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Abstract

The targeted delivery of potent cytotoxic molecules into cancer cells is considered a promising anticancer strategy. The design of clinically effective antibody–drug conjugates (ADCs), in which biologically active drugs are coupled through chemical linkers to monoclonal antibodies, has presented challenges for pharmaceutical researchers. After 30 years of intensive research and development activities, only seven ADCs have been approved for clinical use; two have received fast-track designation and two breakthrough therapy designation from the Food and Drug Administration. There is continued interest in the field, as documented by the growing number of candidates in clinical development. This review aims to summarize the most recent innovations that have been applied to the design of ADCs undergoing early- and late-stage clinical trials. Discovery and rational optimization of new payloads, chemical linkers, and antibody formats have improved the therapeutic index of next-generation ADCs, ultimately resulting in improved clinical benefit for the patients.

Keywords

antibody–drug conjugate cytotoxic payload site-specific conjugation alternative formats

Introduction

Approximately a hundred years have passed since Paul Ehrlich proposed the concept of antibodies as the “magic bullet” for selective targeting of malignant cells.¹ More recently, progress in hybridoma technologies and recombinant DNA techniques has enabled the generation of highly effective monoclonal antibodies (mAbs) ^2–8 to be developed for therapeutic purposes. However, in spite of the conceptual simplicity of antibody–drug conjugates (ADCs) as a therapeutic modality, the design, generation, and clinical use of these therapies have proved challenging. After 30 years of intense research and development activities, only seven ADCs have been granted clinical approval ( Table 1 ), while about 70 ADCs are undergoing clinical trials ( Table 2 ).

Table 1.

Marketed ADCs.

Sponsor	INN or Code name	Target	Antibody	Payload	ConjugationMethod	Approval	Launched Indication	Main Toxicities	References
Pfizer	Mylotarg Gemtuzumab ozogamicin CMA-676	CD33	Humanized IgG4κ	N-acetyl-γ calicheamicin 1,2-dimethyl hydrazine dichloride	Lys, DAR2-3	FDA approval May 17, 2000 FDA withdrawal June 1, 2010 FDA approval September 1, 2017 EMA approval April 19, 2018	Acute myeloid leukemia	Thrombocytopenia, neutropenia, hepatotoxicity including VOD	20–23, 25
Seattle Genetics, Inc.	Adcetris Brentuximab vedotin SGN35	CD30	Chimeric IgG1	mc-vc-PABC-MMAE	Interchain Cys, DAR4	FDA approval August 19, 2011 EMA approval October 25, 2012	Hodgkin lymphoma, anaplastic large cell lymphoma, CD30-expressing mycosis fungoides	Thrombocytopenia, neutropenia, peripheral neuropathy	35
Roche	Kadcyla Trastuzumab emtansine T-DM1	HER2	Humanized IgG1	SMCC-DM1	Lys, DAR3.5	FDA approval February 22, 2013 EMA approval November 15, 2013	HER2+ metastatic breast cancer	Thrombocytopenia, neutropenia, peripheral neuropathy	29–34
Pfizer	Besponsa Inotuzumab ozogamicin CMC-544	CD22	Humanized IgG4	N-acetyl-γ calicheamicin 1,2-dimethyl hydrazine dichloride	Lys, DAR5-7	EMA approval June 29, 2017 FDA approval August 17, 2017	B-cell acute lymphoblastic leukemia	Thrombocytopenia, neutropenia, hepatotoxicity including VOD	26
Roche	Polivy Polatuzumab vedotin-piiq RG-7596	CD79b	Humanized IgG1κ	mc-vc-PABC-MMAE	Interchain Cys, DAR4	FDA approval June 10, 2019	Relapsed or refractory diffuse large B-cell lymphoma	Thrombocytopenia, neutropenia, peripheral neuropathy	36
Astellas Pharma, Inc.	Padcev Enfortumab vedotin ASG-22ME	Nectin4	Human IgG1κ	mc-vc-PABC-MMAE	Interchain Cys, DAR4	FDA approval December 18, 2019	Locally advanced or metastatic urothelial cancer	Peripheral neuropathy	53
Daiichi Sankyo, Inc.	Enhertu Trastuzumab deruxtecan DS-8201	HER2	Humanized IgG1κ	mc-GGFG-DXd	Interchain Cys, DAR8	FDA approval December 20, 2019	HER2+ unresectable or metastatic breast cancer	Thrombocytopenia, neutropenia, interstitial lung disease	41

EMA, European Medicines Agency; INN, international nonproprietary name.

Table 2.

Clinical Stage ADCs.

Sponsor	INN or Code Name	Target	Antibody	Payload	Conjugation Method	Clinical Indications	References and Notes
Phase III
Bio-Thera	BAT8001	HER2	Humanized IgG1	Batansine	Cys, DAR3.5	Metastatic breast cancer	96
ImmunoGen, Inc.	IMGN-853 Mirvetuximab soravtansine	FRα	Humanized IgG1	Sulfo-SPDB-DM4	Lys, DAR3-4	Epithelial ovarian cancer, peritoneal cancer, fallopian tube cancer	87 Fast-track designation
Immunomedics, Inc.	IMMU-132 Sacituzumab govitecan	TROP2	Humanized IgG1κ	Cl2A-SN38	Interchain Cys, DAR7.6	Metastatic breast cancer/triple-negative breast cancer	130–133, 136 Breakthrough designation
Synthon Biopharmaceuticals BV	SYD985 Trastuzumab duocarmazine	HER2	Humanized IgG1κ	vc-PABC-EDA-seco-DUBA	Interchain Cys, DAR2.8	Breast cancer	124, 191 Fast-track designation
Phase II
Abbvie	ABBV-399 Telisotuzumab vedotin	C-Met	Bivalent humanized IgG1κ	mc-vc-PABC-MMAE	Interchain Cys, DAR3.1	Non-small cell lung cancer	55
ADC Therapeutics S.A.	ADCT-402 Loncastuximab tesirine	CD19	Humanized IgG1	SG3249	Interchain Cys, DAR2.3	Diffuse large B-cell lymphoma	113
Astellas Pharma, Inc.	AGS-16C3F	ENPP3	Human IgG2a	mc-MMAF	Interchain Cys, DAR4	Renal cell carcinoma	65
Bayer	BAY94-9343 Anetumab ravtansine	MSLN	Human IgG1λ	SPDB-DM4	Lys, DAR3.2	Non-small cell lung cancer, malignant pleural mesothelioma	83
Debiopharm Group	DEBIO 1562 Naratuximab emtansine	CD37	Humanized IgG1	SMCC-DM1	Lys, DAR3-4	Diffuse large B-cell lymphoma, non-Hodgkin lymphoma	88
GlaxoSmithKline	GSK2857916 Belantamab mafodotin	BCMA	Afucosylated humanized IgG1	mc-MMAF	Cys	Multiple myeloma	66, 67 Breakthrough designation
Genmab	HuMax-TF-ADC Tisotumab vedotin	TF (CD142)	Human IgG1κ	mc-vc-PABC-MMAE	Interchain Cys, DAR4	Cervical cancer, ovarian cancer, solid tumors	56
RemeGen	RC-48-ADC Hertuzumab vedotin	HER2	Humanized IgG1	mc-vc-PABC-MMAE	Interchain Cys, DAR4	Urothelial cancer, gastric cancer, breast cancer	57
Seattle Genetics, Inc. Hoffmann-La Roche	SGN-LIV1A Ladiratuzumab vedotin	LIV1	Humanized IgG1κ	mc-vc-PABC-MMAE	Interchain Cys, DAR4	Breast cancer	58
Phase I/II
Klus Pharma, Inc.	A166	HER2	Humanized IgG1	Duostatin-5	Unknown	HER2-expressing or amplified cancers
ADC Therapeutics S.A.	ADCT-602Epratuzumab-cys-tesirine	CD22	Humanized EC-IgG1	SG3249	Engineered Cys DAR1.7	B-cell acute lymphoblastic leukemia	117
Forbius	AVID100	EGFR	IgG	DM1	Unknown	Advanced epithelial carcinomas
BioAtla LLC	BA3011	AXL	CAB humanized IgG1	Cleavable linker–MMAE	Unknown	Solid tumors	181
BioAtla LLC	BA3021	ROR2	CAB humanized IgG1	Unknown	Unknown	Solid tumors	182
Biotest Pharmaceuticals Corporation	BT-062Indatuximab ravtansine	CD138	Chimeric IgG4	SPDB-DM4	Lys, DAR3.5	Multiple myeloma	84
CytomX Therapeutics	CX-2009	CD166	Probody	SPDB-DM4	Lys	Solid tumors	82
CytomX Therapeutics	CX-2029	CD71	Probody	mc-vc-PABC-MMAE	Cys, DAR2	Solid tumors or diffuse large B-cell lymphoma	54
Genmab	HuMax-AXL-ADCEnapotamab vedotin	AXL	Human IgG1κ	mc-vc-PBAC-MMAE	Interchain Cys, DAR4	Solid tumors	59
Immunomedics, Inc.	IMMU-130Labetuzumab govitecan	CEACAM5	Humanized IgG1κ	Cl2A-SN38	Interchain Cys, DAR7.6	Colorectal cancer	134, 135
MacroGenics	MGC018	B7-H3	Humanized IgG1	vc-PABC-EDA-seco-DUBA	Interchain CysDAR2.7	Solid tumors	125
Tarveda Therapeutics	PEN-221	SSTR2	Pentarin	DM1	NA	Neuroendocrine and small cell lung cancers	187
Tarveda Therapeutics	PEN-866	HSP90	Pentarin	SN38	NA	Solid tumors	189
Sanofi	SAR408701	CEACAM5	Humanized IgG1	SPDB-DM4	Lys, DAR3.5	Solid tumors	85
Daiichi Sankyo, Inc.	U3-1402	HER3	Humanized IgG	mc-GGFG-DXd	Interchain Cys, DAR8	Metastatic breast cancer	42
Pierre Fabre Medicament	W0101	IGF1R	IgG	Auristatin derivative	Unknown	Solid tumors
Phase I
AbbVie	ABBV-011	Unknown	IgG	Unknown	Unknown	Small cell lung cancer
Abbvie	ABBV-085Samrotamab vedotin	LRRC15	Humanized IgG1κ	mc-vc-PABC-MMAE	Interchain Cys, DAR2	Solid tumors	60
Abbvie	ABBV-155	Unknown	IgG	Unknown	Unknown	Solid tumors
Abbvie	ABBV-321	EGFR	IgG	PBD	Unknown	Advanced solid tumors
AbGenomics	ABGn-107	AG7	Humanized IgG	Hydrophilic self-immolative dipeptide linker–dolastatin analog	Unknown	Gastric, colorectal, pancreatic, or biliary cancer
ADC Therapeutics S.A.	ADCT-301Camidanlumab tesirine	CD25	Human Hu-Max IgG1	SG3249	Interchain Cys, DAR2.3	Acute myeloid leukemia, acute lymphoblastic leukemia, Hodgkin and non-Hodgkin lymphoma, advanced solid tumors	114
ADC Therapeutics S.A.	ADCT-601	AXL	Humanized IgG1	SG3249	Glycoconnect Technology, DAR2	Advanced solid tumors	115
Astellas Pharma, Inc.	AGS62P1	FLT3	Engineered (AmbrX technology) human IgG1κ	AGL-0182-30	pAF, DAR2	Acute myeloid leukemia	68
Alteogen, Inc.	ALT-P7	HER2	Engineered (NexMab technology) humanized IgG1κ	MMAE	C-Term Cys, DAR2	Breast cancer	149
Amgen	AMG224	BCMA	Human IgG1	SMCC-DM1	Lys	Multiple myeloma	89
Ambrx, Inc.	ARX788	HER2	Engineered (AmbrX technology) IgG1	AS269	pAF, DAR1.9	HER2-expressing cancers	71
Shanghai Pharmaceuticals Holding Co., Ltd	B003	HER2	Humanized IgG	SMCC-DM1	Unknown	Metastatic breast cancer
Bio-Thera	BAT8003	TROP2	Engineered afucosylated IgG1	Batansine	Engineered Cys	Advanced epithelial cancer	97
Bicycle	BT1718	MT1-MMP	Bicyclic peptide	Hindered disulfide-DM1	NA	Advanced solid tumors	188
Genentech, Inc.	DHES0815A	HER2	IgG	PBD-MA	Unknown	Breast cancer
Daiichi Sankyo, Inc.	DS-1062	TROP-2	Humanized IgG1	mc-GGFG-Dxd	Interchain Cys, DAR4	Advanced solid tumors	43
Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd.	F0002-ADC	CD30	Chimeric IgG1κ	SMCC-DM1	Lys, DAR3.4	Hematologic malignancies	90
Fortis Therapeutics, Inc.	FOR-46	CD46	Human IgG	Unknown	Unknown	Prostate cancer, multiple myeloma
Jiangsu HengRui Medicine Co., Ltd.	HTI-1066	c-Met	Humanized IgG2	Microtubule inhibitor	Unknown	Advanced solid tumors
ImmunoGen, Inc.	IMGN632	CD123	Humanized thiomAb IgG1	DGN549	Engineered Cys, DAR2	Hemathological malignancies	122
ImmunoGen, Inc.	IMGN-779	CD33	Humanized IgG1	Sulfo-SPDB-DGN462	Lys, DAR3	Acute myeloid leukemia	121
Medimmune LLC	MEDI2228	BCMA	Engineered human IgG	Protease cleavable PBD	Site specific	Multiple myeloma
Medimmune LLC	MEDI3726	PSMA	Engineered humanized IgG1κ	SG3249	Interchain Cys, DAR2	Prostate cancer	116
Medimmune LLC	MEDI7247	ASCT2	Human IgG	Protease cleavable PBD	Site specific, DAR2	Hematological malignancies, solid tumors
Menarini Group	MEN1309	CD205	Humanized IgG1	SPDB-DM4	Unknown	Metastatic solid tumors, non-Hodgkin lymphoma	86
Eisai, Inc.	MORAb-202	FRα	Humanized IgG1	mal-PEG2-vc-PABC-Eribulin	Interchain Cys, DAR4	Solid tumors	104
Shangai Miracogen	MRG002	Her2	IgG	Unknown	Unknown	Breast cancer, gastric cancer
Shangai Miracogen	MRG003	Unknown	IgG	Unknown	Unknown	Head and neck cancer, colorectal carcinoma
Novartis Pharmaceuticals	NJH395	HER2	IgG	TLR7 ligand	Engineered Cys, DAR4	Non-breast HER2+ advanced cancer
Pfizer	PF-06647020Cofetuzumab pelidotin	PTK7/CCK4	Humanized IgG1κ	mc-vc-PABC-Aur0101	Interchain Cys, DAR4	Advanced solid tumors, metastatic triple-negative breast cancer	73
Pfizer	PF-06688992	GD3	IgG	PF-06464368	Unknown	Melanoma
Pfizer	PF-06804103	HER2	IgG	mc-vc-PABC-Aur0101	Site specific	Solid tumors	72
Seattle Genetics, Inc.	SGN-CD48A	CD48	Humanized IgG	PEGylated glucuronide-MMAE	Interchain Cys, DAR8	Multiple myeloma	76
Sutro Biopharma, Inc.	STRO-001	CD74	Aglycosylated human IgG1	SC236	pAMF, DAR2	Advanced B-cell malignancies	157
Sutro Biopharma, Inc.	STRO-002	FRα	Human IgG1	SC239	pAMF, DAR4	Ovarian and endometrial cancers	158
Millennium Pharmaceuticals, Inc.	TAK-164	GCC	Human IgG1	DGN549	Unknown	Gastrointestinal cancers	123
Tanabe Research Laboratories USA, Inc.	TR1801-ADC	c-MET	IgG	PBD	Site specific	c-MET-expressing tumors
Triphase R & D III Corp.	TRPH-222	CD22	Engineered (SMARTag technology) humanized IgG	4AP protease-sensitive linker–maytansinoid	fGly, DAR2	B-cell lymphoma	153
Mersana Therapeutics	XMT-1536	NaPi2b	Humanized IgG	Auristatin F-HPA	DAR10-15	Cancers likely to express NaPi2b	79
Zymeworks, Inc.	ZW49	HER2	Biparatopic IgG	Protease cleavable linker–acyl sulfonamide auristatin	Interchain Cys	HER2+ cancers	173
Phase I other therapeutic areas
AbbVie	ABBV-3373	TNF	IgG	Steroid	Unknown	Rheumatoid arthritis
Genentech, Inc.	DSTA4637S	Staphylococcus aureus	THIOMAB engineered human IgG1	vc-dmDNA31	Engineered Cys, DAR2	Staphylococcus aureus bacteremia	147

Information based on pipelines, National Cancer Institute (NCI) definitions, AdisInsight information, databases, clinical trials, press releases, reviews, and bibliographies. INN, international nonproprietary name; NA, not applicable.

The initial appeal of ADCs as a concept was the theoretical improvement they afforded to antibody therapies, in addition to increasing the therapeutic index of highly potent cytotoxic compounds.^9–12 Indeed, the conjugation of small molecules to tumor-selective antibodies is intended to increase their specific retention and cytotoxic activity at the site of tumors, while avoiding activity on healthy tissues. The combination of these actions is to limit undesirable side effects commonly seen with conventional chemotherapeutic drugs.

ADCs are prodrugs ( Fig. 1 ). The antibody is connected to the cytotoxic warhead (drug) via a chemical linker and provides a targeted delivery system to tumors expressing high levels of antibody-recognized cell surface antigens.¹³ ADC prodrugs are supposed to be nontoxic after systemic administration. Upon binding of the antibody to the tumor antigen and internalization of the complex into the cancer cell, the active form of the drug is released in sufficient quantity to kill the cancer cell. A critical attribute of an ADC is the average number of payloads per antibody, defined as the drug-to-antibody ratio (DAR). The DAR influences both the efficacy and toxicity of the ADC. The linker connecting the cytotoxic payload to the antibody is considered a key feature of ADCs and plays a key role in clinical efficacy and ADC safety. The linker must be stable in systemic circulation to avoid release of the cytotoxic payload that is associated with undesired and untargeted toxicities. However, it must also be able to release the cytotoxic warhead in its active form in the cytosol of the target cell following internalization and trafficking to specific subcellular compartments. The intrinsic nature of the linker, together with the cytotoxic warhead, will define the properties of the ADC metabolite that is released inside the cell. The physicochemical properties of the ADC metabolites determine if they have the ability to enter and kill neighboring cells, which are not necessarily antigen-positive. This phenomenon is called the “bystander effect.”¹⁴ Furthermore, the number and nature of the attachment between the chemical linker and the antibody have been shown to influence ADC pharmacokinetics, tumor exposure, and stability in blood.¹⁵ There are two categories of payload conjugation: stochastic or site-specific conjugation (SSC).¹⁶ While the clinical field is still dominated by stochastic conjugates, the functional and analytical advantages of site-specific conjugates have been identified, and ADCs that possess SSC tend to progress further along the clinical value chain.

Figure 1.

Mechanism of action of an ADC.

Today, ADCs have yet to live up to their expected clinical potential, and it is anticipated that a better understanding of the mechanisms of ADC toxicity and efficacy will help in the design of better drugs and improve clinical design, resulting in reduced clinical attrition rates. Key parameters to consider for ADC optimization include:

The presence of a tumor-specific antigen, homogeneously and highly expressed at the tumor site, resulting in efficient internalization and intracellular trafficking of the ADC upon antibody binding. Lower levels of expression may be compensated by increased internalization and trafficking efficiency.

The drug carrier (the antibody) with optimal tumor selectivity and retention properties, as well as sufficient persistence in the blood. As described previously, another key parameter that defines ADC efficacy is the efficiency of drug release. This parameter is dependent on internalization and the trafficking efficiency of the antibody–antigen pair to the appropriate subcellular compartments.

The linker and cytotoxic agent define the properties of the metabolite that is intended to act against the specific cancer type. The metabolite derived from the ADC should have potent cell-killing properties. The indication should also be intrinsically sensitive to the cytotoxic metabolite of the ADC.

The conjugation technology, such as SSC, optimized to improve efficacy, maximize pharmacokinetic (PK) exposure, and limit toxicity and payload metabolism in circulation while attached to the antibody.¹⁷

An adapted clinical trial design, focusing on the dosing schedule and the drug combinations in order to improve the therapeutic index of the agent.

After a brief overview of the ADC design of the Food and Drug Administration (FDA)-approved molecules, this review primarily focuses on innovations that feature in ADCs currently in clinical trials. These innovations comprise the development of new payloads, chemical linkers, and antibody formats that have been used to maximize the therapeutic index of the next-generation ADCs currently showing increased clinical benefit for patients. The success of the targeted drug delivery approach also relies on the identification and validation of appropriate antigenic targets, a subject that is not covered in this article, as several excellent reviews already exist on the topic.^10,13,18,19

FDA-Approved ADCs

To date, seven ADCs have received marketing authorization ( Table 1 ). Four of these ADCs have been approved for hematological malignancies, where the target tends to be more readily accessible, with homogeneous expression of surface antigen, compared with those of solid tumors. The remaining ADCs are approved for the treatment of solid tumors, where poor tumor tissue penetration and heterogeneous antigen distribution are additional challenges for the successful clinical efficacy of ADCs targeting solid tumors.

Mylotarg (gemtuzumab ozogamicin; Pfizer, New York City, NY) was the first ADC to reach the market through accelerated FDA approval in 2000, after it was fast-tracked for the treatment of acute myeloid leukemia (AML).²⁰ This molecule is an anti-CD33 IgG4 antibody covalently linked to the cytotoxic agent N-acetyl γ-calicheamicin, via a pH-sensitive hydrazone linker. N-acetyl γ-calicheamicin is a DNA-damaging agent from the enedyine family. This class of compound induces DNA double-strand breaks and subsequent cancer cell death.²¹ The key insight obtained from the use of this agent is that clinical efficacy can be achieved, with limited side effects, by using a highly cytotoxic compound (100–1000 times more cytotoxic than usual chemotherapeutic agents) coupled with a tumor-selective humanized antibody. In spite of providing these insights, Mylotarg was withdrawn from the market 10 years after its initial release over safety concerns raised during a confirmatory trial.²² This study (SWOG S0106) highlighted increased incidence of mortality during the induction phase, with severe hepatic toxicity, as well as increased risk of potentially fatal veno-occlusive disease (VOD) observed in study patients, particularly those who had undergone hematopoietic stem cell transplant. Ultimately, no clinical benefit was observed for this agent.²³ The limited therapeutic index was the result of the observed toxicities, attributed to inappropriate circulatory release of the free drug,²⁴ due to the instability of the pH-sensitive hydrazone linker in patient blood. This hypothesis has been challenged. In a recent phase III study, clinical benefit and an improved toxicity profile were observed for a subpopulation of acute myeloid lymphoma patients, following fractionated dosing. These observations suggested that toxicity was being driven by a Cmax effect and led to the new approval of this ADC for a restricted CD33-positive AML patient population in 2017.²⁵

Another ADC, Besponsa (inotuzumab ozogamicin; Pfizer), with the same calicheamicin payload, was also approved in 2017. This ADC is composed of a humanized IgG4 mAb against CD22, which is covalently linked to the same cytotoxic agent N-acetyl γ-calicheamicin via an acid-cleavable linker, and stochastically conjugated to the lysines of the antibody. The FDA granted this ADC’s approval for use as a single agent in adults with relapsed or refractory CD22-positive B-cell precursor acute lymphoblastic leukemia (ALL), the most common type of adult ALL.²⁶ Similar adverse events occurred during the clinical trials of both ADCs and suggested that these effects were not target mediated but linked to the calicheamicin-linker platform. Besponsa combination therapies are currently being evaluated in the phase I/II or II setting in ALL and chronic myeloid leukemia and in the phase I setting in Burkitt’s lymphoma.

Adcetris (brentuximab vedotin; Seattle Genetics, Inc., Bothell, WA, and Takeda, Osaka, Japan), Kadcyla (trastuzumab emtansine; Genentech, Inc., South San Fransisco, CA), Polivy (polatuzumab vedotin-piiq; Genentech, Inc.), and Padcev (enfortumab vedotin-ejfv; Astellas Pharma US, Inc., Northbrook, IL) were launched in 2011, 2013, and 2019 (for the last two), respectively. All four ADCs are based on the tubulin binders MMAE (auristatin) and DM1 (maytansinoid). Auristatins are synthetic analogs of dolastatin 10, which inhibits tubulin polymerization, causing cell cycle arrest and eventual death. This agent is 10–50 times more potent than other tubulin-interacting agents such as vinblastine.²⁷ Maytansinoids are maytansine derivatives and share the same destabilizing microtubule assembly²⁸ mechanism of action as the vinca-alkaloids. Kadcyla is composed of a humanized HER2-directed mAb trastuzumab (Herceptin; Genentech, Inc.) randomly conjugated through lysines to maytansinoid cytotoxic DM1 molecules, via a noncleavable thioether linker (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; SMCC), with an average DAR of 3.5.²⁹ Kadcyla is approved as a single agent for the treatment of patients with HER2-positive metastatic breast cancer who have previously received Herceptin and taxane chemotherapy, separately or in combination,^30,31 and for the adjuvant treatment of patients with HER2-positive early breast cancer who have residual invasive disease after neoadjuvant taxane and trastuzumab-based treatment.³² This is the first ADC that has been approved in a solid tumor indication. The superior efficacy of Kadcyla was demonstrated for patients presenting residual invasive HER2-positive breast cancer at surgery, after neoadjuvant therapy containing a taxane and trastuzumab, but not in the neoadjuvant first-line setting.³³ The success of Kadcyla is likely due to its mechanism of action, consisting of the combined antitumor effects of trastuzumab (antibody-dependent cytotoxic activity [ADCC]-mediated cytotoxic effect) and cytotoxic Lys-SMCC-DM1 metabolite released within the target cells upon ADC processing in the lysosome. The reasons for the failure as a front-line agent in neoadjuvant therapy remain unclear.³⁴

Adcetris is a CD30-directed chimeric mAb coupled via a cleavable traceless linker, including a para-amino benzyl carbamate (PABC) self-immolative spacer, to the auristatin derivative MMAE. Adcetris received accelerated approval for the treatment of relapsed or refractory CD30-positive Hodgkin lymphoma (HL) as well as for patients with systemic anaplastic large cell lymphoma (ALCL), after prior failure of at least one multiagent chemotherapy regimen.³⁵ Combinatorial studies of Adcetris are expected to enlarge its use in the treatment of classical Hodgkin lymphoma or other CD30-positive hematological malignancies.

Polivy is a CD79b-directed ADC used in combination with bendamustine and rituximab³⁶ for adult patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL), after at least two prior therapies.

Padcev is a Nectin4-directed ADC used in the treatment of adult patients with locally advanced or metastatic urothelial cancer, who have previously received a programmed cell death receptor-1 (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitor antibody and a platinum-containing chemotherapy. It reached the market through accelerated FDA approval in 2019, after having received breakthrough designation for the treatment of metastatic bladder cancer.

Adcetris, Polivy, and Padcev share the same auristatin-derived payload, namely, mc-vc-PABC-MMAE, whose maleimidocaproyl (mc) cleavable linker is cathepsin B sensitive. The three ADCs are conjugated to interchain cysteines with an average DAR of 4. After internalization of the ADC into target cells, the released drug can elicit bystander effect because of its inherent physicochemical properties.

Enhertu is the second ADC, with Kadcyla, composed of HER2-directed mAb trastuzumab (Herceptin; Genentech, Inc.). The major difference between Kadcyla and Enhertu is the payload consisting of an agent with topoisomerase I inhibitor activity, mc-GGFG-Dxd,³⁷ based on exatecan (or Dxd).^38–43 Dxd is a derivative of DX-8951, a novel topoisomerase I inhibitor that has more potent efficacy than irinotecan against various tumor xenograft models, including irinotecan-resistant tumors, and it was developed up to phase III.^44,45 This payload encompasses a stable protease-sensitive cleavable linker that releases Dxd, having a primary hydroxyl group.³⁹ Moreover, Enhertu is a DAR8 ADC, which is different from other approved ADCs. It is used in the treatment of patients with unresectable or metastatic HER2-positive breast cancer who have received two or more prior anti-HER2-based regimens in the metastatic setting. It is the latest ADC to reach the market through accelerated FDA approval in 2019, after having received breakthrough designation for the treatment of breast cancer.

Clinical trials with several ADCs bearing the same tubulin binder payload and linker have led to the hypothesis that the dose-limiting toxicities are predominantly unrelated to the target antigen, but rather driven by the drug/metabolites of the ADC.⁴⁶ There is a clear association between tubulin binder payloads and the development of reversible ocular side effects, although the frequency and severity of these events are related to the chemical nature of the formed metabolites. For example, MMAE-containing ADCs show less frequent ocular toxicities than MMAF-containing ADCs, a noncleavable tubulin binder ADC derived from auristatin. By contrast, the toxicities of most MMAE conjugates in clinical trials or on the market share a similar toxicity profile characterized by thrombocytopenia, neutropenia, and peripheral neuropathy.⁴⁷

Most of the current ADCs that have reached the clinic still possess a relatively narrow therapeutic window, because of off-target toxicities, competition with unconjugated antibodies, and aggregation or fast clearance of high DAR ADCs. For example, for Adcetris and Kadcyla, there are approximately 5% unconjugated species that compete with the drug-loaded species for binding to the antigen. For Mylotarg, the instability of the linker allows for up to 50% of unconjugated species in circulation.⁴⁸ In specific cases, it has also been demonstrated that species with higher DAR could lead to lower tolerability and higher plasma clearance.^19,49,50

It is becoming clearer that, at least in the case of ADCs with payloads acting primarily on dividing cells, such as tubulin binders, the therapeutic index is primarily influenced by the nature of the payload–linker and the physicochemical properties of its metabolites more so than the expression characteristics of the antigen on the tumor surface versus normal tissues. In addition, stochastic conjugation, the level of unconjugated antibody species in the circulation, and limitations of antibody formats with respect to their biodistribution in solid tumors are other parameters affecting the clinical efficacy of ADCs. In parallel, clinical scientists are working on translational strategies such as the optimization of dosing regimen, the use of biomarkers for the selection of patients who may best benefit from treatment, the early visualization of response signals, and potential synergistic combination therapies (for a review, see Coats et al.⁵¹).

Innovation under Conventional ADC Format

Development of New Cytotoxic Payloads

The first ADCs derived from coupling conventional chemotherapy agents to antibodies (e.g., doxorubicin immunoconjugate BMS-182248-1⁵²) displayed disappointing results in the clinics and were deemed to be insufficiently potent for this approach. As a consequence, much more potent agents (calicheamicin, maytansine, and auristatin) became the payloads of choice. To overcome limitations of the first generation of ADCs based on these agents, two approaches have been deployed to increase payload diversity: First, payloads sharing the mechanisms of action of the two clinically validated warheads, namely, DNA-damaging agents and microtubule-binding agents, were optimized in order to further improve the ADC therapeutic index. Second, payloads with alternative mechanisms of actions were explored, for example, topoisomerase I inhibition ( Fig. 2 ).

Figure 2.

Cytotoxic payloads for ADCs at the clinical stage.

For the auristatin series of tubulin binders, Seattle Genetics developed mc-MMAF, a noncleavable payload based on the auristatin MMAF, as an alternative payload to the first-generation cleavable mc-vc-PABC-MMAE.^53–60 MMAF is less cell permeable than MMAE, and thus less cytotoxic as an unconjugated drug. However, this agent turned out to be highly active as an ADC.⁶¹ In spite of this finding, ABT-414,^62–64 an EGFR-directed ADC based on the mc-MMAF payload, recently failed to meet the primary endpoint of overall survival in phase III trials, and as a result, enrollment in all current studies has been halted. Nevertheless, the payload is still under clinical evaluation in earlier phases, as the payload for GSK2857916 and AGS-16C3F.^65–67 Several other auristatin payloads, currently under clinical evaluation, with DAR values ranging from 2 to 15, have been developed to overcome some limitations of the cathepsine B-sensitive mc-vc-PABC-MMAE payload. These agents are:

AGL-0182-30 consisting of a noncleavable linker and an azido-valine-MMAF that has been modified to contain a N-[2-(aminooxy)acetyl] moiety at the N-terminus, to enable SSC to the antibody at a p-acetyl-phenylalanine (pAF) via an oxime.⁶⁸

AS269 amberstatin consisting of a noncleavable PEG linker designed to improve payload solubility and polarity, and to reduce the export of metabolites by efflux pumps. In addition, the presence of an alkoxyamine-containing MMAF group enables SSC to be carried out via a similar oxime.^69–71

A payload consisting of a cleavable vc-PABC linker and Aur0101, a new microtubule inhibitor selected following N-terminal optimization of dolastatin 10.^72,73 Introduction of α,α-disubstituted amino acids led to auristatin analogs retaining high in vitro potency, while exhibiting differential ADME properties, such as increased liver intrinsic clearance, compared with other auristatin payloads for ADCs. This feature is expected to improve the toxicity profile of the ADC.⁷⁴

A cleavable MMAE payload consisting of a hydrophilic PEGylated glucuronide linker^75,76 that is cleaved by lysosomal β-glucuronidase to achieve efficient drug release.⁷⁷ This improvement of the payload physicochemical properties led to an ADC with a DAR value of 8, which exhibited increased potency without accelerated clearance in plasma compared with the DAR4 ADC. Furthermore, the maleimidocaproyl (mc) spacer, used for mAb conjugation and known to be prone to partial deconjugation, was also replaced by an optimized self-stabilizing maleimide (mDPR) that prevents circulatory payload deconjugation via rapid postconjugation thiosuccinimide hydrolysis.⁷⁸

Dolaflexin (or AF-HPA),⁷⁹ another auristatin payload, consists of several molecules of NMe-MMAF^80,81 covalently linked to alcohol moieties of a water-soluble polyacetal-based polymer. The high level of water solubility of the polymer compensates for the hydrophobicity of the cytotoxic drug and enables DAR values of 12–15 to be reached, while maintaining acceptable plasma clearance.

The second group of tubulin binders are those belonging to the maytansine series. ImmunoGen Inc. (Waltham, MA) also developed disulfide cleavable payloads, SPDB-DM4^82–86 and sulfo-SPDB-DM4,⁸⁷ which, unlike SMCC-DM1,^88–90 elicit bystander killing.⁹¹ The optimal linker turned out to be target dependent for both cleavable and noncleavable classes of linkers.^87,92,93 For disulfide cleavable linkers, varying the steric hindrance at carbon atoms adjacent to the disulfide linkage was one key parameter for balancing conjugate plasma half-life and efficient intracellular release of the cytotoxic drug, resulting in optimal in vivo activity.⁹⁴ The sulfo-SPDB-DM4 payload was explored in addition to more hydrophilic payloads in order to investigate the ability to produce maytansine ADCs with higher DAR values, compared with hydrophobic SPDB and SMCC linkers. As a result of its more polar nature, the active metabolite derived from the sulfo-SPDB-DM4 payload was anticipated to be a poor substrate for MDR efflux pumps, therefore overcoming the multidrug resistance of previous ADCs.⁹⁵ Batansine is another maytansinoid payload constituent in two new ADCs, BAT8001⁹⁶ and BAT8003.⁹⁷ Both of these ADCs are in clinical development and their structures have not been disclosed.

Besides the auristatin and maytansin payloads, other natural product-derived tubulin binder payloads have been developed for ADCs. Many groups have been working on tubulysin given its potent cytotoxicity against a wide range of human cancer cell lines, including significant activity in MDR cancer cell lines.^17,98–100 The main challenge for this tubulysin series was to manage in vivo instability of the acetate,¹⁰¹ a key structural element needed to attain significant potency. The tubulysin AZ13599185 progressed through phase I clinical trials as the payload for MEDI-4276,⁹⁸ a biparatopic HER2-directed ADC, but it was recently discontinued. The reason for discontinuation was a lack of observed benefit as a result of insufficient human exposure, due to the toxicity profile of the ADC’s warhead.¹⁰²

Various linker types and mAb conjugation chemistries were also explored when developing a new ADC payload from eribulin mesylate, marketed as Halaven. This drug is approved in the United States for the treatment of metastatic breast cancer.¹⁰³ The linker investigations led to the identification of maleimido-PEG2-vc-PABC-eribulin payload for MORAb-202.¹⁰⁴

Finally, the cryptophycin series, which is one of the most potent tubulin binder families ever discovered, has also been explored as a tubulin binder payload for ADCs.^105,106 The payload is still in the preclinical stage of discovery. The main challenge with C52 ADCs was to address the deactivating species-dependent payload metabolism that was found to occur in mice. Switching from stochastic to SSC and optimizing the conjugation site reduced this mouse-dependent metabolism.¹⁷ The deactivation was completely abrogated by designing new stable cryptophycin macrocycles, based on its metabolism pathway.¹⁰⁷

DNA-damaging agents, which differ from microtubule-binding agents in their ability to kill cells independently of the cell cycle stage, have also been extensively studied as payloads for ADCs. In addition to calicheamicin, dimers of pyrrolo-benzodiazepines (PBDs) and duocarmycins are the two chemical series that are the most exploited in this category of payload for ADCs. Dimers of PBDs are highly potent DNA minor groove cross-linkers and have been optimized using three different sites for linker introduction: the imine of one PBD monomer, the reactive moiety involved in DNA cross-linking; the exocyclic position of one PBD monomer; and the spacer in between the two PBD monomers. The most advanced representative of this family is Tesirine (SG3249). This agent is derived from the optimization of Talirine (SGD1910),^108,109 which previously failed in clinical trials. The optimization strategy of Talirine aimed at lower payload hydrophobicity, while maintaining cytotoxicity. Optimized Tesirine^110–117 consists of a less hydrophobic PBD dimer, SG3199, in which C2 aryl groups of SGD-1882 have been replaced by methyl groups and the tether between the two PBD monomers is lengthened by two methylene groups to offset the potency drop. The cathepsin B-cleavable valine–alanine–PABC linker was introduced on the imine of one PBD monomer instead of a C2 aryl group to mask the reactivity of the imine. Finally, payload solubility was further enhanced using a PEG8 spacer instead of the caproyl spacer. Tesirine is the payload for Rova-T^111,112 and has reached phase III trials for small cell lung cancer. Rova-T showed no survival benefit compared with placebo in the MERU study, while in the TOHOE study this ADC demonstrated shorter overall survival compared with topotecan. As a result of these findings, Rova-T has been discontinued in clinical trials. However, Tesirine continues to be explored as a payload for other antibodies^113–117 in clinical studies.

Another PBD dimer series still in preclinical studies consists of tomaymycin dimers, tomaymycin being a natural PBD. When using this agent, the linker site for conjugation is introduced on the spacer in between the two PBD monomers,¹¹⁸ providing a symmetrical payload for the ADC. Finally, indolinobenzodiazepine dimers (IGNs), derived from PBDs, display three differentiating structural features:^119,120 (1) the pyrrolidine ring is replaced by an indolino moiety to increase DNA binding and thereby potency, (2) the replacement of the alkyl tether by a phenyl spacer further improves potency and also provides a site for linker introduction, and (3) one imine is selectively reduced, resulting in an asymmetric monoimine DNA-alkylating dimer, in order to reduce payload toxicity, assumed to be related to the DNA-cross-linking mechanism of action. There are two payloads based on this monofunctional DNA-alkylating IGN: (1) DGN462 with a cleavable hydrophilic disulfide sulfo-SPDB linker and, to further improve payload solubility, a branched PEG3 moiety and a sulfonate group on the remaining imine;^119,121 and (2) DGN549 with a cleavable alanine–alanine linker and a maleimido reactive group, enabling SSC to an engineered cysteine instead of stochastic conjugation on lysines.^120,122,123

There are also ADCs containing a DNA-damaging payload belonging to the duocarmycin family^124,125 in clinical trials. Duocarmycins are very potent DNA minor groove alkylators consisting of one DNA-binding and one DNA-alkylating moiety. The main challenges for developing this series as a payload for ADCs are its poor solubility and the stability of both the halogen-containing seco-prodrug used to reduce toxicity and the active cyclopropyl-containing spiro-form.^{124,126–128} Following extensive structure–activity relationship studies aimed at optimizing mainly the hydrophobicity of the core ring structure and the spontaneous elimination of the payload once released from the ADC, an imidazo[1,2-a] pyridine series was selected as the DNA-binding moiety of seco-DUBA (seco-DUocarmycin-hydroxyBenzamide-Azaindole). Linker introduction on seco-DUBA DNA-alkylating moiety was favored over introduction on the DNA-binding moiety as the simplest and most effective way to prevent premature ring closure to the spiro-form and inactivation of the conjugated drug in circulation. Finally, the linker of this optimized seco-DUBA payload consists of the cleavable valine-citrulline dipeptide, two self-immolative spacers—the PABC spacer and an additional bisamine cyclization spacer to allow for a carbamate linkage to seco-DUBA, as this type of linkage is usually more stable than a carbonate equivalent—and PEG2 moieties to further enhance payload solubility.

Topoisomerase I inhibitor-based payloads from the camptothecin series are the only compounds that display a mechanism of action distinct from tubulin and DNA-binding payloads. An alternative payload of this series to mc-GGFG-Dxd^41–43 is Cl2A-SN38. The Cl2A-SN38 payload is derived from SN-38, the active metabolite of irinotecan, a clinically used chemotherapy drug.^129–136 ADCs with the SN-38 payload differ from currently approved ADCs in three main aspects: (1) SN-38 is a moderately cytotoxic drug with nanomolar IC₅₀ compared with the picomolar IC₅₀ of other more potent agents; (2) SN-38 drug loading to the antibody is in the range of 6–8 versus lower substitution ratios for more potent cytotoxics (two to four drugs per antibody); and (3) the pH-sensitive carbonate linkage between the drug and the linker is moderately stable with a half-life in serum of ~24 h, enabling SN-38 to be released slowly at the tumor site once localized at target cells. This phenomenon makes the drug accessible not only to tumor cells internalizing the ADC, but also to surrounding cells. The linker is also composed of a PEG8 moiety to ensure sufficient payload solubility.

Other mechanisms of action for ADC payloads are currently being evaluated at the preclinical stage.¹³⁷ They include RNA splicing inhibition using natural product-derived thailanstatins¹³⁸ and RNA polymerase I inhibition using α-amanitin derivatives.¹³⁹ Both mechanisms are effective on both dividing and quiescent cells. Of note, α-amanitin is a hydrophilic natural product exhibiting potent cytotoxic activity in the ADC format, while, unconjugated, it is unable to enter normal cells via passive uptake. Another interesting feature of this series lies in the fact that hemizygous deletion of p53/Pol2RA increases the susceptibility to α-amanitin ADC and could thus be used as a potential biomarker.

Innovation Related to Antibody Modification

A number of key antibody features have been identified as playing an important role and are currently used in the design of clinical-stage ADCs. A high degree of specificity of the antibody for the tumor antigen is essential. An antibody that cross-reacts with other antigens or displays general nonspecific binding can be taken up into normal tissues in significant amounts, resulting in both toxicity and removal/elimination of the ADC before it can reach the tumor.¹⁴⁰ The antibody must also bind the target antigen with reasonable affinity (Kd < 10 nM) for efficient uptake into target cells, and it should be minimally immunogenic. It is also important to select an antibody with optimal PK properties (longer half-life with slower clearance in plasma¹⁴¹) and desired immune-effector functions. These latter properties can be tailored through the use of different IgG isotypes and mutations in the Fc portion of the antibody.^142,143 More recently, innovations related to the site of conjugation of the payload on the antibody have been addressed.

Site-Specific Conjugation

All the approved ADCs currently on the market exploit stochastic conjugation on preexisting amino acids with chemically activatable side chains, namely, lysine and cysteine. Lysine side chains have been used for the conjugation of calicheamicin and DM1 payloads, whereas those of cysteine have been used for the conjugation of the MMAE payload. More generally, the maytansin platform, including DM4, was developed through lysine conjugation chemistry, while auristatin payloads, including MMAF, were developed through cysteine conjugation. While stochastic conjugation methods have been used for most phase II and III compounds, more recent ADCs entering phase I are primarily conjugated using site-specific methods ( Table 2 ).

Regardless of the conjugated amino acid (cysteine or lysine), ADCs resulting from stochastic conjugation are heterogeneous products ( Fig. 3 ) with DAR distribution displaying Gaussian behavior. This fact raises two potential concerns: (1) a limited amount of unconjugated antibody remaining in the product, binding and occupying ADC binding sites, and thus decreasing efficacy; and (2) ADCs with the highest DARs are more active in vitro, less tolerated in vivo, and display faster elimination when compared with the lowest DARs.⁴⁹ Another difficulty lies in the precise determination of DAR levels for a specific ADC aliquot: any given sample will be composed of mixtures of ADCs that possess distinct structures. In these heterogenous samples, the payload is indiscriminately linked at different sites, with each subtype of molecule possessing a specific in vivo pharmacokinetic, efficacy, and safety profile. To circumvent these difficulties, SSC methods enable more homogeneous and chemically defined ADC populations to be synthesized ( Fig. 3 ), optimizing both the position for conjugation, resulting in improved biological activities, and analytical characterization.

Figure 3.

Conjugation technologies.

SSC on cysteines has been the most widely investigated method. Antibodies engineered to introduce cysteine residues in positions that are different from native interchain disulfides have enabled known and validated maleimide conjugation chemistry to be conducted and existing payloads to be exploited. Limited modification of the conjugation process is needed to ensure maintenance of interchain disulfide bonds and to avoid retro-Michael deconjugation.¹⁴⁴ Different methodologies have been developed to enable the precise introduction of reactive cysteines into antibodies. One such method is THIOMAB technology. This methodology was developed by Genentech, Inc. and led to several site-specific conjugated ADCs currently in clinical trials. The first THIOMAB, described in 2008,^145,146 resulted from phage display screening of reactive cysteines on the light and heavy chains of the constant domains of the antibody Fab region of trastuzumab. The anti-MUC16-targeted antibody, generated using the THIOMAB–drug conjugate (TDC) methodology, demonstrated an improved therapeutic index. The HC-A114C variant emerged from this study as a valuable position for the production of MMAE TDCs and for the conjugation of batansine.⁹⁷ It also led to the first THIOMAB–antibiotic conjugate, DSTA4637S, currently in phase I.¹⁴⁷ TDC demonstrated noticeable, albeit minor, impact on tissue distribution¹⁴⁸ and revealed the importance of the chemical and structural dynamics of the conjugation site through comparison of the HC-A114C and LC-V205C positions for conjugation. The position of the conjugation site modulates the in vivo stability and therapeutic activity of TDCs.¹⁴⁴

The NexMab technology, consisting of introducing peptide motifs containing cysteine residues at the C-terminus of an antibody heavy chain, was also developed for alternative Cys SSC of MMAE.¹⁴⁹ Cysteine engineering was also applied to payloads with other mechanisms of action, with each agent having its optimal position. HC-S239C mutation was optimized to develop several PBD Talirine ADCs.^108,109,150 Alternative methods for SSC on cysteine were also developed and applied to Tesirine. These were (1) a cysteine being inserted one amino acid before or after the three previous selected sites (HC-A114C, LC-V205C, and HC-S239C)¹⁵¹ for conjugation; and (2) an antibody being engineered to LC-C214S, HC-C226V, and HC-C229V, leaving the native interchain disulfide position C220 as the only conjugatable cysteine,¹¹⁶ and therefore enabling SSC on this position. In parallel, a cysteine residue was also engineered at the 442 position of the CH3 domain of the γ1 constant region of a CD123 targeting antibody to allow for SSC of the IGN payload DGN549.¹²²

More recently, de novo conjugation methods have emerged,¹⁹ and the most advanced have led to compounds that are currently in phase I ( Fig. 3 ). GlycoConnect technology utilizes enzymatic modification of N-glycan residing at asparagine-297 that exists across all antibodies and does not require any specific antibody engineering. Antibodies are modified via a two-step, one-pot process using first an enzyme that trims the glycan and a second one that tags it with an azide functional group necessary for subsequent conjugation by click chemistry.¹⁵² SMARTag technology¹⁵³ enables a five-amino-acid consensus sequence (CXPXR) to be genetically encoded at the desired location for conjugation. The resulting Cys residue is recognized by ectopically expressed formylglycine-generating enzyme (FGE), which converts it into a formylglycine (fGly) residue, from which the aldehyde functional group serves as a chemical handle for subsequent conjugation with payloads bearing an aminooxy-functionalized tryptamine to form a stable C–C linkage following Pictet–Spengler ligation.^154,155 A handful of ADCs currently in phase I were built upon antibodies engineered to display unnatural amino acids (UAAs) at a defined site in an antibody sequence. These technologies consist of genetically encoding UAAs displaying biorthogonal chemical reactivity to allow for SSC.^69,156 Using this approach, auristatin payloads, such as AGL-0182-30 and AS269, have been conjugated to pAF-containing antibodies, through an optimized stable oxime linkage.^68,70 These antibodies were expressed in mammalian cells with yields that are similar to those of wild-type proteins. Another UAA, p-azidomethyl-phenylalanine (pAMF), was genetically encoded in antibody sequences in order to obtain either a DAR2 for maytansinoid SC236¹⁵⁷ or a DAR4 for hemiasterlin SC239 ADC.¹⁵⁸ In these instances, antibodies were produced using a cell-free protein expression system and conjugation to pAMF was achieved using a copper-free azide-alkyne cycloaddition, with a dibenzocyclooctyne-bearing payload that provides a stable linkage.¹⁵⁶

Whatever the SSC technology used, ADCs resulting from careful selection of an SSC site—some SSC sites work far worse than stochastic conjugates—demonstrated either an improved tolerability in rodents and nonhuman primates or an increased efficacy in mice versus stochastic ADCs. Some of these include ADCs that are still at the preclinical stage.¹⁵⁹ Strict head-to-head comparisons can be made in the case of Cys conjugation, maintaining all other properties the same (i.e., linkage chemistry, and linker and payload active metabolites). The superiority of the optimized site-specific Cys ADCs versus stochastic Cys ADCs comes only from conjugation site specificity. Direct comparison of stochastic conjugation to SSC is difficult in other cases due to different linkage chemistries, and occasionally different linkers. These linkers impact the nature of active metabolites that are released inside the cell following ADC processing, especially in the case of noncleavable linkers, and this is why the benefit of conjugation site specificity is difficult to evaluate in a systematic head-to-head comparison of SSC technologies. However, SSC enables alternative methods for optimization to be achieved through altering the tethered agent.

Optimization of FcγR Binding Properties of Antibodies

Depending on both the characteristics of the antibody target and the inherent properties of the cytotoxic payload of the ADC, Fcγ receptor (FcγR) binding properties of antibodies are important factors in determining the off-target toxicity of ADCs, such as, for example, neutropenic and thrombocytopenic adverse events. Several hypotheses have been proposed to explain the off-target toxicity of ADCs, one of them relating to the uptake and processing of ADCs by FcγR-expressing cells. Conversely, it has been suggested that FcγR-mediated effector functions may contribute to the clinical efficacy of ADCs.¹⁴²

The efficacy of Kadcyla is in part due to trastuzumab’s capacity to induce ADCC,¹⁶⁰ and it has been suggested that brentuximab’s capacity to cause antibody-dependent cellular phagocytosis (ADCP) in vivo is partly responsible for the efficacy of Adcetris.¹⁶¹ Moreover, it has also been suggested that tumor-associated macrophage-processed MMAE-ADCs can mediate additional “bystander killing” of tumor cells by inducing ADC processing through FcγR interactions.¹⁶² The observed thrombocytopenia side effect induced by Kadcyla is hypothesized to occur as a result of FcγRIIa-mediated internalization of the ADC into megakaryocytes,¹⁶³ resulting in the intracellular release of DM1. To avoid IgG1-mediated effector functions, IgG4 antibodies are used in Mylotarg and Besponsa. The choice of which IgG format to use in ADCs (e.g., IgG1 vs IgG4) may, therefore, define whether to introduce additional efficacy drivers in addition to the payload activity itself,¹⁴² or to minimize side effects.

FcγR interactions between the ADC and the immune cells are important to manage. Because these interactions depend on the Fc isotype of the ADC, Fc engineering could also be exploited to modulate these interactions, particularly when the IgG1 format is used.¹⁶⁴ A method of impairing the FcγR binding is to use aglycosylated antibodies, which lack the Fc effector function, either through a single point mutation, such as N297A or N297Q, or through antibody production in a cell-free protein expression system lacking the glycosylation machinery, such as for STRO-001.¹⁵⁷ Other point mutations could also be used to reduce FcγR binding, as has been demonstrated for MEDI4276, where a L234F point mutation was combined with a S239C mutation for cysteine conjugation in order to reduce ADCC.¹⁶⁵ Finally, as conjugating payloads on the Fc region may impact FcγR binding, some SSC technologies use glycans present on the Fc portion of the mAb as payload attachment sites. The GlycoConnect technology was developed as a way to reduce off-target cellular uptake by FcγR-expressing cells¹⁵² via enzymatic modification and conjugation of N-glycan residues at asparagine-297. More generally, conjugation in these antibody regions may have an impact on effector functions. Less advanced methods are currently under preclinical evaluation, such as conjugation after periodate oxidation of oligosaccharide structures at the fucose residue,¹⁶⁶ conjugation on thiofucose-engineered mAbs,¹⁶⁷ conjugation after remodeling of the glycans of antibodies with azido-containing sialic acid,¹⁶⁸ or conjugation on glutamines at positions 295 ± 297 of aglycosylated mAbs via transglutaminase.^169–172

In contrast to the aim of reducing activity at the FcγR, conjugations that increase the binding affinity of the Fc domain to FcγRIIIa expressed on effector cells have been sought to enhance the ADCC activity of ADCs. To this end, stochastic conjugation on Cys residues has been used on afucosylated mAbs for GSK2857916⁶⁷ and BAT8003,⁹⁷ currently in clinical trials.

Innovation Related to Format

Modified Antibody Formats

Some modifications of mAbs in the Fab region are essentially aimed at optimizing tumor targeting and retention of ADCs in order to reinforce the selectivity and avidity toward the surface antigen, and to maximize ADC intracellular processing from internalization to trafficking to lysosomes. ADCs resulting from some of these technologies have already reached clinical trials.

The most advanced technology relies on biparatopic antibodies. This methodology was applied to HER2-directed ADCs. As HER2 is recycled back to the plasma membrane following spontaneous endocytosis, only a small fraction of Kadcyla is routed to the lysosome, where its active metabolite is released. Thus, it is likely that improving ADC internalization and lysosomal trafficking would significantly enhance HER2-directed ADC efficacy. Biparatopic antibodies targeting two nonoverlapping epitopes on HER2, with one arm binding to the same domain as trastuzumab, were developed. Indeed, these antibodies can induce HER2 receptor clustering, which in turn promotes robust internalization and strong lysosomal trafficking.⁹⁸ Exploiting this finding, ZW25, the antibody component of ZW49,¹⁷³ has been designed to interact with both the trastuzumab and pertuzumab binding domains,¹⁷⁴ already used in combination for treating HER2-positive metastatic breast cancer.¹⁷⁵ The development of the previous biparatopic HER2-directed ADC MEDI4276 was discontinued after phase I completion due to payload-related toxicities. This ADC was designed to combine binding to the same domain as trastuzumab, as well as another unique epitope on HER2 that was different from pertuzumab.^98,176 For similar reasons, the idea of a bispecific antibody targeting both the tumor-specific antigen HER2 and the cell surface antigen APLP2 or PRLR has emerged, but this approach has yet to demonstrate broad utility.^177–179

Another approach aims at reducing nonspecific uptake of ADC as a result of conditional activation of the conjugate at the tumor site, through increased selective ADC binding on tumor cells versus normal tissues. This approach could also enlarge the druggable target space of ADCs when normal tissue expresses the target receptor, in addition to the high level expressed on tumors. One technology takes advantage of the tumor microenvironment (TME) to localize treatment to the tumor more effectively: target-binding regions of antibodies can be activated only in tumor tissue to ensure antitumor activity, while avoiding on-target toxicity in normal tissues. Exploiting this approach, Probody makes use of a N-terminal prodomain that masks the target-binding region of the antibody in circulation, which is removed through the action of proteases, such as matriptase, uPA, and legumain, that are active in the TME.¹⁸⁰ Two Probody drug conjugates are currently under evaluation: CX-2009 targeting CD66 (ALCAM) that is conjugated to SPDB-DM4,⁸² and CX-2029 targeting CD71 (TFRC) conjugated to vc-PABC-MMAE.⁵⁴ More recently, Conditionally Active Biologics (CAB) have been developed as a new kind of antibodies that can bind to their target when in the vicinity of tumors and lose their affinity when less proximal. CAB were identified following library screening in differential conditions (e.g., binding at pH 6 and no binding at pH 7.4), with the objective of increasing selectivity toward tumor cells. Two CAB drug conjugates are currently in phase I/II: BA3011 targeting AXL¹⁸¹ and BA3021 targeting ROR2.¹⁸²

Small Formats

Indications for approved ADCs are mostly for liquid tumors. ADCs targeting solid tumors have usually failed in clinical trials, with low tumor penetration of such large macromolecules being considered as the cause of their limited efficacy in vivo. As an alternative, developing cytotoxic conjugates with smaller targeting moieties has been explored as a strategy to cope with the size of ADCs.^183,184 Different classes of novel entities are still at the preclinical stage, from antibody–fragment drug conjugates (e.g., Fab, diabody, and scFv) to small protein scaffold drug conjugates (e.g., Affibody, DARPin, Abdurin, and Adnectin),^185,186 and a clear benefit from this kind of modality still needs to be demonstrated as superior tumor penetration is usually coupled with faster systemic clearance and subsequent limited in vivo efficacy.¹⁷⁹

To date, only three drug conjugates smaller than antibody–fragment drug conjugates are currently being evaluated in clinical trials, namely, peptide–drug conjugates PEN221¹⁸⁷ and BT1718¹⁸⁸ and a small molecule–drug conjugate PEN866¹⁸⁹ ( Fig. 4 ). PEN221 is the first Pentarin (pen = penetrate, tar = target) drug conjugate designed from a somatostatin receptor-2 (SSTR2), targeting the octreotide peptide analog conjugated to DM1. PEN221 is thought to act as a classical ADC, by inducing significant receptor internalization. The particularity of this construct is the use of a disulfide linker instead of the well-known SMCC noncleavable linker from Kadcyla. The same payload and linker have been used for the construction of BT1718, but in this instance the targeting peptide is a bicyclic peptide (Bicycle) that targets MT1-MMP (MMP14). Bicycle is claimed to behave like an antibody in its targeting, to perform like a small molecule in its efficacy, and to excrete like a peptide. BT1718 is documented as acting not only via target-mediated internalization, but also via extracellular cleavage and release of DM1 within the local tumor environment.¹⁹⁰ Finally, PEN866 consists of a targeting moiety, which is the small molecule binder of HSP90 derived from ganetespib, coupled to the cytotoxic SN-38. PEN866 is reported to exert its activity through high tumor retention and controlled cytotoxic payload release.¹⁸⁹

Figure 4.

Small-format drug conjugates at the clinical stage.

Conclusion and Perspectives

If the story of the first marketed ADC, Mylotarg, was one of ups and downs, the stories of Adcetris and Kadcyla are ones of success, with global market sales for both compounds reaching US$1.87 billion in 2018, with forecasts for 2024 estimated to be US$4.29 billion. Moreover, the forthcoming years may see increased numbers of approvals in the ADC field, which has already begun with the recent approval of Besponsa, Polivy, Padcev, and Enhertu in 2017 and 2019. Recent approval of Enhertu validates topoisomerase I inhibitors as valuable components of future ADCs, with an expectation to enlarge clinical indications to other solid tumors. Looking to the future, four new ADCs are in a position for potential success, with two fast-track designations approved: mirvetuximab-soravtansine in platinum-resistant ovarian cancer and trastuzumab-duocarmazine in HER2+ breast cancer. Furthermore, two breakthrough designations for ADCs described in this review have been granted: sacituzumab-govitecan in triple-negative breast cancer and belantamab-mafodotin in multiple myeloma.

Trastuzumab-duocarmazine¹⁹¹ is built on the same antibody as that of Kadcyla and Enhertu, sharing the same HER2+ breast cancer indication. The major difference between Kadcyla, Enhertu, and this other ADC is the payload consisting of a new DNA binder (duocarmycin based). Sacituzumab-govitecan¹³⁰ is another ADC based on topoisomerase I inhibitor SN-38. This ADC contains a TROP2-directed antibody. The future success of this product may validate the concept of using a moderately potent cytotoxic drug with slow release at the tumor site in addition to expected processing after internalization. Building upon optimization of already approved ADC payloads, mirvetuximab-soravtansine⁸⁷ is composed of an FRα-directed antibody linked to a sulfo-SPDB-DM4 payload. This payload was the result of the optimization of SMCC-DM1 as a cleavable version. Belantamab-mafodotin⁶⁷ is composed of a B-cell maturation antigen (BCMA)-directed antibody linked to mc-MMAF, a noncleavable auristatin developed as an alternative to the cleavable MMAE payload. In the case of this ADC, optimization also involved afucosylation of the antibody as a way to increase effector functions. Taken together, these achievements suggest positive signs after decades of slow progress in developing clinically effective ADCs. The number of positive outcomes may well increase in the coming years as a result of a better understanding of the criteria needed to attain efficacy at the preclinical stage, in addition to current innovations that may offer better cooperative links between the target, mAb, payload, and indication. Additionally, further improvements that have been described in this review and that are still at an early clinical stage (i.e., SSC and modified formats) offer promise for the future of this modality.

The growing promise of the use of immunotherapy to treat cancer could provide a boost to ADCs as a modality for use as therapeutic agents, due to three specific areas of interest. The first is the potential combination of ADC treatments with immunotherapy¹⁹² following the observation that Kadcyla renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade.¹⁹³ Evidence in in vivo preclinical models¹⁹⁴ suggests that there is therapeutic synergy when ADCs and immunotherapy are combined, and combinations of ADCs with anti-PD-1 antibodies are currently being explored in clinical trials.¹⁹⁵ The second option could be the use of immune-stimulant molecules as new payloads for ADCs and the first ADC of this type, NJH395, is already in phase I. This innovative ADC comprises an anti-HER2 antibody conjugated to a TLR7 ligand, where the immune-stimulating moiety is aimed at enhancing the immune-mediated killing of HER2-expressing tumor cells, by activating dendritic cells within the tumor, through the stimulation of toll-like receptors present on immune cells. In addition, targeting receptors present on immune cells for the delivery of cytotoxic or immunomodulatory molecules through ADC modality could also be a field of interest, and first conjugates of that type are emerging.¹⁹⁶ Of note, the tumor-supportive macrophage population is an attractive target for the treatment of macrophage-rich tumors. Indeed, macrophages could be reprogrammed/repolarized to a state where they suppress rather than stimulate tumor growth. This strategy is being explored by an antibody targeting CD163, an effective internalizing receptor, highly expressed in the immunosuppressive macrophage population, conjugated to dexamethasone, a glucocorticoid anti-inflammatory drug.¹⁹⁷

The ADC field may finally be in a position to open the way to a real breakthrough in the fight against cancer. Moreover, some other disease areas could also emerge as new fields of interest for this therapeutic modality. Indeed, some ADCs have already reached phase I for the treatment of rheumatoid arthritis (ABBV-3373) and infectious disease (DSTA4637S¹⁴⁷), and glucorticoïds are being considered as payloads for the treatment of lupus erythematosus.¹⁹⁸ Even if it is too early to tell whether these ADCs will be future clinical successes, these innovations could also enlarge the scope of future ADCs.

Footnotes

Acknowledgements

The authors thank Brigitte Demers and Vincent Mikol for critical reading of the manuscript, Stéphane Cheradame for competitive landscape documentation, and Kwame Amaning for help in proofreading.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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Advances in Antibody–Drug Conjugate Design: Current Clinical Landscape and Future Innovations

Abstract

Keywords

Introduction

FDA-Approved ADCs

Innovation under Conventional ADC Format

Development of New Cytotoxic Payloads

Innovation Related to Antibody Modification

Site-Specific Conjugation

Optimization of FcγR Binding Properties of Antibodies

Innovation Related to Format

Modified Antibody Formats

Small Formats

Conclusion and Perspectives

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

References