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.

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.

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.

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.

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.

Advances in preclinical evaluation of experimental antibody-drug conjugates

The ability to chemically modify monoclonal antibodies with the attachment of specific functional groups has opened up an enormous range of possibilities for the targeted treatment and diagnosis of cancer in the clinic.As the number of such antibody-based drug candidates has increased,so too has the need for more stringent and robust preclinical evaluation of their in vivo performance to maximize the likelihood that time,research effort,and money are only spent developing the most effective and promising candidate molecules for translation to the clinic.Concurrent with the development of antibody-drug conjugate(ADC)technology,several recent advances in preclinical research stand to greatly increase the experimental rigor by which promising candidate molecules can be evaluated.These include advances in preclinical tumor modeling with the development of patient-derived tumor organoid models that far better recapitulate many aspects of the human disease than conventional subcutaneous xenograft models.Such models are amenable to genetic manipulation,which will greatly improve our understanding of the relationship between ADC and antigen and stringently evaluate mechanisms of therapeutic response.Finally,tumor development is often not visible in these in vivo models.We discuss how the application of several preclinical molecular imaging techniques will greatly enhance the quality of experimental data,enabling quantitative pre-and post-treatment tumor measurements or the precise assessment of ADCs as effective diagnostics.In our opinion,when taken together,these advances in preclinical cancer research will greatly improve the identification of effective candidate ADC molecules with the best chance of clinical translation and cancer patient benefit.

CLINICAL APPLICATION OF MONOCLONAL ANTIBODY-DRUG CONJUGATE TO IMMUNOTARGETING CHEMOTHERAPY OF BLADDER CANCER

Monoclonal antibody-drug conjugate was applied in a clinical trial for patients with bladder cancer, Monoclonal antibody HB7A from a mouse splenocyte immunized against human bladder cancer was used as a drug carrier, The anti-cancer drug adriamycin (ADR) was bound to HB7A through a dextran (DEX) bridge to form the conjugate HB7A-DEX-ADR. The in vitro cytotoxic effect of the conjugate on BIU-87 bladder cancer cells was similar to that of free ADR and the mixture of HB7A and ADR, Seven patients with bladder cancer were given HB7A-DEX-ADR intravenously. The immunoperoxidast studies of the resected specimens showed that HB7A was localized specifically in cancer, and histological studies revealed degenerative and necrotic changes of the tumor cells, Patients receiving the conjugate did not experience serious side effects, This study suggests that immunotargeting chemotherapy with HB7A-DEX-ADR is well tolerated by patients and its cytotoxicity on tumor is substantial.

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.

Drug resistance and new therapies in colorectal cancer

Colorectal cancer(CRC) is often diagnosed at an advanced stage when tumor cell dissemination has taken place. Chemo-and targeted therapies provide only a limited increase of overall survival for these patients. The major reason for clinical outcome finds its origin in therapy resistance. Escape mechanisms to both chemo-and targeted therapy remain the main culprits. Here, we evaluate major resistant mechanisms and elaborate on potential new therapies. Amongst promising therapies is α-amanitin antibodydrug conjugate targeting hemizygous p53 loss. It becomes clear that a dynamic interaction with the tumor microenvironment exists and that this dictates therapeutic outcome. In addition, CRC displays a limited response to checkpoint inhibitors, as only a minority of patients with microsatellite instable high tumors is susceptible. In this review, we highlight new developments with clinical potentials to augment responses to checkpoint inhibitors.

Natural Products in Cancer Therapy: Past, Present and Future

Natural products,with remarkable chemical diversity,have been extensively investigated for their anticancer potential for more than a half-century.The collective efforts of the community have achieved the tremendous advancements,bringing natural products to clinical use and discovering new therapeutic opportunities,yet the challenges remain ahead.With remarkable changes in the landscape of cancer therapy and growing role of cutting-edge technologies,we may have come to a crossroads to revisit the strategies to understand nature products and to explore their therapeutic utility.This review summarizes the key advancements in nature product-centered cancer research and calls for the implementation of systematic approaches,new pharmacological models,and exploration of emerging directions to revitalize natural products search in cancer therapy.

Strategies to enhance monoclonal antibody uptake and distribution in solid tumors

Despite the significant resources dedicated to the development of monoclonal antibody(m Ab)therapies for solid tumors,the clinical success,thus far,has been modest.Limited efficacy of m Ab in solid tumors likely relates to unique aspects of tumor physiology.Solid tumors have an aberrant vasculature and a dense extracellular matrix that slow both the convective and diffusive transport of m Abs into and within tumors.For m Abs that are directed against cellular antigens,high antigen expression and rapid antigen turnover can result in perivascular cells binding to and eliminating a significant amount of extravasated m Ab,limiting m Ab distribution to portions of the tumor that are distant from functional vessels.Many preclinical investigations have reported strategies to improve m Ab uptake and distribution;however,to our knowledge,none have translated into the clinic.Here,we provide an overview of several barriers in solid tumors that limit m Ab uptake and distribution and discuss approaches that have been utilized to overcome these barriers in preclinical studies.

Monoclonal antibody-based cancer therapies

Targeted therapy has been widely demonstrated as an effective strategy to treat cancers,the leading cause of death in the world.This minireview summarizes the technical platforms and methodologies utilized to develop and engineer therapeutic monoclonal antibodies and antibody-drug conjugates.First,the USA FDA approved monoclonal antibody(mAb)-based targeted therapies are reviewed.Then the representative innovative chimeric,humanized and fully human anti-cancer antibodies and antibody-drug conjugates are described.Finally,the past and predictive market trend of therapeutic antibodies is discussed.

Human epidermal growth factor receptor 2 targeted therapy in endometrial cancer:Clinical and pathological perspectives

Endometrial cancer is the most common gynecological cancer in developed countries,and its incidence has increased.The majority of patients with endometrial cancer have an early disease and favorable prognosis;however,a significant proportion of endometrial cancer,which mainly comprises high-grade or type II endometrial cancer such as serous,clear cell,and carcinosarcoma,shows advanced/recurrent disease and dismal prognosis.Novel therapeutic development is required for patients with aggressive endometrial cancers.Recent genomic and immunohistochemical analyses revealed human epidermal growth factor receptor 2(HER2)overexpression/gene amplification in 20%-40%of patients with type II endometrial cancer.Historically,HER2 targeted therapy has been developed for various major cancers,including breast and gastric cancer.Notably,recent advances in HER2 targeted therapy for patients with type II endometrial cancer are also expected to change.Simultaneously,an optimized HER2 test for endometrial cancer as companion diagnostics should be established.In this review,we summarize the recent findings on endometrial cancer,current treatment,optimized HER2 testing,key clinical trials on HER2 targeted therapy,and future directions in aggressive endometrial cancer,including serous carcinoma and carcinosarcoma.

Modified Therapeutic Antibodies: Improving Efficacy

The prosperity of the biotherapeutics market reflects the feasibility and effectiveness of therapeutic antibodies for the treatment of cancers,inflammatory disorders,and refractory infections.As drawbacks emerge in clinical trials and practice,such as impeded binding,reduced effector functions,and frequent adverse reactions,modifications of therapeutic antibodies are unprecedently burgeoning in research and development(R&D).These modifications include:①modified glycosylation;②fragment of crystallizable domain(Fc)amino acid alterations;③cross-isotype or cross-subclass exchanges;④antibody–drug conjugates(ADCs);⑤single chain of variable region fragment(scFv)for chimeric antigen receptor T(CAR-T)cells;and⑥bispecific antibodies(bsAbs)in order to promote binding affinity,half-life in circulation,effectiveness toward target cells and,ultimately,to achieve overall improved efficacy.While many achievements have been made around the world in the past decades,China has been playing an active role in this realm,with its great demand for biotherapeutics with R&D potential.This review recapitulates the international progress that has been achieved with modified therapeutic antibodies,and then focuses on that of China in an independent section.

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.