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.

Ultrasonographic assessment of pulmonary and Central venous congestion in experimental heart failure.

Pulmonary and systemic congestion as a consequence of heart failure are clinically recognized as alarm signals for clinical outcome and mortality. Although signs and symptoms of congestion are well detectable in patients, monitoring of congestion in small animals with heart failure lacks adequate noninvasive methodology yet. Here, we developed a novel ultrasonography-based scoring system to assess pulmonary and systemic congestion in experimental heart failure, by using lung ultrasound (LUS) and imaging of the inferior vena cava (Cava), termed CavaLUS. CavaLUS was established and tested in a rat model of supracoronary aortic banding and a mouse model of myocardial infarction, providing high sensitivity and specificity while correlating to numerous parameters of cardiac performance and disease severity. CavaLUS, therefore, provides a novel comprehensive tool for experimental heart failure in small animals to noninvasively assess congestion.NEW & NOTEWORTHY As thorough, noninvasive assessment of congestion is not available in small animals, we developed and validated an ultrasonography-based research tool to evaluate pulmonary and central venous congestion in experimental heart failure models.

Balancing safety and efficacy: tuning the biodistribution and pharmacokinetics of cytokine immunotherapies.

Modulating the immune system shows promise in treating various conditions such as autoimmune, malignant, inflammatory, and infectious diseases. While immunotherapies can provide significant clinical benefits, they can also trigger debilitating immune-related toxicities. Achieving a balance between safety and efficacy of immunotherapy remains a significant engineering challenge. A complex immune response can be simplified into a sequence of coordinated signals with precise spatial and temporal arrangements. Mimicking or inhibiting these signals with protein immunotherapies relies on engineering them with specific biodistribution and pharmacokinetic properties. This review summarizes principles governing the movement of therapeutic proteins (i.e. biologics), focusing on cytokine immunotherapies injected intravenously or locally, and highlights approaches and considerations to balance their efficacy and safety.

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. IL2-MSA increased binding of IL12-MSA by tumor-reactive CD8+ T cells, and combined administration of IL12-MSA and IL2-MSA led to enhanced tumor-reactive CD8+ T cell effector differentiation, decreased numbers of tumor-infiltrating CD4+ regulatory T cells, and increased survival of lung tumor-bearing mice. Predictably, the combination of IL-2 and IL-12 at therapeutic doses led to significant dose-limiting toxicity. Administering IL-12 and IL-2 analogs with preferential binding to cells expressing Il12rb1 and CD25, respectively, led to a significant extension of survival in mice with lung tumors while abrogating dose-limiting toxicity. These findings suggest that IL-12 and IL-2 represent a rational approach to combination cytokine therapy whose dose-limiting toxicity can be overcome with engineered cytokine variants.

Recruited macrophages elicit atrial fibrillation.

Atrial fibrillation disrupts contraction of the atria, leading to stroke and heart failure. We deciphered how immune and stromal cells contribute to atrial fibrillation. Single-cell transcriptomes from human atria documented inflammatory monocyte and SPP1+ macrophage expansion in atrial fibrillation. Combining hypertension, obesity, and mitral valve regurgitation (HOMER) in mice elicited enlarged, fibrosed, and fibrillation-prone atria. Single-cell transcriptomes from HOMER mouse atria recapitulated cell composition and transcriptome changes observed in patients. Inhibiting monocyte migration reduced arrhythmia in Ccr2-∕- HOMER mice. Cell-cell interaction analysis identified SPP1 as a pleiotropic signal that promotes atrial fibrillation through cross-talk with local immune and stromal cells. Deleting Spp1 reduced atrial fibrillation in HOMER mice. These results identify SPP1+ macrophages as targets for immunotherapy in atrial fibrillation.

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. Ten client-owned pet dogs with STS were then enrolled into trial, receiving cytokines at different intervals prior to surgical tumor excision. Tumor tissue was analyzed through IHC and NanoString RNA profiling for dynamic changes within treated tumors. Archived, untreated STS samples were analyzed in parallel as controls. RESULTS: Intratumorally administered collagen-binding IL2 and IL12 were well tolerated by STS-bearing dogs, with only Grade 1/2 adverse events observed (mild fever, thrombocytopenia, neutropenia). IHC revealed enhanced T-cell infiltrates, corroborated by an enhancement in gene expression associated with cytotoxic immune function. We found concordant increases in expression of counter-regulatory genes that we hypothesize would contribute to a transient antitumor effect, and confirmed in mouse models that combination therapy to inhibit this counter-regulation can improve responses to cytokine therapy. CONCLUSIONS: These results support the safety and activity of intratumorally delivered, collagen-anchoring cytokines for inflammatory polarization of the canine STS tumor microenvironment. We are further evaluating the efficacy of this approach in additional canine cancers, including oral malignant melanoma.

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. Using regulatory T (Treg) cell depletion strategies, we found that Treg cells suppressed DC1s in a spatially coordinated manner within tissue-specific microniches within the mLN. Treg cell suppression required MHC II-dependent contact between DC1s and Treg cells. Elevated levels of IFN-γ drove differentiation Treg cells into Th1-like effector Treg cells in the mLN. In patients with cancer, Treg cell Th1 polarization, but not CD8+/Treg cell ratios, correlated with poor responses to checkpoint blockade immunotherapy. Thus, IFN-γ in the mLN skews Treg cells to be Th1-like effector Treg cells, driving their close interaction with DC1s and subsequent suppression of cytotoxic T cell responses.

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. pSer-modified eOD and IL-21 promote enhanced lymphatic drainage and lead to accumulation of the vaccine in B cell follicles in the draining lymph nodes. This in turn promotes enhanced T follicular helper cell priming and robust germinal center responses as well as increased antigen-specific serum IgG titers. This is a general strategy for codelivery of immunostimulatory cytokine with immunogens providing a facile approach to modulate T cell priming and GC reactions toward enhanced protective immunity using the most common clinical vaccine adjuvant.

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 IFNß 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. To this end, we compared the efficacy of IFNα and IFNß that exhibit either brief or sustained retention after intratumoral injection in syngeneic mouse tumor models. Significant enhancement in tumor retention, mediated by anchoring these IFNs to coinjected aluminum-hydroxide (alum) particles, greatly improved both their tolerability and efficacy. The improved efficacy of alum-anchored IFNs could be attributed to sustained pleiotropic effects on tumor cells, immune cells, and nonhematopoietic cells. Alum-anchored IFNs achieved high cure rates of B16F10 tumors upon combination with either anti-PD-1 antibody or interleukin-2. Interestingly however, these alternative combination immunotherapies yielded disparate T cell phenotypes and differential resistance to tumor rechallenge, highlighting important distinctions in adaptive memory formation for combinations of type I IFNs with other immunotherapies.

Cerebrospinal fluid can exit into the skull bone marrow and instruct cranial hematopoiesis in mice with bacterial meningitis.

Interactions between the immune and central nervous systems strongly influence brain health. Although the blood-brain barrier restricts this crosstalk, we now know that meningeal gateways through brain border tissues facilitate intersystem communication. Cerebrospinal fluid (CSF), which interfaces with the glymphatic system and thereby drains the brain's interstitial and perivascular spaces, facilitates outward signaling beyond the blood-brain barrier. In the present study, we report that CSF can exit into the skull bone marrow. Fluorescent tracers injected into the cisterna magna of mice migrate along perivascular spaces of dural blood vessels and then travel through hundreds of sub-millimeter skull channels into the calvarial marrow. During meningitis, bacteria hijack this route to invade the skull's hematopoietic niches and initiate cranial hematopoiesis ahead of remote tibial sites. As skull channels also directly provide leukocytes to meninges, the privileged sampling of brain-derived danger signals in CSF by regional marrow may have broad implications for inflammatory neurological disorders.

Maximizing response to intratumoral immunotherapy in mice by tuning local retention.

Direct injection of therapies into tumors has emerged as an administration route capable of achieving high local drug exposure and strong anti-tumor response. A diverse array of immune agonists ranging in size and target are under development as local immunotherapies. However, due to the relatively recent adoption of intratumoral administration, the pharmacokinetics of locally-injected biologics remains poorly defined, limiting rational design of tumor-localized immunotherapies. Here we define a pharmacokinetic framework for biologics injected intratumorally that can predict tumor exposure and effectiveness. We find empirically and computationally that extending the tumor exposure of locally-injected interleukin-2 by increasing molecular size and/or improving matrix-targeting affinity improves therapeutic efficacy in mice. By tracking the distribution of intratumorally-injected proteins using positron emission tomography, we observe size-dependent enhancement in tumor exposure occurs by slowing the rate of diffusive escape from the tumor and by increasing partitioning to an apparent viscous region of the tumor. In elucidating how molecular weight and matrix binding interplay to determine tumor exposure, our model can aid in the design of intratumoral therapies to exert maximal therapeutic effect.

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. Small-format immunocytokines, constructed with antibody fragments, are hypothesized to improve tumor specificity due to rapid systemic clearance. However, effective design criteria for small-format immunocytokines need further examination. Here, we engineer small interleukin-2 (IL-2) immunocytokines fused to nanobodies with nanomolar to picomolar affinities for the tumor-specific EIIIB domain of fibronectin (also known as EDB). Upon intravenous delivery into immunocompetent mice, such immunocytokines led to similar tumor growth delay as size-matched untargeted IL-2. Intratumoral (i.t.) delivery imparted improved survival dependent on affinity to EIIIB. I.t. administration offers a promising avenue to deliver small-format immunocytokines, given effective affinity for the tumor microenvironment.

Lack of CD8+ T cell effector differentiation during priming mediates checkpoint blockade resistance in non-small cell lung cancer.

In non­small cell lung cancer (NSCLC), response to immune checkpoint blockade (ICB) is associated with programmed cell death ligand 1 expression that is induced by interferon-γ­producing, tumor-infiltrating CD8+ T cells. However, not all tumors with a CD8+ T cell infiltrate respond to ICB, and little is known about the mechanisms governing ICB resistance in T cell­infiltrated NSCLC. We used an orthotopic NSCLC mouse model to study ICB-refractory CD8+ T cell responses. Single-cell RNA sequencing of the NSCLC mouse tumors revealed that lung cancer­specific tumor-infiltrating CD8+ T cells exhibited clonal expansion but lacked expression of genes associated with effector and exhausted T cell responses, indicating that they underwent a differentiation program distinct from conventional T cell exhaustion. This lung cancer­specific T cell dysfunction program was established early during priming in the mediastinal lymph node and was characterized by robust proliferation but a failed up-regulation of effector and exhausted T cell characteristics. Intriguingly, CD8+ T cells from patients with NSCLC expressed an analogous gene expression program, which appeared distinct from conventional T cell exhaustion. Administration of recombinant interleukin-2 (IL-2) and IL-12 was sufficient to restore effector T cell differentiation and induce control of KP lung tumors. These findings imply that a CD8+ T cell differentiation trajectory, activated during T cell priming in the mediastinal lymph node, limits the response of CD8+ T cells to ICB and thereby may contribute to failure of ICB in a subset T cell­infiltrated NSCLC.

Immunotherapy-induced antibodies to endogenous retroviral envelope glycoprotein confer tumor protection in mice.

Following curative immunotherapy of B16F10 tumors, ~60% of mice develop a strong antibody response against cell-surface tumor antigens. Their antisera confer prophylactic protection against intravenous challenge with B16F10 cells, and also cross-react with syngeneic and allogeneic tumor cell lines MC38, EL.4, 4T1, and CT26. We identified the envelope glycoprotein (env) of a murine endogenous retrovirus (ERV) as the antigen accounting for the majority of this humoral response. A systemically administered anti-env monoclonal antibody cloned from such a response protects against tumor challenge, and prophylactic vaccination against the env protein protects a majority of naive mice from tumor establishment following subcutaneous inoculation with B16F10 cells. These results suggest the potential for effective prophylactic vaccination against analogous HERV-K env expressed in numerous human cancers.

Pharmacokinetic tuning of protein-antigen fusions enhances the immunogenicity of T-cell vaccines.

The formulations of peptide-based antitumour vaccines being tested in clinical studies are generally associated with weak potency. Here, we show that pharmacokinetically tuning the responses of peptide vaccines by fusing the peptide epitopes to carrier proteins optimizes vaccine immunogenicity in mice. In particular, we show in immunized mice that the carrier protein transthyretin simultaneously optimizes three factors: efficient antigen uptake in draining lymphatics from the site of injection, protection of antigen payloads from proteolytic degradation and reduction of antigen presentation in uninflamed distal lymphoid organs. Optimizing these factors increases vaccine immunogenicity by up to 90-fold and maximizes the responses to viral antigens, tumour-associated antigens, oncofetal antigens and shared neoantigens. Protein-peptide epitope fusions represent a facile and generalizable strategy for enhancing the T-cell responses elicited by subunit vaccines.

Improved Antitumor Efficacy of Chimeric Antigen Receptor T Cells that Secrete Single-Domain Antibody Fragments.

Chimeric antigen receptor (CAR) T-cell therapy is effective in the treatment of cancers of hematopoietic origin. In the immunosuppressive solid tumor environment, CAR T cells encounter obstacles that compromise their efficacy. We developed a strategy to address these barriers by having CAR T cells secrete single-domain antibody fragments [variable heavy domain of heavy chain antibodies (VHH) or nanobodies] that can modify the intratumoral immune landscape and thus support CAR T-cell function in immunocompetent animals. VHHs are small in size and able to avoid domain swapping when multiple nanobodies are expressed simultaneously-features that can endow CAR T cells with desirable properties. The secretion of an anti-CD47 VHH by CAR T cells improves engagement of the innate immune system, enables epitope spreading, and can enhance the antitumor response. CAR T cells that secrete anti-PD-L1 or anti-CTLA-4 nanobodies show improved persistence and demonstrate the versatility of this approach. Furthermore, local delivery of secreted anti-CD47 VHH-Fc fusions by CAR T cells at the tumor site limits their systemic toxicity. CAR T cells can be further engineered to simultaneously secrete multiple modalities, allowing for even greater tailoring of the antitumor immune response.

Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumors and prime systemic immunity.

Therapies that synergistically stimulate immunogenic cancer cell death (ICD), inflammation, and immune priming are of great interest for cancer immunotherapy. However, even multi-agent therapies often fail to trigger all of the steps necessary for self-sustaining anti-tumor immunity. Here we describe self-replicating RNAs encapsulated in lipid nanoparticles (LNP-replicons), which combine three key elements: (1) an LNP composition that potently promotes ICD, (2) RNA that stimulates danger sensors in transfected cells, and (3) RNA-encoded IL-12 for modulation of immune cells. Intratumoral administration of LNP-replicons led to high-level expression of IL-12, stimulation of a type I interferon response, and cancer cell ICD, resulting in a highly inflamed tumor microenvironment and priming of systemic anti-tumor immunity. In several mouse models of cancer, a single intratumoral injection of replicon-LNPs eradicated large established tumors, induced protective immune memory, and enabled regression of distal uninjected tumors. LNP-replicons are thus a promising multifunctional single-agent immunotherapeutic.

Anchoring of intratumorally administered cytokines to collagen safely potentiates systemic cancer immunotherapy.

The clinical application of cytokine therapies for cancer treatment remains limited due to severe adverse reactions and insufficient therapeutic effects. Although cytokine localization by intratumoral administration could address both issues, the rapid escape of soluble cytokines from the tumor invariably subverts this effort. We find that intratumoral administration of a cytokine fused to the collagen-binding protein lumican prolongs local retention and markedly reduces systemic exposure. Combining local administration of lumican-cytokine fusions with systemic immunotherapies (tumor-targeting antibody, checkpoint blockade, cancer vaccine, or T cell therapy) improves efficacy without exacerbating toxicity in syngeneic tumor models and the BrafV600E /Ptenfl/fl genetically engineered melanoma model. Curative abscopal effects on noncytokine-injected tumors were also observed as a result of a protective and systemic CD8+ T cell response primed by local therapy. Cytokine collagen-anchoring constitutes a facile, tumor-agnostic strategy to safely potentiate otherwise marginally effective systemic immunotherapies.

Combining the Specific Anti-MUC1 Antibody TAB004 and Lip-MSA-IL-2 Limits Pancreatic Cancer Progression in Immune Competent Murine Models of Pancreatic Ductal Adenocarcinoma.

Immunotherapy regimens have shown success in subsets of cancer patients; however, their efficacy against pancreatic ductal adenocarcinoma (PDA) remain unclear. Previously, we demonstrated the potential of TAB004, a monoclonal antibody targeting the unique tumor-associated form of MUC1 (tMUC1) in the early detection of PDA. In this study, we evaluated the therapeutic benefit of combining the TAB004 antibody with Liposomal-MSA-IL-2 in immune competent and human MUC1 transgenic (MUC1.Tg) mouse models of PDA and investigated the associated immune responses. Treatment with TAB004 + Lip-MSA-IL-2 resulted in significantly improved survival and slower tumor growth compared to controls in MUC1.Tg mice bearing an orthotopic PDA.MUC1 tumor. Similarly, in the spontaneous model of PDA that expresses human MUC1, the combination treatment stalled the progression of pancreatic intraepithelial pre-neoplastic (PanIN) lesion to adenocarcinoma. Treatment with the combination elicited a robust systemic and tumor-specific immune response with (a) increased percentages of systemic and tumor infiltrated CD45+CD11b+ cells, (b) increased levels of myeloperoxidase (MPO), (c) increased antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP), (d) decreased percentage of immune regulatory cells (CD8+CD69+ cells), and (e) reduced circulating levels of immunosuppressive tMUC1. We report that treatment with a novel antibody against tMUC1 in combination with a unique formulation of IL-2 can improve survival and lead to stable disease in appropriate models of PDA by reducing tumor-induced immune regulation and promoting recruitment of CD45+CD11b+ cells, thereby enhancing ADCC/ADCP.

Nanobody-based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice.

Chimeric antigen receptor (CAR) T cell therapy has been successful in clinical trials against hematological cancers, but has experienced challenges in the treatment of solid tumors. One of the main difficulties lies in a paucity of tumor-specific targets that can serve as CAR recognition domains. We therefore focused on developing VHH-based, single-domain antibody (nanobody) CAR T cells that target aspects of the tumor microenvironment conserved across multiple cancer types. Many solid tumors evade immune recognition through expression of checkpoint molecules, such as PD-L1, that down-regulate the immune response. We therefore targeted CAR T cells to the tumor microenvironment via the checkpoint inhibitor PD-L1 and observed a reduction in tumor growth, resulting in improved survival. CAR T cells that target the tumor stroma and vasculature through the EIIIB+ fibronectin splice variant, which is expressed by multiple tumor types and on neovasculature, are likewise effective in delaying tumor growth. VHH-based CAR T cells can thus function as antitumor agents for multiple targets in syngeneic, immunocompetent animal models. Our results demonstrate the flexibility of VHH-based CAR T cells and the potential of CAR T cells to target the tumor microenvironment and treat solid tumors.