What are the preclinical assets being developed for PD-1?

Introduction to PD-1 and Its Role in Immunotherapy

PD-1 Pathway and Its Significance
Programmed death‐1 (PD-1) is a critical inhibitory receptor expressed predominantly on activated T cells, B cells, natural killer cells, and other immune‐related cells. Under physiological conditions, PD-1 engagement—via its ligands PD-L1 and PD-L2—serves as a checkpoint for immune responses, preventing excessive or prolonged T cell activation that could cause autoimmunity. In effect, the PD-1 pathway is fundamental to maintaining peripheral tolerance and immune homeostasis. However, in the tumor microenvironment, many cancer cells overexpress PD-L1 to exploit this pathway and dampen anti-tumor immunity, thereby evading immune surveillance. This unique duality, whereby PD-1’s normal regulatory function becomes subverted by tumor cells, underpins the rationale for targeting the pathway in immunotherapy. Detailed structural studies have elucidated the interaction surfaces and binding kinetics between PD-1 and its ligands, providing the groundwork for the rational design of therapeutic agents that either block or modulate this interaction.

Overview of Immunotherapy Targeting PD-1
Immunotherapy has revolutionized cancer treatment by harnessing the body’s immune system to attack malignancies. Among the various strategies, the blockade of immune checkpoints – particularly the PD-1/PD-L1 interaction – has emerged as a leading therapeutic approach. Monoclonal antibodies that inhibit PD-1 or PD-L1 (for example, nivolumab and pembrolizumab) have demonstrated significant clinical efficacy in several tumor types including melanoma, non-small cell lung cancer, and renal cell carcinoma. The success of these agents has spurred a dynamic area of research aimed at refining and enhancing PD-1-targeted immunotherapies. In preclinical settings, innovations ranging from bispecific antibodies to novel fusion proteins and engineered cell therapies are being developed to improve specificity, potency, and safety profiles while reducing systemic toxicity. With an ever-expanding pipeline of assets, researchers are now focusing on both upstream (modulation of PD-1 expression and signaling) and downstream (combinatorial immune activation) strategies to not only block but also fine-tune the immune-evasive mechanisms mediated by PD-1.

Current Preclinical Assets for PD-1

Types of Preclinical Assets
Preclinical assets being developed for PD-1 encompass a broad spectrum of engineered molecules, fusion proteins, cell therapies, and small molecules. Generally, these assets can be classified into several key types:

• Monoclonal Antibodies and Their Derivatives
Traditional fully humanized or human monoclonal antibodies that block PD-1 from engaging with its ligands constitute one of the earliest and most studied asset classes. These assets are being further refined into next-generation antibodies that incorporate modifications – such as improved Fc engineering or altered glycosylation patterns – to optimize their binding efficacy, extend in vivo half-life, and mitigate adverse immune reactions.

• Bispecific Antibodies
An emerging trend is the development of bispecific antibodies that not only target PD-1 but also engage a secondary target relevant to tumor biology (for example, CD19 in the context of hematologic malignancies). Bispecific formats are designed to simultaneously block the inhibitory PD-1 signal while triggering additional anti-tumor immune responses or directly engaging tumor cells. Ono Pharmaceutical, for instance, is executing innovative strategies to design bispecifics that combine PD-1 inhibition with other modalities for enhanced therapeutic benefit.

• Immunocytokine Fusion Proteins
Fusion proteins that combine a PD-1 targeting moiety with immunomodulatory cytokines such as IL-15 or IL-18 are gaining attention. These immunocytokines are engineered to deliver co-stimulatory signals directly to T cells in the tumor microenvironment. Assets such as PD1-IL15 and PD1-IL18 conjugates are preclinically evaluated for their ability to not only block the PD-1 checkpoint but also to locally stimulate T cell proliferation, activation, and cytotoxicity. Such dual-action molecules are aimed at overcoming the limitations of single-agent PD-1 blockade by inducing a more robust anti-tumor immune response.

• Engineered Chimeric Antigen Receptor T Cells
Adoptive cell therapies incorporating modifications to disrupt PD-1 signaling have also emerged as promising preclinical assets. In these innovative CAR-T cell designs, genetic modifications are often introduced to knock out or silence PD-1, thereby circumventing the inhibitory signals from the tumor microenvironment. This enables the engineered T cells to function more effectively even in the presence of high levels of PD-L1 expressed by tumor cells.

• Small Molecule Modulators and Nanoparticle-Based Delivery Systems
While monoclonal antibodies remain predominant, research has also delved into small molecule approaches to interfere with PD-1/PD-L1 interactions. In addition, nanoparticle-based systems are being developed for the controlled release of PD-1 inhibitors, which may enhance tissue penetration and improve pharmacokinetic profiles. These platforms represent a novel modality that could complement or serve as an alternative to antibody-based therapies.

• RNA-Based Therapeutics and Gene Editing Approaches
Recent preclinical advancements include RNA interference (RNAi) techniques to modulate PD-1 expression and CRISPR-Cas9 mediated gene editing strategies. These approaches not only serve as investigative tools to better understand the role of PD-1 in immune regulation but could also be harnessed to create innovative therapeutic constructs that permanently modulate PD-1 signaling pathways.

Key Players and Innovations
A number of world-renowned biopharmaceutical companies and academic laboratories are at the forefront of developing these preclinical assets. Key players include:

• Ono Pharmaceutical Co., Ltd.
Ono Pharmaceutical is notable for developing assets such as ONO-4915, a bispecific antibody that targets both CD19 and PD-1, showcasing their commitment to combining PD-1 blockade with additional immunologic mechanisms to enhance therapeutic utility.

• ImmVira and Shanghai Pharmaceuticals Holdings
In preclinical studies, assets based on oncolytic virus immunotherapies expressing both interleukin-12 and anti-PD-1 antibodies have been engineered. Although primarily designed for intratumoral delivery, these assets exemplify the potential of combining immunocytokines with PD-1 blockade for synergistic effects.

• Innovative Platforms by Akeso Biopharma Co., Ltd.
Preclinical research at Akeso has resulted in the development of bispecific antibodies targeting PD-1 in combination with other immune checkpoints. Their assets are supported by both structural insights and robust preclinical models that evaluate efficacy and safety profiles.

• Excoso as
Excoso as has been actively engaged in preclinical asset development and has repeatedly reached phase time milestones in 2024. Their efforts include optimizing novel scaffolds tailored to modulate PD-1 pathways, both as monotherapy and as components of combination regimens.

• Additional Innovators
Other organizations, such as Compass Therapeutics and Bright Peak Therapeutics, are also pursuing next-generation modalities that leverage PD-1 targeting in innovative conjugate forms (e.g., PD1-IL18 conjugates) with promising preclinical anti-tumor activity. These developments are part of a broader landscape where academic-driven insights are rapidly being translated into clinical candidates via academia-industry collaborations.

Collectively, these innovations illustrate that the preclinical asset landscape for PD-1 is highly diverse, encompassing multiple modalities from classical antibody blockade to cutting-edge fusion proteins and engineered cellular therapies. Each of these assets is designed not only to inhibit the PD-1 pathway but also to overcome inherent resistance mechanisms and augment the overall anti-tumor immune response.

Evaluation of Preclinical Assets

Mechanisms of Action
The preclinical assets targeting PD-1 are meticulously designed to disrupt the inhibitory signals delivered by the PD-1/PD-L1 axis, thereby resuscitating T cell activity against tumor cells across multiple dimensions:

• Blockade of PD-1/PD-L1 Interaction
The primary mechanism is the direct blockade of the PD-1 receptor’s engagement with its ligands, PD-L1 and PD-L2. By preventing this interaction, these agents restore T cell activation and promote cytokine production, ultimately enhancing cytotoxicity against tumor cells. This mechanism has been thoroughly characterized in preclinical models by assessing T cell receptor signaling, cytokine secretion profiles, and downstream effects on tumor regression.

• Dual-Targeting Through Bispecific Formats
Bispecific antibodies combine the blockade of PD-1 with the engagement of alternative targets, such as tumor-associated antigens or other immune checkpoints. For instance, a bispecific antibody that targets both CD19 and PD-1 not only blocks inhibitory signals but also facilitates direct recognition and destruction of malignant B cells. This dual-targeting approach aims to create a ‘one-two punch’ in tumor eradication by orchestrating multiple immune activations concurrently.

• Immunocytokine Fusion for Localized Immune Activation
Fusion proteins that incorporate a PD-1-specific domain with cytokines like IL-15 or IL-18 are engineered to provide localized immunomodulation. The rationale behind these assets is to concentrate immune stimulus within the tumor microenvironment, thereby increasing T cell proliferation and activity at the site of the tumor while potentially reducing systemic side effects. This is achieved by coupling the specificity of PD-1 blockade with the potent secondary signaling provided by the cytokines, leading to improved anti-tumor efficacy.

• Enhancement of CAR-T Cell Therapy
In engineered T cell therapies, genetic modifications—such as PD-1 knockouts or knockdowns—are applied to chimeric antigen receptor (CAR) T cells. This modification renders the CAR-T cells less susceptible to the immunosuppressive signals present in the tumor microenvironment, enabling them to sustain a robust anti-tumor response even in immunologically hostile settings. Such preclinical strategies are validated using syngeneic tumor models and in vitro assays that measure T cell cytotoxicity and persistence.

• Alternative Signaling Modulators and Small Molecule Approaches
In addition to antibody-based well-established mechanisms, novel small molecule modulators and nanoparticle-based delivery systems are being developed to either interfere with the PD-1 protein’s conformation or modulate its downstream signaling. These preclinical assets are designed to offer advantages in terms of manufacturing and scalability, and they often benefit from the rapid pharmacokinetic (PK) profiling afforded by small molecules. Advanced PK/PD modeling approaches have been applied to these assets to predict optimal dosing regimens and therapeutic windows.

Each of these mechanisms is supported by rigorous preclinical research that evaluates molecular interactions, immune cell responses, cytokine milieus, and tumor growth kinetics. Structural biology studies, including crystallographic analyses, have provided critical insights into the atomic-level interactions between PD-1 and its inhibitors, thus guiding the rational design and further refinement of these therapeutic assets.

Preclinical Models and Studies
The evaluation of PD-1 preclinical assets relies on a variety of in vitro and in vivo models, each of which contributes significantly to our understanding of the therapeutic potential and safety profiles of these candidates:

• In Vitro Cellular Assays
Cell culture systems are used to assess the binding affinity, specificity, and functional activity of PD-1 inhibitors. T cell activation assays, cytokine release assays, and cell-based reporter systems provide early evidence of the ability of these agents to disrupt the PD-1/PD-L1 interaction. These in vitro studies are instrumental in optimizing molecular modifications before progressing to in vivo models.

• Syngeneic and Xenograft Mouse Models
Animal models, particularly syngeneic tumor models in immunocompetent mice, are a cornerstone of preclinical evaluation. In these models, the immune system is intact, allowing for a comprehensive assessment of the anti-tumor immune response following PD-1 blockade. Such models are frequently used to study the pharmacodynamic effects, therapeutic efficacy, and potential toxicity of monoclonal antibodies, fusion proteins, and CAR-T cells. Notably, syngeneic models have demonstrated durable tumor regression and enhanced survival rates when treated with PD-1 inhibitors. Additionally, xenograft models (in which human tumors are implanted in immunodeficient mice) are used to gauge direct anti-tumor effects under controlled conditions and to evaluate detailed PK/PD relationships.

• Genetically Engineered and Knockout Models
Genetically engineered mouse models in which PD-1 is manipulated allow researchers to observe the effects of PD-1 deficiency or overexpression. These models serve as valuable tools for dissecting the underlying mechanisms of PD-1 mediated immune inhibition and for testing whether reintroducing PD-1 inhibitors can restore immune function. They also help in understanding the molecular basis of resistance to therapy.

• Advanced PK/PD Modeling and Simulation Studies
Sophisticated pharmacokinetic and pharmacodynamic (PK/PD) analyses are integrated with preclinical studies to predict human dosing regimens and therapeutic windows. Such models utilize plasma, tissue, and regional venous concentration data to simulate the effects of administered agents. PK/PD modeling supports the transition from preclinical asset development to early-phase clinical trials by providing quantitative insights into factors such as tumor exposure, clearance rates, and potential toxicity.

• Immunoimaging and Biomarker Studies
New imaging techniques, including PET and SPECT, are being used in preclinical settings to track the biodistribution and tumor accumulation of PD-1 targeted agents. Concurrently, biomarker studies analyze changes in immune cell populations, cytokine profiles, and gene expression patterns following treatment. These studies are essential for validating mechanism-based endpoints and for optimizing patient stratification in future clinical trials.

Together, these preclinical models offer a multi-layered approach that validates the efficacy and safety of PD-1 inhibitors from molecular, cellular, and organismal perspectives. The data generated from these diverse assays provide robust proof-of-concept and guide iterative improvements in asset design before clinical translation.

Future Directions and Challenges

Emerging Trends in PD-1 Targeting
Looking ahead, several promising trends are likely to shape the future of preclinical asset development for PD-1:

• Combination Immunotherapy Approaches
One of the key future directions is the development of combinatorial strategies that pair PD-1 inhibitors with agents targeting additional immune checkpoints or costimulatory molecules. For example, integrated preclinical studies are investigating combinations of PD-1 blockade with CTLA-4 inhibitors, OX40 agonists, and novel immunocytokines. This approach holds the potential to further amplify anti-tumor immune responses by addressing multiple mechanisms of immune suppression simultaneously.

• Advanced Fusion Proteins and Immunocytokines
The next generation of fusion proteins is expected to improve tumor-specific delivery while minimizing systemic adverse effects. Emerging preclinical assets that link PD-1 binding domains with cytokines (such as IL-15 or IL-18) are designed to create a localized “cytokine burst” within the tumor microenvironment. Such innovations could offer enhanced efficacy over traditional PD-1 monoclonal antibodies, especially in tumors with a highly suppressive microenvironment.

• Cell-Based and Gene Editing Strategies
Recent developments in CAR-T cell engineering, particularly those involving the knockout or silencing of PD-1, represent a significant trend in preclinical asset development. Such strategies enable the generation of cell therapies that are not susceptible to inhibition by the tumor’s PD-L1 expression. Moreover, advances in CRISPR-Cas9 technology hold promise for the precise modulation of immune checkpoints in ex vivo expanded T cells, potentially leading to more potent and durable therapeutic responses.

• Nanotechnology and Novel Delivery Systems
Innovative nanoparticle-based systems and targeted delivery platforms are being explored as methods to improve the localization and controlled release of PD-1 inhibitors. These systems can enhance tissue penetration, reduce the overall dose required, and lower the risk of systemic toxicity. Early preclinical data suggest that these novel delivery vehicles can be engineered to co-deliver multiple agents, thereby enhancing the therapeutic index of PD-1 targeted therapies.

• Biomarker-Driven Personalized Therapy
Improvements in the identification and validation of predictive biomarkers are expected to revolutionize the development of PD-1 assets. Preclinical research is increasingly focused on characterizing the tumor microenvironment, immune cell infiltration patterns, and genetic mutations that influence response to PD-1 inhibition. Such biomarker studies are critical for developing personalized therapeutic regimens and may eventually lead to more precise patient stratification in combination therapy trials.

Challenges in Preclinical Development
Despite the significant progress, several challenges remain in the development of preclinical assets targeting PD-1:

• Translational Discrepancies Between Models and Human Patients
One of the most pressing challenges is the inherent difference between preclinical animal models and human patients. While syngeneic and xenograft mouse models provide valuable insights, they do not fully recapitulate the complexity of the human immune system. Thus, assets that show promise in preclinical evaluations may not always translate into clinical success. Overcoming this gap requires the use of more sophisticated humanized models and ex vivo assays that can better mimic human immunity.

• Immune-Related Adverse Events (IRAEs)
The modulation of immune checkpoints often leads to immune-related adverse events, which can range from mild to severe autoimmune reactions. Designing preclinical studies that effectively predict the incidence and severity of these adverse events remains a challenge. There is a critical need for improved in vivo models and safety biomarkers that can reliably forecast these toxicities and guide the design of safer agents.

• Manufacturing and Scalability of Complex Modalities
Preclinical assets such as bispecific antibodies, fusion proteins, and cell therapies require complex manufacturing processes. Ensuring scalability and standardization of production, while maintaining high purity and biological activity, is a significant challenge that must be addressed before clinical translation. Advances in bioprocessing and quality control are essential to support the transition from the bench to the bedside.

• Defining Optimal Dosing Regimens and PK/PD Parameters
Given the diversity of PD-1 assets—from antibodies to small molecules and cell therapies—optimizing dosing remains a complex challenge. Preclinical PK/PD modeling is crucial, yet the translation of these models to predict human responses accurately often encounters limitations. There is a need for more integrated modeling approaches that combine in vitro, in vivo, and real-time imaging data to refine dose predictions and minimize off-target toxicity.

• Biomarker Validation and Patient Stratification
While the promise of biomarker-driven therapy is immense, the current lack of robust and universally applicable predictive biomarkers hinders the effective translation of preclinical data into clinical practice. Establishing reliable biomarkers that correlate with response and resistance mechanisms is essential for personalizing therapy and improving outcomes. Extensive validation through preclinical studies and subsequent clinical trials is paramount to overcome this challenge.

Conclusion
In summary, the preclinical assets being developed for PD-1 are diverse, innovative, and advancing rapidly. The field encompasses traditional and next-generation monoclonal antibodies, bispecific antibodies, immunocytokine fusion proteins, engineered cell therapies, small molecule modulators, and even RNA-based strategies. These assets are designed to interrupt the PD-1 pathway—thereby releasing T cells from inhibitory signals—and to stimulate a more robust, targeted, anti-tumor immune response.

The detailed mechanistic studies, inclusive of structural biology insights and sophisticated PK/PD modeling, are enhancing our understanding of how best to design these agents. Equally, preclinical models—from in vitro cellular assays to syngeneic and xenograft mouse models—provide comprehensive platforms to evaluate efficacy, safety, and pharmacological profiles. Key players such as Ono Pharmaceutical, ImmVira, Akeso Biopharma, Excoso as, and several emerging innovators are spearheading these developments with novel designs and strategic innovations.

Looking forward, the field is oriented toward combination immunotherapies, the utilization of advanced delivery systems such as nanoparticles, and the integration of biomarker-driven personalized approaches. Despite challenges in translation, adverse event prediction, manufacturing scalability, and biomarker validation, these efforts represent a significant stride towards moving preclinical successes into clinical reality.

The continued convergence of immunological research, bioengineering, and translational science is expected to overcome current limitations and pave the way for more effective, safer, and patient-tailored PD-1-targeted therapies. With concerted multidisciplinary efforts and advancing technologies, the preclinical landscape of PD-1 assets promises to remain at the forefront of cancer immunotherapy innovations, ultimately contributing to improved clinical outcomes and a better understanding of immune regulation in oncological settings.

Ultimately, as we continue to explore and refine these assets through rigorous preclinical evaluation and translational studies, the promise of PD-1 modulation in immunotherapy will become increasingly realized, offering hope for durable responses and enhanced survival in patients across a broad spectrum of cancers.