Gadrich, M. | Search bioRxiv

bioRxiv2026

Dual-Specific Antibody Design Using Artificial Intelligence

Multibodies, or "two-in-one" Immunoglobulin G (IgG) antibodies, are standard symmetrical IgG molecules engineered to competitively bind more than one antigen within a single variable fragment (Fv) binding surface. This format merges the functional advantages of bispecifics, such as multi-target binding and dynamic adaptation to target concentrations, with the superior manufacturing, developability, pharmacokinetics, and avidity of monospecific IgGs. Moreover, the co-accommodation of multiple paratopes on a single set of 6 CDRs introduces new functional possibilities that can improve efficacy and safety. Multibodies can, therefore, be thought of as force multipliers: for any format of antibodies, or fragments thereof, multibodies can bind double the number of epitopes compared to standard antibodies. While these advantages were recognized more than 15 years ago, the systematic design of multibodies has been intractable due to the challenge of optimizing two binding specificities into one Fv region, without having one of them compromising the other and without inducing poly-reactivity. To overcome this engineering barrier, we have developed an artificial intelligence (AI)-assisted computational platform that enables the design of functional multibodies against virtually any pair of targets. We applied the platform to design nine multibodies combining 15 different unrelated targets. We obtained therapeutic-grade multibodies that bind each desired pair of targets. We demonstrate that the generated multibodies possess excellent developability, high affinity, and stringent specificity, comparing favorably to clinical monospecific benchmarks. Critically, we show that these multibodies exhibit superior functional activity across a diverse range of mechanisms of action (MOAs), including internalization, T-cell engagement, and immune system modulation. This capability to reliably engineer versatile multibodies opens a new domain in antibody therapeutics, enabling complex multipharmacology and novel functions within a natural, cost-effective, and highly developable format. Two of these multibodies are currently in IND enabling studies, with first in human studies expected in 2026. The timeline from idea to a fully optimized, developable, lead candidate, ready for IND enabling studies, is 9 months.

bioengineering↗

bioRxiv2019

Prophylactic TLR9 stimulation reduces brain metastasis through microglia activation

Brain metastases are prevalent in various types of cancer, and are often terminal given low efficacy of available therapies. Therefore, preventing them is of outmost clinical relevance and prophylactic treatments are perhaps the most efficient strategy. Here, we show that systemic prophylactic administration of a TLR9 agonist, CpG-C, is effective against brain metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell seeding and growth in the brain in three tumor models in mice, including metastasis of human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the brain, unlike in the periphery, NK cells and monocytes are not involved in controlling metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier integrity and tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as mediators of CpG-C effects through increased tumor killing and phagocytosis, mediated by direct microglia-tumor contact. In vivo, CpG-C-activated microglia displayed elevated mRNA expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging showed that CpG-C-activated microglia cells contact, kill, and phagocytize tumor cells in the early stages of tumor brain invasion more than non-activated microglia. Blocking in vivo activation of microglia with minocycline, and depletion of microglia with a colony-stimulating factor 1 inhibitor, indicated that microglia mediate the anti-tumor effects of CpG-C. Overall, the results suggest prophylactic CpG-C treatment as a new intervention against brain metastasis, through an essential activation of microglia. SummaryBrain metastases are prevalent and often terminal. Thus, reducing their occurrence could markedly improve cancer outcome. We show that systemic prophylactic and perioperative administration of a TLR9 agonist, CpG-C, reduced metastatic growth in experimental and spontaneous brain metastasis models, employing mouse and human tumors. CpG-C was taken up in the brain, without affecting blood-brain barrier integrity and tumor extravasation. In vitro assays, imaging flow cytometry, and intravital imaging pointed to microglia as mediators of CpG-C effects through contact-dependent tumor killing and phagocytosis; corresponding with in vivo mRNA profile. In vivo depletion studies proved that microglia, but not NK cells or monocytes, mediated the beneficial effects of CpG-C; Also hindered by blocking microglial activation. In-toto, perioperative treatment with CpG-C should be considered clinically relevant. SignificancePreventing brain metastases is paramount, as they are considered incurable and their incidence is on the rise due to prolonged survival of cancer patients. Here, we demonstrate that systemic prophylactic treatment with CpG-C reduces peripheral and brain metastasis of mouse and human lung cancers. While traditional therapies are halted during the perioperative period, we found systemic CpG-C treatment during this time frame beneficial in a model of spontaneous brain metastases following excision of a primary melanoma tumor, comprehensively mimicking the clinical setting. Mechanistically, we show microglia activation with CpG-C results in tumor cell eradication, pointing to microglia as potential therapeutic targets. Importantly, CpG-ODNs have negligible toxicity in humans. Therefore, CpG-C may be used prophylactically and during the perioperative period in high-risk cancers.

neuroscience↗