Biology subjects

Phojanakong, P.

Publications and source records attributed to Phojanakong, P..

11 recordsLinked to original sources

bioRxiv2026

Deep Learning-based Modeling Enhances Efficacy of Natural Ligand CAR Binders Targeting CD70

CD70 is well-recognized as a promising "pan-cancer: chimeric antigen receptor (CAR) T-cell target. Prior work has shown that a "natural ligand" (NL)-based CAR targeting CD70, employing its physiological interaction partner CD27, may have therapeutic advantages over antibody-based CARs. Yet while antibody-based CARs are routinely optimized by affinity maturation of their scFv, whether the binding sequence of an NL CAR can be engineered to improve its function remains unexplored. Here, we combined deep learning with physics-based modeling to redesign residues at the CD27:CD70 interface, identifying a CD27 variant, "N88A", which enhances the efficacy of CD70-targeting CAR T-cells across models of acute myeloid leukemia, multiple myeloma, and renal cell carcinoma. Biophysical approaches, including molecular dynamics simulations, support a mechanism of increased binder conformational freedom underlying potency enhancement. Our work presents CD27N88A CAR T-cells as a promising new therapeutic option and proposes that computational modeling could be applied to enhance efficacy of other NL-based immunotherapies.

cancer biology↗

bioRxiv2026

Cellular consequences, citrullination substrates, and antigenicity resulting from wild-type and targeted PAD4 on cell surfaces

Protein arginine deiminase-4 (PAD4) catalyzes hydrolysis of arginine to citrulline in proteins that promotes widespread changes in cellular phenotypes through transcriptional regulation that can induce innate immunity and promote cancer. Overexpression and hyperactivity of PAD4 leads to a form of cell death called NETosis that releases PAD4 to the extracellular space. In excess, release of PAD4 is believed to be a major cause of various autoimmune diseases through the generation of anti-citrulline protein antibodies (ACPAs). Little is known about the specific protein substrates that become citrullinated and lead to autoimmunity, but there is growing evidence that PAD4 can be localized to the cell surface in response to inflammation. Here, we further characterize the cellular consequences for exogenous treatment with PAD4 showing that it induces morphological changes that increase cell migration, a hallmark of cancer. We then devised a more simplified and robust proteomics approach to identify PAD4 substrates. We identified some 1000 endogenously citrullinated peptides from 500 proteins, and 3000 citrullinated peptides from 1300 proteins upon exogenous addition of PAD4 both inside and outside of cells. This extracellular set can be further augmented by targeting PAD4 to a cancer target, HER2, using a binding protein conjugate. Finally, we studied how citrullinated cells can induce a robust humoral response in a syngeneic vaccine model to produce ACPAs. We believe these studies further our understanding of cell phenotypic consequences of extracellular PAD4 and new PAD4 substrates both inside and outside of cells that are potential neoepitopes for generation of ACPAs.

cancer biology↗

bioRxiv2025

Bicistronic CAR T-cells Against CD70 & Active Integrin β2 Overcome Antigen Heterogeneity and Preserve Safety in Acute Myeloid Leukemia

The surface antigen landscape of acute myeloid leukemia (AML) displays significant heterogeneity and overlap with healthy hematopoietic cells. This imparts a substantial hurdle to the development of AML-targeting chimeric antigen receptor (CAR) T-cells that can avoid on- target, off-tumor toxicity. Here, we develop a dual-antigen targeting CAR-T against CD70 and the active conformation of integrin {beta}2 (aITGB2), each previously reported as promising AML targets due to minimal off-tumor expression. We show an OR-gated approach for these antigens significantly increases the proportion of AML blasts that can be targeted, in part using a novel ex vivo co-culture method to restore surface protein homeostasis following a freeze-thaw cycle. We test dual-targeting CAR-T constructs with different combinations of costimulatory domains, identifying constructs with superior anti-tumor cytotoxicity in vitro against AML cell line and patient-derived xenograft models. We further show significantly improved in vivo tumor clearance and survival for a dual-targeting CAR in murine models of AML tumor heterogeneity. Finally, we show that this dual-targeting CAR does not increase off-tumor toxicity, especially against hematopoietic stem and progenitor cells. Together, these findings demonstrate a promising clinically-translatable approach for the treatment of AML without the notable toxicity liabilities associated with other leading CAR-T targets for this disease.

immunology↗

bioRxiv2025

Structure-guided engineering of CCL27 enhances natural ligand CAR T-cells against CCR10 for multiple myeloma

Despite the success of BCMA CAR-Ts, many multiple myeloma patients relapse and require additional therapeutic options. Our group previously identified the chemokine receptor CCR10 as a potential alternate target to address this need. Here, we validated CCR10 expression on primary myeloma tumors and sought to develop CAR T-cells against CCR10, utilizing its natural ligand CCL27 as a CAR binding element. However, CARs based on the native CCL27 sequence were ineffective. We thus utilized computational modeling and structure-guided engineering to inform rational mutations along the CCL27-CCR10 interface, exploiting a hydrophobic pocket on CCR10. This effort identified CCL27 mutants with an additional N-terminal aromatic amino acid that dramatically improved the efficacy of CCL27-based CAR-Ts to near that of current anti-BCMA CAR-Ts. We validated key amino acid contacts at the CCL27-CCR10 interface, which contribute to increased CAR binding avidity, predicted to be influenced by increased Van der Waals interactions. Lastly, we found that the CCL27 mutants have no toxicity in the hematopoietic compartment. This work illustrates the potential of engineering natural ligand CAR-Ts beyond their wild-type sequences and underscores the translational potential of engineered CCL27 mutant CAR-Ts.

cancer biology↗

bioRxiv2025

Affinity-matured CD72-targeting Nanobody CAR T-cells Enhance Elimination of Antigen-Low B-cell Malignancies

BackgroundChimeric antigen receptor (CAR) T-cell therapies are highly efficacious for several different hematologic cancers. However, for most CAR T targets it is observed that low surface antigen density on tumors can significantly reduce therapeutic efficacy. Here, we explore this dynamic in the context of CD72, a surface antigen we recently found as a promising target for refractory B-cell cancers, but for which CD72 low antigen density can lead to therapeutic resistance in preclinical models. MethodsPrimary samples were accessed via institutional review board-approved protocols. Affinity-matured and humanized nanobody clones were previously described in Temple et al.1 CAR T-cells were generated via lentiviral transduction. In vitro cytotoxicity assays were performed using luciferase-labeled cell lines. In vivo studies were performed using cell line- or patient-derived xenografts implanted in NOD scid gamma (NSG) mice. ResultsWe first confirmed ubiquitous CD72 expression across a range of primary B-cell non-Hodgkin lymphomas. We further found that after resistance to CD19-directed therapies, across both B-cell acute lymphoblastic leukemia (B-ALL) models and primary tumor samples, surface CD72 expression was largely preserved while CD22 expression was significantly diminished. Affinity maturation of a nanobody targeting CD72, when incorporated into chimeric antigen receptor (CAR) T-cells, led to more effective elimination in vitro of isogenic models of CD72 low-expressing tumors. These results suggested that nanobody-based CAR T-cells (nanoCARs) may exhibit a similar relationship between binder affinity, antigen expression, and efficacy as previously demonstrated only for scFv-based CAR T-cells. Surprisingly, however, this significantly improved in vitro efficacy only translated to modest in vivo survival benefit. As a parallel strategy to enhance CAR T function, we found that the small molecule bryostatin could also significantly increase CD72 surface antigen density on B-cell malignancy models. Structural modeling and biochemical analysis identified critical residues improving CD72 antigen recognition of our lead affinity-matured nanobody. ConclusionsTogether, these findings support affinity-matured CD72 nanoCARs as a potential immunotherapy product for CD19-refractory B-cell cancers. Our results also suggest that for B-ALL in particular, CD72 may be a preferable second-line immunotherapy target over CD22. What is already known on this topicPrevious work using single chain variable fragment (scFv) based CAR Ts has suggested that improving affinity for target antigen could potentially help mitigate tumor resistance mediated by antigen downregulation, or baseline low antigen density. However, it is unknown whether this same dynamic holds for CAR T-cells that utilize different antigen recognition elements, such as nanobodies. What this study addsHere we show that affinity maturation of nanobody-based CAR T-cells (nanoCARs) targeting CD72 can improve their in vitro efficacy versus CD72-low tumors; however, in vivo efficacy differences are more modest. Furthermore, we show that for refractory B-cell malignancies, surface CD72 appears preserved after CD19 resistance even in situations where CD22 is strongly downregulated. How this study might affect research, practice or policyCD72 warrants further investigation as a preferred immunotherapy target in the context of CD19-refractory B-cell cancers, though nanobody affinity maturation is not a universal solution to the challenge of low tumor surface antigen density.

immunology↗

bioRxiv2024

Chemoproteomics reveals immunogenic and tumor-associated cell surface substrates of ectokinase CK2α

New epitopes for immune recognition provide the basis of anticancer immunity. Due to the high concentration of extracellular adenosine triphosphate in the tumor microenvironment, we hypothesized that extracellular kinases (ectokinases) could have dysregulated activity and introduce aberrant phosphorylation sites on cell surface proteins. We engineered a cell-tethered version of the extracellular kinase CK2, demonstrated it was active on cells under tumor-relevant conditions, and profiled its substrate scope using a chemoproteomic workflow. We then demonstrated that mice developed polyreactive antisera in response to syngeneic tumor cells that had been subjected to surface hyperphosphorylation with CK2. Interestingly, these mice developed B cell and CD4+ T cell responses in response to these antigens but failed to develop a CD8+ T cell response. This work provides a workflow for probing the extracellular phosphoproteome and demonstrates that extracellular phosphoproteins are immunogenic even in a syngeneic system.

biochemistry↗

bioRxiv2024

Targeting high-risk multiple myeloma genotypes with optimized anti-CD70 CAR-T cells

Despite the success of BCMA-targeting CAR-Ts in multiple myeloma, patients with high-risk cytogenetic features still relapse most quickly and are in urgent need of additional therapeutic options. Here, we identify CD70, widely recognized as a favorable immunotherapy target in other cancers, as a specifically upregulated cell surface antigen in high risk myeloma tumors. We use a structure-guided design to define a CD27-based anti-CD70 CAR-T design that outperforms all tested scFv-based CARs, leading to >80-fold improved CAR-T expansion in vivo. Epigenetic analysis via machine learning predicts key transcription factors and transcriptional networks driving CD70 upregulation in high risk myeloma. Dual-targeting CAR-Ts against either CD70 or BCMA demonstrate a potential strategy to avoid antigen escape-mediated resistance. Together, these findings support the promise of targeting CD70 with optimized CAR-Ts in myeloma as well as future clinical translation of this approach. One sentence summaryStructure-optimized CD27-based CAR-T cells targeting CD70 are a promising therapeutic option for high-risk multiple myeloma patients who are most likely to relapse on current BCMA-targeting cellular therapies.

cancer biology↗

bioRxiv2022

Allosteric SHP2 Inhibition Increases Apoptotic Dependency on BCL2 and Synergizes with Venetoclax in FLT3- and KIT- Mutant AML

Mutations in receptor tyrosine kinases (RTKs) FLT3 and KIT are frequent and associated with poor outcomes in acute myeloid leukemia (AML). Although FLT3 inhibitors (FLT3i) are clinically effective, remissions are short-lived due to secondary resistance characterized by acquired mutations constitutively activating the RAS/MAPK pathway. Hereby, we report pre-clinical efficacy of co-targeting SHP2, a critical node in MAPK signaling, and BCL2 in RTK-driven AML. The allosteric SHP2 inhibitor RMC-4550 suppressed proliferation of AML cell lines with FLT3 and KIT mutations, including cell lines with acquired resistance to FLT3i. We demonstrate that SHP2 inhibition unveils an Achilles heel of AML, increasing apoptotic dependency on BCL2 via MAPK-dependent mechanisms, including upregulation of BMF and downregulation of MCL1. Consequently, RMC-4550 and venetoclax are synergistically lethal in FLT3- or KIT-mutant AML cell lines, and in clinically relevant xenograft models. Our results provide new mechanistic rationale and preclinical evidence for co-targeting SHP2 and BCL2 in RTK-driven AML. SignificanceThere is an unmet need for effective therapies targeting the MAPK pathway to overcome resistance in RTK-driven AML. We report that pharmacologic co-inhibition of SHP2 and BCL2 has synergistic anti-leukemia activity in preclinical models of AML with FLT3 and KIT mutations and holds potential clinical utility.

cancer biology↗

bioRxiv2022

Structural surfaceomics reveals an AML-specific conformation of Integrin-β2 as a CAR-T therapy target

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here, we explore the hypothesis that tumor cells express cancer-specific surface protein conformations, invisible to standard target discovery pipelines evaluating gene or protein expression, that can be identified and immunotherapeutically targeted. We term this strategy, integrating cross-linking mass spectrometry (XL-MS) with glycoprotein surface capture, "structural surfaceomics". As a proof of principle, we apply this technology to acute myeloid leukemia, a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin-{beta}2 as a structurally-defined, widely-expressed, AML-specific target. We develop and characterize recombinant antibodies to this protein conformation, and show that chimeric antigen receptor (CAR) T-cells eliminate AML cells and patient-derived xenografts without notable toxicity versus normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen while demonstrating a toolkit for applying these strategies more broadly.

cancer biology↗

bioRxiv2021

Functional multi-omics reveals genetic and pharmacologic regulation of surface CD38 in multiple myeloma

CD38 is a surface ectoenzyme expressed at high levels on myeloma plasma cells and is the target for the monoclonal antibodies (mAbs) daratumumab and isatuximab. Pre-treatment CD38 density on tumor cells is an important determinant of mAb efficacy. Several small molecules have been found to increase tumor surface CD38, with the goal of boosting mAb efficacy in a co-treatment strategy. Numerous other CD38-targeting therapeutics are currently in preclinical or clinical development. Here we sought to extend our currently limited insight into CD38 surface expression by using a multi-omics approach. Genome-wide CRISPR-interference screens integrated with patient-centered epigenetic analysis confirmed known regulators of CD38, such as RARA, while revealing XBP1 and SPI1 as other key transcription factors governing surface CD38 levels. CD38 knockdown followed by cell surface proteomics demonstrated no significant remodeling of the myeloma "surfaceome" after genetically-induced loss of this antigen. Integrated transcriptome and surface proteome data confirmed high specificity of all-trans retinoic acid in upregulating CD38 in contrast to broader effects of azacytidine and panobinostat. Finally, unbiased phosphoproteomics identified inhibition of MAP kinase pathway signaling in tumor cells after daratumumab treatment. Our work provides a resource to design strategies to enhance efficacy of CD38-targeting immunotherapies in myeloma.

cancer biology↗

bioRxiv2018

Proteasome inhibitor-induced modulation reveals the spliceosome as a specific therapeutic vulnerability in multiple myeloma

Enhancing the efficacy of proteasome inhibitors is a central goal in myeloma therapy. We proposed that signaling-level responses after PI would reveal new mechanisms of action that could be therapeutically exploited. Unbiased phosphoproteomics after the PI carfilzomib surprisingly demonstrated the most prominent phosphorylation changes on splicing related proteins. Spliceosome modulation was invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrated broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings led us to evaluate direct spliceosome inhibition in myeloma, which synergized with carfilzomib and showed potent anti-tumor activity. Functional genomics and exome sequencing further supported the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma. SignificanceNew ways to enhance PI efficacy are of major interest. We combine systems-level analyses to discover that PIs specifically interfere with splicing and that myeloma is selectively vulnerable to spliceosome inhibition. We reveal a new approach to advance myeloma therapy and uncover broader roles of splicing modulation in cancer.

cancer biology↗