What are the preclinical assets being developed for OX40?

Introduction to OX40

OX40, also known as CD134 or TNFRSF4, is a member of the tumor necrosis factor receptor superfamily. It is expressed predominantly on activated T cells and plays a central role in mediating costimulatory signals that enhance T-cell survival, proliferation, and differentiation. In recent years, the modulation of OX40 has emerged as a promising strategy both to invigorate antitumor immune responses and to regulate autoimmune conditions. Preclinical assets targeting OX40 have been designed to exploit this powerful costimulatory pathway, with the goal of enhancing immune-mediated clearance of tumors and modulating immunosuppressive environments at sites of inflammation or malignancy.

Definition and Function of OX40

OX40 is defined as a type I transmembrane glycoprotein that appears on T cells after the initial antigen recognition. Its primary role is to act as a costimulatory signal that complements the signals delivered via the T-cell receptor (TCR) and other costimulatory molecules such as CD28. Engagement of OX40 by its natural ligand, OX40L (CD252), provides critical signals that foster the expansion and long-term survival of effector and memory T cells. In addition, OX40 signaling supports the secretion of cytokines and the generation of robust adaptive immune responses by enhancing clonal expansion.

Role of OX40 in Immune Regulation

The role of OX40 in immune regulation extends beyond simply amplifying T-cell responses. By modulating the balance between effector T cells and regulatory T cells (Tregs), OX40 influences both immune activation and tolerance. Activation of OX40 can reduce the suppressive functions of Tregs, thereby enhancing cytotoxic responses against tumors. Moreover, through sustained costimulatory signals, OX40 promotes T-cell survival, increases antibody production, and influences the cytokine milieu in the tumor microenvironment and inflammatory sites. Thus, strategies targeting OX40 provide a dual advantage: they can trigger antitumor immunity by stimulating effector cells while simultaneously modulating suppressive pathways that might otherwise limit therapeutic efficacy.

Current Preclinical Assets Targeting OX40

Preclinical assets being developed for OX40 encompass a wide variety of therapeutic modalities—from conventional monoclonal antibodies and bispecific antibodies to antibody fragments, engineered Fc variants, and novel fusion proteins. These assets are designed to either agonize or inhibit OX40 signaling, intending to fine-tune T-cell responses for oncological or autoimmune applications.

Overview of Preclinical Assets

A diverse array of preclinical assets targeting OX40 reflects the complexity of the receptor's role in immunoregulation:
• Monoclonal antibodies (mAbs) that function as agonists of OX40 are engineered to enhance T-cell activation and sustain their survival. Examples include novel chimeric antibodies developed using recombinant protein, DNA and cell-based immunization strategies that exhibit high specificity for OX40. Some of these candidates have shown potent activity in preclinical models by stimulating memory T-cell functions and enhancing antitumor responses.
• Bispecific antibodies represent another asset class where one arm targets OX40 while the other engages an additional antigen, often expressed within the tumor microenvironment. A prime example is the Anti OX40/FAP-α bispecific antibody developed by Roche. This asset is designed to combine tumor stroma targeting (via FAP inhibition) with immune costimulation (via OX40 activation), thereby focusing the immune-enhancing effects locally within the tumor microenvironment.
• Monoclonal antibodies that inhibit OX40 (as opposed to stimulating it) have also been developed, particularly in the context of treating autoimmune conditions. Patents and preclinical studies suggest that Fc-engineered antibodies, which minimize Fc receptor binding and potential unintended agonistic effects, are in development.
• Chimeric and engineered antibodies with modified Fc regions to either augment or silence effector functions have been developed in order to fine-tune the balance between efficacy and safety. For instance, research comparing Fc-wildtype variants to Fc-silenced versions indicates that selective Fcγ receptor engagement is critical, as seen with assets like IMG-007 that have been engineered to reduce agonistic risks during blockade strategies.
• Furthermore, fusion proteins and targeted delivery systems, such as antibody fragment-mediated delivery of soluble OX40L, have been designed to facilitate tumor-specific activation of OX40. Such approaches aim to overcome the widespread activation of OX40 that may result in systemic side effects, thereby focusing the costimulatory signals in the tumor microenvironment.
• Innovative cell-based approaches such as engineered cellular nanovesicles (NVs) expressing OX40 receptors have also been investigated. These NVs are designed to home in on inflamed tissues, such as rheumatoid arthritis (RA) joints or inflammatory bowel disease (IBD) colons, in order to provide localized blockade of the OX40–OX40L interaction, thus modulating autoaggressive T-cell responses while sparing systemic immunity.

Key Players and Developers

The preclinical development of OX40-targeted assets is pursued by various research organizations and biopharmaceutical companies with expertise in immunomodulatory therapies. Key players include:
• F. Hoffmann-La Roche Ltd. – Known for developing the Anti OX40/FAP-α bispecific antibody, which is currently at the preclinical stage and designed for harnessing both immune costimulation and stromal targeting through dual mechanism action.
• Centenaire Biosciences, Inc. – Developing assets such as CTN-010, a preclinical monoclonal antibody with OX40 inhibitory properties that has been explored in the context of multiple immune-mediated diseases.
• Innolake Pharmaceuticals (Hangzhou) Co., Ltd. – Responsible for the preclinical asset ILB-2107, an anti-OX40 monoclonal antibody currently in the preclinical development stage, with a focus on suppressing immune responses in autoimmune indications.
• JN Biosciences LLC – Their preclinical candidate HuOHX10 is a monoclonal antibody targeting OX40 that has shown promising antitumor activity in early preclinical models.
• Several academic research groups and startups are also actively engaged in developing novel constructs using DNA immunization platforms. These efforts have resulted in chimeric anti-OX40 antibodies that are tailored to activate memory T cells and demonstrate potent antitumor activity in preclinical models.
• Additional entities are exploring Fc-engineered OX40 agonists designed to enhance FcγRIIB binding to drive agonistic activity in tumor infiltrating lymphocytes. Studies using Fc-engineered antibodies have provided evidence that selective Fcγ receptor engagement can markedly boost T-cell responses in the tumor environment.

Mechanisms of Action

Understanding the mechanisms by which OX40 modulation reprograms immune responses is essential for appreciating how different preclinical assets operate. The design of each asset category is driven by the need to precisely modulate the OX40 signaling axis, with subtle differences in mechanism having significant translational implications.

How OX40 Modulation Works

OX40 modulation works by engaging the receptor expressed on activated T cells, thereby providing a second signal that boosts T-cell proliferation, survival, and cytokine production. The engagement of OX40 triggers the activation of NF-κB and other downstream signaling molecules that synergize with TCR signals, resulting in enhanced expansion of effector and memory T-cell populations. This mechanism is particularly effective in the context of cancer, where activating T cells in the tumor microenvironment can overcome immunosuppression and mediate tumor cell killing.
In preclinical models, OX40 agonists have been demonstrated to inhibit the suppressive activity of Tregs, simultaneously enhancing the effector functions of CD4+ and CD8+ T cells. By mitigating Treg-mediated inhibition, these agonists facilitate the recruitment and activation of tumor-infiltrating lymphocytes (TILs), thereby leading to tumor regression in various murine models. Conversely, for autoimmune diseases, OX40 inhibitors or antagonistic antibodies dampen excessive T-cell activation to reduce pathological inflammation.

Differences in Mechanisms Among Assets

Different classes of OX40-targeting assets achieve their objectives via distinct mechanisms:
• Monoclonal antibody agonists predominantly work by binding to OX40 and mimicking the natural ligand (OX40L) function. Some are engineered with enhanced Fc-dependent crosslinking to boost receptor clustering for a stronger agonistic signal. For instance, Fc-engineered antibodies that selectively bind FcγRIIB have demonstrated a pronounced capacity to drive T-cell expansion in tumor tissues.
• Bispecific antibodies, like the Anti OX40/FAP-α bispecific, are designed to simultaneously engage OX40-expressing T cells and FAP-expressing stromal components. This dual engagement not only delivers costimulatory signals to T cells but also localizes the immune activity to the tumor site, reducing systemic side effects.
• Some preclinical assets include antibodies that are designed to be antagonistic. In these cases, the antibodies aim to inhibit OX40 signaling to prevent excessive T-cell activation in diseases where immune overactivity is detrimental. To minimize risks associated with unintentional receptor activation due to clustering, these antibodies are often engineered to have reduced Fc receptor binding capabilities, as seen with the Fc-silenced variant of IMG-007.
• Fusion proteins and targeted delivery systems, including antibody fragment-mediated delivery of soluble OX40L variants, are formulated to retain a low level of systemic activity until the fusion protein is localized to the tumor microenvironment. Once bound to a target antigen, the construct can then efficiently activate OX40 signaling, offering a more targeted therapeutic approach.
• Cell-derived nanovesicles (NVs) represent another innovative mechanism. By displaying OX40 receptors on their surface, these NVs can selectively bind to inflamed or diseased tissues, where they sequester ligands or modulate the local immune response. This approach offers the advantage of using biological membranes to facilitate selective targeting and improved biodistribution.

Therapeutic Potential and Applications

The promise of OX40 as a therapeutic target lies in its versatility. Preclinical studies have demonstrated that manipulation of the OX40 pathway can result in robust antitumor responses as well as beneficial immunomodulation in autoimmune diseases. The development of preclinical assets targeting OX40 is supported by a robust body of evidence from diverse experimental models.

Potential Indications

The potential indications for OX40-targeted therapies extend across a spectrum of diseases:
• Cancer Immunotherapy:
 – Activating OX40 signaling on T cells within the tumor microenvironment has been demonstrated to improve the efficacy of immunotherapy by reinvigorating exhausted T cells and lowering the suppressive effects of Tregs. Preclinical models in melanoma, sarcoma, colon carcinoma, glioma, and several breast cancer models have provided compelling evidence in support of this approach.
 – Combination therapies incorporating OX40 agonists with other immunotherapeutic agents such as anti-PD-1/PD-L1 checkpoint inhibitors have shown enhanced antitumor responses compared to monotherapy, as these combinations offer synergistic effects that maximize T-cell activation and reduce tumor-induced immune suppression.
• Autoimmune Diseases:
 – In conditions like rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), where aberrant T-cell activation drives pathology, blocking or modulating the OX40 pathway offers a route to suppress autoaggressive T-cell activity. Preclinical data on cell-derived nanovesicles (NVs) expressing OX40 receptors have shown promising results in reducing inflammation and modulating cytokine responses locally, thereby mitigating disease severity.
 – OX40 antagonistic approaches are being explored to reduce T-cell mediated tissue damage in autoimmune disorders, making these assets highly relevant for clinical conditions where excessive immune activation is harmful.
• Transplantation and Tolerance Induction:
 – Emerging evidence indicates that controlled modulation of OX40 may also play a role in promoting transplant tolerance by regulating the balance between effector T cells and Tregs, potentially reducing the incidence of graft rejection in transplant patients.

Preclinical Efficacy and Safety Data

The efficacy and safety profiles of these preclinical assets have been extensively evaluated in a variety of in vitro assays and preclinical animal models:
• Efficacy Data:
 – In murine models, treatment with OX40 agonists has resulted in significant tumor regression, increased infiltration of CD8+ cytotoxic T cells, enhanced granzyme B production, and overall improvement in survival rates.
 – Assets such as the bispecific antibody targeting both OX40 and FAP-α have demonstrated notable antitumor activity by mediating dual engagement of immune and stromal targets, leading to highly localized and potent immune activation within the tumor microenvironment.
 – In autoimmune disease models, OX40 blockade via engineered antagonistic antibodies or targeted nanovesicles has shown the ability to reduce proinflammatory cytokines (e.g., TNF-α, IL-1β) and decrease the number of pathogenic CD4+OX40+ T cells, which correlated with reduced disease progression in RA and IBD models.
• Safety Data:
 – Preclinical safety studies have underlined the importance of engineering the Fc regions of OX40-targeting antibodies to mitigate risks such as off-target receptor clustering and unwanted systemic T-cell activation. For example, studies comparing Fc-silenced versus Fc-wildtype variants have demonstrated that minimizing Fc receptor binding can reduce cytokine release and other adverse effects, thereby enhancing the safety profile of these assets.
 – The targeted delivery strategies employed by some assets, such as antibody fragment fusion proteins and targeted nanovesicles, help to confine the biological activity to the tumor or inflamed tissue, thereby further reducing systemic toxicities observed with broadly acting immunomodulators.
 – Many preclinical studies have reported no dose-limiting toxicities at the effective therapeutic doses, which is promising for translating these assets into future clinical trials.

Challenges and Opportunities

While the preclinical landscape for OX40-targeted therapies is robust and filled with diverse assets under development, it also presents unique challenges and opportunities that need to be addressed as the field matures.

Developmental Challenges

Several key challenges have been identified in the development of preclinical assets targeting OX40:
• Optimizing Fc Engineering:
 – One of the primary challenges revolves around fine-tuning the Fc region of monoclonal antibodies to either enhance or silence effector functions. Although Fc-engineered antibodies have demonstrated promising results, ensuring the appropriate balance between efficacy and safety remains critical. Unintended receptor clustering, for instance, may potentiate undesired agonistic signaling in antibodies intended to act as antagonists, as observed in some variants of IMG-007.
• Tumor-Specific Targeting:
 – A significant challenge in the development of OX40 agonists is achieving precise tumor-specific activation. While systemic activation of OX40 can yield robust antitumor responses, it also poses risks for widespread immune activation, which can lead to off-target effects and toxicity. Targeted delivery systems such as bispecific antibodies and antibody fragment-based fusion proteins are designed to overcome these challenges; however, optimizing their pharmacokinetics and biodistribution remains an active area of research.
• Heterogeneity of Immunological Responses:
 – The variability in OX40 expression and downstream signaling among different patients and even within distinct tumor types means that preclinical models must accurately recapitulate human immune heterogeneity. Differences in the tumor microenvironment, immune cell infiltration, and the presence of immunosuppressive factors necessitate the development of sophisticated preclinical models, including organ-on-a-chip systems, to better predict clinical outcomes.
• Translational Gaps:
 – Despite promising data from preclinical studies, translation to clinical success is fraught with uncertainties. Factors such as differences in receptor engagement, the impact of human Fc receptor polymorphisms, and variability in immune system dynamics between animal models and humans can limit the predictive value of preclinical data.
• Manufacturing and Scalability:
 – The complexity inherent in producing bispecific antibodies, Fc-engineered molecules, and cellular nanovesicles means that reproducible manufacturing and ensuring scalability for clinical-grade material is a major practical challenge that preclinical developers must overcome.

Future Opportunities and Directions

Despite these challenges, the future of OX40-targeted therapies offers several exciting opportunities and directions for further research and clinical development:
• Combination Therapies:
 – One of the most promising opportunities lies in the combination of OX40 agonists with other immune checkpoint inhibitors, cytokine therapies, or therapeutic modalities such as chemotherapy and radiotherapy. Combining these agents has the potential to achieve synergistic effects, as preclinical studies have shown that OX40 activation can enhance the efficacy of PD-1/PD-L1 blockade by reinvigorating T-cell responses within the tumor microenvironment.
• Personalized Medicine Approaches:
 – Advances in biomarker discovery and personalized medicine approaches are likely to refine patient selection for OX40-targeted therapies. Companion diagnostics that measure OX40 receptor expression levels, immune cell infiltration profiles, and specific pharmacodynamic biomarkers could help identify patient populations most likely to benefit from these therapies and guide dosing regimens accordingly.
• Innovative Delivery Methods:
 – The development of novel delivery platforms such as antibody fragment-mediated fusion proteins, targeted nanovesicles, and scaffold-based systems offers an opportunity to mitigate systemic side effects while providing localized immune activation. These approaches may revolutionize the way OX40 agonists are administered and further enhance their therapeutic window.
• Next-Generation Fc Engineering:
 – Continued innovations in Fc engineering may allow further customization of antibody properties to maximize therapeutic effects while minimizing adverse effects. Enhanced engagement of specific Fcγ receptors—such as FcγRIIB—has already shown promise in preclinical studies and represents a key area for future development.
• Advanced Preclinical Models:
 – Incorporating cutting-edge preclinical models such as organ-on-a-chip systems and humanized mouse models could reduce the translational gap and improve the predictability of therapeutic outcomes. These models allow researchers to mimic human immune-tumor interactions more closely, thereby refining candidate asset selection and encouraging more robust clinical translation.
• Exploration of Dual-Action Molecules:
 – Future research may focus on developing dual-action molecules that both agonize OX40 and simultaneously modulate secondary pathways involved in T-cell activation or suppression. For example, bispecific antibodies that combine OX40 targeting with inhibition of immunosuppressive molecules could offer a multi-pronged strategy against tumor growth.

Conclusion

In summary, the preclinical assets being developed targeting OX40 embody a diverse array of therapeutic modalities designed to harness the costimulatory power of the OX40 receptor. Starting from the detailed understanding of OX40’s role as a potent costimulatory molecule that influences both effector T-cell responses and the modulation of regulatory T cells, these assets include agonistic monoclonal antibodies, bispecific antibodies, chimeric fusion proteins, and even innovative cell-derived nanovesicles. Each asset class employs unique mechanisms to either activate or inhibit OX40 signaling with the aim of tailoring immune responses for optimal therapeutic outcomes across a range of indications. On the oncology side, assets such as the Anti OX40/FAP-α bispecific antibody developed by Roche and chimeric anti-OX40 antibodies generated by advanced DNA immunization platforms have shown promising antitumor efficacy in preclinical models by reinforcing T-cell proliferation, cytokine production, and overcoming Treg-mediated immune suppression.
For autoimmune diseases, engineered antagonistic antibodies and targeted nanovesicles have been developed to selectively dampen harmful T-cell activity, thereby reducing inflammation and disease progression in conditions like rheumatoid arthritis and inflammatory bowel disease. Moreover, each of these assets is characterized by distinct differences in mechanism of action—ranging from Fc-region modifications that limit off-target effects to bispecific constructs that focus activity within the tumor microenvironment. The efficacy data from multiple preclinical models, combined with encouraging safety profiles that have been optimized through novel engineering techniques, support the clinical promise of these approaches. However, challenges remain, particularly in optimizing targeted delivery, mitigating systemic toxicity, and overcoming the translational gap between animal models and human responses.
Looking forward, opportunities for improved combination therapies, personalized medicine strategies using robust predictive biomarkers, and next-generation delivery systems are poised to enhance the effectiveness of OX40-targeted assets. Continued advancements in Fc engineering and the integration of novel preclinical models will further refine these therapies, ensuring more accurate clinical translation and improved patient outcomes.
In conclusion, the preclinical portfolio for OX40-based therapies is extensive and multifaceted, spanning selective agonists, dual-targeting bispecific antibodies, and targeted delivery vehicles. These assets not only underscore the therapeutic potential of modulating OX40 but also highlight a broader paradigm shift towards precision immunotherapy. With robust preclinical efficacy data and innovative engineering solutions to address key developmental challenges, the future of OX40-targeted therapies appears promising for both oncology and autoimmune indications. Each asset’s unique design and mechanism of action contribute to a comprehensive strategy aimed at enhancing patient outcomes through improved immune modulation, and the continued evolution of these preclinical assets is likely to set the stage for transformative advances in clinical immunotherapy.