Biology subjects

Wittrup, K. D.

Publications and source records attributed to Wittrup, K. D..

7 recordsLinked to original sources

bioRxiv2025

CD45 sequestration lowers the signaling threshold in lymphocytes and enhances anti-tumor immunity

CD45 plays a central role in immune signal regulation by controlling the spatial dynamics of phosphatase activity through steric segregation of its bulky rigid extracellular domain. To modulate CD45 activity, here we develop and characterize protein engineering approaches to induce multivalent clustering of CD45, effectively mimicking the endogenous local receptor sequestration during immune synapse formation. In doing so, we engineer a biologic that enables precise, tunable control over CD45 surface localization and activity. CD45 sequestration exhibited striking synergy when administered in combination with intratumorally anchored IL-12 therapy, markedly delaying tumor progression and extending survival in syngeneic murine melanoma and carcinoma models. Immune profiling revealed that CD8 T cells are essential mediators of this synergistic antitumor response. Mechanistically, IL-12 initiates a wave of antigen generation and T cell priming, while CD45 sequestration subsequently enhances tumor-specific CD8 T cell activation, expansion, and functional states within the tumor-draining lymph node. These findings suggest that CD45 sequestration lowers the activation threshold of T cells, broadens the tumor-reactive T cell repertoire, and therefore promotes more robust tumor-specific T cell responses. Altogether, we establish CD45 as a promising novel target for cancer immunotherapy, capable of potentiating strong anticancer immune responses.

immunology↗

bioRxiv2024

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR (FMC63) as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion and memory development of CD19 CAR-T cells in both syngeneic and humanized mouse models of B-ALL/Lymphoma, and enhanced control of disease progression. This approach thus enables vaccine boosting to be applied to any clinically relevant CAR-T cell product.

bioengineering↗

bioRxiv2024

Tumor-localized interleukin-2 and interleukin-12 combine with radiation therapy to safely potentiate regression of advanced malignant melanoma in pet dogs

The clinical use of interleukin-2 and -12 cytokines against cancer is limited by their narrow therapeutic windows due to on-target, off-tumor activation of immune cells when delivered systemically. Engineering IL-2 and IL-12 to bind to extracellular matrix collagen allows these cytokines to be retained within tumors after intralesional injection, overcoming these clinical safety challenges. While this approach has potentiated responses in syngeneic mouse tumors without toxicity, the complex tumor-immune interactions in human cancers are difficult to recapitulate in mouse models of cancer. This has driven an increased role for comparative oncology clinical trials in companion (pet) dogs with spontaneous cancers that feature analogous tumor and immune biology to human cancers. Here, we report the results from a dose-escalation clinical trial of intratumoral collagen-binding IL-2 and IL-12 cytokines in pet dogs with malignant melanoma, observing encouraging local and regional responses to therapy that may suggest human clinical benefit with this approach.

cancer biology↗

bioRxiv2024

Local delivery of cell surface-targeted immunocytokines programs systemic anti-tumor immunity

Cytokine therapies are potent immunotherapy agents but exhibit severe dose-limiting toxicities. One strategy to overcome this involves engineering cytokines for intratumoral retention following local delivery. Here, we develop a localized cytokine therapy that elicits profound anti-tumor immunity by engineered targeting to the ubiquitous leukocyte receptor CD45. We designed CD45-targeted immunocytokines (CD45-Cyt) that, upon injection, decorated the surface of leukocytes in the tumor and tumor-draining lymph node (TDLN) without systemic exposure. CD45-Cyt therapy eradicated both directly treated tumors and untreated distal lesions in multiple syngeneic mouse tumor models. Mechanistically, CD45-Cyt triggered prolonged pSTAT signaling and reprogrammed tumor-specific CD8+ T cells in the TDLN to exhibit an anti-viral transcriptional signature. CD45 anchoring represents a broad platform for protein retention by host immune cells for use in immunotherapy.

immunology↗

bioRxiv2023

Tregs constrain CD8+ T cell priming required for curative intratumorally anchored anti-4-1BB immunotherapy

Although co-stimulation of T cells with agonist antibodies targeting 4-1BB (CD137) improves antitumor immune responses in preclinical studies, clinical development has been hampered by on-target, off-tumor toxicity. Here, we report the development of a tumor-anchored 4-1BB agonist (4-1BB-LAIR), which consists of an 4-1BB antibody fused to the collagen binding protein LAIR. While combination treatment with an antitumor antibody (TA99) displayed only modest efficacy, simultaneous depletion of CD4+ T cells boosted cure rates to over 90% of mice. We elucidated two mechanisms of action for this synergy: CD4 eliminated tumor draining lymph node Tregs, enhancing priming and activation of CD8+ T cells, and TA99 + 4-1BB-LAIR supported the cytotoxic program of these newly primed CD8+ T cells within the tumor microenvironment. Replacement of CD4 with CTLA-4, a clinically approved antibody that enhances T cell priming, produced equivalent cure rates while additionally generating robust immunological memory against secondary tumor rechallenge. One Sentence SummaryInhibition of nodal Tregs enhances CD8+ T cell priming, improving antitumor responses to collagen-anchored 4-1BB combination therapy.

immunology↗

bioRxiv2022

Reprogramming brain immunosurveillance with engineered cytokines

Immune surveillance of the brain is regulated by resident non-neuronal cells and the blood-brain barrier.1 Dys-regulation of immunosurveillance is a hallmark feature of several diseases2-5 including brain tumors6 that interact with and rely heavily on immune cells,7 suggesting that disrupting the neuroimmunology of tumors could slow their progression. Yet few tools are available to control brain immunology in vivo with local precision, and fewer yet are used for therapeutic intervention. 2 Here, we propose engineered cytokines as a neuroimmune-modulation platform. We demonstrate that the residence time of cytokines in the brain can be tuned by binding them to the extracellular matrix or synthetic scaffolds. We then show that the aluminum hydroxide adjuvant (alum) is retained in the brain >2 weeks. Tethering of inflammatory cytokines such as interleukins (IL) 2 and 12 to alum yields extended neuroinflammation and brain immunosurveillance after intracranial administration, while avoiding systemic toxicity. In mouse models of both immunologically hot and cold brain tumors, the intracranial deposition of alum-tethered cytokines causes significant delay in tumor progression. RNA profiling reveals that engineered cytokines engage both innate and adaptive immunity in the brain. These findings suggest that engineered cytokines can reprogram brain immunosurveillance, informing the development of future therapies for neuroimmune diseases.

bioengineering↗

bioRxiv2022

Identification of highly cross-reactive mimotopes for a public T cell response in murine melanoma

While immune checkpoint blockade results in durable responses for some patients, many others have not experienced such benefits. These treatments rely upon reinvigorating specific T cell-antigen interactions. However, it is often unknown what antigens are being recognized by T cells or how to potently induce antigen-specific responses in a broadly applicable manner. Here, we characterized the CD8+ T cell response to a murine model of melanoma following combination immunotherapy to determine the basis of tumor recognition. Sequencing of tumor-infiltrating T cells revealed a repertoire of highly homologous TCR sequences that were particularly expanded in treated mice and which recognized an antigen from an endogenous retrovirus. While vaccination against this peptide failed to raise a protective T cell response in vivo, engineered antigen mimotopes induced a significant expansion of CD8+ T cells cross-reactive to the original antigen. Vaccination with mimotopes resulted in killing of antigen-loaded cells in vivo yet showed modest survival benefit in a prophylactic vaccine paradigm. Together, this work demonstrates the identification of a dominant tumor-associated antigen and generation of mimotopes which can induce robust functional T cell responses that are cross-reactive to the endogenous antigen across multiple individuals.

immunology↗