1、Antibody-drug conjugates as targeted cancer therapeuticsSUN Yu1, 2*, YU Fei1, SUN Bai-wang1(1. School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China;2. School of Pharmacy, Jiangsu University, Zhenjiang 212013, ChinaAbstract: Traditional chemotherapy has become one

2、 of the essential treatments of cancer. However, cytotoxic agents are not tumor specific, which would cause serious side effects. Antibody-drug conjugates (ADCs, also called immunoconjugates, belong to the “targeted chemotherapeutics” category of anti-cancer drugs. ADCs are composed of three compone

3、nts including the cytotoxic drug, the monoclonal antibody, and the linker connecting the drug to the antibody. With the special-binding between antibody and antigen expressed on the surface of targeted cancer cells, ADCs provide a method to achieve excellent localization of the drug at the desired s

4、ite in the body. The internalization and formation of ADCs are crucial in designing and applying an antibody conjugate to a particular disease model. In this review, we summarize three distinct internalization routes of ADCs and analysis the structure of ADCs. We also discuss in detail the categorie

5、s and interaction of every component, as well as their influence to targeting property, liability and activity.Key words: antibody-drug conjugate; targeted cancer therapeutics; internalization; componentCLC number: R916 Document code: A Article ID: 0513-4870 (2009 09-0943-10靶向癌癥治療試劑抗體-藥物偶聯(lián)物孫玉1, 2*,

6、于菲1, 孫柏旺1(1. 東南大學化學化工學院, 江蘇南京 211189; 2. 江蘇大學藥學院, 江蘇鎮(zhèn)江 212013摘要: 化療已經(jīng)成為癌癥治療的一個必要手段。然而, 細胞毒性試劑對腫瘤細胞缺少特異性, 導致嚴重的副反應?？贵w-藥物偶聯(lián)物 (ADCs, 也被稱為免疫偶聯(lián)物, 屬于靶向抗癌藥物的一種?？贵w-藥物偶聯(lián)物由藥物、抗體以及偶聯(lián)抗體和藥物的連接鍵三部分組成。當抗體和癌細胞表面的抗原特異性結(jié)合時, 抗體-藥物偶聯(lián)物即可將藥物成功靶向體內(nèi)部位?？贵w-藥物偶聯(lián)物的內(nèi)在化過程和組成部分在設計和應用抗體偶聯(lián)物到廣泛的疾病模型中至關重要。本文概述了抗體-藥物偶聯(lián)物內(nèi)在化的三個途徑, 并對它們的

7、結(jié)構(gòu)進行了分析, 詳細討論了各個組成部分的類型、相互作用及其對抗體-藥物偶聯(lián)物的靶向性、穩(wěn)定性和活性的影響。關鍵詞: 抗體-藥物偶聯(lián)物; 靶向癌癥治療; 內(nèi)在化; 構(gòu)成Chemotherapy is an integral to current cancer therapy, because it is effective in many cancers, such as acute childhood leukemia and Hodgkin disease. However, systemic administration of chemotherapeutic drugs not onl

8、y results in the death of cancer cells, but also has the undesired side effects of destroying oral and intestinalm u c o s a,h a i r f o l l i c l e s,a n d b o n e m a r r o w. Received 2009-01-05.*Corresponding author Tel / Fax: 86-25-52090614,E-mail: whsunyu Much attention has been directed to se

9、lective targetingof these agents by conjugation to antibodies against tumor specific markers, so as to improve efficacy and reduce side effects1, 2. Antibody-drug conjugates (ADCs, also called immunoconjugates, belong to the “targeted chemotherapeutics” category of anti-cancer drugs. They are compos

10、ed of one or several cytotoxic drug molecules covalently linked to a monoclonal antibody binding to an antigen expressed on the surfaceof targeted cancer cells. The linkers within theseconjugates are often designed to be cleaved through changes in pH, reduction, or by proteases so that the drug can

11、be preferentially released at the tumor site. As a result, ADCs can increase the effective drug concentration within the targeted area, thereby optimize the therapeutic effect of the agent. In addition, with targeted delivery, the clinician may be able to lower the dose of the therapeutic agent whic

12、h is particularly necessary if the drug payload has associated toxicities orif it is to be used in the treatment of chronic conditions.The concept of arming antibodies by conjugationto protein toxins dated back to 19703, and was followed by antibody conjugates with cytotoxic drugs a few years later.

13、 Antibody therapeutics have come of agein the intervening decades with the advent of hybridoma technology to develop murine monoclonal antibodies (MAb, chimerization and humanization to address the shortcomings of murine MAb as therapeutics, and more recently with direct routes to human antibodies u

14、sing phage display or transgenic mice. The clinical potentialof ADCs has been greatly enhanced by improved choices of targets, conjugated more potent drugs to improve stability and greatly expanded knowledge of ADC cell biology and pharmacology.Numerous preclinical efficacy studies illustrate that A

15、DCs have significant potential for enhancing the antitumor activity of “naked” antibodies and reducing the systemic toxicity of the conjugated drugs4,5. Furthermore, the FDA has granted approval for the first antibody drug conjugate for human therapeutic use in 2000. This conjugate (gemtuzumab ozoga

16、micin (Mylotarg® consists of a humanized anti-CD33 mAb linked to the cytotoxic antibiotic ozogamicin (N-acetyl- calicheamicin. It was licensed for the treatment of acute myeloid leukemia (AML in patients over sixty years of age and deemed unsuitable for cytotoxic chemotherapy6.1 Internalization

17、 of ADCsThe therapeutic concept of ADCs is to use an antibody as a vehicle to deliver a cytotoxic drug to a tumor cell by means of binding to the antigen on targeted cell surface. ADCs are commonly in IgG format with 28 drugs/antibody. After internalization into the targeted cell, ADCs can release d

18、rugs for activation.The major factor that contributes to the selective cytotoxicity of ADCs is the conjugated antibody that targets tumor-associated antigen, so the rates of internalization of ADCs should be compared to their respective antibodies. Some ADCs were found to internalize with rates simi

19、lar to those of their respective antibodies, such as conjugates of an anti-CD30 antibody with auristatin7. However it was reported that some ADCs were internalized more efficiently than the respective antibodies. These ADCs include the mAbs c1F6 (or cAC10 doxorubicin derivatives conjugates8, the ant

20、i-melanotransferrin antibody (or anti-CD20 auristatin conjugates9, the anti-CD79b auristatin F derivatives conjugates10 and so on.The internalization of ADCs are desirable and often indispensable for efficient drug release, depending on the drugs and linkers. Because antibodies cannot penetrate cell

21、ular membranes, they can only get inside mammalian cells via three distinct internalization routes: clathrin-mediated endocytosis, caveolae-mediated uptake, and pinocytosis. The first two types of antibody uptake are antigen-mediated, while the last one is antigen- independent. The antigen-mediated

22、pathways normally result in accumulation of ADCs on the plasma membrane at a local concentration higher than that of the surrounding medium, particularly if the antibody has a high affinity. On the other hand, pinocytosis occurs through sequestration of liquid which contains the ADC at the concentra

23、tion added to the medium. As a result, to achieve equal rates of internalization, a higher concentration of the ADC in the medium would be needed to deliver the same amount of conjugate via pinocytosis inside the cell than via an antigen-mediated pathway.The molecular mechanism of clathrin-mediated

24、endocytosis (Figure 1 can be characterized as follows11: To regain their cytotoxic activity, the cytotoxic agent has to be cleaved from the chemo-immunoconjugate. Uptake of antibodies predominantly occurs via the clathrin-mediated endocytosis pathway. After binding the respective antigen associated

25、with coated pits, antibody-drug conjugates will be readily endocytosed, from where they transit through several stages of transport and endosomal vesicles and finally end up in a lysosome. There, linkers and antibody will be cleaved releasing the cytotoxic agent which-after exit from the lysosomal c

26、ompartment-exerts its cytotoxic effect. For some antibodies and its ADCs, clathrin-mediated endocytosis is a major route for the intracellular uptake. It is reported that the cytotoxic activity of an anti-CD70 antibody and its auristatin conjugates was associatedSUN Yu, et al: Antibody-drug conjugat

27、es as targeted cancer therapeutics·945·with their internalization and subcellular trafficking through the endosomal-lysosomal pathway12. Also, the intracellular uptake of several auristatin conjugates of anti-CD30 mAb cAC10 and of trastuzumab is blocked by inhibitors of clathrin-mediated e

28、ndocytosis, but not by inhibitors of caveolae-mediated uptake. Thus these ADCs do not alter the fate of antibody- antigen complexes11,13. But the conjugation of anti- CD20 antibody and auristatin internalize via both clathrin-mediated endocytosis and the caveolin-mediated uptake, as indicated by co-

29、localization of this conjugate with clathrin and caveolin14. While at high concen-trations, ADCs may kill cells in an antigen-independent manner. Through examining the non-specific killing by gemtuzumab ozogamicin, the authors concluded that the cytotoxicity was derived from the ability of cells to

30、take up conjugate dissolved in the surrounding medium via pinocytosis15. However, an accurate quantification of the amount of pinocytosed ADCs at various concen-trations has not been reported. Figure 1Internalization of antibody-drug conjugates2 Considerations in design of effective ADCsEffective de

31、livery to the targeted cells is a prereq-uisite for high efficacy and low toxicity of any drug substance. Conjugation of a drug to an antibody provides a method to achieve excellent localization of the drug at the desired site in the body. Many kinds of antibodies, drugs, and linkers have been combi

32、ned to prepare ADCs. Cytotoxic drugs used in ADCs generally have potencies several orders of magnitude greater than for conventional chemotherapeutics, making them too potent for systemic delivery. Linkers used to attach the drugs to the antibody delivery vehicles have been designed to exploit intra

33、cellular conditions for drug release including the acidic environment of endosomes (pH 5.56.2 and lysosomes (pH 4.55.0, high thiol concentrations in the cytosol, and proteolytic enzymes in lysosomes. Thus, when designing and applying an antibody conjugate to a particular disease model, we need to ad

34、dress the basic conditions of drugs, antibodies and linkers used in ADCs.2.1 Cytotoxic drugs used in ADCsThe choice of drug payload is critical because it would affect desired efficacy towards the targeted disease and its stoichiometry, orientation, or associated chemistry of conjugation to the anti

35、body would hinder biological activity of the antibody. Up to now, about six categories of highly cytotoxic drugs have been con-jugated to monoclonal antibodies: anthracycline drugs, CC-1065 analogs and duoearmycin analogs, taxoids derivatives, calicheamicin derivatives, maytansinoids, dolastatin der

36、ivatives. Early ADCs exploited conven-tional anti-cancer drugs (e.g. doxorubicinand16, tax-ane17 because of their ready availability, amenabilityto chemical manipulation and their well known toxico-logical properties. But these ADCs were moderately potent and usually less cytotoxic for the targeted

37、tumor cells than the corresponding unconjugated drugs. Nowadays, many novel ADCs focus on such highly potent drugs as calicheamicins, maytansinoids and auristatins. Because new cytotoxic agents possess the following properties18: high potency in vitro toward tumor cell lines, with IC50 values in the

38、 range of 0.010.1 nmol·L1 (i.e., active in the concentration range of antibody binding to tumor cells; a suitable functional group for linkage to an antibody (if a functional group is not already present, the desired substituent has to be introduced at a suitable site to retain potency of the p

39、arent drug; reasonable solubility in aqueous solutions to enable the reaction with antibodies, and prolonged stability in aqueous formulations commonly used for antibodies.The following three tables list the components and applications in cancer therapy of some representative ADCs (antibody calichea

40、micin conjugates, antibody maytansinoid conjugates, antibody auristatin conjugates. The particular properties and the internalization routes will be further elaborated in the following parts.2.2 Antibody engineering contribute to ADCsAntibodies are extremely versatile targeting proteins. Molecular e

41、ngineering has been used to modify the size and pharmacokenetic properties of antibodies, alter the valency of antigen binding, and even alter effector activity. The main purpose of this·946·藥學學報Acta Pharmaceutica Sinica 2009, 44 (9: 943952Table 1 Antibody calicheamicin conjugatesConjugate

42、 name Drug Antibody Linker classMean Drug/Antibody ratioCancer ReferenceAnti-CD33 antibody calicheamicin conjugates CalichDMH,CalichDMAP67.6, CMA676Hydrazone linker,amide linker23 Acutemyeloidleukemia(AML19Anti-CD20 antibody calicheamicin conjugates CalichDMH,CalichDMARituximab AcButlinker,amide lin

43、ker23 non-HodgkinsB-celllymphoma13Anti-MUC1 antibody calicheamicin conjugates CalichDMH,CalichDMAhCTM01 Hydrazonelinker,amide linker23 Breast and ovarian tumor 20Anti-CD22 antibody calicheamicin conjugates CalichDMH CMC-544 AcButlinker 57 B-lymphomamalignancies 21Anti-Lewis y antibody calicheamicin

44、conjugates CalichDMH hu3S193 AcBut linker 2, 3 Cancer cells with highlyexpressed Lewis y22CalichDMH: N-acetyl calicheamicin dimethylhydrazide; CalichDMA: N-acetyl calicheamicin dimethyl acid; AcBut linker: an acid-labile 4-(4-acetylphenoxy butanoic acid linker including a hydrazone linker and a disu

45、lfide linkerTable 2 Antibody maytansinoid conjugatesConjugate name Drug Antibody Linker classMean Drug/Antibody ratioCancer ReferencehuC242 Antibodymaytansinoid conjugatesDM1, DM4 huC242 Disulfide linker 34 Cancer with CanAg antigen 23Anti-CD138+ antibody maytansinoid conjugates DM1 B-B4 Disulfideli

46、nker 3.5CD138+ Multiple myelomacells24Anti-CD56 antibody maytansinoid conjugate DM1 huN901 Disulfidelinke 34CD56+ Multiple myelomacells25DM1: N-deacetyl-N-(3-mercapto-1-oxopentyl-maytansine; DM4: N-deacetyl-N-(4-mercapto-4-methyl-1-oxopropyl-maytansineTable 3Antibody auristatin conjugatesConjugate n

47、ame Drug Antibody Linker classMean Drug/Antibody ratioCancer ReferenceCR011-vcMMAE MMAE CR011 Peptidelinker 2.7 Melanoma 26 Anti-CD70 auristatinconjugatesMMAF, AFP m1F6, c1F6 Peptide linker 4, 8 Renal cell carcinoma 27Anti-CD79 auristatin conjugates MMAF CD79a,CD79bMaleimidocaproyllinkerNA non-Hodgk

48、inlymphoma(NHL10Anti-CD30 auristatin conjugates MMAE,MMAFcAc10 Hydrazonelinker,dipeptides linker2, 4, 8 CD30+ malignant cells 28Anti-MUC16 THIOMABauristatin conjugatesMMAE THIOMAB Disulfide linker 2 Ovarian cancer 29 MMAE: monomethyl auristatin E; MMAF: monomethyl auristatin F; AFP: auristatin pheny

49、lalanine phenylenediamine; NA: not available; THIOMAB: engineered antibodies which are created by replacing Ala114 at the junction of the CH1 domain and the variable heavy chain domain with cysteinework is to produce new mAbs, mAb fragments, and mAb constructs that have both high tumor to non-tumor

50、binding ratios and high intratumoral localization characteristics. The research show that ADCs prepared with these Fab fragments demonstrate a more homogeneous affinity than any conjugates prepared with the whole antibody30, 31. Another feasible engi-neering approach was to replace the solvent-acces

51、sible cysteines forming the interchain disulfide bonds in cAC10 with serine, and in this way, to reduce the eight potential conjugation sites down to 2 or 4. These CysSer antibody variants were conjugated to MMAE in near quantitative yield (89%96% with defined stoichiometries (2 or 4 drugs/antibody

52、and sites of drug attachment. This strategy for generating antibody- drug conjugates with defined sites and stoichiometries of drug loading didnt change the antigen-binding affinities, in vitro cytotoxic activities and in vivo properties28. A novel method was used to improve the therapeutic index of

53、 ADCs by engineering cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups. This THIOMAB approach provided reactive thiol groups and did not perturb immunoglobulin folding and assembly, or alter antigen binding29. So we can conclude that antibody engineerin

54、g will undoubtedly play an extremelySUN Yu, et al: Antibody-drug conjugates as targeted cancer therapeutics·947·important role in the new generation of gradually developing drug conjugates. Until now functional antibodies that are currently approved for the treatmentof cancer include Ritux

55、an (rituximab for B-cell lymphomas, Herceptin (trastuzumab for breast cancer, Campath (alemtuzumab for certain leukemias, and Erbitux (cetuximab, Vectibix (panitumumab, and Avastin (bevacizumab for colorectal cancers.2.3 Linker category for conjugationThe linker between the antibody and drug has to

56、be designed in a manner that not only ensures stability during circulation in blood but allows the rapid releaseof an active form of the cytotoxic drug inside the targeted tumor cells. Furthermore, the conjugate must remain intact during storage in aqueous solution to allow formulations for convenie

57、nt intravenous admini-stration. So, conjugation technology is a critical aspect in generating effective ADCs and optimization strategies which are varied with the drug, linker, and the antibody used. Nowadays, there are largely three classes of linkers that used in ADCs: hydrazone linker, disulfide

58、linker and peptide linker. And the data of research indicate that each type of drug might require its own class of linker. The acid-labile hydrazone linker mainly exists in antibody-calicheamicin conjugate, the optimal linker for maytansinoid-antibody conjugateis found to be a hindered disulfide moiety and the optimal linker for auristatin-antibody conjugate appearsto be a peptide linker that is readily cleaved in lysosomal compartments. The following discussion talks mainly about the characters of linkers and their influence to drug release and internalization.The antibody