Modeling next generation AR pathway inhibitors in prostate cancer

Bispecific antibodies represent a groundbreaking innovation in cancer immunotherapy. By binding to two distinct antigens simultaneously, these molecules direct immune cells to target cancer cells with precision, enhancing immune-mediated killing. The development of these therapies has opened new frontiers in oncology, with preclinical platforms playing a pivotal role in evaluating their efficacy, safety, and mechanisms of action before clinical trials. History and Challenges of Bispecific Antibodies Research The concept of bispecific antibodies originated in the 1960s, with the first experimental bispecific constructs developed in the 1980s using chemical cross-linking. Early versions were limited by instability and production challenges. Advances in genetic engineering in the 1990s led to the creation of more stable and functional bispecific molecules. Over time, the field has evolved to include trispecific, tetraspecific, and even pentaspecific antibodies, each designed to address specific therapeutic needs by engaging multiple targets or pathways simultaneously. The structural complexity of these molecules has grown, incorporating modular designs like dual-variable domain antibodies and Fc-engineered formats to enhance therapeutic efficacy and half-life. [1, 2] The dual mechanisms of bispecific antibodies pose unique challenges in preclinical research. Traditional cell line-based models often fail to replicate the intricate interactions within the tumor microenvironment (TME), limiting their predictive power. Advanced models that closely mimic human tumor biology are required to fully understand the therapeutic potential and risks of bispecific antibodies. Testing Bispecific Antibodies: In Vivo and Ex Vivo Approaches Advanced preclinical models have been used to evaluate the performance of bispecific antibodies. Among the pre-clinical models, patient-derived xenografts (PDX) are invaluable for maintaining the genetic and histological fidelity of patient tumors. Representing the testing platform closer to the clinic, these models allow researchers to study tumor-specific responses and resistance mechanisms in vivo. Syngeneic models, using murine tumors in immunocompetent mice, provide insights into the immune system’s role in therapy efficacy. Yet, murine tumors and immune responses are very different from their human counterpart. While syngeneic models are a good proof of concept, they do not provide clinically translatable reliable data sets. [3] Humanized mice incorporating human tumors together with human immune cells are very important in the field. The standard models used to test bispecific are adoptive transfer models leveraging peripheral blood mononuclear cells (PBMCs) or CD34+ stem cells. These systems replicate the interactions within the TME, enabling the study of immune cell recruitment, activation, and cytotoxicity driven by bispecific antibodies. The integration of humanized mouse models, engrafted with PBMCs or CD34+ stem cells, enhances the translational relevance of these studies by mimicking human immune responses more accurately. The use of humanized mice models can be challenging; however, the right expertise can help the selection of the best strategy for each bispecific MoA or combinatorial treatment of interest with CPI. [4] Organoid models offer a controlled ex vivo environment to study complex cellular interactions, preserving the molecular diversity and histological characteristics of original patient tumors. Adding immune cells to organoids generating a co-culture assay enables researchers to analyze interactions between cancer cells and immune cells within a reconstructed TME. These assays provide critical insights into immune cell recruitment and cytotoxicity, key to understanding bispecific antibody mechanisms of action. Co-culture systems offer a high-throughput, controlled environment for testing bispecific antibodies, maintaining tumor heterogeneity and complexity, making them ideal for pre-clinical interrogation of different strategies to address the complexities of bispecific antibody research. In the hematologic malignancies field, leveraging primary, never-passaged, well-characterized patient material allows the evaluation of bispecific antibody efficacy in a clinically relevant ex vivo system where immune cell viability and function are retained. Conclusion The complexity of bispecific antibody mechanisms needs advanced preclinical platforms that accurately replicate human tumor biology, such as PDX in humanized in vivo hosts, organoids-based co-culture assays, and hematological platforms supporting immune cells, offering unparalleled tools for translational research. By integrating these platforms into bispecific antibody development, researchers can gain deeper insight into mechanisms of action, better predict therapeutic outcomes, and ultimately enhance clinical success. The future of bispecific, trispecific, and even pentaspecific antibody therapies and the combination with checkpoint inhibitors hinges on the optimal selection and execution of specific translational strategies leveraging these innovative preclinical models.

Cancer research is constantly evolving, seeking new methods to better understand and treat this complex disease. Among the most groundbreaking developments in this field are patient-derived xenograft (PDX) models. These models have revolutionized our approach to studying tumors by preserving the heterogeneity and molecular characteristics of a patient's tumor. This blog post aims to provide an in-depth look at the heterogeneity of PDX models and why it’s important to have diversity in your preclinical in vivo studies. The Importance of PDX Models Unlike traditional cell line-based models, PDX models retain the genetic diversity and molecular complexity of the original tumor. PDX models offer researchers a closer approximation of how human tumors behave in vivo, with different cell populations responding differently to therapies. They provide a dynamic and clinically relevant platform for studying tumor growth, metastasis, and response to treatment, making them a powerful tool in the fight against cancer. (Read our blog "The Ultimate Guide to Designing a Mouse Clinical Trial and Data Analysis") Models pretreated with targeted therapies are ideal to test next-generation agents. For example, when developing a drug that aims to overcome resistance to the standard of care therapy in a given tumor type, choosing models pretreated with that standard of care agent for our efficacy study will reveal true therapeutic effects in the target patient population. (Read our blog "Accelerating Innovation & Drug Development with Pre-treated PDX Models") Tumor Heterogeneity of PDX models is important for translational oncology studies One of the most significant advantages of PDX models is their ability to preserve tumor heterogeneity. Tumors are not uniform; they consist of various cell types with different genetic mutations and behaviors. On the contrary, traditional cell lines are developed in vitro by clonal selection, leading to a less accurate representation of the human disease.[1] PDX models also retain the molecular characteristics of the original tumor. This includes genetic mutations, epigenetic modifications, and gene expression patterns. By maintaining these features, PDX models provide a more accurate representation of the human tumor genetic background, which is crucial for developing effective therapies.[1] Advancing Cancer Research with PDX Models The ability of PDX models to preserve tumor heterogeneity and molecular characteristics has significantly advanced cancer research. Researchers can study the behavior of different tumor subpopulations, identify potential targets for therapy, and evaluate the efficacy of new treatments in a more realistic setting. PDX models have heterogeneous biomarker expression. The figure below, for example, shows the expression of KRAS in Champions’ PDX models varying across and within tumor types. Selecting PDX models with a higher KRAS expression will be paramount to successfully test KRAS inhibitors and could provide more clinically relevant results. Nonetheless, a PDX model with elevated KRAS expression will still present with heterogeneous expression across the different cell clones within the tumor, a setting that realistically reproduces the human tumor heterogeneous cell composition. In this setting, a KRAS inhibitor might only be partially effective at killing the tumor because cell clones with lower KRAS expression within the tumor might not be affected, resulting in lower overall therapeutic efficacy. Similarly, in the clinic, we could observe a partial response followed by tumor progression, due to the selection of the low KRAS-expressing cell clone within the tumor. For this reason, studies that use PDX models instead of cell line-derived models will produce more reliable and clinically relevant results. KRAS gene expression across Champions' PDX models of different tumor types. Patient-derived xenograft models have revolutionized cancer research by providing a more accurate representation of human tumors. Their ability to preserve tumor heterogeneity and molecular characteristics makes them an invaluable tool for studying cancer biology, testing new therapies, and personalizing treatment plans. By continuing to develop and refine these PDX models, researchers can accelerate the discovery of new treatments and bring them to patients faster. Champions Oncology has over 1500 clinically relevant PDX models that can accelerate your oncology research program. Our models have broad clinical annotation and have been deeply characterized using NGS (WES & RNA-seq), proteomic, and phospho-proteomic datasets to interrogate the heterogeneity of the tumor. To learn more about our PDX tumor models, access Lumin Analytics.

Chronic lymphocytic leukemia (CLL) is one of the most common forms of adult leukemia, and its chronic nature has made it a challenging blood cancer to completely cure. CLL affects B cells and is typically classified into two categories: little or no somatic hypermutation in the immunoglobulin heavy chain variable region (IGHV), called unmutated CLL, or high mutation levels in the IGHV gene, called mutated CLL.[1] Unmutated CLL is more aggressive than mutated CLL, and the presence of these abnormal IGHV sequences, a part of B cell receptors (BCR), leads to abnormal BCR signaling and the uncontrolled proliferation of leukemic cells.  Bruton’s tyrosine kinase (BTK) is an essential enzyme downstream from the BCR and is responsible for propagating the signaling cascade initiated by BCR engagement with pathogen-associated antigens.[2] Notably, BTK has been shown to be constitutively active in CLL and drives both leukemic cell proliferation and lymph node homing.[3] As antigen binds to the BCR, multiple protein tyrosine kinases are activated via interactions with the cytoplasmic domain of the BCR, including Lyn and Syk, as well as translocation of BTK to the plasma membrane through interactions with phosphatidylinositol-3,4,5 (PIP3).[4] Lyn and Syk phosphorylate BTK to activate multiple non-receptor protein tyrosine kinase signaling pathways, including NF-κB, MAPK, and phospholipase C gamma (PLCγ). Under normal conditions, this pathway leads to controlled B cell proliferation and differentiation, however, this same pathway leads to uncontrolled B cell proliferation in malignancies, such as CLL. The role of BTK in CLL has drawn researchers to develop BTK inhibitors, such as ibrutinib - an FDA (Food and Drug Administration)[5] and EMA (European Medicines Agency)[6] - approved CLL treatment. Ibrutinib forms a covalent bond with a cysteine residue (C481) at BTK’s active site, and thus inhibits kinase activity, including autoactivation.[3] This kinase inhibition shuts down BCR signaling, reduces B cell proliferation, and promotes apoptosis of leukemic cells. Ibrutinib is effective as a standalone treatment for mutated and unmutated CLL and when combined with rituximab is an effective therapy for relapsed CLL.[1] In general, ibrutinib is well tolerated and shows continued efficacy during extended treatment periods.[7]   While ibrutinib has been a clinical success, there are a subset of CLL patients who develop resistance to this therapeutic. This resistance has been mapped to a C481S mutation that prevents ibrutinib from covalently binding to BTK resulting in continuous BCR signaling within leukemic cells.[3] Next-generation, reversible BTK inhibitors, such as ARQ 531, are currently being developed for relapsed/refractory CLL. ARQ-531 is a non-selective BTK inhibitor that suppresses signaling in cells with C481S BTK and PLCγ mutations. ARQ-531 also has an additional inhibitory activity against ERK signaling.[8] Furthermore, the FDA has recently approved LOXO-305, a non-covalent, reversible BTK inhibitor. LOXO-305 inhibits signaling in cells with wild-type or C481S-mutated BTK.[9, 10] In contrast, acalabrutinib, a second-generation irreversible BTK inhibitor has also been approved by the FDA. Acalabrutinib has a shorter half-life, allowing for variable dosages, and has increased C481 specificity that enhances its BTK inhibitory effects. The biochemical properties of acalabrutinib make it a suitable option for patients with treatment naïve CLL.[11] In January 2023, the FDA and EMA approved an additional second-generation irreversible BTK inhibitor, zanubrutinib, for CLL.[12] Zanubrutinib’s design was guided by a structure-activity strategy to generate sustained BTK occupancy. Zanubrutinib exhibits reduced ITK and EGFR inhibition and has 4x longer half-life than acalabrutinib.[13] As such, it persists at high concentrations within the body making it available to re-inhibit newly synthesized BTK proteins, a unique difference from ibrutinib and acalabrutinib.[14] In a phase I/II study, all CLL patients treated with zanubrutinib had complete and sustained BTK occupancy in peripheral blood mononuclear cells and lymph nodes.[15] Zanubrutinib’s performance supports the hypothesis that increased BTK selectivity maximizes BTK inhibition and drug efficacy. Additionally, zanubrutinib’s enhanced BTK specificity reduces the incidence of off-target toxicities which may reduce side effects commonly associated with ibrutinib, such as cardiac events, subdural hematomas, and gastrointestinal bleeding.[10, 16, 17] This is particularly relevant to CLL as this class of BTK inhibitors are used by CLL patients indefinitely. BTK inhibitors have become an invaluable CLL treatment and will continue to improve and provide therapeutic benefits. Additional studies such as NCT03734016 are underway to evaluate the effects of zanubrutinib and BTK inhibitors with other targeted therapies, which may improve the efficacy and durability of these treatments.