ABSTRACT
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). We identify bivalent recruitment of SHP2 by bioPROTACs as a broadly applicable strategy to improve potency. Moreover, we demonstrate that SHP2-targeted bioPROTACs can effectively counteract gain-of-function SHP2 mutants present in cancer, which are otherwise challenging to selectively target with small molecule constructs. Overall, this study demonstrates the utility of bioPROTACs for challenging targets, and further explicates design principles for therapeutic bioPROTACs.
ABSTRACT
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. In the present study, we report a localized cytokine therapy that safely elicits systemic antitumor immunity by targeting the ubiquitous leukocyte receptor CD45. CD45-targeted immunocytokines have lower internalization rates relative to wild-type counterparts, leading to sustained downstream cis and trans signaling between lymphocytes. A single intratumoral dose of αCD45-interleukin (IL)-12 followed by a single dose of αCD45-IL-15 eradicated treated tumors and untreated distal lesions in multiple syngeneic mouse tumor models without toxicity. Mechanistically, CD45-targeted cytokines reprogrammed tumor-specific CD8+ T cells in the tumor-draining lymph nodes to have an antiviral transcriptional signature. CD45 anchoring represents a broad platform for protein retention by host immune cells for use in immunotherapy.
ABSTRACT
PURPOSE: Interleukin-2 and -12 cytokines have potent anti-cancer 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 with in dogs with advanced melanoma. EXPERIMENTAL DESIGN: This study examined fifteen client-owned dogs with histologically- or cytologically-confirmed malignant melanoma who received a single 9 Gray fraction of radiation therapy, followed by six cycles of combined collagen-anchored IL-2 and IL-12 therapy Q2W. Cytokine dosing followed a 3+3 dose escalation design, with the initial cytokine dose chosen from prior evaluation in canine sarcomas. No exclusion criteria for tumor stage or metastatic burden, age, weight, or neuter status were applied for this trial. RESULTS: Median survival regardless of tumor stage or dose level was 256 days and 10/13 (76.9%) dogs that completed treatment had CT-measured tumor regression at the treated lesion. In dogs with metastatic disease, 8/13 (61.5%) dogs had partial responses across their combined lesions, evidence of locoregional response. Profiling by Nanostring of treatment-resistant dogs revealed that B2m loss was predictive of poor response to this therapy. CONCLUSIONS: Collectively, these results confirm the ability of locally administered tumor-anchored cytokines to potentiate responses at regional disease sites when combined with radiation. This evidence supports the clinical translation of this approach and highlights the utility of comparative investigation in canine cancers.
ABSTRACT
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. Here, we describe CLN-617, a first-in-class therapeutic for intratumoral (IT) injection that co-delivers IL2 and IL12 on a single molecule in a safe and effective manner. CLN-617 is a single-chain fusion protein comprised of IL2, leukocyte-associated immunoglobulin-like receptor 2 (LAIR2), human serum albumin (HSA), and IL12. LAIR2 and HSA function to retain CLN-617 in the treated tumor by binding collagen and increasing molecular weight, respectively. We found that IT administration of a murine surrogate of CLN-617, mCLN-617, eradicated established treated and untreated tumors in syngeneic models, significantly improved response to anti-PD1 checkpoint therapy, and generated a robust abscopal response dependent on cellular immunity and antigen cross-presentation. CLN-617 is being evaluated in a clinical trial in patients with advanced solid tumors (NCT06035744).
Subject(s)
Interleukin-12 , Interleukin-2 , Animals , Female , Humans , Mice , Cell Line, Tumor , Interleukin-12/metabolism , Interleukin-2/therapeutic use , Interleukin-2/pharmacology , Mice, Inbred C57BL , Neoplasms/immunology , Neoplasms/therapy , Neoplasms/drug therapy , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
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 of both murine CD19 CAR-T cells in a syngeneic model and human CAR-T cells in a humanized mouse model of B cell acute lymphoblastic leukemia (B-ALL), and enhanced control of leukemia progression. This approach thus enables vaccine boosting to be applied to any clinically-relevant CAR-T cell product.
ABSTRACT
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.
ABSTRACT
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 (αCD45-Cyt) that, upon injection, decorated the surface of leukocytes in the tumor and tumor-draining lymph node (TDLN) without systemic exposure. αCD45-Cyt therapy eradicated both directly treated tumors and untreated distal lesions in multiple syngeneic mouse tumor models. Mechanistically, αCD45-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.
ABSTRACT
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. Unlike traditional reporter assays that utilize loss-of-signal readouts to detect degradation, our assay platform expresses a robust fluorescence signal in response to the depletion of a target protein and incorporates additional measures intended to prevent undesirable false positives. Using this gain-of-signal assay, we successfully identified novel macromolecule SHP2 degraders from a screen of 192 candidates and proposed design principles for further development of macromolecule degraders. This work demonstrates a proof of concept for gain-of-signal assays as a tool for screening targeted degrader candidates.
Subject(s)
Proteins , ProteolysisABSTRACT
ABSTRACT: 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. CLEC2D protein was detected by flow cytometry on a panel of cell lines representing a diverse set of hematological malignancies. We, therefore, used yeast display to generate a panel of high-affinity, fully human CD161 monoclonal antibodies (mAbs) that blocked CLEC2D binding. These mAbs were specific for CD161 and had a similar affinity for human and nonhuman primate CD161, a property relevant for clinical translation. A high-affinity CD161 mAb enhanced key aspects of T-cell function, including cytotoxicity, cytokine production, and proliferation, against B-cell lines originating from patients with acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and Burkitt lymphoma. In humanized mouse models, this CD161 mAb enhanced T-cell-mediated immunity, resulting in a significant survival benefit. Single cell RNA-seq data demonstrated that CD161 mAb treatment enhanced expression of cytotoxicity genes by CD4 T cells as well as a tissue-residency program by CD4 and CD8 T cells that is associated with favorable survival outcomes in multiple human cancer types. These fully human mAbs, thus, represent potential immunotherapy agents for hematological malignancies.
Subject(s)
Hematologic Neoplasms , Neoplasms , Animals , Mice , Humans , CD4-Positive T-Lymphocytes , Immunity, Cellular , CD8-Positive T-Lymphocytes , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Hematologic Neoplasms/genetics , Hematologic Neoplasms/therapy , NK Cell Lectin-Like Receptor Subfamily B/geneticsABSTRACT
IL-12 is a potent cytokine that can promote innate and adaptive anticancer immunity, but its clinical development has been limited by toxicity when delivered systemically. Intratumoral (i.t.) administration can expand the therapeutic window of IL-12 and other cytokines but is in turn limited by rapid drug clearance from the tumor, which reduces efficacy, necessitates frequent administration, and increases systemic accumulation. To address these limitations, we developed an anchored IL-12 designated ANK-101, composed of an engineered IL-12 variant that forms a stable complex with the FDA-approved vaccine adjuvant aluminum hydroxide (Alhydrogel). Following i.t. administration of murine ANK-101 (mANK-101) in early intervention syngeneic mouse tumors, the complex formed a depot that was locally retained for weeks as measured by IVIS or SPECT/CT imaging, while unanchored protein injected i.t. was cleared within hours. One or 2 i.t. injections of mANK-101 induced single-agent antitumor activity across a diverse range of syngeneic tumors, including models resistant to checkpoint blockade at doses where unanchored IL-12 had no efficacy. Local treatment with mANK-101 further induced regressions of noninjected lesions, especially when combined with systemic checkpoint blockade. Antitumor activity was associated with remodeling of the tumor microenvironment, including prolonged IFN-γ and chemokine expression, recruitment and activation of T and NK cells, M1 myeloid cell skewing, and increased antigen processing and presentation. Subcutaneous administration of ANK-101 in cynomolgus macaques was well tolerated. Together, these data demonstrate that ANK-101 has an enhanced efficacy and safety profile and warrants future clinical development.
Subject(s)
Interleukin-12 , Neoplasms , Mice , Animals , Aluminum Hydroxide/pharmacology , Tumor Microenvironment , CytokinesABSTRACT
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.
Subject(s)
Interleukin-12 , Lung Neoplasms , Mice , Animals , Interleukin-12/genetics , Interleukin-2/genetics , Immunotherapy , Cytokines , Lung Neoplasms/genetics , Lung Neoplasms/therapyABSTRACT
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. Comparison of b1s1e2-Fc to 9d9, an antagonistic anti-CTLA-4 antibody, allowed for interrogation of the separate contributions of CTLA-4 antagonism and Treg depletion to efficacy. Despite equivalent levels of intratumoral Treg depletion, 9d9 achieved more long-term cures than b1s1e2-Fc in MC38 tumors, demonstrating that CTLA-4 antagonism provided additional survival benefit. Consistent with prior reports that CTLA-4 antagonism enhances priming, treatment with 9d9, but not b1s1e2-Fc, increased the percentage of activated T cells in the tumor-draining lymph node (tdLN). Treg depletion with either construct was restricted to the tumor due to insufficient surface CTLA-4 expression on Tregs in other compartments. Through intratumoral administration of diphtheria toxin in Foxp3-DTR mice, we show that depletion of both intratumoral and nodal Tregs provided even greater survival benefit than 9d9, consistent with Treg-driven restraint of priming in the tdLN. Our data demonstrate that anti-CTLA-4 therapies require both CTLA-4 antagonism and intratumoral Treg depletion for maximum efficacy-but that potential future therapies also capable of depleting nodal Tregs could show efficacy in the absence of CTLA-4 antagonism.
Subject(s)
Neoplasms , T-Lymphocytes, Regulatory , Mice , Animals , Neoplasms/drug therapy , Neoplasms/genetics , CTLA-4 Antigen , Lymphocyte DepletionABSTRACT
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-É (IFNÉ) 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. Furthermore, cytokines' ability to stimulate multiple cell types, often with paradoxical effects, may present significant challenges for their translation into effective therapies. Recently, protein engineering has emerged as a tool to address the shortcomings of first-generation cytokine therapies. In this perspective, we contextualize cytokine engineering strategies such as partial agonism, conditional activation and intratumoral retention through the lens of spatiotemporal regulation. By controlling the time, place, specificity, and duration of cytokine signaling, protein engineering can allow exogenous cytokine therapies to more closely approach their endogenous exposure profile, ultimately moving us closer to unlocking their full therapeutic potential.
Subject(s)
Cytokines , Neoplasms , Humans , Cytokines/metabolism , Neoplasms/drug therapy , Protein Engineering , ImmunotherapyABSTRACT
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. We studied the therapeutic effect of tumor-localized catalases with the aim to further elucidate the mechanism of action. To do this, we engineered two approaches to maximize intratumoral catalase exposure: 1) an injected extracellular catalase with enhanced tumor retention, and 2) tumor cell lines that over-express intracellular catalase. Both approaches were characterized for functionality and tested for therapeutic efficacy and mechanism in 4T1 and CT26 murine syngeneic tumor models. The injected catalase was confirmed to have enzyme activity >30,000 U/mg and was retained at the injection site for more than one week in vivo. The engineered cell lines exhibited increased catalase activity and antioxidant capacity, with catalase over-expression that was maintained for at least one week after gene expression was induced in vivo. We did not observe a significant difference in tumor growth or survival between catalase-treated and untreated mice when either approach was used. Finally, bulk RNA sequencing of tumors was performed, comparing the gene expression of catalase-treated and untreated tumors. Gene expression analysis revealed very few differentially expressed genes as a result of exposure to catalase and notably, we did not observe changes consistent with an altered state of hypoxia or oxidative stress. In conclusion, we observe that sustained intratumoral catalase neither has therapeutic benefit nor triggers significant differential expression of genes associated with the anticipated therapeutic mechanism in the subcutaneous syngeneic tumor models used. Given the lack of effect observed, we propose that further development of catalase as a cancer therapeutic should take these findings into consideration.
Subject(s)
Antioxidants , Neoplasms , Animals , Mice , Catalase/genetics , Catalase/metabolism , Antioxidants/metabolism , Neoplasms/genetics , Oxidative Stress , Hypoxia/genetics , Hydrogen Peroxide/metabolism , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Melanoma , Sarcoma , Mice , Animals , Dogs , Interleukin-12/genetics , Interleukin-2 , Tumor Microenvironment , Cytokines , Sarcoma/drug therapy , CollagenABSTRACT
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. This study aimed to determine whether supplementing anti-PD-1 ICB with engineered extended half-life IL2, a potent lymphoproliferative cytokine, could improve outcomes. This combination therapy, subsequently referred to as enhanced checkpoint blockade (ECB), delivered intraperitoneally, reliably cures approximately 50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extends median survival of the treated cohort. In the CT2A model, characterized as being resistant to CBI, ECB caused a decrease in CT2A tumor volume in half of measured animals similar to what was observed in GL261-bearing mice, promoting a trending survival increase. ECB generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Through employment of depletion strategies, ECB's efficacy was shown to be independent of host MHC class I-restricted antigen presentation but reliant on CD4 T cells. These results demonstrate ECB is efficacious against the GL261 glioma model through an MHC class I-independent mechanism and supporting further investigation into IL2-supplemented ICB therapies for tumors of the central nervous system.
Subject(s)
Glioblastoma , Glioma , Mice , Animals , Interleukin-2/pharmacology , Interleukin-2/therapeutic use , Half-Life , Mice, Inbred C57BL , Glioma/pathology , Cell Line, TumorABSTRACT
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 É4-1BB agonist (É4-1BB-LAIR), which consists of an É4-1BB 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: ÉCD4 eliminated tumor draining lymph node Tregs, enhancing priming and activation of CD8+ T cells, and TA99 + É4-1BB-LAIR supported the cytotoxic program of these newly primed CD8+ T cells within the tumor microenvironment. Replacement of ÉCD4 with ÉCTLA-4, a clinically approved antibody that enhances T cell priming, produced equivalent cure rates while additionally generating robust immunological memory against secondary tumor rechallenge.
ABSTRACT
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.
Subject(s)
Lung Neoplasms , T-Lymphocytes, Regulatory , Humans , CD8-Positive T-Lymphocytes , Interferon-gamma , T-Lymphocytes, CytotoxicABSTRACT
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.
ABSTRACT
The carboxyl groups of a protein can be esterified by reaction with a diazo compound, 2-diazo-2-(p-methylphenyl)-N,N-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. Solubility screening revealed that ß-cyclodextrin (ß-CD) is an optimal solubilizing agent for esterified green fluorescent protein (est-GFP). Its addition can increase the recovery of est-GFP by 10-fold. α-CD, γ-CD, and cucurbit-7-uril are less effective excipients. 1H NMR titration experiments revealed that ß-CD encapsulates the hydrophobic tolyl group of ester conjugates with Ka = 321 M-1. Combining l-arginine and sucrose with ß-CD enables the nearly quantitative recovery of est-GFP. Thus, the insolubility of esterified proteins can be overcome with excipients.