Driven by increasingly mature technologies, ADC payloads have undergone three generations of innovation. In the first generation of ADC drugs, traditional chemotherapy agents such as methotrexate, vincristine, and doxorubicin were used as cytotoxic payloads. However, due to the insufficient cytotoxicity of these first-generation ADCs against cancer cells, lack of tumor selectivity, and limited accumulation in target cells, their efficacy was even inferior to their parent drugs, leading to clinical failures. Most second-generation ADCs utilized significantly more effective microtubule inhibitors as payloads. Unfortunately, although microtubule inhibitors are highly effective against actively dividing tumor cells, they are less effective against quiescent cancer cells. To overcome this limitation, there has been a shift towards using DNA-damaging agents capable of targeting the entire cell cycle, including topoisomerase I inhibitors, as the cytotoxic payloads of most third-generation ADCs.
Despite ADCs having gone through three generations, current payloads still have clinical limitations such as severe side effects and the development of resistance. Therefore, there is a need to develop more effective ADC payloads with better therapeutic windows.
Novel ADC payloads include RNA inhibitors, Bcl-xL inhibitors, NAMPT inhibitors, and cartilaginous compounds. Additionally, immunomodulators, protein degraders, photodynamic agents, and dual toxin strategies have also been applied.
Targeting Microtubule Proteins with Toxins
1.Maytansinoids
DM1 and DM4 are the two most commonly used maytansinoid-like ADC payloads in clinical practice. For instance, trastuzumab-SMCC-DM1 is the first ADC drug based on a maytansinoid derivative approved for marketing.
2. Auristatin
Dolastatin 10 was isolated from sea hare in 1987 and exhibits strong anti-proliferative activity against various cancer cells. It strongly inhibits microtubule assembly, leading to cell cycle arrest and apoptosis, making it a promising anticancer agent. Water-soluble synthetic analogs of dolastatin 10 are called auristatins. Among them, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are the most widely used.
3. Eribulin
Eribulin is a naturally occurring polyether macrolide, with Halichondrin B initially isolated from Okadai et al., demonstrating good anti-proliferative activity. Eribulin has potent anti-mitotic activity in tumor biology, making it a promising payload for ADCs. Earl F. Albone’s team designed an ADC with a C-35 amine group that had a strong impact on ovarian cancer cells IGBOV1 (half-maximal inhibitory concentration of 20 pmol/L). In a xenograft model of non-small cell lung cancer cells NCI-H2110, a 5 mg/kg ADC induced complete tumor eradication, indicating that microtubule-targeting agent eribulin could serve as an effective payload for ADCs.
4. Tubulysins
Tubulysins are natural anti-mitotic peptides isolated from myxobacterial culture by Hofle et al. They inhibit microtubule polymerization, induce cell apoptosis, and exhibit strong anti-proliferative activity against multidrug-resistant KB-V1 cells (half-maximal inhibitory concentration = 0.08 nmol/L), offering promising prospects for anticancer drug development.
5. Cryptophycins
Cryptophycins are natural cyclic peptides produced by cyanobacteria, consisting of a 16-membered ring. It contains two hydroxy acids (A and D units) and two amino acids (B and C units). Cryptophycins irreversibly inhibit β-tubulin polymerization during mitosis, causing cell cycle arrest in the G2/M phase, activating apoptosis pathways, thus exhibiting picomolar anti-proliferative potency in vitro.
6. EG5 inhibitors
Eg5, also known as kinesin spindle protein (KSP) or kinesin family member 11 (KIF11), is an ATP-dependent motor protein involved in centrosome separation and bipolar spindle formation during the G2/M phase of the cell cycle, playing a crucial role in mitosis. EG5 is overexpressed in hematologic malignancies (such as AML progenitor cells and diffuse large B-cell lymphoma (DLBCL)) and solid tumors (such as breast cancer, bladder cancer, and pancreatic cancer), correlating with poor prognosis, making it an attractive cancer therapy target.
Targeting DNA with Toxins
Compared to microtubule inhibitors, DNA inhibitors can disrupt DNA through mechanisms such as double-strand breaks, alkylation, intercalation, and cross-linking, acting throughout the cell cycle, exerting cytotoxic effects, and showing promising therapeutic effects against solid tumors. Additionally, DNA inhibitors have fewer targets compared to microtubule inhibitors, and when ADCs carry the same amount of payload into cells, DNA inhibitors can demonstrate better cytotoxic effects. Furthermore, ADCs carrying DNA inhibitors as payloads can target tumors with low antigen expression, which also explains why DNA inhibitors have been selected as payloads in many next-generation ADCs.
1.Calicheamicins
Calicheamicins target minor grooves binding with DNA, specifically inducing double-strand breaks in DNA, leading to apoptosis. Calicheamicin γ1 (46) exhibits strong cellular effects, and Mylotarg and Besponsa utilize this toxin as a payload. The Nicolaou group synthesized Calicheamicin θ1 (48), a calicheamicin γ1 analog, with half-maximal inhibitory concentrations of <1 pmol/L against various tumor cells, making it suitable as an ADC payload.
2. Topoisomerase I Inhibitors
Topoisomerase I (TOPO-I) is an essential enzyme that affects genome stability and DNA structural preservation, becoming a hot target for ADCs. The binding mode of topotecan involves stacking with DNA, hydrogen bonding with Asp-533, and water bridging with the active site between phosphotyrosine and Asn-722. Natural pentacyclic alkaloid camptothecin (CPT), similar to topotecan, is a particularly attractive ADC payload. It forms stable complexes with TOPO-I and DNA, inducing double-strand DNA breaks in S-phase cells, leading to apoptosis. However, the extremely low solubility of camptothecin hinders its widespread use as a cancer therapeutic. SN-38 is the active component of irinotecan (CPT-11), a semi-synthetic camptothecin. While SN-38 possesses the desired potency, the preparation of active conjugates is challenging due to its highly hydrophobic nature and limited available coupling sites. TRODELVY® (sacituzumab govitecan) is an ADC utilizing SN-38 as the payload. Daiichi Sankyo’s DXd (DX-8951 derivative) is a novel, highly membrane-permeable topoisomerase I inhibitor with cell half-maximal inhibitory concentrations ranging from 2.05 to 17.88 nmol/L, overcoming P-glycoprotein-mediated multidrug resistance and demonstrating toxicity against various tumor xenograft models, including CPT-11-resistant tumors in vivo, potent toxin DS-8201a.
3. PBD
[1,4]-Benzodiazepine drugs (PBD) discovered in Streptomyces are a class of natural products with anti-tumor activity. These compounds consist of an aromatic A ring, a 1-4-diaza-5-1 B ring, and a pyrrolidine C ring. Their mode of action involves selective alkylation in DNA minor grooves, where the N2 of purine forms a covalent bond with the electrophilic N10/C11 imine of PBD. PBD dimers have the potential to be used as ADC payloads, such as ror1-targeting ADC CS5001 designed with PBD as the payload.
4. Duocarmycins
Duocarmycin A is a potent DNA alkylating agent isolated from Streptomyces, consisting of a DNA alkylating portion and a binding portion. The first discovered natural member of duocarmycins, CC-1065, binds to DNA minor grooves via its highly active propane ring and alkylates adenine at the N3 position, ultimately leading to cell death. ADC SYD985 demonstrates good performance both in vitro and in vivo, utilizing the duocarmycin DUBA (75) as the payload prodrug seco-DIBA.
Targeting RNA with Toxins
There are still many tumors that do not respond to ADCs or develop resistance to drugs. Slow-growing tumor cells typically do not rely on microtubule-mediated cellular processes as much as rapidly dividing cells. Therefore, to further expand the range of ADC payloads and identify other types of ADC payloads effective against both rapidly proliferating and slow-growing cells, and capable of overcoming MDR-mediated resistance, attention has shifted to small molecule payloads targeting RNA.
Small molecule inhibitors targeting RNA have the potential to kill dividing and dormant tumor cells, and as ADC payloads, they are expected to address the issues of tumor resistance and recurrence caused by ineffective targeting of dormant tumor cells.
Currently, there are two main types of RNA inhibitors available for ADC payloads: RNA splicing inhibitors (Thailanstatin) and RNA polymerase II inhibitors (Amatoxins).
1.Thailanstatin
RNA splicing primarily controls metabolism, angiogenesis, cancer cell proliferation, and metastasis by removing introns and exons, complex cellular mechanisms responsible for converting RNA into mRNA. They also directly control transcription initiation, elongation, and termination, which are biological targets for cancer inhibition.
2. Amatoxins
Amatoxins were first isolated by Heinrich Wieland and Rudolf Hallermayer in 1941. They are toxic bicyclic octapeptides synthesized by ribosomes and are selective inhibitors of eukaryotic RNA polymerase II, leading to apoptosis.
Immune ADC Payloads
Immune checkpoint inhibitors enhance the anti-cancer immune response of T cells by eliminating tumor suppression. One focus of immunotherapy research and development is to develop agents that can stimulate patients’ innate immune responses, turning “cold” tumors into “hot” tumors, and promoting the recruitment of tumor-specific T cells. On one hand, antibody-conjugated immune agonists act to enhance the immune response. On the other hand, tumor-targeting antibodies can reduce the toxicity of small molecule immune agonists. In terms of target selection, traditional ADCs must strictly limit tumor-specific antigens due to the toxicity of the payload, while immune-modulating ADCs can target a wider range of tumor-associated antigens.
1.Toll-like receptor agonists
Toll-like receptors (TLRs) are a class of crucial protein molecules involved in innate immunity, serving as the body’s first line of defense against infectious diseases. Activation of TLR receptors can enhance antigen presentation by dendritic cells and macrophages in the tumor microenvironment, promoting the proliferation of CD8+ T cells. Toll-like receptor agonists, as immune enhancers, can directly or indirectly induce enhanced anti-tumor immune responses in lung cancer, melanoma, leukemia, and glioblastoma. The application of TLR agonists in tumor immunotherapy is expected to activate innate immune responses, mediate acquired immune responses, convert cold tumors into hot tumors, address the low response rate to single immune checkpoint inhibitors, improve the effectiveness of immunotherapy, and reshape the tumor microenvironment.
Bolt’s BCC-1001 utilizes TLR7/8 agonist conjugation. Sutro’s IADC technology can be used to conjugate antibodies with TLR agonists. Tallac’s TRAAC technology is used for coupling with TLR9 agonists. ALX Oncology’s SIRPa antibody, developed in collaboration with Tallac, can be conjugated with TLR9 agonists. Hengrui Pharmaceutical utilizes TLR7 agonists, BeiGene utilizes TLR7/8 agonists, Innovent Biologics adopts Bolt’s technology and utilizes TLR7/8 agonist conjugation. Silverback’s ImmunoTAC technology selects TLR8 agonists as payloads.
2. STING agonists
Stimulator of interferon genes (STING) is a critical regulatory factor in the natural immune signaling pathway, initiating the body’s natural immune defense responses and promoting the formation of adaptive immune responses by T cells. Activation of the STING pathway can induce the expression and secretion of various cytokines, including type I interferons, activating innate immune responses, promoting anti-tumor immune responses, and achieving the goal of treating tumors. In addition to inducing natural immune responses, they also play a role in other DNA or RNA sensor signaling pathways, autophagy, endoplasmic reticulum stress, and apoptosis. STING agonists possess anti-tumor and immunogenic characteristics and have become new immunotherapeutic investigational drugs and vaccine adjuvants.
3. Glucocorticoid receptor modulators
Glucocorticoid receptor modulators (GRMs) are commonly used to treat inflammation associated with various diseases. ABBV-3373, being developed by AbbVie, is an ADC drug composed of α-TNF and GRM, which can precisely target and regulate activated immune cells, modulate the TNF-mediated inflammatory signaling pathway, significantly reduce systemic side effects associated with glucocorticoids, and may treat rheumatoid arthritis (RA).
Immune agonists can activate nonspecific immune responses when administered systemically, and in severe cases, can trigger lethal cytokine storms. Conjugating immune agonists with tumor-targeting antibodies can deliver the immune agonists to the tumor microenvironment and release them locally, mitigating the severe toxicity of systemic immune stimulation and helping to avoid systemic side effects in anti-tumor therapy. However, while the idea of immune-stimulating ADC drugs achieving anti-tumor effects through immune mechanisms is very clear, preclinical data are relatively good, but addressing dose-dependent toxicity issues in clinical trials is challenging. As the efficacy of agonists increases, so does toxicity, making it difficult to ensure that efficacy can be improved in a safe and reliable manner. Therefore, in subsequent research, the most important consideration is the balance between toxicity and efficacy. Redesigning to reduce toxicity and improve delivery windows is also highly advantageous.
New types of ADC payloads
1.Bcl-xL inhibitors
Bcl-xL is an anti-apoptotic protein that plays a crucial role in tumor formation, metastasis, and drug resistance. Targeting Bcl-xL as an ADC drug payload can retain the activity of Bcl-xL inhibitors while reducing their toxicity to platelets.
2. NAMPT inhibitors
Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme that converts nicotinamide into nicotinamide mononucleotide, controlling the concentration of NAD+ within cells. When NAMPT is inhibited, NAD+ levels decrease to below the metabolic requirement, leading to an energy crisis and eventual cell death.
3. Carmaphycins
Proteasome activity inhibitors are an emerging class of anticancer drugs with potent cytotoxicity against certain cancer cells, with epoxyketone proteasome inhibitors being representative of this class. Carmaphycins can be divided into four distinct parts (P1-P4).
Novel strategies guiding ADC payloads
1.PROTAC molecules as ADC payloads
One of the primary challenges in ADC development is balancing efficacy with off-target toxicity, often indicated by dose-limiting toxicities (DLTs). PROTACs are bifunctional compounds consisting of a ligand targeting the protein of interest (POI), a ligand targeting an E3 ligase, and a linker. PROTACs bring the POI and E3 ligase closer together, leading to ubiquitination of the POI via a proteasome-mediated degradation process. PROTACs have catalytic activity, enabling effective degradation of the target protein at lower doses.
2. NIR-PIT drugs as ADC payloads
Near-infrared photoimmunotherapy (NIR-PIT) drugs typically consist of a tumor-specific monoclonal antibody and a light-activatable chemical substance connected via a linker, essentially functioning as ADC drugs. NIR-PIT drugs represent a new targeted cancer therapy platform, utilizing device-administered infrared light to irradiate tumor sites. This platform enables highly tumor-specific antibody-mediated delivery while accurately inducing rapid cancer cell death through the biophysical mechanisms of photoactivated drugs without harming surrounding normal tissues.
In 2020, Rakuten’s Cetuximab Sarotalocan was the first approved photodynamic therapy drug, comprising the anti-EGFR antibody Cetuximab and the water-soluble silicon phthalocyanine derivative IRDye700DX, targeting locally advanced or locally recurrent head and neck cancer that is not amenable to curative treatment. Administered 24 hours before, the drug specifically accumulates on the surface of EGFR-positive tumor cells. Illuminating the tumor site with near-infrared light at a wavelength of 690 nm induces cell death and activates an immune response.
3. ADCs with dual payloads
With the emergence of ADC therapy resistance, combining an antibody with two or more distinct cytotoxic payloads offers an attractive option for the development of next-generation ADCs.
4. PDCs
Peptide-drug conjugates (PDCs) represent a novel targeted therapeutic approach comprising a linker, a homing peptide, and a cytotoxic payload. Compared to ADC drugs, PDC drugs offer advantages such as small molecular weight, strong tumor penetration, low immunogenicity, large-scale solid-phase synthesis, low production costs, favorable pharmacokinetics, and relatively uniform batch products.
As of April 2023, only two PDC drugs have been approved globally: Lutathera and Pepaxto. Lutathera is a PDC targeting somatostatin receptors developed by a subsidiary of Novartis. Upon entering cells, it releases the radioactive isotope 177Lu, leading to radiation-induced damage to tumor cells. Pepaxto is a PDC targeting aminopeptidases, consisting of a DNA alkylating agent, melphalan, covalently linked to a targeting peptide for aminopeptidases. Melphalan has high lipophilicity and is rapidly absorbed by multiple myeloma cells.