几种抗CD20抗体偶联药物的制备与生物学活性初步研究
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摘要
细胞毒类抗肿瘤药物能杀伤肿瘤细胞,但这些药物对正常的细胞同样有较大杀伤力,从而极大的限制了该类药物的进一步应用和发展。单克隆抗体具有良好的靶向性,但是其对肿瘤细胞的杀伤力往往较弱甚至没有。抗体偶联药物(antibody-drug conjugates, ADCs),即毒性药物与单抗相连接组成化学免疫偶联物,将药物“精确”地运送到靶细胞,既有效地提高了肿瘤局部的药物浓度,又极大地降低了体内其他组织、器官的药物浓度,从而达到增效减毒的作用。1958年Mathe首次将抗鼠白细胞免疫球蛋白与甲氨蝶呤偶联用于靶向治疗,拉开了抗体偶联药物的研究序幕。但是几十年的研究里,没有一个抗体偶联药物的疗效和安全性达到足够要求成为治疗性的药物,直至2000年美国FDA批准Mylotarg上市,抗体偶联药物的研究与开发再次成为生物技术药物的新热点。多种抗体如anti-her2抗体Herceptin、BR96、anti-CD30、anti-CD33、anti-CD79等和多种毒性药物海兔毒素(MMAE)、阿霉素(DOX)、 Calicheamicin、美登素(Maytansine, AP3为其中一种)等被用来做抗体偶联药物的研究,包括对连接抗体的选择改造,连接方式的改进,连接药物的选择改造等。到目前,临床研究中的抗体偶联药物有20余种,2011年brentuximab vedotin上市,T-DM1也于2013年2月获FDA批准上市。
CD20是一种在成熟B细胞表面高表达的蛋白,同时也在95%以上的B细胞性淋巴瘤细胞表面高表达,这个特性使之成为靶向单克隆治疗的重要靶点。事实上,抗CD20抗体已经被广泛用来治疗B细胞性淋巴瘤。抗体与CD20结合以后,通过引起细胞凋亡以及抗体依赖的细胞介导的细胞毒作用(Antibody Dependent Cell Mediated Cytotoxicity, ADCC)、补体依赖的细胞毒作用(complement dependentcytotoxicity, CDC)发挥杀伤作用。但是,抗CD20抗体常因温和的杀伤能力在治疗CD20阳性B细胞淋巴瘤上存在缺陷：只对部分病人有治疗效果,对一些CD20阳性病人起初就无效或者很快使其产生耐药；治愈率低；躲避抗体杀死的肿瘤细胞很快造成病情复发。因此,提高抗CD20抗体杀伤肿瘤靶细胞的能力成了临床研究中迫切需要解决的问题,而抗体偶联药物的理论似乎可以满足这一需求。的确有研究将MMAE或者Calicheamicin偶联抗CD20抗体Rituximab (RTX)使其活性得到了改善,但是,此方法是否也适用于其他众多的抗CD20抗体并未见报道。
为了提高抗CD20抗体直接杀伤CD20阳性细胞的能力及确认方法的适应性,本文将选择多种抗CD20抗体,即Rituximab (RTX)、 Ofatumumab (OFA)和TGLA三种抗体进行研究。RTX是第一个上市用来治疗非霍奇金淋巴瘤(B-cell non-Hodgkin lymphoma, B-NHL)的抗CD20嵌合抗体；OFA是第一个上市用来治疗慢性淋巴细胞白血病(CLL)的全入源化抗CD20抗体；而TGLA是一种具有RTX同样靶点、近似活性的抗CD20单克隆抗体。本文首先选择了TGLA抗体和DOX、MMAE、AP3(一种美登素)三种药物进行偶联探索和活性观察；同时为了验证方法的适应性,我们将选择一种药物再和其他两种抗CD20抗体RTX、OFA进行偶联及活性研究。本研究不仅采用抗体赖氨酸的侧链胺基或二硫键还原的巯基形式结合药物,也选择了一种抗CD20抗体进行恒定区的半胱氨酸突变以定点定量的方式偶联毒性药物,使抗体能够在保持亲和性、CDC、ADCC活性的提前下提高直接杀伤肿瘤细胞的活性,从而为高效低毒的抗CD20抗体偶联药物临床开发提供理论和实验基础。
一、抗肿瘤小分子的改造
目前抗体偶联药物研究中所使用的小分子主要有海兔毒素(MMAE),美登素(Maytansine),刺包霉素(Calicheamicin),阿霉素(DOX)等。这些小分子往往不能直接和抗体偶联,需要借助连接物或者连接基团才能达到偶联的目的。本章我们选用具有代表性的毒性药物阿霉素(DOX)和Maytansine (AP3)进行连接前改造,为后续实验垫定物质基础。
DOX所采用是抗体偶联药物临床研究中较常使用的连接物maleimidocaproyl-valine-citrulline-p-aminobenzylcarbonyl (MC-Val-Cit-PABC)。 MC能够和抗体被还原出的巯基反应,PABC部分可以连接药物分子的—OH或—NH2基团(若药物分子中无—OH或—NH2基团,则要进行相应改造),肽链在血液中较稳定,但当抗体偶联药物进入肿瘤细胞时便被溶酶体酶酶切释放出游离药物,发挥药效。本研究参考Gene M. Dubowchik等报道的合成路线,并通过改进合成条件缩短了反应时间,以Fmoc-Val-OSu、瓜氨酸(Cit)、对氨基苯甲醇(PABOH)、6.(马来酰亚胺基)已酸琥珀酰亚胺酯(MC)等为原料经过六步反应合成阿霉素的连接物MC-Val-Cit-PABC-DOX,各步产物采用核磁和质谱确定,终产物经过酶切鉴定能释放出药物DOX.
AP3常以高毒性的DM1形式被用于抗体偶联药物的研究中。本研究参照专利里报道过的DM1合成路线,以AP3为起始原料,还原成AP0,然后再经过四步反应合成带有巯基的DM1,DM1可以和经过修饰后的抗体偶联。
二、传统抗体偶联药物的合成及生物学活性研究
为了建立传统以抗体赖氨酸的侧链胺基或二硫键还原的巯基结合药物的连接方式,并观测各种药物对抗CD20抗体杀伤活性的直接影响,我们选用新型的抗CD20抗体TGLA为载体进行了与MC-Val-Cit-PABC-DOX (vcDOX)、 MC-Val-Cit-PABC-MMAE (vcMMAE)和DM1的偶联实验。
TGLA经过DTT还原出巯基以后,与vcDOX进行偶联。理论上总共可以结合8个药物,两条轻链各结合一个,两条重链各结合三个。经过条件摸索,实验中DOX/TGLA偶联数经紫外分光光度法检测最多能达到约6个。SDS-PAGE上显示轻链大部分已经结合上药物,而重链因为本身分子量大的原因在SDS-PAGE上没有分开。抗体偶联药物对靶细胞Raji细胞的杀伤能力相对Anti-CD20单抗略有提高。
以vcDOX反应条件合成TGLA-vcMMAE,获得药物／抗体偶联比约6.4。
TGLA和DM1的偶联采用可断开的二硫键SPDP形式及不可断开的硫醚SMCC形式。经过SPDP修饰,与DM1偶联的产物DM1/TGLA禺联数约在3.5,偶联物能够在二硫苏糖醇(DTT)作用下还原出游离药物DM1；以SPDP反应条件合成TGLA-SMCC-DM1,获得偶联比约2.9。
对这些高毒性的抗体偶联药物进行细胞水平的活性比较,MMAE、DM1能够显著提高TGLA的活性。相比较DOX因为毒性较低只微弱的提高了TGLA的活性,提示我们选用高毒性药物作为偶联对象的重要性。
三、不同抗CD20抗体的MMAE偶联物研究
以往报道中对ADCs的研究通常是选定一种抗体进行多种药物或多种连接物的筛选,几乎没有文章讲述针对同一抗原的不同抗体偶联相同药物时是否也有区别。本章节中我们选用高毒性的vcMMAE作为连接药物,合成了三种抗CD20抗体OFA、RTX、TGLA的抗体偶联药物,并对这些偶联物的亲和性、吞噬、杀伤活性进行了评价。三种抗CD20抗体经过三(2-羧乙基)膦盐酸盐(TCEP)还原再与MMAE偶联,5,5-二硫二硝基苯甲酸(DTNB)方法检测偶联数量约为7.5-7.6,流式细胞仪检测偶联后抗体结合细胞的能力有略微下降。流式和共聚焦结果显示抗体偶联药物都能够被CD20阳性肿瘤细胞WIL2-S吞噬,进一步对CD20抗原检测,发现CD20抗原也有相似程度的减少,说明偶联物是通过CD20抗原介导抗体偶联药物的吞噬。活性实验表明,抗CD20抗体偶联药物以后活性能够得到专一性的提高,只对CD20阳性细胞起作用。AN/PI双染和caspase, parp蛋白的WB显示偶联物通过诱导细胞凋亡来发挥杀死作用。我们的研究还表明,即使是对同一种细胞表面的抗原,不同的抗体作为载体制造偶联物时活性也是不同的。在细胞水平有较高活性的OFA偶联物在小鼠体内也表现出明显的抗肿瘤活性,所以选其作为突变偶联的抗体。
四、2F2和突变体Thio-2F2的构建和表达
采用传统的以抗体赖氨酸的侧链胺基或二硫键还原的巯基结合药物的连接方式带来不均一的产物,而且改变了抗体的结构降低了其亲和性。Jagath R Junutula等人在抗MUC16抗体的特定部位设计一个具有活性的半胱氨酸残基,能以确定的比例结合药物而不改变抗体对靶细胞的亲和性,并在体内外实验中证明了该抗体偶联药物的高效、低毒和长半衰期等种种优点。但是这个方法是否适合抗CD20抗体并未有过报道,因此,我们利用Jagath R Junutula报道的方法,对OFA(原名2F2)抗体进行类似改造,即在其特定部位设计一个具有活性的半胱氨酸残基,希望能以确定的比例结合药物提高杀伤能力而不改变抗体对靶细胞的亲和性、CDC、 ADCC等活性。
将带有2F2DNA序列的表达载体导入到CHO细胞中稳定表达,获得了2F2。同时,对2F2进行突变,把重链恒定区第一位的丙氨酸A变成了半胱氨酸C,获得突变体Thio-2F2,并且初步证明突变体Thio-2F2保持了原抗体2F2的体外亲和性和CDC活性及体内抗肿瘤活性。
五、Thio-2F2-vc的合成和生物学活性研究
把Thio-2F2和vcMMAE进行定点定量的偶联,亲和性实验证实了2F2突变、偶联之后,亲和性、CDC、ADCC活性几乎没有受到影响,而抗体的体外杀伤CD20阳性细胞的能力得到了提高,对CD20阴性细胞不造成杀伤,说明了偶联物的良好靶向性。在Ramos淋巴瘤的裸鼠皮下移植瘤实验中,证明了其相对于原抗体和突变抗体抗肿瘤活性的提高。
主要创新点：
1.利用多种抗CD20抗体与多种毒性小分子偶联,获得了多种未经报道的新型高活性抗CD20抗体偶联药物,验证了抗CD20抗体偶联高毒性的药物能提高其杀伤CD20阳性肿瘤细胞的能力,初步搭建针对CD20靶点的抗体偶联药物技术平台。
2.探索了多种anti-CD20-vcMMAE禺联物活性机理,阐明针对同一靶点的不同抗体偶联同样药物也具有活性差异,拓宽了抗体偶联药物的设计思路；其中所构建的全新抗体偶联药物OFA-vcMMAE在体内外实验中表现出极好的抗肿瘤活性,甚至在两种小鼠淋巴瘤模型中完全治愈肿瘤,且复发率极低。
3.设计了2F2抗体的突变体,能够定点定量的偶联小分子药物,保证产物的均一性；更为重要的是该突变体与MMAE的偶联物能在保持原有抗体亲和性、CDC和ADCC活性的基础上提高其体内外抗肿瘤活性。该方法也为其他抗体或蛋白的偶联设计提供了参考。
Cytotoxic anticancer drugs can not only kill tumor cells, but also kill normal cells, resulting in obvious limitations of their further application and developments. However, biotherapeutics such as antibodies against tumor-specific antigens often lack therapeutic activity. Antibody-drug conjugates (ADCs), which consist of toxic drugs and mAbs, deliver the drug to the target cells specifically. Thus, ADCs can effectively improve the drug concentration in the site of tumor, and greatly reduces the drug concentrations in other organizations so as to reduce side effects. In1958Mathe coupled anti-rat leukocyte immunoglobulin protein and methotrexate for targeted therapy for the first time, that opened the prelude of the development of antibody-drug conjugates (ADCs). But in the past few decades, none of ADCs in esearch is effective and safe enough to get FDA approved until recently. Since the US FDA approved the first ADCs, that is a humanized anti-CD33conjugated to calicheamicin (gemtuzumab ozogamicin;Mylotarg), for the treatment of acute myeloid leukemia in2000, many ADCs are being actively pursued to combat different types of cancer. A variety of antibodies such as anti-Her2mAb Herceptin, BR96, anti-CD20mAb Rituximab (RTX), anti-CD30mAb, anti-CD33mAb, anti-CD79mAb and various cytotoxic drugs such as Dolastatins(MMAE), doxorubicin (DOX),Calicheamicin, Maytansine(DMl, DM3, DM4) are used to construct different kinds of ADCs. Researches on antibody modification,development of innovative cytotoxic drugsand use of various linkers are currently under progress. Until now, more than20ADCs are in various phases of clinical trials. brentuximab vedotin and T-DM1were approved by FDA in2011, and2013, respectively.
CD20is a B-cell specific surface protein expressed on mature B lymphocytes. CD20is an appealing target for monoclonal antibody (mAb) therapy, because of its overexpression on the cell membrane of most of the B-lymphocytic lymphomas. In fact, efficacy of several anti-CD20mAbs for the treatment of B-cell lymphoma has been studied for long to be achieved. The binding of mAbs to the CD20antigen on the cell surface may directly induce apoptosis, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)
However, anti-CD20mAb showed some limitations for CD20-positive B-lymphoma cell lines due to its modest efficacy:only some patients respond to the treatment, and some are initially unresponsive to it or later resist to it; Complete esponses are low; Easy to relapse. Therefore, improving the ability of anti-CD20antibody to achieve effective tumor cell killing becomes an urgent problem in clinical research, and antibody-drug conjugates seem to meet this demand. Indeed, it has been reported that conjugation of RTX with MMAE or Calicheamicin could improve the activity of RTX. However, whether this method is also applicable to many other anti-CD20antibodies is unknown to our best knowledge.
To improve the activity of anti-CD20antibody and confirm the suitability of the method, we chose several anti CD20antibodies, Rituximab (RTX), Ofatumumab (OFA) and TGLA for conjugation studies. RTX (Chimeric Antibody) was the first anti-CD20mAb approved by FDA for the treatment of non-Hodgkin's B-cell lymphomas (B-NHL).Ofatumumab (OFA) is the first fully human anti-CD20mAb for patients with chronic lymphocytic leukemia (CLL).TGLA, which shares the same epitope with RTX, exhibiting similar efficacy with rituximab on CDC, cell growth arrest and so on. We first chose TGLA and DOX, MMAE, AP3(a kind of maytansine) for the study of conjugation and activity. For the validation of the method, we also chose one drug and the two above mentioned anti-CD20Abs (RTX and OFA). Toxic drugs were conjugated to antibodies either through lysine side-chain amines or through cysteine sulfhydryl groups which were activated by reducing interchain disulfide bonds. A cysteine was then introduced by site-directed mutagenesis in the constant region of Ab for defined stoichiometry of the conjugated drugs site-specifically, so that affinity, CDC and ADCC activity is well kept. Our researches provide theoretical and experimental basis for the preclinical discovery of anti-CD20ADCs with high efficiency and low toxicity.
1. Modification of anti-tumor small molecules
At present, the drugs used in ADCs are focused on MMAE, Maytansine (AP3), Calicheamicin, doxorubicin (DOX) and so on. Before conjugation, those drugs need to be modified. We chose the toxic drugs doxorubicine and AP3which were commonly used in ADCs researches as the conjugated drugs, and modification were then performed.
MC-VC-PABC-DOX uses the linker MC-VC-PABC.This linker is stable in blood and gets cleaved by lysosomal enzymes, therefore releasing active drug in target cancer cells. The PABC part could couple to-OH group or-NH2group of drugs, and the MC part could react with-SH group of an antibody. MC-VC-PABC-DOX was synthetized by a method reported by Gene M. Dubowchik et al. Fmoc-Val-OSu, L-Citrulline (L-Cit), p-aminobenzyl alcohol (PABOH),6-Maleimidohexanoic acid N-hydroxysuccinimide ester (MC-OSu) and so on were used to construct MC-VC-PABC-DOX. The reactions were monitored by MS and H-NMR and the end-product could release free drug DOX after enzymolysis.
Maytansinol DM1was synthetized according to patents. Firstly, AP3was reduced to APO. After re-esterification and reduction, APO was transformed into DM1. DM1can be conjugated to Abs through several linkers.
2. Synthesis and biological activity of traditional antibody-drug conjugates
After the modification of small molecule drugs, we chose anti-CD20antibody TGLA as the carrier,-vcDOX/-vcMMAE/DM1as the conjugated drugs to explore the conjugation conditions and cytotoxicity of conjugates.
Cysteine sulfhydryl groups were activated by reducing interchain disulfide bonds of TGLA with DTT. In theory, there would be8-SH groups,2light chains each with1-SH group and2heavy chains each with3-SH groups. In this research, about6DOX were conjugated to each antibody in average after the reaction. SDS-PAGE indicated that most of the light chains had been conjugated with DOX, nevertheless, the heavy chains were separated on SDS-PAGE for the heavy molecule weight. Compared with Anti-CD20Ab, the anti-CD20-vcDOX conjugates exhibited an enhanced effect on killing the CD20-positive cells (Raji cells). Since the cytotoxicity of DOX is not potent enough, the increased cytotoxicity of ADCs is not significant.
The conjugation condition for vcMMAE was the same with vcDOX, and MMAE/Ab ratio was about6.4.
The ratio of DM1/TGLA for TGLA-SPDP-DM1was about3.5. The DM1was released from ADCs in the presence of DTT. The ratio of DM1/TGLA for TGLA-MCC-DM1was about2.9. MMAE and DM1ADCs significantly improved the activity of TGLA in vitro, while DOX which owns lower toxicity than MMAE or DM1only resulted in a slight increase in cytotoxicity of TGLA. This result suggested us to choose highly toxic drugs as conjugated drugs.
3. Study of different anti-CD20-vcMMAE conjugates
Studies on ADCs usually focus on a selected Ab and various drugs or linkers.There is almost no report demonstrating the difference caused by different antibodies against the same antigen. Here, we synthesized three anti-CD20-vcMMAE conjugates: Ofatumumab-vcMMAE, Rituximab-vcMMAE and TGLA-vcMMAE, and then evaluated the affinity, CDC, internalization and cytotoxicity of these three types against CD20-positive human B lymphoma cells, namely, Raji, Daudi and WIL2-S cells.
As a result, conjugation of anti-CD20mAbs with MMAE caused a modest decrease in the binding affinity to cell surface CD20. All anti-CD20-vcMMAE conjugates were modulated from the cell surface and subsequently entered the lysosomes through the CD20receptor-mediated endocytosis. In addition, the conjugation of MMAE significantly improved the cytotoxic activity of all anti-CD20mAbs against CD20-positive cell lines. The results of Annexin V/PI staining and WB demonstrate that treatment of CD20-positive tumor cells with anti-CD20-vcMMAE conjugates could induce apoptotic cell death through a caspase-3-like protease-dependent pathway. We also observed variable cytotoxicity between the anti-CD20mAbs (i.e., OFA, RTX and TGLA) with the same MMAE conjugated. OFA-vcMMAE exhibited great anti-tumor activity in xenograft models of CD20-positive lymphoma where OFA, equivalent free MMAE or irrelevant Herceptin-vcMMAE was ineffective. These results stimulated us to choose OFA as the carrier to carry out mutation and conjugation.
4. Construction and expression of2F2and its mutant Thio-2F2
In the traditional study, ADCs were synthetized by conjugating cytotoxic drugs to antibodies either through lysine side-chain amines or through cysteine sulfhydryl groups activated by reducing interchain disulfide bonds. Both procedures bring some disadvantages:1) The reaction of interchain disulfide bonds or surface amine groups may result in structural changes in Abs, sabotaging binding affinity of the antibody.2) Traditional procedures yield heterogenous products, containing a mixture of species with different molar ratios of drug to antibody. To solve the problems Jagath R Junutula et al. in Genentech have engineered reactive cysteine residues at specific sites in the constant region of antibodies to allow drugs to be conjugated with defined stoichiometry without disrupting the interchain disulfide bonds. One such ADCs exhibits high efficacy, low side effect and long half-time in vivo. Here we used the method reported by Jagath R Junutula to modify and conjugate OFA, hoping to improve the direct toxic effect under the condition of keeping the affinity, CDC and ADCC activity of antibody on tumor cells.
We found DNA sequences of OFA in the US2004/0167319A15patents (namely2F2) and then construct the2F2gene carriers and stably express the Ab in CHO. Meanwhile, we performed mutation on the heavy chain constant region of2F2, that is changing an alanine (A) into a cysteine (C)(Thio-2F2). At last we proved the affinity and CDC activity of2F2and Thio-2F2preliminarily.
5Synthesis and biological activity of Thio-2F2-vcN
We conjugated vcMMAE to the specific sites in the constant region of Thio-2F2with defined stoichiometry. Experiments confirmed the mutation and conjugation had little influence in affinity, CDC, ADCC activity of2F2. In addition, the conjugation of MMAE significantly improved the cytotoxic activity of Thio-2F2against CD20-positive cell lines. Thio-2F2-vcMMAE exhibited great anti-tumor activity in xenograft models of CD20-positive lymphoma at doses where primary antibody and mutant antibody were ineffective.
The most important discoveries in this paper
1. We synthesized various anti-CD20-drug conjugates and obtained several unreported high-activity conjugates, and demonstrated that the conjugation of high-toxic drugs could significantly improve the cytotoxic activity of anti-CD20mAbs against CD20-positive cell lines. Here, we built a technology platform of constructing anti-CD20-drug conjugates.
2. We explored the mechanism of various anti-CD20-vcMMAE conjugates and found that the cytotoxicity was different between the anti-CD20mAbs (i.e., OFA, RTX and TGLA) after conjugation with the same toxic drug, broadening the design ideas of the ADCs. The new ADC OFA-vcMMAE showed excellent anti-tumor activity in vitro and in vivo. It completely inhibited the growth of two lymphoma xenografts and the recurrence rates were very low.
3. We designed a2F2mutant which could conjugate drugs at specific sites and with defined stoichiometry, ensuring the homogeneity of the products; Experiments confirmed the mutation and conjugation had little influence in affinity, CDC, ADCC activity of2F2. In addition, the conjugation of MMAE significantly improved the cytotoxic activity of Thio-2F2. This method provides a reference for the designs of other antibodies or proteins-drugs conjugates.
CD20是一种在成熟B细胞表面高表达的蛋白,同时也在95%以上的B细胞性淋巴瘤细胞表面高表达,这个特性使之成为靶向单克隆治疗的重要靶点。事实上,抗CD20抗体已经被广泛用来治疗B细胞性淋巴瘤。抗体与CD20结合以后,通过引起细胞凋亡以及抗体依赖的细胞介导的细胞毒作用(Antibody Dependent Cell Mediated Cytotoxicity, ADCC)、补体依赖的细胞毒作用(complement dependentcytotoxicity, CDC)发挥杀伤作用。但是,抗CD20抗体常因温和的杀伤能力在治疗CD20阳性B细胞淋巴瘤上存在缺陷：只对部分病人有治疗效果,对一些CD20阳性病人起初就无效或者很快使其产生耐药；治愈率低；躲避抗体杀死的肿瘤细胞很快造成病情复发。因此,提高抗CD20抗体杀伤肿瘤靶细胞的能力成了临床研究中迫切需要解决的问题,而抗体偶联药物的理论似乎可以满足这一需求。的确有研究将MMAE或者Calicheamicin偶联抗CD20抗体Rituximab (RTX)使其活性得到了改善,但是,此方法是否也适用于其他众多的抗CD20抗体并未见报道。
为了提高抗CD20抗体直接杀伤CD20阳性细胞的能力及确认方法的适应性,本文将选择多种抗CD20抗体,即Rituximab (RTX)、 Ofatumumab (OFA)和TGLA三种抗体进行研究。RTX是第一个上市用来治疗非霍奇金淋巴瘤(B-cell non-Hodgkin lymphoma, B-NHL)的抗CD20嵌合抗体；OFA是第一个上市用来治疗慢性淋巴细胞白血病(CLL)的全入源化抗CD20抗体；而TGLA是一种具有RTX同样靶点、近似活性的抗CD20单克隆抗体。本文首先选择了TGLA抗体和DOX、MMAE、AP3(一种美登素)三种药物进行偶联探索和活性观察；同时为了验证方法的适应性,我们将选择一种药物再和其他两种抗CD20抗体RTX、OFA进行偶联及活性研究。本研究不仅采用抗体赖氨酸的侧链胺基或二硫键还原的巯基形式结合药物,也选择了一种抗CD20抗体进行恒定区的半胱氨酸突变以定点定量的方式偶联毒性药物,使抗体能够在保持亲和性、CDC、ADCC活性的提前下提高直接杀伤肿瘤细胞的活性,从而为高效低毒的抗CD20抗体偶联药物临床开发提供理论和实验基础。
一、抗肿瘤小分子的改造
目前抗体偶联药物研究中所使用的小分子主要有海兔毒素(MMAE),美登素(Maytansine),刺包霉素(Calicheamicin),阿霉素(DOX)等。这些小分子往往不能直接和抗体偶联,需要借助连接物或者连接基团才能达到偶联的目的。本章我们选用具有代表性的毒性药物阿霉素(DOX)和Maytansine (AP3)进行连接前改造,为后续实验垫定物质基础。
DOX所采用是抗体偶联药物临床研究中较常使用的连接物maleimidocaproyl-valine-citrulline-p-aminobenzylcarbonyl (MC-Val-Cit-PABC)。 MC能够和抗体被还原出的巯基反应,PABC部分可以连接药物分子的—OH或—NH2基团(若药物分子中无—OH或—NH2基团,则要进行相应改造),肽链在血液中较稳定,但当抗体偶联药物进入肿瘤细胞时便被溶酶体酶酶切释放出游离药物,发挥药效。本研究参考Gene M. Dubowchik等报道的合成路线,并通过改进合成条件缩短了反应时间,以Fmoc-Val-OSu、瓜氨酸(Cit)、对氨基苯甲醇(PABOH)、6.(马来酰亚胺基)已酸琥珀酰亚胺酯(MC)等为原料经过六步反应合成阿霉素的连接物MC-Val-Cit-PABC-DOX,各步产物采用核磁和质谱确定,终产物经过酶切鉴定能释放出药物DOX.
AP3常以高毒性的DM1形式被用于抗体偶联药物的研究中。本研究参照专利里报道过的DM1合成路线,以AP3为起始原料,还原成AP0,然后再经过四步反应合成带有巯基的DM1,DM1可以和经过修饰后的抗体偶联。
二、传统抗体偶联药物的合成及生物学活性研究
为了建立传统以抗体赖氨酸的侧链胺基或二硫键还原的巯基结合药物的连接方式,并观测各种药物对抗CD20抗体杀伤活性的直接影响,我们选用新型的抗CD20抗体TGLA为载体进行了与MC-Val-Cit-PABC-DOX (vcDOX)、 MC-Val-Cit-PABC-MMAE (vcMMAE)和DM1的偶联实验。
TGLA经过DTT还原出巯基以后,与vcDOX进行偶联。理论上总共可以结合8个药物,两条轻链各结合一个,两条重链各结合三个。经过条件摸索,实验中DOX/TGLA偶联数经紫外分光光度法检测最多能达到约6个。SDS-PAGE上显示轻链大部分已经结合上药物,而重链因为本身分子量大的原因在SDS-PAGE上没有分开。抗体偶联药物对靶细胞Raji细胞的杀伤能力相对Anti-CD20单抗略有提高。
以vcDOX反应条件合成TGLA-vcMMAE,获得药物／抗体偶联比约6.4。
TGLA和DM1的偶联采用可断开的二硫键SPDP形式及不可断开的硫醚SMCC形式。经过SPDP修饰,与DM1偶联的产物DM1/TGLA禺联数约在3.5,偶联物能够在二硫苏糖醇(DTT)作用下还原出游离药物DM1；以SPDP反应条件合成TGLA-SMCC-DM1,获得偶联比约2.9。
对这些高毒性的抗体偶联药物进行细胞水平的活性比较,MMAE、DM1能够显著提高TGLA的活性。相比较DOX因为毒性较低只微弱的提高了TGLA的活性,提示我们选用高毒性药物作为偶联对象的重要性。
三、不同抗CD20抗体的MMAE偶联物研究
以往报道中对ADCs的研究通常是选定一种抗体进行多种药物或多种连接物的筛选,几乎没有文章讲述针对同一抗原的不同抗体偶联相同药物时是否也有区别。本章节中我们选用高毒性的vcMMAE作为连接药物,合成了三种抗CD20抗体OFA、RTX、TGLA的抗体偶联药物,并对这些偶联物的亲和性、吞噬、杀伤活性进行了评价。三种抗CD20抗体经过三(2-羧乙基)膦盐酸盐(TCEP)还原再与MMAE偶联,5,5-二硫二硝基苯甲酸(DTNB)方法检测偶联数量约为7.5-7.6,流式细胞仪检测偶联后抗体结合细胞的能力有略微下降。流式和共聚焦结果显示抗体偶联药物都能够被CD20阳性肿瘤细胞WIL2-S吞噬,进一步对CD20抗原检测,发现CD20抗原也有相似程度的减少,说明偶联物是通过CD20抗原介导抗体偶联药物的吞噬。活性实验表明,抗CD20抗体偶联药物以后活性能够得到专一性的提高,只对CD20阳性细胞起作用。AN/PI双染和caspase, parp蛋白的WB显示偶联物通过诱导细胞凋亡来发挥杀死作用。我们的研究还表明,即使是对同一种细胞表面的抗原,不同的抗体作为载体制造偶联物时活性也是不同的。在细胞水平有较高活性的OFA偶联物在小鼠体内也表现出明显的抗肿瘤活性,所以选其作为突变偶联的抗体。
四、2F2和突变体Thio-2F2的构建和表达
采用传统的以抗体赖氨酸的侧链胺基或二硫键还原的巯基结合药物的连接方式带来不均一的产物,而且改变了抗体的结构降低了其亲和性。Jagath R Junutula等人在抗MUC16抗体的特定部位设计一个具有活性的半胱氨酸残基,能以确定的比例结合药物而不改变抗体对靶细胞的亲和性,并在体内外实验中证明了该抗体偶联药物的高效、低毒和长半衰期等种种优点。但是这个方法是否适合抗CD20抗体并未有过报道,因此,我们利用Jagath R Junutula报道的方法,对OFA(原名2F2)抗体进行类似改造,即在其特定部位设计一个具有活性的半胱氨酸残基,希望能以确定的比例结合药物提高杀伤能力而不改变抗体对靶细胞的亲和性、CDC、 ADCC等活性。
将带有2F2DNA序列的表达载体导入到CHO细胞中稳定表达,获得了2F2。同时,对2F2进行突变,把重链恒定区第一位的丙氨酸A变成了半胱氨酸C,获得突变体Thio-2F2,并且初步证明突变体Thio-2F2保持了原抗体2F2的体外亲和性和CDC活性及体内抗肿瘤活性。
五、Thio-2F2-vc的合成和生物学活性研究
把Thio-2F2和vcMMAE进行定点定量的偶联,亲和性实验证实了2F2突变、偶联之后,亲和性、CDC、ADCC活性几乎没有受到影响,而抗体的体外杀伤CD20阳性细胞的能力得到了提高,对CD20阴性细胞不造成杀伤,说明了偶联物的良好靶向性。在Ramos淋巴瘤的裸鼠皮下移植瘤实验中,证明了其相对于原抗体和突变抗体抗肿瘤活性的提高。
主要创新点：
1.利用多种抗CD20抗体与多种毒性小分子偶联,获得了多种未经报道的新型高活性抗CD20抗体偶联药物,验证了抗CD20抗体偶联高毒性的药物能提高其杀伤CD20阳性肿瘤细胞的能力,初步搭建针对CD20靶点的抗体偶联药物技术平台。
2.探索了多种anti-CD20-vcMMAE禺联物活性机理,阐明针对同一靶点的不同抗体偶联同样药物也具有活性差异,拓宽了抗体偶联药物的设计思路；其中所构建的全新抗体偶联药物OFA-vcMMAE在体内外实验中表现出极好的抗肿瘤活性,甚至在两种小鼠淋巴瘤模型中完全治愈肿瘤,且复发率极低。
3.设计了2F2抗体的突变体,能够定点定量的偶联小分子药物,保证产物的均一性；更为重要的是该突变体与MMAE的偶联物能在保持原有抗体亲和性、CDC和ADCC活性的基础上提高其体内外抗肿瘤活性。该方法也为其他抗体或蛋白的偶联设计提供了参考。
Cytotoxic anticancer drugs can not only kill tumor cells, but also kill normal cells, resulting in obvious limitations of their further application and developments. However, biotherapeutics such as antibodies against tumor-specific antigens often lack therapeutic activity. Antibody-drug conjugates (ADCs), which consist of toxic drugs and mAbs, deliver the drug to the target cells specifically. Thus, ADCs can effectively improve the drug concentration in the site of tumor, and greatly reduces the drug concentrations in other organizations so as to reduce side effects. In1958Mathe coupled anti-rat leukocyte immunoglobulin protein and methotrexate for targeted therapy for the first time, that opened the prelude of the development of antibody-drug conjugates (ADCs). But in the past few decades, none of ADCs in esearch is effective and safe enough to get FDA approved until recently. Since the US FDA approved the first ADCs, that is a humanized anti-CD33conjugated to calicheamicin (gemtuzumab ozogamicin;Mylotarg), for the treatment of acute myeloid leukemia in2000, many ADCs are being actively pursued to combat different types of cancer. A variety of antibodies such as anti-Her2mAb Herceptin, BR96, anti-CD20mAb Rituximab (RTX), anti-CD30mAb, anti-CD33mAb, anti-CD79mAb and various cytotoxic drugs such as Dolastatins(MMAE), doxorubicin (DOX),Calicheamicin, Maytansine(DMl, DM3, DM4) are used to construct different kinds of ADCs. Researches on antibody modification,development of innovative cytotoxic drugsand use of various linkers are currently under progress. Until now, more than20ADCs are in various phases of clinical trials. brentuximab vedotin and T-DM1were approved by FDA in2011, and2013, respectively.
CD20is a B-cell specific surface protein expressed on mature B lymphocytes. CD20is an appealing target for monoclonal antibody (mAb) therapy, because of its overexpression on the cell membrane of most of the B-lymphocytic lymphomas. In fact, efficacy of several anti-CD20mAbs for the treatment of B-cell lymphoma has been studied for long to be achieved. The binding of mAbs to the CD20antigen on the cell surface may directly induce apoptosis, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)
However, anti-CD20mAb showed some limitations for CD20-positive B-lymphoma cell lines due to its modest efficacy:only some patients respond to the treatment, and some are initially unresponsive to it or later resist to it; Complete esponses are low; Easy to relapse. Therefore, improving the ability of anti-CD20antibody to achieve effective tumor cell killing becomes an urgent problem in clinical research, and antibody-drug conjugates seem to meet this demand. Indeed, it has been reported that conjugation of RTX with MMAE or Calicheamicin could improve the activity of RTX. However, whether this method is also applicable to many other anti-CD20antibodies is unknown to our best knowledge.
To improve the activity of anti-CD20antibody and confirm the suitability of the method, we chose several anti CD20antibodies, Rituximab (RTX), Ofatumumab (OFA) and TGLA for conjugation studies. RTX (Chimeric Antibody) was the first anti-CD20mAb approved by FDA for the treatment of non-Hodgkin's B-cell lymphomas (B-NHL).Ofatumumab (OFA) is the first fully human anti-CD20mAb for patients with chronic lymphocytic leukemia (CLL).TGLA, which shares the same epitope with RTX, exhibiting similar efficacy with rituximab on CDC, cell growth arrest and so on. We first chose TGLA and DOX, MMAE, AP3(a kind of maytansine) for the study of conjugation and activity. For the validation of the method, we also chose one drug and the two above mentioned anti-CD20Abs (RTX and OFA). Toxic drugs were conjugated to antibodies either through lysine side-chain amines or through cysteine sulfhydryl groups which were activated by reducing interchain disulfide bonds. A cysteine was then introduced by site-directed mutagenesis in the constant region of Ab for defined stoichiometry of the conjugated drugs site-specifically, so that affinity, CDC and ADCC activity is well kept. Our researches provide theoretical and experimental basis for the preclinical discovery of anti-CD20ADCs with high efficiency and low toxicity.
1. Modification of anti-tumor small molecules
At present, the drugs used in ADCs are focused on MMAE, Maytansine (AP3), Calicheamicin, doxorubicin (DOX) and so on. Before conjugation, those drugs need to be modified. We chose the toxic drugs doxorubicine and AP3which were commonly used in ADCs researches as the conjugated drugs, and modification were then performed.
MC-VC-PABC-DOX uses the linker MC-VC-PABC.This linker is stable in blood and gets cleaved by lysosomal enzymes, therefore releasing active drug in target cancer cells. The PABC part could couple to-OH group or-NH2group of drugs, and the MC part could react with-SH group of an antibody. MC-VC-PABC-DOX was synthetized by a method reported by Gene M. Dubowchik et al. Fmoc-Val-OSu, L-Citrulline (L-Cit), p-aminobenzyl alcohol (PABOH),6-Maleimidohexanoic acid N-hydroxysuccinimide ester (MC-OSu) and so on were used to construct MC-VC-PABC-DOX. The reactions were monitored by MS and H-NMR and the end-product could release free drug DOX after enzymolysis.
Maytansinol DM1was synthetized according to patents. Firstly, AP3was reduced to APO. After re-esterification and reduction, APO was transformed into DM1. DM1can be conjugated to Abs through several linkers.
2. Synthesis and biological activity of traditional antibody-drug conjugates
After the modification of small molecule drugs, we chose anti-CD20antibody TGLA as the carrier,-vcDOX/-vcMMAE/DM1as the conjugated drugs to explore the conjugation conditions and cytotoxicity of conjugates.
Cysteine sulfhydryl groups were activated by reducing interchain disulfide bonds of TGLA with DTT. In theory, there would be8-SH groups,2light chains each with1-SH group and2heavy chains each with3-SH groups. In this research, about6DOX were conjugated to each antibody in average after the reaction. SDS-PAGE indicated that most of the light chains had been conjugated with DOX, nevertheless, the heavy chains were separated on SDS-PAGE for the heavy molecule weight. Compared with Anti-CD20Ab, the anti-CD20-vcDOX conjugates exhibited an enhanced effect on killing the CD20-positive cells (Raji cells). Since the cytotoxicity of DOX is not potent enough, the increased cytotoxicity of ADCs is not significant.
The conjugation condition for vcMMAE was the same with vcDOX, and MMAE/Ab ratio was about6.4.
The ratio of DM1/TGLA for TGLA-SPDP-DM1was about3.5. The DM1was released from ADCs in the presence of DTT. The ratio of DM1/TGLA for TGLA-MCC-DM1was about2.9. MMAE and DM1ADCs significantly improved the activity of TGLA in vitro, while DOX which owns lower toxicity than MMAE or DM1only resulted in a slight increase in cytotoxicity of TGLA. This result suggested us to choose highly toxic drugs as conjugated drugs.
3. Study of different anti-CD20-vcMMAE conjugates
Studies on ADCs usually focus on a selected Ab and various drugs or linkers.There is almost no report demonstrating the difference caused by different antibodies against the same antigen. Here, we synthesized three anti-CD20-vcMMAE conjugates: Ofatumumab-vcMMAE, Rituximab-vcMMAE and TGLA-vcMMAE, and then evaluated the affinity, CDC, internalization and cytotoxicity of these three types against CD20-positive human B lymphoma cells, namely, Raji, Daudi and WIL2-S cells.
As a result, conjugation of anti-CD20mAbs with MMAE caused a modest decrease in the binding affinity to cell surface CD20. All anti-CD20-vcMMAE conjugates were modulated from the cell surface and subsequently entered the lysosomes through the CD20receptor-mediated endocytosis. In addition, the conjugation of MMAE significantly improved the cytotoxic activity of all anti-CD20mAbs against CD20-positive cell lines. The results of Annexin V/PI staining and WB demonstrate that treatment of CD20-positive tumor cells with anti-CD20-vcMMAE conjugates could induce apoptotic cell death through a caspase-3-like protease-dependent pathway. We also observed variable cytotoxicity between the anti-CD20mAbs (i.e., OFA, RTX and TGLA) with the same MMAE conjugated. OFA-vcMMAE exhibited great anti-tumor activity in xenograft models of CD20-positive lymphoma where OFA, equivalent free MMAE or irrelevant Herceptin-vcMMAE was ineffective. These results stimulated us to choose OFA as the carrier to carry out mutation and conjugation.
4. Construction and expression of2F2and its mutant Thio-2F2
In the traditional study, ADCs were synthetized by conjugating cytotoxic drugs to antibodies either through lysine side-chain amines or through cysteine sulfhydryl groups activated by reducing interchain disulfide bonds. Both procedures bring some disadvantages:1) The reaction of interchain disulfide bonds or surface amine groups may result in structural changes in Abs, sabotaging binding affinity of the antibody.2) Traditional procedures yield heterogenous products, containing a mixture of species with different molar ratios of drug to antibody. To solve the problems Jagath R Junutula et al. in Genentech have engineered reactive cysteine residues at specific sites in the constant region of antibodies to allow drugs to be conjugated with defined stoichiometry without disrupting the interchain disulfide bonds. One such ADCs exhibits high efficacy, low side effect and long half-time in vivo. Here we used the method reported by Jagath R Junutula to modify and conjugate OFA, hoping to improve the direct toxic effect under the condition of keeping the affinity, CDC and ADCC activity of antibody on tumor cells.
We found DNA sequences of OFA in the US2004/0167319A15patents (namely2F2) and then construct the2F2gene carriers and stably express the Ab in CHO. Meanwhile, we performed mutation on the heavy chain constant region of2F2, that is changing an alanine (A) into a cysteine (C)(Thio-2F2). At last we proved the affinity and CDC activity of2F2and Thio-2F2preliminarily.
5Synthesis and biological activity of Thio-2F2-vcN
We conjugated vcMMAE to the specific sites in the constant region of Thio-2F2with defined stoichiometry. Experiments confirmed the mutation and conjugation had little influence in affinity, CDC, ADCC activity of2F2. In addition, the conjugation of MMAE significantly improved the cytotoxic activity of Thio-2F2against CD20-positive cell lines. Thio-2F2-vcMMAE exhibited great anti-tumor activity in xenograft models of CD20-positive lymphoma at doses where primary antibody and mutant antibody were ineffective.
The most important discoveries in this paper
1. We synthesized various anti-CD20-drug conjugates and obtained several unreported high-activity conjugates, and demonstrated that the conjugation of high-toxic drugs could significantly improve the cytotoxic activity of anti-CD20mAbs against CD20-positive cell lines. Here, we built a technology platform of constructing anti-CD20-drug conjugates.
2. We explored the mechanism of various anti-CD20-vcMMAE conjugates and found that the cytotoxicity was different between the anti-CD20mAbs (i.e., OFA, RTX and TGLA) after conjugation with the same toxic drug, broadening the design ideas of the ADCs. The new ADC OFA-vcMMAE showed excellent anti-tumor activity in vitro and in vivo. It completely inhibited the growth of two lymphoma xenografts and the recurrence rates were very low.
3. We designed a2F2mutant which could conjugate drugs at specific sites and with defined stoichiometry, ensuring the homogeneity of the products; Experiments confirmed the mutation and conjugation had little influence in affinity, CDC, ADCC activity of2F2. In addition, the conjugation of MMAE significantly improved the cytotoxic activity of Thio-2F2. This method provides a reference for the designs of other antibodies or proteins-drugs conjugates.
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9. Burke PJ, Toki BE, Meyer DW, Miyamoto JB, Kissler KM, et al. (2009) Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system. Bioorg Med Chem Lett 19:2650-2653.
10. Breccia M, Lo-Coco F (2011) Gemtuzumab ozogamicin for the treatment of acute promyelocytic leukemia:mechanisms of action and resistance, safety and efficacy. Expert Opin Biol Ther 11:225-234.
11. Adair JR, Howard PW, Hartley JA, Williams DG, Chester KA (2012) Antibody-drug conjugates-a perfect synergy. Expert Opin Biol Ther 12:1191-1206.
12.李菁等(2009).基因工程抗体研究进展.生物技术通报10：40-44.
13. Sharkey RM, Goldenberg DM (2006) Targeted therapy of cancer:new prospects for antibodies and immunoconjugates. CA Cancer J Clin 56:226-243.
14.邵荣光(2010).高效小型化抗肿瘤抗体药物的研究展望.医学研究杂志39：3-4
15. Omelyanenko V, Kopeckova P, Gentry C, Shiah JG, Kopecek J (1996) HPMA copolymer-anticancer drug-OV-TL16 antibody conjugates.1. influence of the method of synthesis on the binding affinity to OVCAR-3 ovarian carcinoma cells in vitro. J Drug Target 3:357-373.
16.甄永苏(2007).抗体药物与肿瘤靶向治疗.医学研究杂志36：1-2.
17. Zahnd C, Kawe M, Stumpp MT, de Pasquale C, Tamaskovic R, et al. (2010) Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size. Cancer Res 70:1595-1605.
18. Law CL, Cerveny CG, Gordon KA, Klussman K, Mixan BJ, et al. (2004) Efficient elimination of B-lineage lymphomas by anti-CD20-auristatin conjugates. Clin Cancer Res 10:7842-7851.
19. Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, et al. (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10:7063-7070.
20. McDonagh CF, Turcott E, Westendorf L, Webster JB, Alley SC, et al. (2006) Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment. Protein Eng Des Sel 19:299-307.
21. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, et al. (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26:925-932.
22. Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, et al. (2010) Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin Cancer Res 16:4769-4778.
23. Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, et al.(2012) Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A.109:16101-16106
24. Dubowchik GM, Firestone RA, Padilla L, Willner D, Hofstead SJ, et al. (2002) Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates:model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug Chem 13:855-869.
25. Ojima I, Geng X, Wu X, Qu C, Borella CP, et al. (2002) Tumor-specific novel taxoid-monoclonal antibody conjugates. J Med Chem 45:5620-5623.
26. Senter PD (2009) Potent antibody drug conjugates for cancer therapy. Curr Opin Chem Biol 13:235-244.
27. Lambert JM (2005) Drug-conjugated monoclonal antibodies for the treatment of cancer. Curr Opin Pharmacol 5:543-549.
28. Chari RV (1998) Targeted delivery of chemotherapeutics:tumor-activated prodrug therapy. Adv Drug Deliv Rev 31:89-104.
29. Singh R, Erickson HK (2009) Antibody-cytotoxic agent conjugates:preparation and characterization. Methods Mol Biol 525:445-467, xiv.
30. Takeshita A, Yamakage N, Shinjo K, Ono T, Hirano I, et al. (2009) CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells. Leukemia 23: 1329-1336.
31. Isakoff SJ, Baselga J (2011) Trastuzumab-DM1:building a chemotherapy-free road in the treatment of human epidermal growth factor receptor 2-positive breast cancer. J Clin Oncol 29:351-354.
32. Krop IE, Beeram M, Modi S, Jones SF, Holden SN, et al. (2010) Phase I study of trastuzumab-DM1, an HER2 antibody-drug conjugate, given every 3 weeks to patients with HER2-positive metastatic breast cancer. J Clin Oncol 28: 2698-2704.
33. Burris HA,3rd, Rugo HS, Vukelja SJ, Vogel CL, Borson RA, et al. (2011) Phase II study of the antibody drug conjugate trastuzumab-DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy. J Clin Oncol 29:398-405.
34. Gerber HP, Kung-Sutherland M, Stone I, Morris-Tilden C, Miyamoto J, et al. (2009) Potent antitumor activity of the anti-CD 19 auristatin antibody drug conjugate hBU12-vcMMAE against rituximab-sensitive and -resistant lymphomas. Blood 113:4352-4361.
35. Dornan D, Bennett F, Chen Y, Dennis M, Eaton D, et al. (2009) Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma. Blood 114:2721-2729.
36. Sun Y, Yu F, Sun BW (2009) Antibody-drug conjugates as targeted cancer therapeutics. Yao Xue Xue Bao 44:943-952.
37. Amadori S, Stasi R (2006) Monoclonal antibodies and immunoconjugates in acute myeloid leukemia. Best Pract Res Clin Haematol 19:715-736.
38. Ichikawa M, Hangaishi A, Nannya Y, Kurokawa M (2010) Postremission gemtuzumab ozogamicin for elderly patients with acute myelogenous leukemia with favorable characteristics and comorbid conditions. Int J Hematol 92: 673-674.
39. Mahato R, Tai W, Cheng K(2011) Prodrugs for improving tumor targetability and efficiency. Adv Drug Deliv Rev.63:659-670.
40. S Suttur M, R Mysore S, Krishnamurthy B, B Nallur R (2009) Rare association of Turner syndrome with neurofibromatosis type 1 and tuberous sclerosis complex. Indian J Hum Genet 15:75-77.
41. Chari RV (2008) Targeted cancer therapy:conferring specificity to cytotoxic drugs. Acc Chem Res 41:98-107.
42. Stack GD, Walsh JJ (2012) Optimising the delivery of tubulin targeting agents through antibody conjugation. Pharm Res 29:2972-2984.
43. Ishitsuka K, Jimi S, Goldmacher VS, Ab O, Tamura K (2008) Targeting CD56 by the maytansinoid immunoconjugate IMGN901 (huN901-DM1):a potential therapeutic modality implication against natural killer/T cell malignancy. Br J Haematol 141:129-131.
44. Yoneda Y, Steiniger SC, Capkova K, Mee JM, Liu Y, et al. (2008) A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy. Bioorg Med Chem Lett 18:1632-1636.
45. Doronina SO, Toki BE, Torgov MY, Mendelsohn BA, Cerveny CG, et al. (2003) Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat Biotechnol 21:778-784.
46. Wahl AF, Klussman K, Thompson JD, Chen JH, Francisco LV, et al. (2002) The anti-CD30 monoclonal antibody SGN-30 promotes growth arrest and DNA fragmentation in vitro and affects antitumor activity in models of Hodgkin's disease. Cancer Res 62:3736-3742.
47. Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, et al. (2008) Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res 68:9280-9290.
1.戴垚,刘秀均,甄永苏(2006)抗IV型胶原酶单抗与平阳霉素新型免疫偶联物的抗肿瘤作用.药学学报41：41-46.
2. Burke PJ, Toki BE, Meyer DW, Miyamoto JB, Kissler KM, et al. (2009) Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system. Bioorg Med Chem Lett 19:2650-2653.
3. Breccia M, Lo-Coco F (2011) Gemtuzumab ozogamicin for the treatment of acute promyelocytic leukemia:mechanisms of action and resistance, safety and efficacy. Expert Opin Biol Ther 11:225-234.
4. Takeshita A, Yamakage N, Shinjo K, Ono T, Hirano I, et al. (2009) CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells. Leukemia 23: 1329-1336.
5. Isakoff SJ, Baselga J (2011) Trastuzumab-DM1:building a chemotherapy-free road in the treatment of human epidermal growth factor receptor 2-positive breast cancer. J Clin Oncol 29:351-354.
6. Krop IE, Beeram M, Modi S, Jones SF, Holden SN, et al. (2010) Phase Ⅰ study of trastuzumab-DM1, an HER2 antibody-drug conjugate, given every 3 weeks to patients with HER2-positive metastatic breast cancer. J Clin Oncol 28: 2698-2704.
7. Burris HA, Rugo HS, Vukelja SJ, Vogel CL, Borson RA, et al. (2011) Phase Ⅱ study of the antibody drug conjugate trastuzumab-DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy. J Clin Oncol 29:398-405.
8. Gerber HP, Kung-Sutherland M, Stone I, Morris-Tilden C, Miyamoto J, et al. (2009) Potent antitumor activity of the anti-CD 19 auristatin antibody drug conjugate hBU12-vcMMAE against rituximab-sensitive and -resistant lymphomas. Blood 113:4352-4361.
9. Dornan D, Bennett F, Chen Y, Dennis M, Eaton D, et al. (2009) Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma. Blood 114:2721-2729.
10. Sun Y, Yu F, Sun BW (2009) Antibody-drug conjugates as targeted cancer therapeutics. Yao Xue Xue Bao 44:943-952.
11. Dubowchik GM, Firestone RA, Padilla L, Willner D, Hofstead SJ, et al. (2002) Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates:model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug Chem 13:855-869.
12.张铂,吴梧桐(2004)海洋抗肿瘤活性蛋白与多肽的研究进展.中国海洋药物2：32-39
13. Attard G, Greystoke A, Kaye S, De BJ (2006) Update on tubulin-binding agents. Pathol Biol 54:72-84
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18. Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, et al. (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10:7063-7070.
19. McDonagh CF, Turcott E, Westendorf L, Webster JB, Alley SC, et al. (2006) Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment. Protein Eng Des Sel 19:299-307.
20. Braslawsky GR, Kadow K, Knipe J, McGoff K, Edson M, et al. (1991) Adriamycin(hydrazone)-antibody conjugates require internalization and intracellular acid hydrolysis for antitumor activity. Cancer Immunol Immunother 33:367-374.
21. Sapra P, Allen TM (2002) Internalizing antibodies are necessary for improved therapeutic efficacy of antibody-targeted liposomal drugs. Cancer Res 62: 7190-7194.
22. Goulet AC, Goldmacher VS, Lambert JM, Baron C, Roy DC, et al. (1997) Conjugation of blocked ricin to an anti-CD19 monoclonal antibody increases antibody-induced cell calcium mobilization and CD 19 internalization. Blood 90: 2364-2375.
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24. Dijoseph JF, Dougher MM, Armellino DC, Kalyandrug L, Kunz A, et al. (2007) CD20-specific antibody-targeted chemotherapy of non-Hodgkin's B-cell lymphoma using calicheamicin-conjugated rituximab. Cancer Immunol Immunother 56:1107-1117.
25. Michel RB, Mattes MJ (2002) Intracellular accumulation of the anti-CD20 antibody 1F5 in B-lymphoma cells. Clin Cancer Res 8:2701-2713.
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27. Doronina SO, Toki BE, Torgov MY, Mendelsohn BA, Cerveny CG, et al. (2003) Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat Biotechnol 21:778-784.
28. Teeling JL, Mackus WJ, Wiegman LJ, van den Brakel JH, Beers SA, et al. (2006) The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20. J Immunol 177:362-371.
29. Wierda WG, Padmanabhan S, Chan GW, Gupta IV, Lisby S, et al. (2011) Ofatumumab is active in patients with fludarabine-refractory CLL irrespective of prior rituximab:results from the phase 2 international study. Blood 118: 5126-5129.
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32. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, et al. (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26:925-932.
33. Ostergaard M, Baslund B, Rigby W, Rojkovich B, Jorgensen C, et al. (2010) Ofatumumab, a human anti-CD20 monoclonal antibody, for treatment of rheumatoid arthritis with an inadequate response to one or more disease-modifying antirheumatic drugs:results of a randomized, double-blind, placebo-controlled, phase Ⅰ/Ⅱ study. Arthritis Rheum 62:2227-2238.
34. Taylor PC, Quattrocchi E, Mallett S, Kurrasch R, Petersen J, et al. (2011) Ofatumumab, a fully human anti-CD20 monoclonal antibody, in biological-naive, rheumatoid arthritis patients with an inadequate response to methotrexate:a randomised, double-blind, placebo-controlled clinical trial. Ann Rheum Dis 70:2119-2125.
35. Zhang B (2009) Ofatumumab. MAbs 1:326-331.
36. Li B, Shi S, Qian W, Zhao L, Zhang D, et al. (2008) Development of novel tetravalent anti-CD20 antibodies with potent antitumor activity. Cancer Res 68: 2400-2408.
2. Berger M, Shankar V, Vafai A (2002) Therapeutic applications of monoclonal antibodies. Am J Med Sci 324:14-30.
3. Alley SC, Okeley NM, Senter PD (2010) Antibody-drug conjugates:targeted drug delivery for cancer. Curr Opin Chem Biol 14:529-537.
4.刘煜等(2003).对有效的抗肿瘤单抗药物的探索.药物生物技术10：194-198.
5.甄永苏(2000).单克隆抗体药物治疗肿瘤的研究现状与展望.中国医学科学院学报22：9-13.
6. Sapra P, Stein R, Pickett J, Qu Z, Govindan SV, et al. (2005) Anti-CD74 antibody-doxorubicin conjugate, IMMU-110, in a human multiple myeloma xenograft and in monkeys. Clin Cancer Res 11:5257-5264.
7. Moolten FL, Cooperband SR (1970) Selective destruction of target cells by diphtheria toxin conjugated to antibody directed against antigens on the cells. Science 169: 68-70.
8.戴垚,刘秀均,甄永苏(2006)抗IV型胶原酶单抗与平阳霉素新型免疫偶联物的抗肿瘤作用.药学学报41：41-46.
9. Burke PJ, Toki BE, Meyer DW, Miyamoto JB, Kissler KM, et al. (2009) Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system. Bioorg Med Chem Lett 19:2650-2653.
10. Breccia M, Lo-Coco F (2011) Gemtuzumab ozogamicin for the treatment of acute promyelocytic leukemia:mechanisms of action and resistance, safety and efficacy. Expert Opin Biol Ther 11:225-234.
11. Adair JR, Howard PW, Hartley JA, Williams DG, Chester KA (2012) Antibody-drug conjugates-a perfect synergy. Expert Opin Biol Ther 12:1191-1206.
12.李菁等(2009).基因工程抗体研究进展.生物技术通报10：40-44.
13. Sharkey RM, Goldenberg DM (2006) Targeted therapy of cancer:new prospects for antibodies and immunoconjugates. CA Cancer J Clin 56:226-243.
14.邵荣光(2010).高效小型化抗肿瘤抗体药物的研究展望.医学研究杂志39：3-4
15. Omelyanenko V, Kopeckova P, Gentry C, Shiah JG, Kopecek J (1996) HPMA copolymer-anticancer drug-OV-TL16 antibody conjugates.1. influence of the method of synthesis on the binding affinity to OVCAR-3 ovarian carcinoma cells in vitro. J Drug Target 3:357-373.
16.甄永苏(2007).抗体药物与肿瘤靶向治疗.医学研究杂志36：1-2.
17. Zahnd C, Kawe M, Stumpp MT, de Pasquale C, Tamaskovic R, et al. (2010) Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size. Cancer Res 70:1595-1605.
18. Law CL, Cerveny CG, Gordon KA, Klussman K, Mixan BJ, et al. (2004) Efficient elimination of B-lineage lymphomas by anti-CD20-auristatin conjugates. Clin Cancer Res 10:7842-7851.
19. Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, et al. (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10:7063-7070.
20. McDonagh CF, Turcott E, Westendorf L, Webster JB, Alley SC, et al. (2006) Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment. Protein Eng Des Sel 19:299-307.
21. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, et al. (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26:925-932.
22. Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, et al. (2010) Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin Cancer Res 16:4769-4778.
23. Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, et al.(2012) Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A.109:16101-16106
24. Dubowchik GM, Firestone RA, Padilla L, Willner D, Hofstead SJ, et al. (2002) Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates:model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug Chem 13:855-869.
25. Ojima I, Geng X, Wu X, Qu C, Borella CP, et al. (2002) Tumor-specific novel taxoid-monoclonal antibody conjugates. J Med Chem 45:5620-5623.
26. Senter PD (2009) Potent antibody drug conjugates for cancer therapy. Curr Opin Chem Biol 13:235-244.
27. Lambert JM (2005) Drug-conjugated monoclonal antibodies for the treatment of cancer. Curr Opin Pharmacol 5:543-549.
28. Chari RV (1998) Targeted delivery of chemotherapeutics:tumor-activated prodrug therapy. Adv Drug Deliv Rev 31:89-104.
29. Singh R, Erickson HK (2009) Antibody-cytotoxic agent conjugates:preparation and characterization. Methods Mol Biol 525:445-467, xiv.
30. Takeshita A, Yamakage N, Shinjo K, Ono T, Hirano I, et al. (2009) CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells. Leukemia 23: 1329-1336.
31. Isakoff SJ, Baselga J (2011) Trastuzumab-DM1:building a chemotherapy-free road in the treatment of human epidermal growth factor receptor 2-positive breast cancer. J Clin Oncol 29:351-354.
32. Krop IE, Beeram M, Modi S, Jones SF, Holden SN, et al. (2010) Phase I study of trastuzumab-DM1, an HER2 antibody-drug conjugate, given every 3 weeks to patients with HER2-positive metastatic breast cancer. J Clin Oncol 28: 2698-2704.
33. Burris HA,3rd, Rugo HS, Vukelja SJ, Vogel CL, Borson RA, et al. (2011) Phase II study of the antibody drug conjugate trastuzumab-DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy. J Clin Oncol 29:398-405.
34. Gerber HP, Kung-Sutherland M, Stone I, Morris-Tilden C, Miyamoto J, et al. (2009) Potent antitumor activity of the anti-CD 19 auristatin antibody drug conjugate hBU12-vcMMAE against rituximab-sensitive and -resistant lymphomas. Blood 113:4352-4361.
35. Dornan D, Bennett F, Chen Y, Dennis M, Eaton D, et al. (2009) Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma. Blood 114:2721-2729.
36. Sun Y, Yu F, Sun BW (2009) Antibody-drug conjugates as targeted cancer therapeutics. Yao Xue Xue Bao 44:943-952.
37. Amadori S, Stasi R (2006) Monoclonal antibodies and immunoconjugates in acute myeloid leukemia. Best Pract Res Clin Haematol 19:715-736.
38. Ichikawa M, Hangaishi A, Nannya Y, Kurokawa M (2010) Postremission gemtuzumab ozogamicin for elderly patients with acute myelogenous leukemia with favorable characteristics and comorbid conditions. Int J Hematol 92: 673-674.
39. Mahato R, Tai W, Cheng K(2011) Prodrugs for improving tumor targetability and efficiency. Adv Drug Deliv Rev.63:659-670.
40. S Suttur M, R Mysore S, Krishnamurthy B, B Nallur R (2009) Rare association of Turner syndrome with neurofibromatosis type 1 and tuberous sclerosis complex. Indian J Hum Genet 15:75-77.
41. Chari RV (2008) Targeted cancer therapy:conferring specificity to cytotoxic drugs. Acc Chem Res 41:98-107.
42. Stack GD, Walsh JJ (2012) Optimising the delivery of tubulin targeting agents through antibody conjugation. Pharm Res 29:2972-2984.
43. Ishitsuka K, Jimi S, Goldmacher VS, Ab O, Tamura K (2008) Targeting CD56 by the maytansinoid immunoconjugate IMGN901 (huN901-DM1):a potential therapeutic modality implication against natural killer/T cell malignancy. Br J Haematol 141:129-131.
44. Yoneda Y, Steiniger SC, Capkova K, Mee JM, Liu Y, et al. (2008) A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy. Bioorg Med Chem Lett 18:1632-1636.
45. Doronina SO, Toki BE, Torgov MY, Mendelsohn BA, Cerveny CG, et al. (2003) Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat Biotechnol 21:778-784.
46. Wahl AF, Klussman K, Thompson JD, Chen JH, Francisco LV, et al. (2002) The anti-CD30 monoclonal antibody SGN-30 promotes growth arrest and DNA fragmentation in vitro and affects antitumor activity in models of Hodgkin's disease. Cancer Res 62:3736-3742.
47. Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, et al. (2008) Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res 68:9280-9290.
1.戴垚,刘秀均,甄永苏(2006)抗IV型胶原酶单抗与平阳霉素新型免疫偶联物的抗肿瘤作用.药学学报41：41-46.
2. Burke PJ, Toki BE, Meyer DW, Miyamoto JB, Kissler KM, et al. (2009) Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system. Bioorg Med Chem Lett 19:2650-2653.
3. Breccia M, Lo-Coco F (2011) Gemtuzumab ozogamicin for the treatment of acute promyelocytic leukemia:mechanisms of action and resistance, safety and efficacy. Expert Opin Biol Ther 11:225-234.
4. Takeshita A, Yamakage N, Shinjo K, Ono T, Hirano I, et al. (2009) CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells. Leukemia 23: 1329-1336.
5. Isakoff SJ, Baselga J (2011) Trastuzumab-DM1:building a chemotherapy-free road in the treatment of human epidermal growth factor receptor 2-positive breast cancer. J Clin Oncol 29:351-354.
6. Krop IE, Beeram M, Modi S, Jones SF, Holden SN, et al. (2010) Phase Ⅰ study of trastuzumab-DM1, an HER2 antibody-drug conjugate, given every 3 weeks to patients with HER2-positive metastatic breast cancer. J Clin Oncol 28: 2698-2704.
7. Burris HA, Rugo HS, Vukelja SJ, Vogel CL, Borson RA, et al. (2011) Phase Ⅱ study of the antibody drug conjugate trastuzumab-DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy. J Clin Oncol 29:398-405.
8. Gerber HP, Kung-Sutherland M, Stone I, Morris-Tilden C, Miyamoto J, et al. (2009) Potent antitumor activity of the anti-CD 19 auristatin antibody drug conjugate hBU12-vcMMAE against rituximab-sensitive and -resistant lymphomas. Blood 113:4352-4361.
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