Local delivery of cell surface-targeted immunocytokines programs systemic antitumor immunity.

Systemically administered cytokines are potent immunotherapeutics but can cause severe dose-limiting toxicities. To overcome this challenge, cytokines have been engineered for intratumoral retention after local delivery. However, despite inducing regression of treated lesions, tumor-localized cytokines often elicit only modest responses at distal untreated tumors.

Bivalent target-binding bioPROTACs induce potent degradation of oncogenic SHP2.

Targeted protein degradation is an emergent and rapidly evolving therapeutic strategy. In particular, biologics-based targeted degradation modalities (bioPROTACs) are relatively under explored compared to small molecules. Here, we investigate how target affinity, cellular localization, and valency of bioPROTACs impact efficacy of targeted degradation of the oncogenic phosphatase src-homology 2 containing protein tyrosine phosphatase-2 (SHP2).

Tumor-Localized Interleukin-2 and Interleukin-12 Combine with Radiation Therapy to Safely Potentiate Regression of Advanced Malignant Melanoma in Pet Dogs.

Purpose: Cytokines IL2 and IL12 exhibit potent anticancer activity but suffer a narrow therapeutic window due to off-tumor immune cell activation. Engineering cytokines with the ability to bind and associate with tumor collagen after intratumoral injection potentiated response without toxicity in mice and was previously safe in pet dogs with sarcoma. Here, we sought to test the efficacy of this approach in dogs with advanced melanoma.

CLN-617 Retains IL2 and IL12 in Injected Tumors to Drive Robust and Systemic Immune-Mediated Antitumor Activity.

Despite clinical evidence of antitumor activity, the development of cytokine therapies has been hampered by a narrow therapeutic window and limited response rates. Two cytokines of high interest for clinical development are interleukin 2 (IL2) and interleukin 12 (IL12), which potently synergize to promote the activation and proliferation of T cells and NK cells. However, the only approved human IL2 therapy, Proleukin, is rarely used in the clinic due to systemic toxicities, and no IL12 product has been approved to date due to severe dose-limiting toxicities.

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.

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.

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.

A Novel Gain-of-Signal Assay to Detect Targeted Protein Degradation.

Targeted protein degradation offers a promising avenue for expanding therapeutic development to previously inaccessible proteins of interest by regulating the target abundance rather than activity. However, current methods to screen for effective degraders serve as major bottlenecks for the development of degrader therapies. Here, we develop a novel assay platform for identification and characterization of macromolecules capable of inducing targeted degradation of oncogenic phosphatase SHP2.

Targeting of the CD161 inhibitory receptor enhances T-cell-mediated immunity against hematological malignancies.

The CD161 inhibitory receptor is highly upregulated by tumor-infiltrating T cells in multiple human solid tumor types, and its ligand, CLEC2D, is expressed by both tumor cells and infiltrating myeloid cells. Here, we assessed the role of the CD161 receptor in hematological malignancies. Systematic analysis of CLEC2D expression using the Cancer Cell Line Encyclopedia revealed that CLEC2D messenger RNA was most abundant in hematological malignancies, including B-cell and T-cell lymphomas as well as lymphocytic and myelogenous leukemias.

Overcoming lung cancer immunotherapy resistance by combining nontoxic variants of IL-12 and IL-2.

Engineered cytokine-based approaches for immunotherapy of cancer are poised to enter the clinic, with IL-12 being at the forefront. However, little is known about potential mechanisms of resistance to cytokine therapies. We found that orthotopic murine lung tumors were resistant to systemically delivered IL-12 fused to murine serum albumin (MSA, IL12-MSA) because of low IL-12 receptor (IL-12R) expression on tumor-reactive CD8+ T cells.

Both intratumoral regulatory T cell depletion and CTLA-4 antagonism are required for maximum efficacy of anti-CTLA-4 antibodies.

Anti-CTLA-4 antibodies have successfully elicited durable tumor regression in the clinic; however, long-term benefit is limited to a subset of patients for select cancer indications. The incomplete understanding of their mechanism of action has hindered efforts at improvement, with conflicting hypotheses proposing either antagonism of the CTLA-4:B7 axis or Fc effector-mediated regulatory T cell (Treg) depletion governing efficacy. Here, we report the engineering of a nonantagonistic CTLA-4 binding domain (b1s1e2) that depletes intratumoral Tregs as an Fc fusion.

Spatiotemporally programming cytokine immunotherapies through protein engineering.

Cytokines have long been considered promising cancer immunotherapy agents due to their endogenous role in activating and proliferating lymphocytes. However, since the initial FDA approvals of Interleukin-2 (IL-2) and Interferon-ɑ (IFNɑ) for oncology over 30 years ago, cytokines have achieved little success in the clinic due to narrow therapeutic windows and dose-limiting toxicities. This is attributable to the discrepancy between the localized, regulated manner in which cytokines are deployed endogenously versus the systemic, untargeted administration used to date in most exogenous cytokine therapies.

Tumor-localized catalases can fail to alter tumor growth and transcriptional profiles in subcutaneous syngeneic mouse tumor models.

Catalase is an antioxidant enzyme that catalyzes the rapid conversion of hydrogen peroxide to water and oxygen. Use of catalase as a cancer therapeutic has been proposed to reduce oxidative stress and hypoxia in the tumor microenvironment, both activities which are hypothesized to reduce tumor growth. Furthermore, exposing murine tumors to exogenous catalase was previously reported to have therapeutic benefit.

Collagen-Anchored Interleukin-2 and Interleukin-12 Safely Reprogram the Tumor Microenvironment in Canine Soft-Tissue Sarcomas.

Purpose: Cytokine therapies such as IL2 and IL12 suffer from impractically small therapeutic windows driven by their on-target, off-tumor activity, limiting their clinical potential despite potent antitumor effects. We previously engineered cytokines that bind and anchor to tumor collagen following intratumoral injection, and sought to test their safety and biomarker activity in spontaneous canine soft-tissue sarcomas (STS).

Experimental Design: Collagen-binding cytokines were canine-ized to minimize immunogenicity and were used in a rapid dose-escalation study in healthy beagles to identify a maximum tolerated dose.

Anti-PD-1 and Extended Half-life IL2 Synergize for Treatment of Murine Glioblastoma Independent of Host MHC Class I Expression.

Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite preclinical successes, most interventions have failed to extend patient survival by more than a few months. Treatment with anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint blockade (ICB) monotherapy has been beneficial for malignant tumors such as melanoma and lung cancers but has yet to be effectively employed in GBM.

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 ɑ4-1BB agonist (ɑ4-1BB-LAIR), which consists of an ɑ4-1BB 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.

Tissue-specific abundance of interferon-gamma drives regulatory T cells to restrain DC1-mediated priming of cytotoxic T cells against lung cancer.

Local environmental factors influence CD8 T cell priming in lymph nodes (LNs). Here, we sought to understand how factors unique to the tumor-draining mediastinal LN (mLN) impact CD8 T cell responses toward lung cancer. Type 1 conventional dendritic cells (DC1s) showed a mLN-specific failure to induce robust cytotoxic T cells responses.

Intratumoral nanobody-IL-2 fusions that bind the tumor extracellular matrix suppress solid tumor growth in mice.

Confining cytokine exposure to the tumors would greatly enhance cancer immunotherapy safety and efficacy. Immunocytokines, cytokines fused to tumor-targeting antibodies, have been developed with this intention, but without significant clinical success to date. A critical limitation is uptake by receptor-expressing cells in the blood, that decreases the dose at the tumor and engenders toxicity.

Co-Anchoring of Engineered Immunogen and Immunostimulatory Cytokines to Alum Promotes Enhanced-Humoral Immunity.

Protein antigens are often combined with aluminum hydroxide (alum), the most commonly used adjuvant in licensed vaccines; yet the immunogenicity of alum-adjuvanted vaccines leaves much room for improvement. Here, the authors demonstrate a strategy for codelivering an immunostimulatory cytokine, the interleukin IL-21, with an engineered outer domain (eOD) human immunodeficiency virus gp120 Env immunogen eOD, bound together to alum to bolster the humoral immune response. In this approach, the immunogen and cytokine are co-anchored to alum particles via a short phosphoserine (pSer) peptide linker, promoting stable binding to alum and sustained bioavailability following injection.

Alum-anchored intratumoral retention improves the tolerability and antitumor efficacy of type I interferon therapies.

Effective antitumor immunity in mice requires activation of the type I interferon (IFN) response pathway. IFNα and IFNβ therapies have proven promising in humans, but suffer from limited efficacy and high toxicity. Intratumoral IFN retention ameliorates systemic toxicity, but given the complexity of IFN signaling, it was unclear whether long-term intratumoral retention of type I IFNs would promote or inhibit antitumor responses.

Host-Guest Complexation by β-Cyclodextrin Enhances the Solubility of an Esterified Protein.

The carboxyl groups of a protein can be esterified by reaction with a diazo compound, 2-diazo-2-(-methylphenyl)-,-dimethylacetamide. This esterification enables the entry of the protein into the cytosol of a mammalian cell, where the nascent ester groups are hydrolyzed by endogenous esterases. The low aqueous solubility of the ensuing esterified protein is, however, a major practical challenge.

An affinity threshold for maximum efficacy in anti-PD-1 immunotherapy.

Monoclonal antibodies targeting the programmed cell death protein 1 (PD-1) remain the most prevalent cancer immunotherapy both as a monotherapy and in combination with additional therapies. Despite the extensive success of anti-PD-1 monoclonal antibodies in the clinic, the experimental relationship between binding affinity and functional potency for anti-PD-1 antibodies in vivo has not been reported. Anti-PD-1 antibodies with higher and lower affinity than nivolumab or pembrolizumab are entering the clinic and show varied preclinical efficacy.

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.