["Lead" enjoy] Nature Reviews Clinical Oncology: Exploring the next generation of antibody conjugates

Introduction Antibody drug conjugates (ADCs) are composed of monoclonal antibodies targeting specific antigens and small molecule cytotoxic drugs linked through linkers. They combine the powerful killing effect of traditional small molecule chemotherapy and the tumor targeting of antibody drugs. Since ADCs first entered clinical trials in the mid-1990s, after nearly 30 years of development, they have become a very successful oncology platform. In 2009, calimycins, marigolds, and maytansinoids were the main cytotoxic drugs used in ADC development. A decade on, these molecules are still being used as payloads to be optimized for better stability and hydrophilicity. New cytotoxic substances have also been developed, such as PBDs, ducarmycin and camptothecin derivatives. Antibody engineering has also made considerable progress in the past 10 years, allowing more site-specific conjugation and improving the uniformity and stability of ADCs. New second- and third-generation ADCs have entered clinical practice in the hope of achieving better therapeutic effects and safety. Dozens of bioconjugation techniques based on cysteine residues, unnatural amino acids, or molecularly engineered patterns have also been validated in preclinical studies. However, to fully realize the potential of ADC platforms, innovative molecular design, as well as the identification of tumor-specific target antigens and effective biomarkers, are necessary to address several clinical challenges such as drug resistance, tumor heterogeneity, and treatment-related adverse reactions. Currently, multiple emerging ADC forms have entered early stages of preclinical and clinical development, including bispecific ADCs, conditionally activated ADCs, immunostimulatory ADCs, protein-degrading ADCs, and dual-loaded ADCs. Each ADC provides unique capabilities to address these various challenges and provide more and better treatment options for cancer patients. First and Second Generation ADCs A successful ADC drug depends on two key factors. The first is the need for a stable and reliable linker to connect the antibody and payload that remains stable in plasma circulation and is rapidly cleaved after tumor cell endocytosis to selectively deliver the payload into the tumor and limit adverse reactions due to off-target toxicity. The linker needs to be sensitive to lysosomal conditions (proteases, acidic and reducing mediators). The second key factor for success is that a potent cytotoxic agent must be conjugated to the antibody. In fact, the first ADCs were characterized by a low therapeutic index due to the low potency of the payload (e.g., anthracyclines), resulting in a very limited therapeutic effect up to the maximum tolerated dose (MTD).

· Mylotarg, Besponsa and first-generation cleavable linkers

Mylotarg was approved by the FDA in 2000 to treat acute myeloid leukemia (AML). It is composed of calicheamicin conjugated to gemtuzumab (a mutated anti-CD33 IgG4 subtype monoclonal antibody) through a cleavable linker containing a hydrazone bond. This ADC has an average drug-to-antibody ratio (DAR) of only 1.5 and contains approximately 50% unconjugated monoclonal antibody. After ADC is internalized, the hydrazone bond can be hydrolyzed in the acidic environment of the endosome to release the precursor of calicheamicin, which is then reduced by glutathione to free and active calicheamicin. The latter binds to DNA minor grooves and undergoes Bergman cyclization, thereby generating highly reactive diradicals that cause sequence-selective DNA double-strand cleavage.

Theoretically, hydrazones should remain stable in the blood circulation at physiological pH and undergo selective hydrolysis after internalization under acidic conditions. However, the linker of Mylotarg showed certain instability, resulting in the premature release of calicheamicin in the plasma circulation. Severe toxicity led Pfizer to subsequently withdraw Mylotarg from the market in 2010. Thanks to the clinical experience accumulated in recent years and the advancement of technology, Mylotarg was re-approved in 2017. After optimization, the stability of the linker was improved, it was used at a lower dose, and the dosing plan was modified to be suitable for different patient groups.

· Kadcyla and second generation non-cleavable linkers

In light of these findings, alternative strategies for linker design continue to be developed. However, a serendipitous discovery allowed Immunogen to identify an unexpectedly effective ADC. DM1 is conjugated to the lysine residue of trastuzumab via a non-cleavable linker containing N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), an ADC (T-DM1, Kadcyla) approved by the FDA in 2013 for use in patients with HER2-positive breast cancer.

This new type of ADC is very effective in HER2-positive breast cancer models. Only after the ADC is completely digested by enzymes in lysosomes after internalization, the original structure becomes active to obtain the active metabolite Lys-MCC-DM1.

· Adcetris, Polivy and second generation cleavable linkers

At the same time, Seattle Genetics designed its own conjugation technology to bioconjugate marigold (MMAE) to the cysteine residues of anti-CD30 antibodies through the cleavable linker mc-VC-PABC, which contains a maleimide spacer, a standard Val Cit dipeptide sequence as a cathepsin substrate, and a PABC self-degrading spacer. This was approved by the FDA in 2011 for the treatment of anaplastic large cell lymphoma and Hodgkin lymphoma.

After Adcetris is internalized by tumor cells, it can cleave the linker and degrade it. The released MMAE can destroy the target cells and diffuse to the cell membrane, reaching and killing adjacent cancer cells. This phenomenon, known as the bystander effect, allows the released MMAE to kill both CD30-positive and CD30-negative tumor cells.

Third generation ADC

Due to the interference related to the internalization, transport or recycling of monoclonal antibodies in the first two generations of ADCs, the shedding of antigens, and the lysosomal degradation defects of ADCs, drug release will be reduced, thereby affecting the efficacy of ADCs. Therefore, there is an urgent need to develop new technologies related to bioconjugation, carrier formats, linkers or toxic drugs to broaden the application fields of ADCs, and the third generation of ADCs emerged at the historic moment.

Among the many companies developing ADCs, Immunomedics engineered a surprising ADC by making a triple bet: building an ADC against a slightly overexpressed target, using a system that mixes intracellular and extracellular release, and a less toxic drug than conventionally used payloads. Sacituzumab-govitecan (IMMU-132) is an anti-TROP-2 mAb conjugated to SN-38 (the active metabolite of irinotecan) via a cleavable maleimide linker with a short pegylation unit.

Approved by the FDA in April 2020, this achievement is impressive because this ADC is used in refractory or drug-resistant triple-negative breast cancer (TNBC) for which there are no effective treatments. Another interesting feature of this ADC is that the optimization of the linker structure including the PEGylation unit allows the ADC to have a DAR as high as 7.6 without compromising its tolerance or efficiency. DAR=4 has long been considered optimal, but this statement now only applies to known approved ADCs whose payloads are DM1 or MMAE second-generation headers.

Similarly, to combine irinotecan derivatives with carefully designed linkers, Japan's Daiichi Sankyo developed DXd (exatecan or DX-8951). DXd is a cytotoxic agent 10 times more active against cancer cells in vitro than SN-38. DXd has better safety and optimal solubility, and can induce a bystander killing effect to kill adjacent cancer cells, which is an advantage in heterogeneous tumors, but has a short half-life to avoid off-target toxicity.

Bioconjugation of DXd to anti-HER2 trastuzumab cysteine residues via a proteolytically sensitive maleimide linker resulted in conjugated fam-trastuzumab-deruxtecan nxki (DS-8201a) with a uniform DAR of 7.7. Despite its high DAR, Daiichi Sankyo's DS-8201a was very well tolerated in rats and monkeys and was very stable in plasma.

DS-8201a is able to effectively deliver DXd into heterogeneous tumors in vivo and shows high therapeutic efficacy. Last year, DS-8201a was successfully compared with T-DM1 in a phase III clinical study of metastatic HER2-positive breast cancer and was finally approved by the FDA at the end of December 2019.

Bispecific ADC

Tumor heterogeneity and drug resistance often limit the antitumor activity of single-target therapies. To address this challenge, bispecific antibodies are an approach capable of simultaneously binding to two different target molecules and/or cells. Bispecific ADCs utilizing this technology have become a recent hot topic as a potential way to enhance anti-tumor efficacy. The designs explored so far can be divided into two types: bispecific ADCs that target different epitopes of the same antigen, and bispecific ADCs that target two different antigens.

MEDI4276

It is a quadrivalent HER2-targeting ADC that fuses the scFv of trastuzumab to the N-terminus of 39S, another anti-HER2 IgG1 antibody. MEDI4276 showed significant activity in mouse xenograft models of refractory HER2+ cancers, but did not demonstrate a favorable efficacy-safety balance in clinical testing. In patients with breast cancer, the overall response rate (ORR) was low (9.4%), and the maximum tolerated dose (MTD) was determined to be 0.75 mg/kg every 3 weeks.

ZW49

It is another ADC targeting HER2 dual targets, and its asymmetric structure can achieve bivalent HER2 binding. In the Phase I dose-finding study testing ZW49, the recommended Phase II dose (RP2D) was 2.5 mg/kg every 3 weeks. Among 29 efficacy-evaluable patients treated with ZW49 under this dosing regimen, the confirmed ORR was 28% and the disease control rate was 72% across multiple HER2+ advanced cancer types. Only 9% of patients experienced grade ≥3 treatment-related adverse events (TRAEs), and three patients experienced clinically serious events. These results demonstrate that ZW49 has a manageable safety profile and promising antitumor activity in heavily pretreated patients. Both MEDIO276 and ZW49 are designed to recognize HER2 and promote receptor aggregation, internalization and lysosomal trafficking.

AZD9592

It is an EGFR/c-Met ADC developed by AstraZeneca. It is coupled to a new topoisomerase 1 payload through a connectable linker and mainly solves osimertinib resistance. AZD9592 has a higher affinity for c-MET compared to EGFR, with the goal of reducing EGFR-driven toxicity to normal tissues. In PDX and drug resistance models, osimertinib alone or in combination showed good anti-tumor activity.

M1231

It is a MUC1/EGFR dual anti-ADC jointly developed by Sutro and EMD Serono, a subsidiary of Merck. It uses non-natural amino acid site-directed coupling technology and is coupled to hemiasterlin derivatives (microtubule inhibitors) through a cleavable VC linker. It has a DAR of 4. Preclinical studies have shown strong anti-tumor activity in ESCC and NSCLC patient-derived xenograft models.

BL-B01D1

It is China's first dual-antibody ADC to enter Phase I clinical trials. It targets EGFR and HER3. The linker uses its own Ac linker, which has better stability, better hydrophilicity and less aggregation than the Mc linker. The toxin is its own camptothecin analogue ED04. Its phase I clinical drug safety is relatively good, with no drug-related patient deaths. Among the 10 evaluable end-line NSCLC patients with good safety profile, the ORR was 60% and the DCR was 90%.

Conditional activation of ADC

Traditional ADCs targeting receptors expressed not only on tumor cells but also on certain non-malignant tissues are often associated with unavoidable on-target non-tumor toxicities, leading to dose reduction or treatment interruption. To address this issue, novel ADC designs featuring conditionally active antibodies have been developed.

This design concept is inspired by the prodrug design of small molecules, whereby a pharmacologically inactive form of the drug is conferred, which is then metabolized to its active form in the circulation or certain organs, thereby improving in vivo stability and/or specificity. Several conditionally activated ADCs have been developed through a cleavable peptide sequence (LSGRSDNH) that is sensitive to multiple proteases and are currently in preclinical and clinical development, including praluzatamab ravtansine (NCT03149549 and NCT04596150) and CX-2029 (NCT03543813).

In addition, ADCs with pH-responsive antigen-binding sites have also been developed. The TME is generally slightly more acidic than most non-malignant tissues, and this pH difference can be used to achieve conditional activation of ADCs due to reversible conformational changes in the antigen-binding site. To date, various pH-dependent ADCs have been developed including those targeting EGFR, HER2, AXL, and ROR2. Demonstrated promising antitumor activity in multiple preclinical mouse xenograft models.

immune agonist ISAC

Transformative advances in cancer immunotherapy over the past decade have spurred renewed interest in the field. In the context of innate immune activation triggered by the release of damage-associated molecular patterns (DAMPs) from tumor cells, immune adjuvant molecules that interact with pattern-recognition receptors (PRRs) have become a focus of cancer drug development. Among various attempts to specifically deliver PRR agonists to tumors, antibodies conjugated to specific PRR agonists have emerged as a promising method for local activation of innate immunity.

Preclinical studies have demonstrated that ISACs have unique potential advantages compared with traditional ADCs carrying cytotoxic payloads: first, ISAC-mediated anti-tumor responses can target a variety of tumor-associated DAMPs; second, ISAC-mediated immune stimulation ultimately activates not only antigen-presenting cells (APCs), but may also activate other tumor-infiltrating immune cells, such as T cells; third, ISACs trigger immune memory effects in the entire cellular immune response, providing long-lasting anti-tumor effects and reducing the risk of recurrence.

Of all TLRs characterized to date, TLR7, TLR8, and TLR9 are the primary targets of most ISACs developed to date. The anti-HER2 TLR8 ISAC SBT6050, pertuzumab zuvotolimod, consists of a TLR8 agonist conjugated to pertuzumab via a cleavable linker. It was tested as monotherapy in combination with the anti-PD-1 antibodies pembrolizumab or cemiplimab in a Phase I study (NCT04460456) and in combination with other HER2-targeted therapies in a Phase I/II study (NCT05091528). However, these studies were ultimately terminated due to cytokine-related adverse events and lack of adequate single-agent activity. The efficacy and safety of immune agonistic ISACs still need further clinical testing.

PROTAC-based DAC

DACs carrying proteolytic targeting chimeras (PROTACs) instead of traditional cytotoxic payloads are a new class of targeted therapies. The DAC format has the potential to further enhance the clinical utility of this new paradigm by leveraging the ability of antibody-based drug delivery to provide high levels of tumor specificity and durable activity.

The monoclonal antibody portion of DAC recognizes tumor-associated antigens, triggering the internalization of the DAC-antigen complex. The linker is degraded under proteolytic, acidic and/or reducing conditions, thereby releasing the bound PROTAC molecule into the cytoplasm. Ubiquitination of the protein of interest (POI) occurs through the engagement of E3 ligases, leading to degradation of the POI. Several DACs are in early clinical development, such as a DAC targeting bromodomain-containing protein 4 (BRD4) being tested in patients with CLL1+ acute myeloid leukemia (AML); and ORM-5029 targeting the G1 to S phase transition 1 protein (GSPT1), which is currently being tested in patients with HER2+ breast cancer (NCT05511844).

Dual load ADC

Most solid tumors consist of heterogeneous cancer cell subpopulations with different gene expression profiles and levels of sensitivity to drugs with different mechanisms of action. Therefore, combination regimens involving multiple drugs with different modes of action are often used in clinical practice. Dual-payload ADCs have the potential to elicit additive or synergistic effects as a single agent and overcome drug resistance in patients with treatment-refractory tumors while maintaining a simple dosing envelope.

A method to produce dual-loaded ADCs using a branched chemical linker containing two orthogonally masked cysteine residues was reported in 2017. Sequential conjugation of the payload enables uniform conjugation of MMAE and MMAF to anti-CD30 antibodies with a DAR of 16. This dual-loaded ADC showed potent activity in a mouse xenograft model of CD30+MDR-expressing anaplastic large cell lymphoma (ALCL).

In addition to ADCs with MMAE and MMAF payloads, the clinical potential of dual-payload ADCs has been further explored with the development of ADCs that combine two different payload classes. For example, the combination of asterlin plus a TLR agonist conjugated to an anti-FolRα antibody showed synergistic antitumor activity and immune memory in mouse models. Currently, research into the potential of dual-load ADCs is still in the early stages of exploration.

Summary

The therapeutic potential of ADCs is huge, but to realize this potential, several key challenges need to be overcome, such as drug resistance, intra- and inter-tumor heterogeneity, and the risk of TRAEs. Emerging ADC modalities, including bispecific and dual-loaded ADCs, show potential to address drug resistance and tumor heterogeneity, while conditionally activated ADCs may increase tumor specificity and reduce the incidence of adverse events. Combining ADC platforms with other intervention strategies, such as immunomodulation and degradation of traditionally untreatable targets, offers the opportunity to implement multimodal cancer treatments alongside chemotherapy, radiotherapy, immunotherapy, and other targeted therapies.

The development of ADCs is on the verge of transformative growth and promises to dramatically change the cancer treatment landscape. As we better understand tumor biology and improve ADC design, we will get closer to the goal of truly effective, safe and personalized cancer treatments, which will ultimately bring new hope to patients with intractable cancers.

References:

1.Exploring the next generation of antibody-drug conjugates. Nat Rev Clin Oncol.2024 Jan 8.2. Antibody–Drug Conjugates: The Last Decade. Pharmaceuticals 2020, 13, 245