The ABC of ADCs (Antibody-Drug Conjugates): A Comprehensive Review of Technical, Regulatory, and Clinical Challenges

Over the past several decades, there has been a significant surge in the development of Antibody-Drug Conjugates (ADCs). Designing an ideal ADC presents a multifaceted challenge, requiring the precise orchestration of various elements such as antigens, antibodies, linkers, and payloads. While ADCs aim to target tumor cells specifically, several antigens can also be found in regular tissues, potentially compromising the specificity of ADCs in therapeutic applications. The complexity extends to antibody selection, necessitating effective targeting of the desired antigen and ensuring compatibility with linkers for effective payload delivery. Additionally, the linker and payload combination are critical for the ADC’s therapeutic efficiency, balancing stability in circulation and timely payload release upon target binding. ADC doses must be safe for normal tissues while ensuring the released payloads are effective. The success of ADCs is attributed to their unmatched efficacy compared to traditional chemotherapy agents. The current research article aims to provide a technical review of Antibody-Drug Conjugates (ADCs) for cancer therapies. A brief discussion on the basics of ADCs, regulatory approach, overview, and technical complexities for quantification is presented. This review also summarizes recently approved ADCs and introduces the concepts of antibodies, linkers, and payloads. The article also outlines cancer-specific ADCs currently in late-stage clinical trials for cancer treatment.

Antibody-Drug Conjugates (ADCs): Navigating Four Pillars of Safety, Development, Supply Chain and Manufacturing Excellence

Antibody-drug conjugates (ADCs) are pioneering biologics that merge antibodies’ specificity with small molecules’ potency. With a handful of FDA-approved ADCs in the market and many under development, ADCs are poised to revolutionize therapeutics. This paper examines the complexities of ADC production, emphasizing the importance of process characterization and the pivotal role of supply chain characteristics, safety requirements, and Contract Manufacturing Organizations (CMOs) with proficiency. The swift transition of antibody-drug conjugate (ADC) programs from early to advanced clinical stages underscores the urgency for quick and efficient commercial launch preparation. This article delves into strategies to hasten commercial readiness, supply chain strategy, the significance of partnering with adept contract development and manufacturing organizations (CDMOs), and the challenges of ADC production.

TCR-mimic antibody-drug conjugates targeting intracellular tumor-specific mutant antigen KRAS G12V mutation

Limited clinical application of antibody-drug conjugates(ADCs)targeting tumor associated antigens(TAAs)is usually caused by on-target off-tumor side effect.Tumor-specific mutant antigens(TSMAs)only expressed in tumor cells which are ideal targets for ADCs.In addition,intracellular somatic mutant proteins can be presented on the cell surface by human leukocyte antigen class I(HLA I)molecules forming tumor-specific peptide/HLA I complexes.KRAS G12 V mutation frequently occurred in varied cancer and was verified as a promising target for cancer therapy.In this study,we generated two TCR-mimic antibodydrug conjugates(TCRm-ADCs),2E8-MMAE and 2 A5-MMAE,targeting KRAS G12 V/HLAA*0201 complex,which mediated specific antitumor activity in vitro and in vivo without obvious toxicity.Our findings are the first time validate the strategy of TCRm-ADCs targeting intracellular TSMAs,which improves the safety of antibody-based drugs and provides novel strategy for precision medicine in cancer therapy.

Current LC-MS-based strategies for characterization and quantification of antibody-drug conjugates

The past few years have witnessed enormous progresses in the development of antibody-drug conjugates(ADCs).Consequently,comprehensive analysis of ADCs in biological systems is critical in supporting discovery,development and evaluation of these agents.Liquid chromatography-mass spectrometry(LC-MS)has eme rged as a promising and versatile tool for ADC analysis across a wide range of scenarios,owing to its multiplexing ability,rapid method development,as well as the capability of analyzing a variety of targets ranging from small-molecule payloads to the intact protein with a high,molecular resolution.However,despite this tremendous potential,challenges persist due to the high complexity in both the ADC molecules and the related biological systems.This review summarizes the up-to-date LC-MS-based strategies in ADC analysis and discusses the challenges and opportunities in this rapidly-evolving field.

Enzyme-linked immunosorbent assays for quantification of MMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosorbent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were successfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation between serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias%ranged from -12.2% to -5.2%,precision ranged from -12.4% to -1.4%,and the relative standard deviation(RSD)was less than 6.6% and 8.7%,respectively.The total error was less than 20.4%.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.

Disitamab vedotin,a HER2-directed antibody-drug conjugate,in patients with HER2-overexpression and HER2-low advanced breast cancer:a phase I/Ib study

Background:Disitamab vedotin(DV;RC48-ADC)is an antibody-drug conjugate comprising a human epidermal growth factor receptor 2(HER2)-directed antibody,linker and monomethyl auristatin E.Preclinical studies have shown that DV demonstrated potent antitumor activity in preclinical models of breast,gastric,and ovarian cancers with different levels of HER2 expression.In this pooled analysis,we report the safety and efficacy of DV in patients with HER2-overexpression and HER2-low advanced breast cancer(ABC).Methods:In the phase I dose-escalation study(C001 CANCER),HER2-overexpression ABC patients received DV at doses of 0.5-2.5 mg/kg once every two weeks(Q2W)until unacceptable toxicity or progressive disease.The dose range,safety,and pharmacokinetics(PK)were determined.The phase Ib dose-range and expansion study(C003 CANCER)enrolled two cohorts:HER2-overexpression ABC patients receiving DV at doses of 1.5-2.5 mg/kg Q2W,with the recommended phase 2 dose(RP2D)determined,andHER2-lowABC patients receiving DV at doses of 2.0 mg/kg Q2W to explore the efficacy and safety of DV in HER2-low ABC.Results:Twenty-four patients with HER2-overexpression ABC in C001 CANCER,46 patients with HER2-overexpressionABCand 66 patients with HER2-low ABC in C003 CANCER were enrolled.At 2.0 mg/kg RP2D Q2W,the confirmed objective response rates were 42.9%(9/21;95%confidence interval[CI]:21.8%-66.0%)and 33.3%(22/66;95%CI:22.2%-46.0%),with median progression-free survival(PFS)of 5.7 months(95%CI:5.3-8.4 months)and 5.1 months(95%CI:4.1-6.6 months)for HER2-overexpression and HER2-low ABC,respectively.Common(≥5%)grade 3 or higher treatment-emergent adverse events included neutrophil count decreased(17.6%),gamma-glutamyl transferase increased(13.2%),asthenia(11.0%),white blood cell count decreased(9.6%),peripheral neuropathy such as hypoesthesia(5.9%)and neurotoxicity(0.7%),and pain(5.9%).Conclusion:DV demonstrated promising efficacy in HER2-overexpression and HER2-low ABC,with a favorable safety profile at 2.0 mg/kg Q2W.

Trastuzumab-Doxorubicin Conjugate Provides Enhanced Anti-Cancer Potency and Reduced Cardiotoxicity

Since trastuzumab monotherapy for treatment of breast cancer with HER2/ErbB2 over-expression has been shown to have limited efficacy, combined treatment of trastuzumab with chemotherapy is widely practiced in clinic. However, certain combination treatments of trastuzumab and chemotherapy (i.e. doxorubicin) are not recommended due to high risk of cardiotoxicity. Antibody-drug conjugates (ADCs) offer selective delivery of cytotoxic agents into targeted cancer cells, thereby allowing for reduced general cellular cytotoxicity caused by chemotherapeutic agents through antibody mediated specific recognition of tumor antigens. In this study, we constructed a trastuzumab-doxorubicin conjugate (T-Dox) using a thioether linkage and characterized both biophysical stability and anti-cancer potency of the T-Dox using a panel of HER2 expressing cancer cell lines. The T-Dox conjugate showed significantly improved anti-cancer potency in comparison with trastuzumab. The results demonstrated for the first time that there were significant differences in the uptake of T-Dox among high HER2 expression cancer cells and higher T-Dox uptake also showed stronger anti-cancer potency. Similar to trastuzumab, T-Dox selectively bound to HER2 overexpressing cancer cells and low HER2 expression cells had no detectable uptake of T-Dox. Consistent to the uptake data, human cardiomyocyte cells had no detectable HER2 expression and T-Dox showed minimal cytotoxic effects. On the contrary, a treatment with combination of trastuzumab and doxorubicin showed severe cytotoxicity to human cardiomyocytes (>90% cell death after 3 day exposure). This study demonstrated that trastuzumab conjugated with doxorubicin (T-Dox) can provide valuable alternative to the combination treatment with doxorubicin and trastuzumab for high HER2 expressing cancer patients.

2024年欧洲肿瘤内科学会乳腺癌年会研究进展

2024年5月举行的欧洲肿瘤内科学会乳腺癌年会(European Society for Medical Oncology Breast Cance,ESMO BC)在德国柏林召开。本次年会展示了一系列针对乳腺癌治疗的最新进展,包括创新疗法和诊治策略。特别值得关注的是,细胞周期蛋白依赖性激酶4和6(cyclin-dependent kinases 4 and 6,CDK4/6)抑制剂联合内分泌治疗(endocrine therapy,ET)在早期乳腺癌新辅助治疗中的研究尚未达到主要终点,而探索性分析结果表明,该领域仍有待进一步研究。CDK4/6抑制剂联合ET继续作为高危患者辅助治疗的标准。同时,阿贝西利在携带BRCA1/2突变的患者亚组中显示出初步的一致性获益。瑞波西利虽然不良反应可控,但仍需进行密切监测。新型CDK4选择性抑制剂、AKT抑制剂、抗体药物偶联物(antibody-drug conjugates,ADC)以及免疫治疗为晚期乳腺癌患者提供了新的治疗选项。本次年会所展现的研究成果,不仅代表了乳腺癌治疗领域的进步,也为未来的研究方向和患者治疗策略提供了新的视野。本文就此次年会上重点的研究进展进行综述。

乳腺癌受体阳转阴的发生机理与治疗进展

乳腺癌因其高度异质性,给乳腺癌精准治疗带来挑战。雌激素受体(estrogen receptor, ER)、孕激素受体(progesterone receptor, PR)和人表皮生长因子受体-2(human epidermal growth factor receptor-2, HER2)状态是乳腺癌精准诊疗的重要依据。对于复发或转移性乳腺癌患者,既往治疗主要基于原发病灶受体状态。但研究发现部分患者复发/转移灶受体表达与原发病灶不同,推测可能与肿瘤异质性和治疗后的克隆选择效应相关。相较于受体表达“阴转阳”,受体“阳转阴”的发生率更高,且受体表达的缺失可能导致对原有疗法耐药且预后不良。重新评估乳腺癌复发/转移灶受体状态对调整治疗策略和判断预后具有显著临床意义,但能否基于受体变化情况指导临床决策,在临床研究或实践层面仍存在较大争议。随着靶向药物、免疫疗法及抗体偶联药物(antibody-drug conjugates, ADC)等新型抗肿瘤药物的应用,如何优化受体“阳转阴”乳腺癌患者的治疗方案以提高疗效成为未解决的临床需求。该文旨在深入探讨乳腺癌受体“阳转阴”的机理、预后影响以及治疗现状,为这类患者的治疗提供理论支持和实践指导。

抗体偶联药物在晚期胃癌治疗中的研究进展

胃癌发生隐匿,恶性程度较高,对传统放化疗不敏感,预后差,因此,研发新型抗胃癌药物迫在眉睫。抗体偶联药物(Antibody-drug conjugate,ADC)是由单克隆抗体经特定的连接子与细胞毒药物连接而成的一类靶向生物制剂,具有高效、靶向、精准、低毒等优势。目前,ADC已广泛用于治疗血液系统肿瘤及实体肿瘤,并在晚期胃癌的治疗中展现出良好疗效,具有广阔的应用前景。

Resistance to antibody-drug conjugates in breast cancer:mechanisms and solutions

Antibody-drug conjugates(ADCs)are a rapidly developing therapeutic approach in cancer treatment that has shown remarkable activity in breast cancer.Currently,there are two ADCs approved for the treatment of human epidermal growth factor receptor 2-positive breast cancer,one for triple-negative breast cancer,and multiple investigational ADCs in clinical trials.However,drug resistance has been noticed in clinical use,especially in trastuzumab emtansine.Here,the mechanisms of ADC resistance are summarized into four categories:antibodymediated resistance,impaired drug trafficking,disrupted lysosomal function,and payload-related resistance.To overcome or prevent resistance to ADCs,innovative development strategies and combination therapy options are being investigated.Analyzing predictive biomarkers for optimal therapy selection may also help to prevent drug resistance.

抗体偶联药物诱导的间质性肺病的发病机制

药物性间质性肺病(drug-induced interstitial lung disease,DI-ILD)是药物所致肺部不良事件的最常见形式,其最常见原因是抗癌药物。DI-ILD首次被确定为一种需要特别关注的不良事件是因抗体偶联药物(antibody-drug conjugates,ADC)。随着研发药物数量和种类的增多、多款ADC药物被批准或即将被批准用于临床治疗,DI-ILD引发了越来越多的关注。然而,ADC药物引发DI-ILD的机制尚未明确,本文就目前常见且应用广泛的抗人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)ADC药物DI-ILD的发生机制进行综述。

A mild phenoxysilyl linker for self-immolative release of antibody-drug conjugates

Self-immolative linkers have been widely used to construct prodrugs to improve their efficacy and safety.In this study,we report the use of phenoxysilyl linker as a self-immolative unit to prepare antibody-drug conjugates(ADCs).Phenoxysily based ADC Ate-PPS-CA4 was prepared and its release was systematically investigated by mass spectrometry.Biological evaluation showed that Ate-PPS-CA4 displayed the ability to target delivery and self-immolative release the active payload CA4 on PD-L1 positive cells MDA-MB-231.As the same with its payload CA4,it could arrest the cell cycle to the G2/M phase and induced changes in cell morphology at the dose of its IC_(50).The development of this linker with novel drug release mechanisms will expand the methodology to construct ADCs,especially for non-internalizing ADCs by extracellular cleavage.

HER3-targeted therapeutic antibodies and antibody-drug conjugates in non-small cell lung cancer refractory to EGFR-tyrosine kinase inhibitors

Human epidermal growth factor receptor 3(HER3)is a unique member of the human epidermal growth factor receptor(HER/EGFR)family,since it has negligible kinase activity.Therefore,HER3 must interact with a kinase-proficient receptor to form a heterodimer,leading to the activation of signaling cascades.Overexpression of HER3 is observed in various human cancers,including non-small cell lung cancer(NSCLC),and correlates with poor clinical outcomes in patients.Studies on the underlying mechanism demonstrate that HER3-initiated signaling promotes tumor metastasis and causes treatment failure in human cancers.Upregulation of HER3 is frequently observed in EGFR-mutant NSCLC treated with EGFR-tyrosine kinase inhibitors(TKIs).Increased expression of HER3 triggers the so-called EGFR-independent mechanism via interactions with other receptors to activate“by-pass signaling pathways”,thereby resulting in resistance to EGFR-TKIs.To date,no HER3-targeted therapy has been approved for cancer treatment.In both preclinical and clinical studies,targeting HER3 with a blocking an-tibody(Ab)is the only strategy being examined.Recent evaluations of an anti-HER3 Ab-drug conjugate(ADC)show promising results in patients with EGFR-TKI-resistant NSCLC.Herein,we summarize our understanding of the unique biology of HER3 in NSCLC refractory to EGFR-TKIs,with a focus on its dimerization partners and subsequent activation of signaling pathways.We also discuss the latest development of the therapeutic Abs and ADCs targeting HER3 to abrogate EGFR-TKI resistance in NSCLC.

靶向Trop2的抗体偶联药物治疗三阴性乳腺癌的研究进展

乳腺癌是目前全球发病率最高的恶性肿瘤之一,其中三阴性乳腺癌(triple-negative breast cancer,TNBC)占浸润性乳腺癌的10%~20%。TNBC是一种高度异质性和侵袭性的恶性肿瘤,与其他乳腺癌亚型相比,TNBC治疗手段相对匮乏,预后较差,临床上亟待寻找可用于精准治疗及提高预后的新靶点。人滋养层细胞表面抗原2(trophoblast cell surface antigen 2,Trop2)在三阴性乳腺癌及多种恶性肿瘤中高表达,其通过细胞表面受体信号在肿瘤细胞的增殖、黏附、侵袭、转移中发挥重要作用,以其为靶点的抗体偶联药物(antibody-drug conjugate,ADC)在临床上中具有广阔的应用前景。本文对靶向Trop2的ADC治疗TNBC的临床研究进展予以综述,为靶向Trop2的ADC在TNBC治疗中的临床应用和提高患者生存预后方面提供参考。

Targeting triple-negative breast cancer:A clinical perspective

Triple-negative breast cancer(TNBC)is a disease with often an aggressive course and a poor prognosis compared to other subtypes of breast cancer.TNBC accounts for approximately 10%–15%of all diagnosed breast cancer cases and represents a high unmet need in the field.Up to just a few years ago,chemotherapy was the only systemic treatment option for this subtype(1).To date,TNBC is considered a heterogeneous disease.One of the existing classifications is based on the analysis of mRNA expression in 587 TNBC cases,in which Lehman et al.proposed six subtypes of TNBC as follows:two basal-like(BL1 and BL2)subtypes,a mesenchymal(M)subtype,a mesenchymal stem-like(MSL)subtype,an immunomodulatory(IM)subtype,and a luminal androgen receptor(LAR)subtype(2).Later studies have demonstrated that the IM and MSL subtypes do not correlate with independent subtypes but reflect background expression by dense infiltration of tumor-infiltrating lymphocytes(TILs)or stromal cells.According to this finding,the classification of TNBC has been revised into the following four subtypes:basal 1,basal 2,LAR,and mesenchymal subtypes(3).Over the last years,several new strategies have been investigated for the treatment of patients with TNBC.Among them,immunotherapy,antibody drug conjugates,new chemotherapy agents,and targeted therapy have been and are currently being developed.The present article aims to provide an updated overview on the different treatment options that are now available or are still under investigation for patients with TNBC.

Bispecific antibodies targeting immunomodulatory checkpoints for cancer therapy

Advances in antibody engineering have led to the generation of more innovative antibody drugs,such as bispecific antibodies(bs Abs).Following the success associated with blinatumomab,bs Abs have attracted enormous interest in the field of cancer immunotherapy.By specifically targeting two different antigens,bs Abs reduce the distance between tumor and immune cells,thereby enhancing tumor killing directly.There are several mechanisms of action upon which bs Abs have been exploited.Accumulating experience on checkpoint-based therapy has promoted the clinical transformation of bs Abs targeting immunomodulatory checkpoints.Cadonilimab(PD-1×CTLA-4)is the first approved bs Ab targeting dual inhibitory checkpoints,which confirms the feasibility of bs Abs in immunotherapy.In this review we analyzed the mechanisms by which bs Abs targeting immunomodulatory checkpoints and their emerging applications in cancer immunotherapy.

Antibody-drug conjugates： recent advances in conjugation and linker chemistries

The antibody-drug conjugate （ADC）, a humanized or human monoclonal antibody conjugated with highly cytotoxic small molecules （payloads） through chemical linkers, is a novel therapeutic format and has great potential to make a paradigm shift in cancer chemother- apy. This new antibody-based molecular platform enables selective delivery of a potent cytotoxic payload to target cancer cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics （PK）I pharmacodynamics （PD） and biodistribution compared to traditional chemotherapy. Boosted by the successes of FDA-approved Adcetris~ and Kadcyla~, this drug class has been rapidly growing along with about 60 ADCs cur- rently in clinical trials. In this article, we briefly review molecular aspects of each component （the antibody, payload, and linker） of ADCs, and then mainly discuss traditional and new technologies of the conjugation and linker chemistries for successful construction of clini- cally effective ADCs. Current efforts in the conjugation and linker chemistries will provide greater insights into molecular design and strategies for clinically effective ADCs from medicinal chemistry and pharmacology standpoints. The development of site-specific conjuga- tion methodologies for constructing homogeneous ADCs is an especially promising path to improving ADC design, which will open the way for novel cancer therapeutics.

Antibody-drug conjugates:Recent advances in linker chemistry

Antibody-drug conjugates(ADCs)are gradually revolutionizing clinical cancer therapy.The antibody-drug conjugate linker molecule determines both the efficacy and the adverse effects,and so has a major influence on the fate of ADCs.An ideal linker should be stable in the circulatory system and release the cytotoxic payload specifically in the tumor.However,existing linkers often release payloads nonspecifically and inevitably lead to off-target toxicity.This defect is becoming an increasingly important factor that restricts the development of ADCs.The pursuit of ADCs with optimal therapeutic windows has resulted in remarkable progress in the discovery and development of novel linkers.The present review summarizes the advance of the chemical trigger,linker-antibody attachment and linker-payload attachment over the last 5 years,and describes the ADMET properties of ADCs.This work also helps clarify future developmental directions for the linkers.

Antibody-drug conjugates in HER2-positive breast cancer

Antibody-drug conjugates(ADCs)combine the high specificity of monoclonal antibodies with the high anti-tumor activity of small molecular cytotoxic payloads.The anti-tumor activity of ADCs is mainly achieved by the direct blocking of the receptor by monoclonal antibodies,direct action and bystander effect of cytotoxic drugs,and antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.ADCs have been used in adjuvant therapy and rescue treatment of human epidermal receptor 2(HER2)-positive breast cancer,greatly improving the prognosis of breast cancer patients.Several ongoing clinical trials of ADC for breast cancer and other solid tumors proved the potential of ADCs will provide more promising treatment options for patients with malignant tumors.This review introduces the mechanism and latest clinical progress of ADC drugs approved for HER2-positive breast cancer to guide clinical practice and conduct research.