Researchers at Vanderbilt University have developed a novel nanobody platform that significantly enhances cancer immunotherapy by leveraging albumin hitchhiking to deliver STING agonists directly to tumor sites. The approach addresses a critical limitation in current immunotherapy treatments, where only a small fraction of cancer patients benefit from existing therapies.
The research team, led by John T. Wilson, associate professor of chemical and biomolecular engineering and biomedical engineering, engineered nanobodies derived from llamas that bind to serum albumin, the most abundant protein in blood. This albumin-binding strategy dramatically extends the circulation time of therapeutic molecules from approximately 5 minutes to 55 hours, matching albumin's natural half-life.
Enhanced Tumor Targeting Through Albumin Hitchhiking
The nanobody platform achieved remarkable tumor accumulation, with approximately 11% of the injected dose per gram of tissue reaching tumor sites within 24 hours. This represents a significant improvement over conventional approaches, as the researchers demonstrated through biodistribution studies in mouse models of breast cancer and melanoma.
"We discovered this approach inhibited tumor growth in mouse models of breast cancer and melanoma, as well as improved response to currently approved immunotherapies such as immune checkpoint and inhibitors and adoptive T cell therapy," said Wilson, who also co-leads the Host-Tumor Interactions Program of the Vanderbilt-Ingram Cancer Center.
The nanobodies preferentially accumulated at tumor sites and were internalized primarily by cancer cells and tumor-associated myeloid cells through micropinocytosis. Flow cytometry analysis revealed that approximately 8% of all live cells in tumors were positive for the nanobody conjugate, with the highest uptake observed in macrophages, dendritic cells, and cancer cells.
STING Pathway Activation Drives Antitumor Immunity
The researchers conjugated their albumin-binding nanobodies to diABZI, a STING agonist that activates the stimulator of interferon genes pathway. STING activation has emerged as a promising target for cancer immunotherapy, but previous approaches have been limited by rapid clearance and poor tumor penetration.
The nanobody-STING agonist conjugate (nAlb-diABZI) demonstrated potent antitumor effects at doses as low as 1.25 μg of diABZI content. In the challenging B16.F10 melanoma model, all tested doses significantly inhibited tumor growth and extended survival time, with the 5 μg dose showing enhanced efficacy compared to a three-fold higher dose of free diABZI.
Gene expression analysis revealed that nAlb-diABZI treatment increased expression of STING-associated genes including Ifnb1, Cxcl10, Cxcl9, and Tnf in tumor tissue. The treatment also elevated plasma levels of antitumor cytokines including type I interferons and IL-12 within 4 hours of administration.
Bivalent Design Achieves Complete Tumor Elimination
To further enhance therapeutic efficacy, the researchers engineered a bivalent nanobody fusion protein that targets both albumin and PD-L1, termed AP-diABZI. This dual-targeting approach combines STING activation with immune checkpoint inhibition in a single molecule.
The AP-diABZI conjugate demonstrated superior tumor accumulation compared to the albumin-binding nanobody alone, achieving 2.19% injected dose per gram of tumor tissue. More importantly, the bivalent construct achieved complete tumor elimination in 100% of EMT6 breast cancer models (10/10 mice), compared to a 30% complete response rate with the albumin-binding nanobody alone.
Mice that achieved complete responses with AP-diABZI treatment were largely resistant to tumor rechallenge 80 days later, with only 1 out of 9 mice developing tumors upon re-exposure to cancer cells. This demonstrates the generation of immunological memory capable of preventing recurrent disease.
Cellular Mechanisms of Antitumor Activity
Flow cytometric immunophenotyping revealed that AP-diABZI treatment significantly increased infiltration of activated CD8+ T cells and natural killer cells in the tumor microenvironment. The treatment enhanced the ratio of CD8+ T cells to regulatory T cells, indicating a shift toward a more immunogenic "hot" tumor profile.
Depletion studies confirmed that both CD8+ T cells and NK cells are essential for the therapeutic efficacy of AP-diABZI. When either cell population was depleted using specific antibodies, the antitumor response was almost completely inhibited, with CD8+ T cell depletion having a slightly stronger effect.
The platform also stimulated antigen-specific CD8+ T cell responses. In B16.F10-OVA tumor models, AP-diABZI treatment generated a strong peripheral ovalbumin-specific CD8+ T cell response, with approximately 60% displaying an effector memory phenotype capable of targeting tumor-associated antigens.
Efficacy Against Metastatic Disease
The researchers extended their investigations to evaluate therapeutic efficacy in an aggressive model of lung metastatic melanoma. Mice treated with AP-diABZI showed nearly complete elimination of metastatic tumor burden compared to high metastatic burden in control groups.
This finding is particularly significant as it suggests potential for treating micrometastases, which typically lack the leaky vasculature required for conventional tumor accumulation strategies. Albumin-binding molecules have been shown to accumulate in micrometastases, offering advantages over nanoparticle-based approaches.
Applications in Adoptive Cell Therapy
The versatility of the platform extends to adoptive cellular immunotherapy applications. The researchers demonstrated that AP-diABZI treatment can precondition the tumor microenvironment to enhance adoptive T cell therapy outcomes.
In B16.F10-OVA tumor models, treatment with three doses of AP-diABZI followed by adoptive transfer of OVA-specific CD8+ T cells resulted in a 25% complete response rate. This provides evidence that the nanobody-STING agonist conjugates can establish an inflammatory milieu that supports T cell infiltration and function.
Safety Profile and Clinical Translation Potential
Preclinical toxicity analysis revealed a favorable safety profile for the nanobody conjugates. Mice treated with the therapeutic dose experienced only mild and transient weight loss similar to that described for nanoparticle-based STING agonist delivery systems.
Histological evaluation by a board-certified veterinary pathologist observed no clinically notable changes between untreated control mice and treated mice, with only minor changes in blood biochemistry and cellular composition. The researchers also found no evidence of anti-nanobody antibody responses that could lead to accelerated clearance.
The modular design of the nanobody platform offers significant advantages for clinical translation. Nanobodies are molecularly well-defined, amenable to scalable industrial manufacturing, and are components of approved therapeutics including ozoralizumab, which contains an anti-albumin nanobody domain.
This research represents a significant advancement in cancer immunotherapy, offering a programmable platform that can potentially be adapted for various therapeutic combinations and cancer types. The ability to achieve complete tumor elimination while generating lasting immunological memory positions this approach as a promising strategy for improving patient outcomes in cancer treatment.
