodies or antigen-binding fragments thereof under appropriate conditions. The antibodies or antigen-binding fragments thereof have stronger specificity, longer half-life and higher efficacy, and can deliver antibody drugs to target cells at a lower dosage, thereby achieving effective treatment or prevention of PSMA-mediated diseases, having low toxic side effects and higher safety.
需要说明的是，“适合条件”，是指适合本发明所述抗体或抗原结合片段、重组蛋白或者多特异性抗体达的条件。本领域技术人员容易理解的是，适合所述抗体或抗原结合片段、重组蛋白或者多特异性抗体表达的条件包括但不限于合适的转化或转染方式、合适的转化或转染条件、健康的宿主细胞状态、合适的宿主细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适合条件”不受特别限制，本领域技术人员可根据实验室的具体环境，优化最适的上述抗体或抗原结合片段、重组蛋白或者多特异性抗体表达的条件。It should be noted that "suitable conditions" refer to conditions suitable for the expression of the antibody or antigen-binding fragment, recombinant protein or multi-specific antibody of the present invention. It is easy for those skilled in the art to understand that conditions suitable for the expression of the antibody or antigen-binding fragment, recombinant protein or multi-specific antibody include but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the above-mentioned antibodies or antigen-binding fragments, recombinant proteins or multi-specific antibodies according to the specific environment of the laboratory.
根据本发明的实施例，所述重组细胞是通过将第四方面所述的表达载体引入至宿主细胞中而获得的。According to an embodiment of the present invention, the recombinant cell is obtained by introducing the expression vector described in the fourth aspect into a host cell.
需要注意的是，本发明所述重组细胞不受特别限制，可以为原核细胞、真核细胞或噬菌体。所述原核细胞可以为大肠杆菌、枯草杆菌、链霉菌或奇异变形菌等。所述真核细胞包括巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉等真菌，草地粘虫等昆虫细胞，烟草等植物细胞，BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞等哺乳动物细胞。在一些实施例中，本发明所述重组细胞优选为哺乳动物细胞，包括BHK细胞、CHO细胞、NSO细胞或COS细胞，且不包括动物生殖细胞、受精卵或胚胎干细胞。It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.
根据本发明的实施例，所述重组细胞为真核细胞，优选为哺乳动物细胞。According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell, preferably a mammalian cell.
偶联物、药物组合物和试剂盒Conjugates, pharmaceutical compositions and kits
在本发明的第六方面，本发明提出了一种偶联物。根据本发明的实施例，所述偶联物包含第一方面所述的抗体或抗原结合片段、或者第二方面所述的多特异性抗体；以及偶联部分，所述偶联部分与所述抗体或抗原结合片段或多特异性抗体相连。本发明的偶联物具有高PSMA结合亲和力，可用于检测PSMA、或者用于预防和/或治疗PSMA介导的相关疾病，尤其是用于检测前列腺癌、或预防和/或治疗前列腺癌。In the sixth aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect; and a coupling portion, wherein the coupling portion is connected to the antibody or antigen-binding fragment or multispecific antibody. The conjugate of the present invention has a high PSMA binding affinity and can be used to detect PSMA, or to prevent and/or treat PSMA-mediated related diseases, especially to detect prostate cancer, or to prevent and/or treat prostate cancer.
根据本发明的实施例，所述偶联部分包括选自为载体、药物、毒素、细胞因子、蛋白标签和修饰物中的至少之一。According to an embodiment of the present invention, the coupling part includes at least one selected from a carrier, a drug, a toxin, a cytokine, a protein tag and a modifier.
在本文中，所述载体可以为能够在液相中悬浊或分散的物质(例如，粒子、磁珠等固相载体)，或者为能够收容或搭载液相的固相(例如，板、膜、试管等支持体，以及孔板、微流路、玻璃毛细管、纳米柱、整体柱等的容器)；也可以为用于对抗体或抗原结合片段、重组蛋白或多特异性抗体进行标记的标记载体，例如酶(例如，过氧化物酶、碱性磷酸酶、虫萤光素酶(luciferin)、β-半乳糖苷酶)、亲和性物质(例如，链霉亲和素和生物素中的一者，相互互补的正义链和反义链的核酸中的一者)、荧光物质(例如，荧光素、异硫氰酸荧光素、罗丹明、绿色荧光蛋白质、红色荧光蛋白质)、发光物质(例如，虫萤光素、水母发光蛋白(Aequorin)、吖啶酯、三(2,2'-联吡啶)钌、鲁米诺)、放射性同位素(例如，3H、14C、32P、35S、125I)以及金胶体等。Herein, the carrier can be a substance that can be suspended or dispersed in a liquid phase (for example, solid phase carriers such as particles and magnetic beads), or a solid phase that can contain or carry a liquid phase (for example, supports such as plates, membranes, test tubes, and containers such as well plates, microfluidics, glass capillaries, nanocolumns, and monolithic columns); it can also be a labeling carrier for labeling antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies, such as enzymes (for example, peroxidase, alkaline phosphatase, luciferin, β-galactosidase), affinity substances (for example, one of streptavidin and biotin, one of mutually complementary positive and antisense nucleic acids), fluorescent substances (for example, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (for example, luciferin, aequorin, acridinium esters, tris(2,2'-bipyridine)ruthenium, luminol), radioactive isotopes (for example,3 H,14 C,32 P,35 S,125 I) and gold colloid, etc.
根据本发明的实施例，所述药物为可与抗体或抗原结合片段、重组蛋白或多特异性抗体结合的小分子药物。According to an embodiment of the present invention, the drug is a small molecule drug that can bind to an antibody or antigen-binding fragment, a recombinant protein or a multi-specific antibody.
根据本发明的实施例，所述蛋白标签包括但不限His标签、Flag标签、GST标签、MBP标签、SUMO标签和C-Myc标签等。According to an embodiment of the present invention, the protein tag includes but is not limited to a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, a C-Myc tag, and the like.
根据本发明的实施例，所述修饰物应作广义理解，可以指用于修饰蛋白的物质。示例性地，可以为聚乙二醇或其衍生物。According to an embodiment of the present invention, the modifier should be understood in a broad sense, and may refer to a substance used to modify a protein. For example, it may be polyethylene glycol or a derivative thereof.
需要说明的是，偶联部分和抗体或抗原结合片段、重组蛋白或多特异性抗体的结合方法，可以使用本领域公知的方法。例如，可以举出物理吸附法、共价结合法、利用亲和性物质(例如，生物素、链霉亲和素)的方法及离子结合法。It should be noted that the binding method of the coupling part and the antibody or antigen-binding fragment, recombinant protein or multispecific antibody can be a method known in the art. For example, physical adsorption, covalent binding, methods using affinity substances (e.g., biotin, streptavidin) and ion binding methods can be mentioned.
在本发明的第七方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体、第三方面所述的核酸分子、第四方面所述的表达载体、第五方面所述的重组细胞或第六方面所述的偶联物。本发明的药物组合物具有高PSMA结合亲和力和肿瘤杀伤力，可有效预防和/或治疗PSMA介导的相关疾病，尤其是用于预防和/或治疗前列腺癌。In the seventh aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the conjugate described in the sixth aspect. The pharmaceutical composition of the present invention has high PSMA binding affinity and tumor killing ability, and can effectively prevent and/or treat PSMA-mediated related diseases, especially for preventing and/or treating prostate cancer.
根据本发明的实施例，所述药物组合物进一步包括药学上可接受的辅料。According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
本发明药物组合物的施用可以通过任何可接受施用方式进行。本发明的药物组合物可以配制成固体、半固体、液体或气体形式的制剂，例如注射剂、冻干粉剂，制备这些剂型的现行的方法是已知的，或者对于本领域技术人员是显而易见的。施用这类药物组合物的典型途径，包括但不限于皮下注射，静脉内、肌内、皮内、胸骨内注射或输注技术。配制本发明的药物组合物以便允许经过对患者施用该组合物后其中含有的生物活性成分是生物可利用的。The administration of the pharmaceutical composition of the present invention can be carried out by any acceptable mode of administration. The pharmaceutical composition of the present invention can be formulated into a solid, semisolid, liquid or gaseous form of preparation, such as an injection, a lyophilized powder, and the current methods for preparing these dosage forms are known or obvious to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical composition of the present invention is formulated so as to allow the biologically active ingredients contained therein to be bioavailable after the composition is administered to a patient.
在本发明的第八方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体、第三方面所述的核酸分子、第四方面所述的表达载体、第五方面所述的重组细胞或第六方面所述的偶联物。本发明的试剂盒具有高PSMA结合亲和力，可有效检测PSMA，可进一步诊断肿瘤或癌症(尤其是前列腺癌)。In the eighth aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit comprises the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the conjugate described in the sixth aspect. The kit of the present invention has high PSMA binding affinity, can effectively detect PSMA, and can further diagnose tumors or cancers (especially prostate cancer).
如前所述，本发明一些具体实施方式的抗体或抗原结合片段能够有效与人PSMA蛋白进行结合，因此，包含所述抗体或抗原结合片段的试剂盒能够有效的对人PSMA蛋白进行定性或定量检测。本发明提供的试剂盒，例如可以用于免疫印迹、免疫沉淀等涉及到利用人PSMA和抗体特异性结合性来检测的试剂盒等。这些试剂盒可包含下列中的任意一种或多种：拮抗剂、抗PSMA抗体或者药物参照材料；蛋白纯化柱；免疫球蛋白亲和纯化缓冲剂；细胞的测定稀释剂；说明书或者文献等。抗PSMA抗体可被用于不同类型的诊断测试，例如可以在体外或者体内检测各种各样的疾病或者药物、毒素或者其他蛋白等的存在，例如可以通过对受试者的血清或者血液进行检测，用来测试相关疾病，也可以进行科研研究，利用所述试剂盒检测待测样品中的人PSMA蛋白。这种相关疾病可包括PSMA相关疾病，例如癌症。当然本文提供的抗体或抗原结合片段也可以用于上述疾病的放射免疫检测和放射免疫治疗等等。针对于上述应用场景，所述结合分子同样适用，此处不再累述。As mentioned above, the antibodies or antigen-binding fragments of some specific embodiments of the present invention can effectively bind to human PSMA protein, so the kit containing the antibody or antigen-binding fragment can effectively detect human PSMA protein qualitatively or quantitatively. The kit provided by the present invention can be used, for example, for immunoblotting, immunoprecipitation, etc., which involve the use of human PSMA and antibody specific binding to detect the kit. These kits may contain any one or more of the following: antagonists, anti-PSMA antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-PSMA antibodies can be used for different types of diagnostic tests, such as in vitro or in vivo detection of various diseases or the presence of drugs, toxins or other proteins, for example, by testing the serum or blood of the subject to test related diseases, and can also be used for scientific research, using the kit to detect human PSMA protein in the sample to be tested. Such related diseases may include PSMA-related diseases, such as cancer. Of course, the antibodies or antigen-binding fragments provided herein can also be used for radioimmunoassay and radioimmunotherapy of the above-mentioned diseases, etc. For the above-mentioned application scenarios, the binding molecules are also applicable and will not be repeated here.
根据本发明的一些具体实施例，所述试剂盒还可以包括常规用于检测PSMA，如包被液等。According to some specific embodiments of the present invention, the kit may further include conventional materials for detecting PSMA, such as coating fluid, etc.
用途use
在本发明的第九方面，本发明提出了一种第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体、第三方面所述的核酸分子、第四方面所述的表达载体、第五方面所述的重组细胞、第六方面所述的偶联物或第七方面所述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗PSMA介导的相关疾病，尤其是预防和/或治疗前列腺癌。In the ninth aspect of the present invention, the present invention proposes a use of the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect, the conjugate of the sixth aspect or the pharmaceutical composition of the seventh aspect in the preparation of a drug, wherein the drug is used for preventing and/or treating PSMA-mediated related diseases, especially preventing and/or treating prostate cancer.
根据本发明的实施例，所述PSMA介导的相关疾病包括肿瘤或癌症According to an embodiment of the present invention, the PSMA-mediated related diseases include tumors or cancers.
根据本发明的实施例，所述癌症为前列腺癌、肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌和头颈癌中的至少一种。According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.
根据本发明的实施例，所述癌症为前列腺癌。According to an embodiment of the present invention, the cancer is prostate cancer.
在本发明的第十方面，本发明提出了一种第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体、第三方面所述的核酸分子、第四方面所述的表达载体、第五方面所述的重组细胞或第六方面所述的偶联物在制备试剂盒中的用途，所述试剂盒用于检测PSMA、检测PSMA介导的相关疾病、诊断PSMA介导的相关疾病、对PSMA介导的相关疾病进行分期、或者评估PSMA介导的相关疾病预后。In the tenth aspect of the present invention, the present invention proposes a use of the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect or the conjugate of the sixth aspect in preparing a kit, wherein the kit is used to detect PSMA, detect PSMA-mediated related diseases, diagnose PSMA-mediated related diseases, stage PSMA-mediated related diseases, or evaluate the prognosis of PSMA-mediated related diseases.
根据本发明的实施例，所述PSMA介导的相关疾病包括肿瘤和/或癌症。According to an embodiment of the present invention, the PSMA-mediated related diseases include tumors and/or cancers.
根据本发明的实施例，所述癌症为前列腺癌、肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌和头颈癌中的至少一种。According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.
根据本发明的实施例，所述癌症为前列腺癌。According to an embodiment of the present invention, the cancer is prostate cancer.
方法method
在本发明的第十二方面，本发明提出了一种检测PSMA的方法。根据本发明的实施例，所述方法包括：采用第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体或第六方面所述的偶联物与待检测样本进行接触，形成免疫复合物。本发明的方法具有检测PSMA，还可进一步检测前列腺癌，尤其是用于体外检测，具有检测准确性高等优点。In the twelfth aspect of the present invention, the present invention proposes a method for detecting PSMA. According to an embodiment of the present invention, the method comprises: contacting the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, or the conjugate described in the sixth aspect with the sample to be detected to form an immune complex. The method of the present invention has the advantages of detecting PSMA and further detecting prostate cancer, especially for in vitro detection, and has the advantages of high detection accuracy.
根据本发明的实施例，基于所述免疫复合物的信号，确定所述待检测样本中是否含有新型冠状病毒的含量。According to an embodiment of the present invention, based on the signal of the immune complex, it is determined whether the sample to be tested contains the content of the new coronavirus.
根据本发明的实施例，所述免疫复合物还包括第二抗体，所述第二抗体与所述抗体或抗原结合片段结合。According to an embodiment of the present invention, the immune complex further comprises a second antibody, which binds to the antibody or antigen-binding fragment.
根据本发明的实施例，所述免疫复合物还包括第二抗体，所述第二抗体与新型冠状病毒结合。According to an embodiment of the present invention, the immune complex also includes a second antibody, which binds to the new coronavirus.
在本发明的第十三方面，本发明提出了一种预防和/或治疗癌症的方法。根据本发明的实施例，所述方法包括：向受试者施用药学上可接受量的第一方面所述的抗体或抗原结合片段、第二方面所述的多特异性抗体或第六方面所述的偶联物。本发明的方法可有效治疗或预防肿瘤和/或癌症，尤其是预防和/或治疗前列腺癌。In the thirteenth aspect of the present invention, the present invention provides a method for preventing and/or treating cancer. According to an embodiment of the present invention, the method comprises: administering to a subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, or the conjugate of the sixth aspect. The method of the present invention can effectively treat or prevent tumors and/or cancer, especially prevent and/or treat prostate cancer.
本发明所述的抗体或抗原结合片段、多特异性抗体、药物组合物或偶联物的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the antibody or antigen-binding fragment, multispecific antibody, pharmaceutical composition or conjugate of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.
本发明的抗体或抗原结合片段、多特异性抗体、药物组合物或偶联物可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物中。这些药物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)或冻干粉。典型的药物为注射溶液或输注溶液形式。前述抗体或抗原结合片段、多特异性抗体、药物组合物或偶联物可通过静脉输注或注射或肌肉内或皮下注射来施用。The antibodies or antigen-binding fragments, multispecific antibodies, pharmaceutical compositions or conjugates of the present invention can be incorporated into drugs suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injection solutions or infusion solutions. The aforementioned antibodies or antigen-binding fragments, multispecific antibodies, pharmaceutical compositions or conjugates can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
在本文中，术语“受试者”是指脊椎动物，优选地是哺乳动物，最优选地是人类。哺乳动物包括但不限于鼠类、猿类、人类、家畜、竞技动物和宠物。在体内获得的或在体外培养的生物实体的组织、细胞及其子代也包括在内。As used herein, the term "subject" refers to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, competitive animals, and pets. Tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro are also included.
根据本发明的实施例，所述方法的给药途径采用皮下注射或静脉注射。According to an embodiment of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.
根据本发明的实施例，所述癌症为前列腺癌、肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌和头颈癌中的至少一种。According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.
在本发明的第十四方面，本发明提出了一种诊断PSMA介导的相关疾病的方法。根据本发明的实施例，所述方法包括：采用第一方面所述的抗体或抗原结合片段、第三方面所述的核酸分子、第四方面所述的表达载体或第五方面所述的重组细胞对待测样品中的PSMA进行检测；基于所述PSMA的检测结果，确定所述待测样品中PSMA的含量。本发明提出的所述抗体或抗原结合片段，或核酸分子、表达载体、重组细胞表达的抗体或抗原结合片段，均可以有效与人PSMA蛋白进行结合，因此，采用本发明的方法可以有效检测来源于受试个体的待测样品中PSMA的含量，并可以对PSMA引起的相关疾病进行有效诊断。In the fourteenth aspect of the present invention, the present invention proposes a method for diagnosing PSMA-mediated related diseases. According to an embodiment of the present invention, the method comprises: using the antibody or antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell described in the fifth aspect to detect PSMA in the sample to be tested; based on the detection result of the PSMA, determining the content of PSMA in the sample to be tested. The antibody or antigen-binding fragment, or the nucleic acid molecule, expression vector, or antibody or antigen-binding fragment expressed by the recombinant cell proposed in the present invention can effectively bind to the human PSMA protein. Therefore, the method of the present invention can effectively detect the content of PSMA in the sample to be tested from the subject, and can effectively diagnose the related diseases caused by PSMA.
根据本发明的实施例，上述诊断疾病的方法还可以进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above method for diagnosing a disease may further include at least one of the following additional technical features:
根据本发明的实施例，所述待测样品中PSMA的含量不低于患病的最低标准是待测样品来源于患有PSMA引起的相关疾病的患者的指示。所述最低标准的值可以通过对大量患有所述PSMA引起的相关疾病的个体和大量健康个体的待测样本中的PSMA的含量进行差异比较分析、以及验证，而确定下来。According to an embodiment of the present invention, the PSMA content in the test sample is not less than the minimum standard for disease, which indicates that the test sample is derived from a patient suffering from a PSMA-related disease. The value of the minimum standard can be determined by performing differential analysis and verification on the PSMA content in the test samples of a large number of individuals suffering from the PSMA-related disease and a large number of healthy individuals.
根据本发明的实施例，所述待测样品包括下列中的至少之一：血液、唾液、汗液、组织、细胞、血液、血清、血浆、粪便和尿液。According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.
根据本发明的实施例，所述PSMA介导的相关疾病包括癌症。According to an embodiment of the present invention, the PSMA-mediated related diseases include cancer.
根据本发明的实施例，所述癌症为前列腺癌、肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌和头颈癌中的至少一种。According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.
在本发明的第十五方面，本发明提出了一种对PSMA介导的相关疾病进行分期的方法。根据本发明的实施例，所述方法包括：采用第一方面所述的抗体或抗原结合片段、第三方面所述的核酸分子、第四方面所述的表达载体或第五方面所述的重组细胞对待测样品中的PSMA进行检测；基于所述PSMA的检测结果，确定所述待测样品中PSMA的含量。本发明提出的所述抗体或抗原结合片段，或核酸分子、表达载体、重组细胞表达的抗体或抗原结合片段，均可以有效与人PSMA蛋白进行结合，因此，采用发明的方法可以有效检测来源于受试个体的待测样品中PSMA的含量，并基于所述PSMA的含量对PSMA引起的相关疾病所处的时期进行评估。In the fifteenth aspect of the present invention, the present invention proposes a method for staging PSMA-mediated related diseases. According to an embodiment of the present invention, the method comprises: using the antibody or antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell described in the fifth aspect to detect PSMA in the sample to be tested; based on the detection result of the PSMA, determining the content of PSMA in the sample to be tested. The antibody or antigen-binding fragment, or the nucleic acid molecule, expression vector, or antibody or antigen-binding fragment expressed by the recombinant cell proposed in the present invention can effectively bind to the human PSMA protein. Therefore, the method of the invention can effectively detect the content of PSMA in the sample to be tested from the subject, and evaluate the stage of the related disease caused by PSMA based on the content of PSMA.
根据本发明的实施例，上述对疾病进行分期的方法还可以进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above method for staging a disease may further include at least one of the following additional technical features:
根据本发明的实施例，所述待测样品中PSMA的含量不低于肿瘤IV期患病的标准水平是待测样品来源于患有肿瘤IV期的患者的指示，所述待测样品中PSMA的含量位于肿瘤IV期和III期患病的标准水平之间是待测样品来源于患有肿瘤III期的患者的指示；所述待测样品中PSMA的含量处于肿瘤III期和II期患病的标准水平之间是待测样品来源于患有肿瘤II期的患者的指示；所述待测样品中PSMA的含量处于I期和II期患病的标准水平之间是待测样品来源于患有肿瘤I期的患者的指示。本领域技术人员可以理解，所述的肿瘤I期、II期、III期、IV期患病时PSMA的水平根据肿瘤种类的不同而变化，判断肿瘤所述时期，只要将所述待测样品中PSMA的含量与对应的该肿瘤阶段PSMA的标准水平进行对比即可知晓，或将所述待测样品中PSMA的含量与已知患病时期的个体或群体来源的样品PSMA的含量进行对比。所述肿瘤I期、II期、III期、IV期标准水平的值可以通过对大量所述患有PSMA引起的相关疾病的个体和大量健康个体的待测样本中的PSMA的含量的差异进行比较分析、以及验证，而确定下来。According to an embodiment of the present invention, the content of PSMA in the sample to be tested is not less than the standard level of tumor stage IV, which indicates that the sample to be tested is derived from a patient with tumor stage IV; the content of PSMA in the sample to be tested is between the standard levels of tumor stage IV and stage III, which indicates that the sample to be tested is derived from a patient with tumor stage III; the content of PSMA in the sample to be tested is between the standard levels of tumor stage III and stage II, which indicates that the sample to be tested is derived from a patient with tumor stage II; the content of PSMA in the sample to be tested is between the standard levels of stage I and stage II, which indicates that the sample to be tested is derived from a patient with tumor stage I. Those skilled in the art can understand that the level of PSMA in the tumor stage I, stage II, stage III, and stage IV varies according to the type of tumor, and the stage of the tumor can be determined by comparing the content of PSMA in the sample to be tested with the standard level of PSMA in the corresponding tumor stage, or comparing the content of PSMA in the sample to be tested with the content of PSMA in samples from individuals or groups with known disease stages. The values of the standard levels of tumor stages I, II, III, and IV can be determined by comparative analysis and verification of the differences in PSMA content in test samples from a large number of individuals suffering from PSMA-related diseases and a large number of healthy individuals.
根据本发明的实施例，所述待测样品包括下列中的至少之一：血液、唾液、汗液、组织、细胞、血液、血清、血浆、粪便和尿液。According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.
根据本发明的实施例，所述PSMA介导的相关疾病包括癌症。According to an embodiment of the present invention, the PSMA-mediated related diseases include cancer.
根据本发明的实施例，所述癌症为前列腺癌、肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌和头颈癌中的至少一种。According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.
在本发明的第十五方面，本发明提出了一种评估PSMA介导的相关疾病预后的方法。根据本发明的实施例，所述方法包括：采用第一方面所述的抗体或抗原结合片段、第三方面所述的核酸分子、第四方面所述的表达载体或第五方面所述的重组细胞对待测样品中的PSMA进行检测；基于所述PSMA的检测结果，确定所述待测样品中PSMA的含量。如前所述，PSMA的含量对癌症具有重要影响，患有相关疾病个体进行治疗后，通过监测其组织或排泄物，如外周血、尿液等中PSMA的含量可以有效评估该类疾病的预后，例如，将治疗前后的受试者体内PSMA的含量进行比较，或将治疗后的受试者体内PSMA的含量与正常个体或患病个体的PSMA水平进行比较等方式，本发明第一方面的抗体或抗原结合片段，或核酸分子、表达载体、重组细胞表达的抗体或抗原结合片段，均可以有效与人PSMA进行结合，因此，采用发明的方法可以有效检测来源于受试个体的待测样品中PSMA的含量，并基于所述PSMA的含量对PSMA引起的相关疾病的预后进行评估。In the fifteenth aspect of the present invention, the present invention provides a method for evaluating the prognosis of PSMA-mediated related diseases. According to an embodiment of the present invention, the method comprises: using the antibody or antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell described in the fifth aspect to detect PSMA in the sample to be tested; based on the detection result of PSMA, determining the content of PSMA in the sample to be tested. As mentioned above, the content of PSMA has an important influence on cancer. After treatment of individuals with related diseases, the prognosis of such diseases can be effectively evaluated by monitoring the content of PSMA in their tissues or excreta, such as peripheral blood, urine, etc. For example, the content of PSMA in the subject before and after treatment is compared, or the content of PSMA in the subject after treatment is compared with the PSMA level of a normal individual or a diseased individual. The antibody or antigen-binding fragment of the first aspect of the present invention, or the nucleic acid molecule, expression vector, or antibody or antigen-binding fragment expressed by a recombinant cell can effectively bind to human PSMA. Therefore, the method of the invention can effectively detect the content of PSMA in a test sample derived from a test individual, and evaluate the prognosis of a related disease caused by PSMA based on the content of PSMA.
根据本发明的实施例，上述评估疾病预后的方法还可以进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above method for evaluating disease prognosis may further include at least one of the following additional technical features:
根据本发明的实施例，所述待测样品来源于治疗前或治疗后的患有PSMA介导的相关疾病的患者。According to an embodiment of the present invention, the sample to be tested is derived from a patient suffering from a PSMA-mediated related disease before or after treatment.
根据本发明的实施例，所述待测样品包括下列中的至少之一：血液、唾液、汗液、组织、细胞、血液、血清、血浆、粪便和尿液。According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.
根据本发明的实施例，基于所述治疗前或治疗后的患有PSMA介导的相关疾病的患者的待测样品中的PSMA的含量，确定PSMA介导的相关疾病的预后效果。According to an embodiment of the present invention, the prognostic effect of a PSMA-mediated related disease is determined based on the PSMA content in the test sample of a patient suffering from a PSMA-mediated related disease before or after the treatment.
根据本发明的实施例，所述PSMA介导的相关疾病包括癌症。According to an embodiment of the present invention, the PSMA-mediated related diseases include cancer.
根据本发明的实施例，所述癌症为前列腺癌、肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌和头颈癌中的至少一种。According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.
本文中涉及的氨基酸序列如表B所示：The amino acid sequences involved in this article are shown in Table B:
表B：氨基酸序列Table B: Amino Acid Sequences
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.
除非另外指明，否则实践本公开将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术，所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术，如《分子克隆：实验室手册(Molecular Cloning:ALaboratory Manual)》，第二版(Sambrook等人，1989)；《寡核苷酸合成(OligonucleotideSynthesis)》(M.J.Gait编，1984)；《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编，1987)；《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press，Inc.)；《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编)；《哺乳动物细胞用基因转移载体(Gene Transfer Vectors forMammalian Cells)》(J.M.Miller和M.P.Calos编，1987)；《当代分子生物学方法(CurrentProtocols in Molecular Biology)》(F.M.Ausubel等人编，1987)；《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编，1994)；以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编，2011)，所述文献中的每个文献均通过引用明确并入本文中。Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (Current Protocols in Molecular Biology, 1996). "PCR: The Polymerase Chain Reaction" (Mullis et al., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., 2011), each of which is expressly incorporated herein by reference.
在本发明实施例中，制备表达载体用到的核苷酸序列可根据其氨基酸序列采用常规方法或常规的软件(例如在线程序Vectorbuilder(网址：https://www.vectorbuilder.cn/tool/codon-optimization.html)、GeneOptimizer在线程序等)得到。In the embodiments of the present invention, the nucleotide sequence used to prepare the expression vector can be obtained according to its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder (website: https://www.vectorbuilder.cn/tool/codon-optimization.html), GeneOptimizer online program, etc.).
实施例1：抗体的制备Example 1: Preparation of antibodies
抗体的制备步骤如下：The steps for preparing the antibody are as follows:
(1)利用ExpiCHO Expression Medium培养基(购自Thermo Fisher)培养ExpiCHO细胞(购自Thermo Fisher)，调整细胞浓度为6×106/mL，以获得ExpiCHO细胞溶液。(1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×106 /mL to obtain an ExpiCHO cell solution.
(2)当抗体为单克隆抗体时，将含有编码抗体重链和抗体轻链的核苷酸序列的pcDNA3.4载体(委托南京金斯瑞合成)按照1：1的比例加入2mL OptiSFM培养基(购自ThermoFisher)中，获得溶液A；(2) When the antibody is a monoclonal antibody, a pcDNA3.4 vector (commissioned by Nanjing GenScript for synthesis) containing nucleotide sequences encoding the antibody heavy chain and the antibody light chain was added to 2 mL of OptiSFM medium (purchased from ThermoFisher) at a ratio of 1:1 to obtain solution A;
当抗体为双特异抗体时，将含有编码CD3抗体、PSMA抗体重链和PSMA抗体轻链的核苷酸序列的pcDNA3.4载体(委托南京金斯瑞合成)按照1：1：1的比例加入2mL OptiSFM培养基(购自Thermo Fisher)中，获得溶液A。When the antibody is a bispecific antibody, a pcDNA3.4 vector (commissioned to Nanjing GenScript for synthesis) containing nucleotide sequences encoding CD3 antibody, PSMA antibody heavy chain and PSMA antibody light chain was added to 2 mL of OptiSFM culture medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution A.
(3)将160μL ExpiFectamineCHO转染试剂(购自Thermo Fisher)加入2mL OptiSFM培养基(购自Thermo Fisher)中，获得溶液B。(3) Add 160 μL of ExpiFectamine CHO transfection reagent (purchased from Thermo Fisher) to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain solution B.
(4)然后将溶液A和溶液B混合，获得转染混合物，并在5分钟内将转染混合物全部加入50mL ExpiCHO细胞溶液中。(4) Solution A and Solution B were then mixed to obtain a transfection mixture, and the entire transfection mixture was added to 50 mL of the ExpiCHO cell solution within 5 minutes.
(5)在37℃、5％ CO2条件下培养1天后，加入8mL Feed、300μL Enhancer(购自Thermo Fisher)，并转入32℃、5％ CO2条件下培养9天后收获培养上清，其中第5天添加8mLFeed。(5) After culturing for 1 day at 37°C and 5%CO2 , 8 mL of Feed and 300 μL of Enhancer (purchased from Thermo Fisher) were added, and the cells were transferred to 32°C and 5% CO2 for 9days . The culture supernatant was harvested, with 8 mL of Feed added on the 5th day.
(6)利用Protein A纯化柱(购自纳微)从培养上清中亲和纯化，获得目的抗体。(6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from Nano-Micro).
实施例2:抗体亲和力成熟Example 2: Antibody Affinity Maturation
天然抗体在亲和力成熟过程中，体细胞高频突变主要集中在CDR区。通过体外实验在CDR区每个位点上进行单点饱和突变，获得足够的突变多样性，同时不会破坏蛋白质结构，该途径可以实现与天然抗体在体内的体细胞高频突变最相似的体外重现。During the affinity maturation process of natural antibodies, somatic high-frequency mutations are mainly concentrated in the CDR region. Through in vitro experiments, single-point saturation mutations are performed at each site in the CDR region to obtain sufficient mutation diversity without destroying the protein structure. This approach can achieve in vitro reproduction that is most similar to the somatic high-frequency mutations of natural antibodies in vivo.
对CDR区的每一个氨基酸位点进行单点饱和突变，构建出母本抗体的无偏差单点饱和突变质粒库。利用ELISA筛选出与抗原特异性结合增强的突变位点，再将这些位点进行组合筛选得到候选的抗体突变序列。Single-point saturation mutations were performed on each amino acid site in the CDR region to construct an unbiased single-point saturation mutation plasmid library of the parent antibody. Mutation sites that enhance antigen-specific binding were screened using ELISA, and these sites were combined to obtain candidate antibody mutation sequences.
通过该技术路线对母本PSMA抗体parental mAb(通过实施例1所述方法制备得到，重链可变区SEQ ID NO:23和轻链可变区SEQ ID NO:24，重链的氨基酸序列如SEQ ID NO:19所示、轻链的氨基酸序列如SEQ ID NO:20所示)进行亲和力优化，得到解离速度更慢的PSMA单抗PSMAvll(通过实施例1所述方法制备得到，重链的氨基酸序列如SEQ ID NO:11所示、轻链的氨基酸序列如SEQ ID NO:12所示)，其具有重链可变区(氨基酸序列如SEQ ID NO:7所示)和轻链可变区(氨基酸序列如SEQ ID NO:8所示)的序列。母本抗体parental mAb和亲和力成熟抗体PSMAvll的CDR序列的比较如图1所示。The parental PSMA antibody parental mAb (prepared by the method described in Example 1, heavy chain variable region SEQ ID NO: 23 and light chain variable region SEQ ID NO: 24, heavy chain amino acid sequence as shown in SEQ ID NO: 19, light chain amino acid sequence as shown in SEQ ID NO: 20) was affinity optimized to obtain a PSMA monoclonal antibody PSMAvll (prepared by the method described in Example 1, heavy chain amino acid sequence as shown in SEQ ID NO: 11, light chain amino acid sequence as shown in SEQ ID NO: 12) with a slower dissociation rate, which has a heavy chain variable region (amino acid sequence as shown in SEQ ID NO: 7) and a light chain variable region (amino acid sequence as shown in SEQ ID NO: 8). The comparison of the CDR sequences of the parental antibody parental mAb and the affinity matured antibody PSMAvll is shown in Figure 1.
进一步，采用本实施例制备的parental mAb抗体和PSMAvll抗体进行下述实施例3～实施例6：Furthermore, the parental mAb antibody and PSMAvll antibody prepared in this example were used to carry out the following examples 3 to 6:
实施例3:抗体亲和力检测Example 3: Antibody Affinity Detection
Biacore是一种基于光学表面等离子共振(SPR)原理的分析生物分子相互作用的方法，不仅可以检测抗原和抗体之间的特异性结合，还可以获得分子间的结合速率常数(Ka)、解离速率常数(Kd)、平衡解离常数(KD)等在药物研发中非常重要的数据，从而计算得到抗体的亲和力。Biacore is a method for analyzing biomolecular interactions based on the principle of optical surface plasmon resonance (SPR). It can not only detect the specific binding between antigens and antibodies, but also obtain the intermolecular binding rate constant (Ka), dissociation rate constant (Kd), equilibrium dissociation constant (KD) and other very important data in drug development, thereby calculating the affinity of the antibody.
在Biacore 1K((Cytiva)系统中，用运行缓冲液(HBS-EP)将抗体稀释至10μg/mL，在流速10μL/min的条件下将抗体偶联到protein A((Cytiva,29127556)芯片上。在流速30μL/min的条件下，检测抗原与抗体结合的动力学和亲和力数据，设定的结合时间为120s，解离时间为800s。In the Biacore 1K ((Cytiva)) system, the antibody was diluted to 10 μg/mL with running buffer (HBS-EP) and coupled to the protein A ((Cytiva, 29127556)) chip at a flow rate of 10 μL/min. The kinetics and affinity data of antigen-antibody binding were detected at a flow rate of 30 μL/min, with a set binding time of 120 s and a dissociation time of 800 s.
检测母本抗体parental mAb和亲和力优化抗体PSMAvll与PSMA结合的动力学和亲和力数据，结果如图2所示，与母本抗体parental mAb相比，亲和力优化抗体PSMAvll与PSMA亲和力提高约1.8倍，解离速率减缓4.6倍。The kinetics and affinity data of the binding of the parental mAb and the affinity-optimized antibody PSMAvll to PSMA were detected. The results are shown in Figure 2. Compared with the parental mAb, the affinity-optimized antibody PSMAvll had an approximately 1.8-fold increase in affinity for PSMA and a 4.6-fold decrease in dissociation rate.
注：Ka代表结合速率常数(数值越大代表亲和力越强)；Kd代表解离速率常数(数值越小亲和力越强)，反映的是化合物对靶标的亲和力大小；KD代表Kd/Ka，为平衡解离常数(亲和力常数)，KD越小说明解离越少，代表亲和力越强。Note: Ka represents the binding rate constant (the larger the value, the stronger the affinity); Kd represents the dissociation rate constant (the smaller the value, the stronger the affinity), which reflects the affinity of the compound for the target; KD represents Kd/Ka, which is the equilibrium dissociation constant (affinity constant). The smaller the KD, the less dissociation, which means the stronger the affinity.
实施例4：PSMA抗体ELISA结合实验Example 4: PSMA antibody ELISA binding experiment
利用ELISA检测母本抗体parental mAb和亲和力优化抗体PSMAvll和PSMA的结合特性。发明人将PSMA蛋白包被到96孔板中，抗体加入后信号的强弱用于判断抗体和PSMA的结合特性。ELISA was used to detect the binding properties of parental mAb and affinity-optimized antibody PSMAvll and PSMA. The inventors coated PSMA protein into a 96-well plate, and the strength of the signal after the addition of the antibody was used to determine the binding properties of the antibody and PSMA.
用PBS缓冲液将PSMA蛋白(购自Acro)稀释为1μg/ml，以100μL/孔的体积加于96孔板中，于4℃放置过夜。将96孔板中PBS缓冲液吸掉，用PBST(即pH7.2的PBS中含0.1体积％的Tween 20)缓冲液洗板6次后，加入200μL/孔含有10％ BSA的PBS，37℃孵育2h进行封闭。移去封闭液，用PBST洗板6次后，加入100μL/孔用含有0.05％ BSA的PBST梯度稀释(最高工作浓度20000ng/ml，5倍稀释，8个梯度)的母本抗体parental mAb、亲和力优化抗体PSMAvll、对照IgG1(购自百英生物)，37℃孵育1h。吸取孔中的反应体系，用PBST洗板6次后，以100μL/孔用含有0.05％ BSA的PBST稀释HRP(辣根过氧化物酶)标记的抗人抗体二抗(Fab特异性)(购自Sigma)，37℃孵育1h。吸去孔中的二抗，用PBST洗板6次后，加入80μL/孔TMB(四甲基联苯胺)，于室温孵育3min，加入80μL/孔4M硫酸终止反应。用酶标仪在450mm处读取吸光值。PSMA protein (purchased from Acro) was diluted to 1 μg/ml with PBS buffer, added to a 96-well plate at a volume of 100 μL/well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (i.e., PBS with pH 7.2 containing 0.1% by volume Tween 20) buffer, and then 200 μL/well of PBS containing 10% BSA was added, and incubated at 37°C for 2h for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST, and then 100 μL/well of parental mAb, affinity-optimized antibody PSMAvll, and control IgG1 (purchased from Bio-Bio) diluted with PBST containing 0.05% BSA (maximum working concentration 20000 ng/ml, 5-fold dilution, 8 gradients) were added, and incubated at 37°C for 1h. The reaction system in the well was aspirated, and the plate was washed 6 times with PBST. Then, 100 μL/well of HRP (horseradish peroxidase)-labeled anti-human antibody secondary antibody (Fab specific) (purchased from Sigma) was diluted with PBST containing 0.05% BSA, and incubated at 37°C for 1 hour. The secondary antibody in the well was aspirated, and the plate was washed 6 times with PBST. Then, 80 μL/well of TMB (tetramethylbenzidine) was added, and the plate was incubated at room temperature for 3 minutes. 80 μL/well of 4M sulfuric acid was added to terminate the reaction. The absorbance value was read at 450 mm using an ELISA reader.
结果表明本发明的亲和力优化抗体PSMAv11能够结合PSMA蛋白。The results show that the affinity-optimized antibody PSMAv11 of the present invention can bind to the PSMA protein.
实施例5：PSMA抗体流式细胞术结合实验Example 5: PSMA antibody flow cytometry binding experiment
用PBS将肿瘤细胞(22RV1或LNCaP人前列腺癌)稀释为2×106/mL，以100μL/管的体积加于1.5ml EP管中，向其中加入10μL/管山羊血清，于4℃封闭30min。加入梯度稀释(最高工作浓度为50μg/ml，5倍稀释，10个梯度)的母本抗体parental mAb、亲和力优化抗体PSMAvll、对照IgG1(购自百英生物)，于4℃孵育30min。向EP管中加入1mL PBS，在4℃、3500rpm条件下离心5min，弃尽上清，再用PBS洗一遍。离心后弃尽上清，用100μl/管PBS重悬细胞，向其中加入1μL/管Alexa-647标记的山羊抗人抗体二抗(购自Jackson lab)，4℃避光孵育30min。用PBS洗两遍，离心后弃尽上清。用200μL/管PBS重悬细胞，用流式细胞仪进行检测。Tumor cells (22RV1 or LNCaP human prostate cancer) were diluted to 2×106 /mL with PBS, added to 1.5ml EP tubes at a volume of 100μL/tube, and 10μL/tube goat serum was added to it, and blocked at 4℃ for 30min. Gradient dilutions (maximum working concentration is 50μg/ml, 5-fold dilution, 10 gradients) of parental mAb, affinity-optimized antibody PSMAvll, and control IgG1 (purchased from Bio-Bio) were added and incubated at 4℃ for 30min. 1mL PBS was added to the EP tube, centrifuged at 4℃ and 3500rpm for 5min, the supernatant was discarded, and then washed with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended with 100μl/tube PBS, and 1μL/tube Alexa-647-labeled goat anti-human antibody secondary antibody (purchased from Jackson lab) was added to it, and incubated at 4℃ for 30min in the dark. Wash twice with PBS, and discard the supernatant after centrifugation. Resuspend the cells with 200 μL/tube PBS and detect them using flow cytometry.
结果如图3、4所示，显示本发明的亲和力优化抗体PSMAv11结合22RV1、LNCaP人前列腺癌细胞荧光强度高于母本抗体，表明亲和力优化抗体PSMAv11结合活性更高。The results are shown in Figures 3 and 4, which show that the affinity-optimized antibody PSMAv11 of the present invention binds to 22RV1 and LNCaP human prostate cancer cells with higher fluorescence intensity than the parent antibody, indicating that the affinity-optimized antibody PSMAv11 has higher binding activity.
实施例6:PSMA抗体促进PBMC杀伤肿瘤细胞Example 6: PSMA antibody promotes PBMC to kill tumor cells
检测PSMA单抗促进PBMC杀伤22RV1、LNCaP人前列腺癌细胞的能力。The ability of PSMA monoclonal antibody to promote PBMC to kill 22RV1 and LNCaP human prostate cancer cells was detected.
(1)按照50μL/孔的体积向16孔RTCA板中加入完全RPMI-1640培养基，上机校准；(1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL/well and calibrate the plate.
(2)用完全RPMI-1640培养基将肿瘤细胞稀释为2×105/mL，按照50μL/孔的体积分别单独添加至加入步骤(1)获得的RTCA板中，然后于37℃、5％ CO2条件下使用xCELLigenceRTCA MP设备检测细胞系数24h；(2) diluting the tumor cells to 2×105 /mL with complete RPMI-1640 medium, adding them to the RTCA plates obtained in step (1) at a volume of 50 μL/well, and then detecting the cell coefficient using the xCELLigence RTCA MP device at 37°C and 5% CO2 for 24 hours;
(3)用完全RPMI-1640培养基将母本抗体parental mAb、亲和力优化抗体PSMAvll进行梯度稀释，加入步骤(2)获得的RTCA板中，添加体积为20μL/孔；(3) using complete RPMI-1640 medium to grade dilute the parental mAb and affinity-optimized antibody PSMAvll, and add them to the RTCA plate obtained in step (2) at a volume of 20 μL/well;
(4)用完全RPMI-1640培养基将PBMC(购自赛笠生物)稀释为1.25×106个/mL，加入步骤(3)获得的RTCA板中，添加体积为80μL/孔；(4) PBMC (purchased from Sai Li Biotechnology) was diluted to 1.25×106 cells/mL with complete RPMI-1640 medium and added to the RTCA plate obtained in step (3) at a volume of 80 μL/well;
(5)将步骤(4)获得的反应体系于37℃，5％ CO2使用xCELLigence RTCA MP设备检测细胞系数24h。(5) The reaction system obtained in step (4) was incubated at 37°C and 5%CO2 for 24 hours using an xCELLigence RTCA MP instrument to detect the cell coefficient.
如图5、6所示，本发明的PSMAv11抗体促进PBMC杀伤肿瘤，促杀伤功能强于母本抗体parental mAb。As shown in Figures 5 and 6, the PSMAv11 antibody of the present invention promotes PBMC to kill tumors, and its killing function is stronger than that of the parental mAb.
实施例7：CD3×PSMA抗体流式细胞术结合实验Example 7: CD3×PSMA antibody flow cytometry binding experiment
按照实施例1的方法分别制备实验组CD3×PSMAv11双特异抗体(其中，CD3抗体的氨基酸序列如SEQ ID NO:17所示、重链的氨基酸序列如SEQ ID NO:18所示、轻链的氨基酸序列如SEQ ID NO:12所示)和对照组CD3×parental mAb双特异抗体(其中，CD3抗体的氨基酸序列如SEQ ID NO:17所示、重链的氨基酸序列如SEQ ID NO:21所示、轻链的氨基酸序列如SEQ ID NO:20所示)。然后进行流式细胞术结合实验，具体实验步骤如下：According to the method of Example 1, the experimental group CD3×PSMAv11 bispecific antibody (wherein the amino acid sequence of the CD3 antibody is shown in SEQ ID NO: 17, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain is shown in SEQ ID NO: 12) and the control group CD3×parental mAb bispecific antibody (wherein the amino acid sequence of the CD3 antibody is shown in SEQ ID NO: 17, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 21, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20) were prepared respectively. Then, a flow cytometry binding experiment was performed, and the specific experimental steps were as follows:
用PBS将肿瘤细胞稀释为2×106/mL，以100μL/管的体积加于1.5ml EP管中，向其中加入10μL/管山羊血清，于4℃封闭30min。加入梯度稀释的CD3×parental PSMA双特异抗体、CD3×PSMAvll双特异抗体、对照IgG1LALA(购自百英生物)，于4℃孵育30min。向EP管中加入1mL PBS，4℃3500rpm×5min离心，弃尽上清，再用PBS洗一遍。离心后弃尽上清，用100μl/管PBS重悬细胞，向其中加入1μL/管Alexa-647标记的山羊抗人抗体二抗(购自Jacksonlab)，4℃避光孵育30min。用PBS洗两遍，离心后弃尽上清。用200μL/管PBS重悬细胞，用流式细胞仪进行检测。Tumor cells were diluted to 2×106 /mL with PBS, added to a 1.5ml EP tube at a volume of 100μL/tube, and 10μL/tube of goat serum was added to it, and blocked at 4℃ for 30min. Gradient dilutions of CD3×parental PSMA bispecific antibody, CD3×PSMAvll bispecific antibody, and control IgG1LALA (purchased from Baiying Bio) were added, and incubated at 4℃ for 30min. 1mL PBS was added to the EP tube, centrifuged at 4℃3500rpm×5min, the supernatant was discarded, and then washed with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended with 100μl/tube PBS, and 1μL/tube Alexa-647-labeled goat anti-human antibody secondary antibody (purchased from Jacksonlab) was added, and incubated at 4℃ for 30min in the dark. Wash twice with PBS, and discard the supernatant after centrifugation. Resuspend the cells with 200μL/tube PBS and detect them using a flow cytometer.
结果如图7、8所示，结果表明本发明的CD3×PSMAv11结合22RV1、LNCaP人前列腺癌细胞强于CD3×parental PSMA双特异抗体。The results are shown in Figures 7 and 8, which indicate that the CD3×PSMAv11 of the present invention binds to 22RV1 and LNCaP human prostate cancer cells more strongly than the CD3×parental PSMA bispecific antibody.
实施例8:CD3×PSMA抗体促进PBMC杀伤肿瘤细胞Example 8: CD3×PSMA antibody promotes PBMC to kill tumor cells
检测实施例7中得到的CD3×PSMA双特异抗体和CD3×parental mAb双特异抗体促进PBMC杀伤22RV1、LNCaP人前列腺癌细胞的能力。具体步骤如下：The ability of the CD3×PSMA bispecific antibody and CD3×parental mAb bispecific antibody obtained in Example 7 to promote PBMC to kill 22RV1 and LNCaP human prostate cancer cells was tested. The specific steps are as follows:
(1)按照50μL/孔的体积向16孔RTCA板中加入完全RPMI-1640培养基，上机校准；(1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL/well and calibrate the plate.
(2)用完全RPMI-1640培养基将肿瘤细胞稀释为2×105/mL，按照50μL/孔的体积分别单独添加至加入步骤(1)获得的RTCA板中，然后于37℃，5％ CO2条件下使用xCELLigenceRTCA MP设备检测细胞系数24h；(2) diluting the tumor cells to 2×105 /mL with complete RPMI-1640 medium, adding them to the RTCA plates obtained in step (1) at a volume of 50 μL/well, and then detecting the cell coefficient using the xCELLigence RTCA MP device at 37°C and 5% CO2 for 24 hours;
(3)用完全RPMI-1640培养基将CD3×parental PSMA双特异抗体、CD3×PSMAvll双特异抗体、对照IgG1LALA(购自百英生物)进行梯度稀释，加入步骤(2)获得的RTCA板中，添加体积为20μL/孔；(3) CD3×parental PSMA bispecific antibody, CD3×PSMAvll bispecific antibody, and control IgG1LALA (purchased from Bio-Bio) were graded diluted in complete RPMI-1640 medium and added to the RTCA plate obtained in step (2) at a volume of 20 μL/well;
(4)用完全RPMI-1640培养基将PBMC(购自赛笠生物)稀释为1.25×106个/mL，加入步骤(3)获得的RTCA板中，添加体积为80μL/孔；(4) PBMC (purchased from Sai Li Biotechnology) was diluted to 1.25×106 cells/mL with complete RPMI-1640 medium and added to the RTCA plate obtained in step (3) at a volume of 80 μL/well;
(5)将步骤(4)获得的反应体系于37℃，5％ CO2使用xCELLigence RTCA MP设备检测细胞系数24h。(5) The reaction system obtained in step (4) was incubated at 37°C and 5%CO2 for 24 hours using an xCELLigence RTCA MP instrument to detect the cell coefficient.
如图9、10所示，本发明的CD3×PSMAv11抗体促进PBMC杀伤肿瘤远强于CD3×parental PSMA双特异抗体。As shown in Figures 9 and 10, the CD3×PSMAv11 antibody of the present invention promotes PBMC to kill tumors much more effectively than the CD3×parental PSMA bispecific antibody.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.
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| CN202311699412.4ACN118027203A (en) | 2023-12-11 | 2023-12-11 | PSMA antibodies and uses thereof |
| PCT/CN2024/136743WO2025124255A1 (en) | 2023-12-11 | 2024-12-04 | Psma antibody and use thereof |
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| CN101124249B (en)* | 2005-02-18 | 2011-06-29 | 米德列斯公司 | Human monoclonal antibodies to prostate specific membrane antigen(PSMA) |
| US20180016346A1 (en)* | 2015-01-08 | 2018-01-18 | Kyowa Hakko Kirin Co., Ltd | Bispecific antibody binding to trailr2 and psma |
| CA3093078A1 (en)* | 2018-03-06 | 2019-09-12 | The Trustees Of The University Of Pennsylvania | Prostate-specific membrane antigen cars and methods of use thereof |
| CN118027203A (en)* | 2023-12-11 | 2024-05-14 | 合肥天港免疫药物有限公司 | PSMA antibodies and uses thereof |
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| WO2025124255A1 (en)* | 2023-12-11 | 2025-06-19 | 合肥天港免疫药物有限公司 | Psma antibody and use thereof |
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