The challenge of tumor heterogeneity has long been a significant obstacle in cancer treatment, particularly in cases like breast cancer where diverse cell populations within a tumor can lead to drug resistance, recurrence, and metastasis. Traditional chemotherapy agents often struggled with these complexities, revealing their limited efficacy against the varied cell types within tumors. In response to this challenge, antibody-drug conjugates (ADCs) have emerged as a promising solution.

ADCs are a class of targeted cancer therapies combining the specificity of antibodies with the cytotoxic power of chemotherapeutic agents. This synergy allows for selective delivery of drugs to cancer cells, potentially reducing the side effects associated with traditional chemotherapy. Since their inception, ADCs have undergone significant advancements, improving their safety profile and therapeutic efficacy.

Here, we’ll focus on how recent advancements in ADCs, particularly those involving innovative payloads, are addressing the challenge of tumor heterogeneity. We’ll explore the scientific advancements in ADC payloads, including dual-action drugs and immune system modulators, and their impact on treating heterogeneous tumors to offer a clear informative view of the current state and future potential of ADCs in cancer treatment.

The evolution of ADCs

The journey of ADCs in cancer therapy has been marked by significant evolution and innovation. Initially, ADCs faced challenges such as limited potency, suboptimal targeting, and off-target effects. However, advancements in engineering and biochemistry have led to the development of more effective and safer ADCs.

The cornerstone of this evolution lies in the refinement of an ADCs’ three key components: the antibody, the linker, and the payload. Antibodies in ADCs are now selected and engineered for higher specificity and affinity to tumor antigens, ensuring that the drug is delivered more precisely to cancer cells. The linker technology, which attaches the payload to the antibody, has also undergone advancements. Modern linkers are often more stable in circulation, reducing premature release of the cytotoxic payload and thereby minimizing damage to healthy cells (Wang et al. 2023).

Payloads, the component responsible for inducing cell death, have seen a shift from traditional cytotoxic agents to more potent and diverse molecules (Esapa et al. 2023). These include novel agents capable of inducing immunogenic cell death or targeting specific cellular pathways unique to cancer cells, increasing the therapeutic window of ADCs. Moreover, recent developments have focused on addressing the heterogeneity of tumors (Aggarwal et al. 2023). Tumors are not uniform; they comprise a variety of cell types with different molecular characteristics. This diversity often leads to treatment resistance and disease recurrence. The new generation of ADCs aims to tackle this challenge by combining different mechanisms of action (MoAs) within a single molecule or by developing ADCs that can simultaneously target multiple antigens present in various tumor cells.

The advancements in ADC technology not only enhance their efficacy but also expand their potential applications. Initially limited to certain types of cancers, such as breast and hematological malignancies, ADCs are now being explored for a broader range of tumors, thanks to the development of antibodies targeting a wider array of antigens. The field of ADCs is, thankfully, advancing rapidly, fueled by a deeper understanding of cancer biology and technological innovation. These developments promise to overcome some of the historical limitations of cancer therapy, offering a more effective and targeted approach to treating this complex disease.

Tackling tumor heterogeneity

Tumor heterogeneity remains a significant challenge in cancer therapy. Cancer’s dynamic nature often leads to increased heterogeneity over the disease’s progression, and so a single tumor can contain myriad cells, each with unique molecular signatures and varying sensitivities to treatment (Dagogo-Jack and Shaw 2018). Such diversity within the tumor, known as spatial heterogeneity, involves the uneven distribution of genetically distinct tumor-cell groups across and within affected sites.

Additionally, there’s temporal heterogeneity, which refers to changes in the molecular composition of cancer cells over time. This complex mosaic of cell types not only complicates the accurate diagnosis and effective treatment of cancer but also fuels drug resistance, making it imperative to thoroughly understand tumor heterogeneity to develop successful therapies.

ADCs represent a promising approach to tackle this issue. By using their ability to deliver cytotoxic agents directly to cancer cells, ADCs can target specific cell populations within a heterogeneous tumor. A key advancement has been the design of ADCs with dual payloads (Yamazaki et al. 2021). These ADCs carry two different cytotoxic agents, each targeting different aspects of cancer cell biology. This combination allows for the targeting of different aspects of cancer cell physiology, potentially overcoming resistance mechanisms that tumors develop against single-agent payloads (Conilh et al. 2023; Wang et al. 2023).

ADC payloads that use the immune system

Recent developments in ADC technology have focused on enhancing this ability to address the varied nature of tumor cells and while traditional payloads were primarily focused on agents that disrupt cell division, the new era of ADC research is exploring more diverse and potent options. A significant advancement is the integration of payloads that modulate the immune system. This approach not only focuses on the direct eradication of cancer cells but also harnesses the body’s immune system to combat the disease.

When it comes to immunomodulatory ADCs, we’ve seen toll-like receptors (TLRs) gaining momentum as therapeutic targets. TLRsa re key players in immune surveillance and might be pivotal in turning the tables on cancer. Agonists that activate TLRs, particularly TLR7, TLR8, and TLR9, have been under the spotlight for their potential to intensify the body’s immune response against various cancer types, including lung cancer, melanoma, leukemia, and glioma (Sun et al. 2022). This is because activating TLRs not only impedes tumor growth but also primes the immune system for a more robust attack (Wang et al. 2023).

However, TLR8 agonists, while effective in enhancing anti-tumor responses through improved antigen presentation and CD8+ T cell proliferation (Kim et al. 2018; Sun et al. 2022) often cause adverse side effects when administered systemically, which limits their clinical application. To address this, ADC scientists proposed a solution: conjugating TLR agonists with antibodies to minimize side effects. In 2021, a breakthrough was achieved with an immune-stimulating antibody conjugate (ISAC) combining a dual-acting TLR7/8 agonist with a HER2-specific antibody (Ackerman et al. 2021). This ADC initiated a potent myeloid-driven response against tumor cells, followed by T-cell activation, and showed promising results in mouse models, with well-tolerated systemic administration and tumor regression.

Expanding on the concept of ADCs, the integration with checkpoint inhibitors has been a significant development in oncology. While checkpoint inhibitors, like anti-PD-L1 antibodies, have been successful (Akinleye and Rasool 2019), their effectiveness is uncertain in tumors lacking immune cell infiltration. ADCs can enhance this by inducing immunogenic cell death (ICD), which activates antigen-presenting cells and facilitates T-cell cross-presentation, thereby bolstering antitumor immunity (Rosenberg et al. 2022; Hoimes et al. 2023).

The 2021 introduction of HE-S2, an ADC linking an anti-PD-L1 THIOMAB to the immunomodulator D18, marked another advancement (He et al. 2021). This conjugate not only obstructs PD-L1 but also stimulates immune cell infiltration and TLR activation directly at tumor sites, potentially offering a new avenue for treating chronic viral infections like HIV or HBV.

However, clinical trials combining ADCs with checkpoint inhibitors have yielded mixed results, and although the integration of immune modulation into ADC design is an incredibly promising step in cancer therapy, we need to continue research and trials to better understand this complex system.

Overcoming resistance and enhancing efficacy

If ADCs are going to have a significant impact on cancer therapy, they need to overcome drug resistance and enhance treatment efficacy. Drug resistance, a major hurdle in cancer treatment, often leads to therapy failure and disease progression. ADCs, with their targeted approach and novel payloads, are being developed to address this challenge.

A key strategy in enhancing the efficacy of ADCs involves improving target antigen selection. The selection of appropriate antigens on cancer cells for targeting by ADCs is crucial. It ensures that the drug is delivered specifically to cancer cells, minimizing off-target effects, and improving treatment outcomes. An ideal target antigen for ADCs needs to be extracellular for accessible antibody binding and capable of efficient internalization upon binding. This internalization is crucial for delivering the cytotoxic payload directly inside cancer cells, enhancing the ADC’s therapeutic effect while limiting damage to normal cells.

If we look at HER2-positive breast cancer as an example, the heterogeneity of the disease is evident in the varied expression of HER2 itself, ranging from high in HER2-E subtypes to low in other subtypes. This variation has led to the development of ADCs like trastuzumab deruxtecan, which show activity even in tumors with low HER2 expression (Schettini and Prat 2021). These advancements in ADC technology and target antigen selection are crucial in addressing the diverse molecular profiles and treatment responses seen in this type of cancer.

Additionally, advancements in linker technology play a crucial role in enhancing the efficacy of ADCs. The stability of the linker, which connects the antibody to the payload, is paramount in ensuring that the cytotoxic agent is released specifically at the tumor site. Modern linkers are designed to be stable in the bloodstream and release the payload in the acidic environment of the tumor or upon internalization into cancer cells, leading to a targeted release that minimizes systemic toxicity and enhances the efficacy of the payload.

Clinical trials have shown that ADCs with improved targeting and novel payloads can lead to better treatment responses, even in cancers known for their resilience to standard therapies. For instance, ADCs targeting novel antigens or utilizing innovative payloads have demonstrated improved clinical outcomes in patients with various types of resistant cancers (Conilh et al. 2023).

Ongoing advancements in ADCs, from antigen selection to payload and linker development, are pivotal in overcoming drug resistance and enhancing the efficacy of cancer therapies. These innovations not only promise improved treatment outcomes but also pave the way for more personalized and effective cancer treatment strategies.

Abzena’s contribution to ADC development

At Abzena, we specialize in the development and production of these complex biologics and bioconjugates and have extensive experience with different conjugation technologies. As already mentioned, linker chemistry is incredibly important for ADC efficacy and stability, so we created ThioBridge technology to address some of the key challenges in ADC development.

Traditional conjugation technologies often result in heterogeneous DARs and linker instability, limiting the therapeutic potential of ADCs. ThioBridge overcomes this with a more uniform DAR profile, stable linker attachment, and improved pharmacokinetic (PK) properties. This technology enables precise and targeted modification of disulfide bonds, preserving the covalent integrity of the disulfide bridge without extensive antibody re-engineering. The ThioBridge platform can be used for the conjugation of many different modalities including antibodies, bispecifics, radioconjugates, oligonucleotides, immunomodulators, and peptides. The site-specific attachment and the stability of the ThioBridge linker ensure the integrity of the conjugate, leading to improved potency and efficacy (Huang et al. 2016).

Future directions and challenges

The field of ADCs continues to evolve, with new research and technological advancements continually emerging. As we look towards the future, several key directions and challenges become apparent, shaping the trajectory of ADC development and their application in cancer therapy. These include:

• Expanding the scope of targetable antigens: one of the foremost areas of focus is the identification and targeting of new antigens. The effectiveness of ADCs largely depends on their ability to recognize and bind to specific antigens on cancer cells. Expanding the repertoire of targetable antigens will broaden the applicability of ADCs to a wider range of cancers, including those currently lacking effective targeted therapies.

• Enhancing payload diversity and specificity: another area of significant interest is the development of more diverse and specific payloads. While traditional payloads have focused on agents that disrupt cell division, there is a growing trend towards payloads that target specific cellular pathways, induce ICD, or modulate the immune response. The exploration of these novel payload types promises to improve the efficacy of ADCs, particularly in treating tumors with high heterogeneity and drug resistance.

• Improving delivery and reducing toxicity: the continued improvement of linker technology and antibody engineering is essential for improving the delivery of ADCs to tumor cells while minimizing off-target effects. Enhancements in linker stability and the development of antibodies with higher specificity will reduce systemic toxicity and improve patient outcomes. Technologies like Abzena’s ThioBridge support these efforts, offering more stable and specific delivery of payloads.

• Addressing regulatory and manufacturing challenges: finally, the regulatory and manufacturing processes for ADCs present ongoing challenges. Ensuring the safety, efficacy, and quality of these complex biopharmaceuticals requires stringent regulatory oversight and sophisticated manufacturing capabilities. Streamlining these processes while maintaining high standards will be key to the successful development and commercialization of new ADCs.

Conclusion

As we reflect on the current state and future of ADCs in cancer treatment, it’s clear that this field stands to hold incredible potential and has already opened new pathways in the fight against cancer, particularly in addressing the complexities of tumor heterogeneity and drug resistance.

The specificity and efficacy of ADCs have been significantly improved through advancements in antibody engineering, linker technology, and the exploration of diverse payload mechanisms. These enhancements, like ThioBridge, have broadened the therapeutic window of ADCs, making them applicable to a wider range of cancer types and more effective against traditionally hard-to-treat tumors. The integration of immune-modulating payloads into ADCs is a particularly interesting advancement and benefits from the growing body of research in the field of immuno-oncology.

The evolution of ADCs embodies the dynamic nature of cancer research and treatment. With continued advancements in technology and a deeper understanding of tumor biology, ADCs stand as a testament to the potential of targeted therapies in oncology. The role of CDMOs like Abzena in this progress underscores the importance of collaborative efforts in advancing medical science. Research will continue to push the boundaries of what is possible in cancer therapy, and we have no doubt ADCs will play a central role in shaping that future.

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Louise Duffy is focused on the scientific and technical aspects associated with Abzena’s portfolio from early development to commercial production. Louise is responsible for leading and driving Abzena’s technical CMC strategy, providing technical and CMC regulatory leadership and guidance to project teams while working collaboratively with clients for project success. Prior to joining Abzena in 2020 she was an independent consultant in biopharmaceuticals and cell and gene therapies and has held senior leadership roles in GlaxoSmithKline as VP & Global Supply Chain Head, Biopharmaceuticals and Janssen R&D (J&J) as VP & Global Head, Strategic Sciences.

References
• Ackerman, Shelley E., Cecelia I. Pearson, Joshua D. Gregorio, Joseph C. Gonzalez, Justin A. Kenkel, Felix J. Hartmann, Angela Luo, et al. 2021. ‘Immune-Stimulating Antibody Conjugates Elicit Robust Myeloid Activation and Durable Antitumor Immunity’. Nature Cancer 2 (1): 18–33. https://doi.org/10.1038/s43018-020-00136-x.
• Aggarwal, Devesh, Jie Yang, Md. Abdus Salam, Sagnik Sengupta, Md. Yusuf Al-Amin, Saad Mustafa, Mohammad Aasif Khan, Xun Huang, and Jogendra Singh Pawar. 2023. ‘Antibody-Drug Conjugates: The Paradigm Shifts in the Targeted Cancer Therapy’. Frontiers in Immunology 14. https://www.frontiersin.org/articles/10.3389/fimmu.2023.1203073.
• Akinleye, Akintunde, and Zoaib Rasool. 2019. ‘Immune Checkpoint Inhibitors of PD-L1 as Cancer Therapeutics’. Journal of Hematology & Oncology 12 (1): 92. https://doi.org/10.1186/s13045-019-0779-5.
• Conilh, Louise, Lenka Sadilkova, Warren Viricel, and Charles Dumontet. 2023. ‘Payload Diversification: A Key Step in the Development of Antibody–Drug Conjugates’. Journal of Hematology & Oncology 16 (1): 3. https://doi.org/10.1186/s13045-022-01397-y.
• Dagogo-Jack, Ibiayi, and Alice T. Shaw. 2018. ‘Tumour Heterogeneity and Resistance to Cancer Therapies’. Nature Reviews Clinical Oncology 15 (2): 81–94. https://doi.org/10.1038/nrclinonc.2017.166.
• Esapa, Benjamina, Jiexuan Jiang, Anthony Cheung, Alicia Chenoweth, David E. Thurston, and Sophia N. Karagiannis. 2023. ‘Target Antigen Attributes and Their Contributions to Clinically Approved Antibody-Drug Conjugates (ADCs) in Haematopoietic and Solid Cancers’. Cancers 15 (6): 1845. https://doi.org/10.3390/cancers15061845.
• He, Lei, Liangliang Wang, Zhisong Wang, Tiantian Li, Hui Chen, Yaning Zhang, Zeping Hu, Dimiter S. Dimitrov, Juanjuan Du, and Xuebin Liao. 2021. ‘Immune Modulating Antibody-Drug Conjugate (IM-ADC) for Cancer Immunotherapy’. Journal of Medicinal Chemistry 64 (21): 15716–26. https://doi.org/10.1021/acs.jmedchem.1c00961.
• Hoimes, Christopher J., Thomas W. Flaig, Matthew I. Milowsky, Terence W. Friedlander, Mehmet Asim Bilen, Shilpa Gupta, Sandy Srinivas, et al. 2023. ‘Enfortumab Vedotin Plus Pembrolizumab in Previously Untreated Advanced Urothelial Cancer’. Journal of Clinical Oncology 41 (1): 22–31. https://doi.org/10.1200/JCO.22.01643.
• Huang, Lei, Bob Veneziale, Mark Frigerio, George Badescu, Xiaoming Li, Qiping Zhao, Jesse Bahn, et al. 2016. ‘Abstract 1217: Preclinical Evaluation of a next-Generation, EGFR Targeting ADC That Promotes Regression in KRAS or BRAF Mutant Tumors’. Cancer Research 76 (14_Supplement): 1217. https://doi.org/10.1158/1538-7445.AM2016-1217.
• Kim, Hyunjoon, Lin Niu, Peter Larson, Tamara A. Kucaba, Katherine A. Murphy, Britnie R. James, David M. Ferguson, Thomas S. Griffith, and Jayanth Panyam. 2018. ‘Polymeric Nanoparticles Encapsulating Novel TLR7/8 Agonists as Immunostimulatory Adjuvants for Enhanced Cancer Immunotherapy’. Biomaterials 164 (May): 38–53. https://doi.org/10.1016/j.biomaterials.2018.02.034.
• Rosenberg, J. E., M. Milowsky, C. Ramamurthy, N. Mar, R. R. McKay, T. Friedlander, C. Ferrario, et al. 2022. ‘LBA73 Study EV-103 Cohort K: Antitumor Activity of Enfortumab Vedotin (EV) Monotherapy or in Combination with Pembrolizumab (P) in Previously Untreated Cisplatin-Ineligible Patients (Pts) with Locally Advanced or Metastatic Urothelial Cancer (La/mUC)’. Annals of Oncology 33 (September): S1441. https://doi.org/10.1016/j.annonc.2022.08.079.
• Schettini, Francesco, and Aleix Prat. 2021. ‘Dissecting the Biological Heterogeneity of HER2-Positive Breast Cancer’. The Breast : Official Journal of the European Society of Mastology 59 (August): 339–50. https://doi.org/10.1016/j.breast.2021.07.019.
• Sun, Hao, Yingmei Li, Peng Zhang, Haizhou Xing, Song Zhao, Yongping Song, Dingming Wan, and Jifeng Yu. 2022. ‘Targeting Toll-like Receptor 7/8 for Immunotherapy: Recent Advances and Prospectives’. Biomarker Research 10 (December): 89. https://doi.org/10.1186/s40364-022-00436-7.
• Wang, Zhijia, Hanxuan Li, Lantu Gou, Wei Li, and Yuxi Wang. 2023. ‘Antibody-Drug Conjugates: Recent Advances in Payloads’. Acta Pharmaceutica Sinica. B 13 (10): 4025–59. https://doi.org/10.1016/j.apsb.2023.06.015.
• Yamazaki, Chisato M., Aiko Yamaguchi, Yasuaki Anami, Wei Xiong, Yoshihiro Otani, Jangsoon Lee, Naoto T. Ueno, Ningyan Zhang, Zhiqiang An, and Kyoji Tsuchikama. 2021. ‘Antibody-Drug Conjugates with Dual Payloads for Combating Breast Tumor Heterogeneity and Drug Resistance’. Nature Communications 12 (1): 3528. https://doi.org/10.1038/s41467-021-23793-7.