Blood-brain barrier crossing biopolymer targeting c-Myc and anti-PD-1 activate primary brain lymphoma immunity: Artificial intelligence analysis

Journal of Controlled Release · 2025-03-13 · 5 citations

Two-component T-cell immunotherapy enables antigen pre-targeting to reduce cytokine release without forfeiting efficacy

Nanomedicine Nanotechnology Biology and Medicine · 2025-04-30 · 2 citations

Contemporary T-cell immunotherapies, despite impressive targeting precision, are hindered by aberrant cytokine release and restrictive targeting stoichiometry. We introduce a two-component T-cell immunotherapy targeting B-cell malignancies: Multi-Antigen T-Cell Hybridizers (MATCH). This split antibody technology differs from current therapies by separating cancer cell-targeting components from T cell-engaging components. We demonstrate that this two-component structure facilitates tunable T-cell activation. αCD19 and αCD20 MATCH, administered in two steps, are both compared to the clinical standard bispecific antibody, blinatumomab. In vitro two-dimensional dose analysis and cytokine release data indicate MATCH improves cancer clearance with reduced cytokine release. Cytolytic mechanisms of action are evaluated. αCD20 MATCH anti-cancer efficacy is assayed using a human lymphoma murine model. Decreasing T-cell engager dose 10-fold yields comparable efficacy to non-reduced doses. Ultimately, this split-antibody paradigm may enhance antigen targeting while reducing cytokine release, with such safety and efficacy advantages augmented by the future possibility of multi-antigen targeting with MATCH.

DDEL-09. Therapeutic activation of primary CNS lymphoma immunity by BBB-penetrant c-Myc targeted biopolymer, and anti-PD-1: artificial intelligence guided pathway insights

Neuro-Oncology · 2025-11-01

Abstract Primary central nervous system lymphoma (PCNSL) is a lethal cancer with poor survival, especially in its recurrent form. This is mainly due to low penetrance of therapeutic agents across the blood brain barrier (BBB). We created a novel nano immunodrug (nanodrug) that can cross the BBB and deliver anti-cancer agents directly to the tumor. The RNA therapeutics based on N-(2-hydroxypropyl)methacrylamide copolymer nanoplatform that was designed to block lymphoma’s cell c-Myc protein synthesis and to ensure brain tumor targeting and BBB crossing. Inhibiting of c-Myc protein synthesis played a dual role as anti-tumor proliferative factor and immune stimulator. Angiopep-2 peptide is conjugated to the nano platform to provide BBB crossing and brain lymphoma cell targeting via LRP-1 receptor. The nanodrug also contains H6 (6 histidines for endosome escape) to release into lymphoma cell cytoplasm. We tested the nanodrug in a A20 intracranial brain lymphoma mouse model alone, and in combination with anti-PD-1 antibody. Treatment with nanodrug resulted in a significant survival advantage compared to control. Survival was significantly enhanced when the nanodrug was co-injected with anti-PD-1. Spectral flow cytometry and RNA-seq analysis of treated tumors showed robust activation of tumor-infiltrating T lymphocytes with enhanced interferon γ signaling and polarization to M1-type macrophages. Artificial Intelligence-assisted analysis of gene expression data from RNA-seq revealed novel immune pathways, molecular targets, and suggested effective multifunctional drugs. Overall, we created a novel nano therapeutic drug delivery system that inhibits tumor c-Myc protein to treat PCNSL. When used in conjunction with anti-PD-1 checkpoint inhibitor, the treatment results in enhanced survival of tumor bearing animals by activating both adaptive and immune responses. SUPPORT NIH grants: R01 CA246716, R01 CA206220, R01 CA209921, R01 CA284247

Self‐Assembling Multi‐Antigen T Cell Hybridizers for Precision Immunotherapy of Multiple Myeloma

Advanced Healthcare Materials · 2025-08-01 · 3 citations

Abstract Bispecific T‐cell engagers show promise in treating multiple myeloma (MM), but challenges remain in adaptability and targeting flexibility. This paper presents a novel T‐cell based immunotherapy, M ulti‐ A ntigen TC ell H ybridizers (MATCH), a modular, self‐assembling T‐cell engager designed for versatile and patient‐specific cancer targeting. MATCH consists of two components: a B‐cell‐targeting Fab’ fragment conjugated to a 25‐base morpholino oligonucleotide (Fab’ B cell antigen ‐MORF1) and a T‐cell engaging anti‐CD3 Fab’ fragment conjugated to the complementary morpholino oligonucleotide (Fab’ CD3 ‐MORF2). Upon hybridization of MORF1 and MORF2, MATCH enables pre‐targeting of malignant cells followed by in situ post‐assembly of the bispecific complex, facilitating targeted T‐cell recruitment. To enhance antigen specificity based on MM patient expression profile, a panel of Fab’‐MORF1 conjugates targeting key MM surface markers (Fab’ BCMA ‐MORF1, Fab’ SLAMF7 ‐MORF1, Fab’ CD38 ‐MORF1) is developed, which pairs interchangeably with Fab’ CD3 ‐MORF2 for T‐cell engagement. MATCH effectively induces immune synapse formation and exhibits potent, antigen‐specific cytotoxicity across MM cells. Ex vivo validation in patient‐derived bone marrow samples confirms significant tumor cell depletion. Preliminary in vivo studies in humanized mouse model demonstrated effective cancer inhibition along with favorable pharmacokinetics and distribution profiles. These findings support MATCH as a flexible and customizable immunotherapy platform with strong translational potential for the treatment of MM.

Immune cell tumor engagers (SITE) via morpholino-guided self-assembly for precision immunotherapy of multiple myeloma

Blood · 2025-11-03

Abstract Introduction: Bispecific T cell engagers have shown significant clinical efficacy and are currently FDA approved for the treatment of relapsed and refractory multiple myeloma (RRMM). However, challenges remain, including targeting flexibility, immune-related toxicity, and durability of response. To overcome these limitations, we developed Self-Assembled Immune Cell-Tumor Engagers (SITE), a two-component system that targets both immune cell and MM cell surface antigens; each component is tagged with a complementary morpholino oligonucleotide (M1 or M2). Upon administration, these components hybridize in situ via M1-M2 pairing to form bispecific complexes to redirect immune cells to MM cells. Utilization of this pre-targeting approach enables the sequential engagement of immune cells, including T cells, NK cells, and macrophages. Additionally, by simultaneously targeting of multiple antigens (i.e. BCMA, GPRC5D and CD38), this strategy minimizes the risk of antigen loss and immune escape. Herein, we report the efficacy of SITE across multiple models, including in vitro cell lines, ex vivo patient-derived samples, and in vivo mouse models, demonstrating its potential as an innovative, cost-effective platform for orchestrating a coordinated and robust anti-tumor immune response. Methods: Anti-MM antibodies were digested enzymatically to F(ab')2 fragments, further reduced with tris(2-carboxyethyl)phosphine to generate Fab'MM-thiols. A pair of 25 bp M1/M2 was customized from Genetools. Following 3'-end maleimido modification, a panel of Fab'MM-M1 targeting BCMA, GPRC5D and CD38 was obtained. Similarly, α-hCD3 and α-hCD314 were used to generate immune cell targeting Fab'immune-M2. Stability of M1-M2 hybrids was assessed using circular dichroism (CD) spectroscopy in PBS, and size-exclusion chromatography (SEC) following incubation in mouse serum. Primary human T cells and NK cells were isolated from healthy donor blood. In vitro cytotoxicity and immune activation of SITE were assessed by flow cytometry on MM.1S and RPMI-8226 cells. To assess therapeutic efficacy, both patient-derived bone marrow mononuclear cells and a preclinical NRG mouse model were utilized. Mice were sub-lethally irradiated and i.v. injected with 3×106 MM.1S-Luc cells. Human T cells (9×106,3×106, or1×106) were administered to study the influence of T cells. Different dosing regimens (premix vs. consecutive) of T cell-specific SITE constructs (Fab'BCMA-M1/Fab'CD3-M2) were evaluated and compared with conventional teclistamab treatment. Cytokine production (IL-2, IFN-γ, TNF-α) was analyzed, and immune cell subsets were profiled by flow cytometry. Results: Conjugates Fab'MM-M1 and Fab'immune-M2 were successfully synthesized; hybridization of Fab'MM-M1/Fab'immune-M2 upon 1:1 mixing was confirmed by a shift in elution profiles in SEC. Complexes remained stable in 10% mouse serum for over 24 h. CD spectra showed a positive peak at 260 nm and a negative minimum at 210 nm, indicating formation of A-form double helices under physiological conditions. In vitro, SITE constructs successfully engaged T cells and NK cells, triggering potent cytotoxicity towards MM.1S and RPMI-8226 cells. Flow cytometry demonstrated increased expression of activation markers and induction of apoptosis in target cells. Ex vivo studies using patient-derived samples confirmed significant clearance of MM cells by patient's own T cells. In vivo, NRG mice bearing MM.1S-Luc and treated with SITE-T cell therapy extended survival and inhibited tumor growth compared to teclistamab-treated groups. Flow cytometric analysis of bone marrow confirmed a marked reduction in MM.1S cell populations in SITE-treated mice. T cell-related toxicity was found to be associated with injected T-cell numbers: reducing the dose from 9×106 to 3×106 or lower, no body weight loss occurred, highlighting the advantageous flexibility of two-component SITE. Overall, SITE demonstrated a superior anti-tumor effect compared to teclistamab, highlighting its potent efficacy in controlling MM progression in vivo. Conclusion: The SITE platform represents a promising advance in immunotherapy for MM, offering a versatile, potent, and modular approach to address the disease's complex antigenic landscape. By enabling multi-antigen targeting and the orchestration of diverse immune effectors, the platform effectively overcomes challenges such as antigenic heterogeneity, immune escape, and cytokine release syndrome.

Polymer-based chemo-immunotherapy: Combining immunogenic cell death induction and PD-L1 blockade enhances antitumor immunity in melanoma

Journal of Controlled Release · 2025-09-02 · 3 citations

Melanoma remains a challenging malignancy despite the significant outcomes achieved with immune checkpoint inhibitor (ICI) monotherapy. Here, we investigated a polymer-based chemo-immunotherapy strategy combining KT-1, a backbone-degradable N -(2-hydroxypropyl)methacrylamide (HPMA) copolymer–epirubicin conjugate that induces immunogenic cell death (ICD), with MPPA, a multivalent HPMA copolymer–peptide antagonist of PD-L1 (PPA: (NYSKPTDRQYHF). In B16F10 melanoma, a 3-day dosing schedule significantly outperformed 7-day dosing. KT-1 monotherapy induced CD8 + T cell–mediated immunity through increased infiltration and upregulation of effector genes (Prf1, Gzmk, Eomes, Xcl1, Cxcl10), with depletion studies confirming CD8 + T cell dependence. Concurrent KT-1 + MPPA administration proved superior to sequential dosing. Single-cell RNA sequencing revealed that KT-1 promoted dendritic cell maturation and CD8 + T cell activation, while MPPA selectively reversed KT-1–induced PD-L1 upregulation on tumor cells. The combination enhanced dendritic cell activation, CD8 + T cell cytotoxicity, and reduced regulatory T cell immunosuppression. Importantly, MPPA did not induce autoimmune diabetes in NOD mice, in contrast to conventional anti–PD-L1 antibodies, and showed no observed immune-related adverse effects highlighting the safety. These findings support HPMA copolymer-based chemo-immunotherapy as a safer, effective alternative to traditional ICI regimens for treating immune-excluded tumors.

Hydrophilic biomaterials: From crosslinked and self-assembled hydrogels to polymer-drug conjugates and drug-free macromolecular therapeutics

Journal of Controlled Release · 2024-05-17 · 13 citations

Human serum albumin-based drug-free macromolecular therapeutics induce apoptosis in chronic lymphocytic leukemia patient cells by crosslinking of CD20 and/or CD38 receptors

Drug Delivery and Translational Research · 2024-05-27 · 4 citations

Multiantigen T-Cell Hybridizers: A Two-Component T-Cell-Activating Therapy

ACS Nano · 2024 · 4 citations

• Cancer research
• Biology
• Medicine

model, insights regarding optimal T-cell-to-target cell ratio were gleaned when a ratio of 5:1 T-cell-to-target cell MATCH-treated mice significantly delayed the onset of disease compared to higher and lower ratios.

Obinutuzumab‐Based Drug‐Free Macromolecular Therapeutics Synergizes with Topoisomerase Inhibitors

Macromolecular Bioscience · 2023-10-15 · 3 citations

Abstract Drug‐free macromolecular therapeutics (DFMT) utilizes modified monoclonal antibodies (or antibody fragments) to generate antigen‐crosslinking‐induced apoptosis in target cells. DFMT is a two‐component system containing a morpholino oligonucleotide (MORF1) modified antibody (Ab‐MORF1) and human serum albumin conjugated with multiple copies of complementary morpholino oligonucleotide (MORF2), (HSA‐(MORF2) x ). The two components recognize each other via the Watson–Crick base pairing complementation of their respective MORFs. One HSA‐(MORF2) x molecule can hybridize with multiple Ab‐MORF1 molecules on the cell surface, thus serving as the therapeutic crosslink‐inducing mechanism of action. Herein, various anti‐neoplastic agents in combination with the anti‐CD20 Obinutuzumab (OBN)‐based DFMT system are examined. Three different classes of chemotherapies are examined: DNA alkylating agents; proliferation pathway inhibitors; and DNA replication inhibitors. Chou–Talalay combination index mathematics is utilized to determine which drugs engaged synergistically with OBN‐based DFMT. It is determined that OBN‐based DFMT synergizes with topoisomerase inhibitors and DNA nucleotide analogs but is antagonistic with proliferation pathway inhibitors. Cell mechanism experiments are performed to analyze points of synergism or antagonism by investigating Ca 2+ influx, mitochondrial health, lysosomal stability, and cell cycle arrest. Finally, the synergistic drug combinatorial effects of OBN‐based DFMT with etoposide in vivo are demonstrated using a human xenograft non‐Hodgkin's lymphoma mouse model.