Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles.

Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.

Oxidation and reduction analysis of therapeutic recombinant human interleukin-15 by HPLC and LC-MS.

Being an important immune stimulant of T lymphocytes and NK cells, the recombinant human interleukin-15 (rhIL-15) has been extensively researched in tumor immunotherapy or as a vaccine adjuvant. However, the rhIL-15 manufacturing level lags far behind its growing clinical demand due to the lack of efficient and exact analysis methodologies to characterize the trace by-products, typically redox and deamidation. In order to improve the production and quality control of rhIL-15, here we developed an expanded resolution reverse-phase high-performance liquid chromatography (ExRP-HPLC) approach to quickly and accurately analyze the oxidation and reduction by-products of rhIL-15, which may appear during the purification processes. Firstly, we developed RP-HPLC methods which can separate rhIL-15 fractions with different levels of oxidization or reduction, respectively, and the redox status of each peak was then determined by measuring the intact mass with a high-resolution mass spectrometer (UPLC-MS). To further clarify the complex pattern of oxidization of specific residues, the peaks with various oxidation levels were digested into pieces for peptide mapping to pinpoint the exact changes of oxygen and hydrogen atoms in the rhIL-15 by-products. In addition, we performed the ExRP-HPLC and UPLC-MS analysis of partially deamidated rhIL-15 to characterize their oxidation and reduction. Our work is the first in-depth characterization of the redox by-products of rhIL-15, even for deamidated impurities. The ExRP-HPLC method we reported can facilitate the rapid and accurate quality analysis of rhIL-15, which is substantially helpful for streamlining the industrial manufacturing of rhIL-15 to better meet the demands of clinical applications. KEYPOINTS: • The oxidization and reduction rhIL-15 by-products were characterized for the first time. • The changes of oxygen and hydrogen atoms in rhIL-15 redox by-products were accurately determined by UPLC-MS. • Oxidation and reduction by-products of deamidated rhIL-15 were further analyzed.

Human IL-2Rɑ subunit binding modulation of IL-2 through a decline in electrostatic interactions: A computational and experimental approach.

Although high-dose IL-2 has clear antitumor effects, severe side effects like severe toxicity and activation of Tregs by binding of IL-2 to high-affinity IL-2R, hypotension, and vascular leak syndrome limit its applications as a therapeutic antitumor agent. Here in this study, a rational computational approach was employed to develop and design novel triple-mutant IL-2 variants with the aim of improving IL-2-based immunotherapy. The affinity of the mutants towards IL-2Rα was further computed with the aid of molecular dynamic simulations and umbrella sampling techniques and the obtained results were compared to those of wild-type IL-2. In vitro experiments by flow cytometry showed that the anti-CD25 mAb was able to bind to PBMC cells even after mutant 2 preincubation, however, the binding strength of the mutant to α-subunit was less than of wtIL-2. Additionally, reduction of IL-2Rα subunit affinity did not significantly disturb IL-2/IL2Rßγc subunits interactions.

Enhancing the Pharmacokinetic Profile of Interleukin 2 through Site-Specific Conjugation to a Selective Small-Molecule Transthyretin Ligand.

Protein drugs hold great promise as therapeutics for a wide range of diseases. Unfortunately, one of the greatest challenges to be addressed during clinical development of protein therapeutics is their short circulation half-life. Several protein conjugation strategies have been developed for half-life extension. However, these strategies have limitations and there remains room for improvement. Here, we report a novel nature-inspired strategy for enhancing the in vivo half-life of proteins. Our strategy involves conjugating proteins to a hydrophilic small molecule that binds reversibly to the plasma protein, transthyretin. We show here that our strategy is effective in enhancing the pharmacokinetic and pharmacodynamic properties of human interleukin 2 in rats, potentially opening the door for more effective and safer cancer immunotherapies. To our knowledge, this is the first example of successful use of a small-molecule that not only extends the half-life but also maintains the smaller size, binding potency, and hydrophilicity of proteins.

An engineered IL-2 partial agonist promotes CD8+ T cell stemness.

Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+ T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential.

An engineered IL-2 reprogrammed for anti-tumor therapy using a semi-synthetic organism.

The implementation of applied engineering principles to create synthetic biological systems promises to revolutionize medicine, but application of fundamentally redesigned organisms has thus far not impacted practical drug development. Here we utilize an engineered microbial organism with a six-letter semi-synthetic DNA code to generate a library of site-specific, click chemistry compatible amino acid substitutions in the human cytokine IL-2. Targeted covalent modification of IL-2 variants with PEG polymers and screening identifies compounds with distinct IL-2 receptor specificities and improved pharmacological properties. One variant, termed THOR-707, selectively engages the IL-2 receptor beta/gamma complex without engagement of the IL-2 receptor alpha. In mice, administration of THOR-707 results in large-scale activation and amplification of CD8+ T cells and NK cells, without Treg expansion characteristic of IL-2. In syngeneic B16-F10 tumor-bearing mice, THOR-707 enhances drug accumulation in the tumor tissue, stimulates tumor-infiltrating CD8+ T and NK cells, and leads to a dose-dependent reduction of tumor growth. These results support further characterization of the immune modulatory, anti-tumor properties of THOR-707 and represent a fundamental advance in the application of synthetic biology to medicine, leveraging engineered semi-synthetic organisms as cellular factories to facilitate discovery and production of differentiated classes of chemically modified biologics.

Immune Modulatory Activities of Arginyl-Fructose (AF) and AF-Enriched Natural Products in In-Vitro and In-Vivo Animal Models.

The immune system plays an important role in maintaining body homeostasis. Recent studies on the immune-enhancing effects of ginseng saponins have revealed more diverse mechanisms of action. Maillard reaction that occurs during the manufacturing processes of red ginseng produces a large amount of Amadori rearrangement compounds (ARCs), such as arginyl-fructose (AF). The antioxidant and anti-hyperglycemic effects of AF have been reported. However, the possible immune enhancing effects of non-saponin ginseng compounds, such as AF, have not been investigated. In this study the effects of AF and AF-enriched natural product (Ginofos, GF) on proliferation of normal mouse splenocytes were evaluated in vitro and male BALB/c mice models. The proliferation of splenocytes treated with mitogens (concanavalin A, lipopolysaccharide) were further increased by addition of AF (p < 0.01) or GF (p < 0.01), in a dose dependent manner. After the 10 days of oral administration of compounds, changes in weights of spleen and thymus, serum immunoglobulin, and expression of cytokines were measured as biomarkers of immune-enhancing potential in male BALB/c mice model. The AF or GF treated groups had higher weights of the thymus (0.94 ± 0.25 and 0.86 ± 0.18, p < 0.05, respectively) than that of cyclophosphamide treated group (0.59 ± 0.18). This result indicates that AF or AF-enriched extract (GF) increased humoral immunity against CY-induced immunosuppression. In addition, immunoglobulin contents and expression of cytokines including IgM (p < 0.01), IgG (p < 0.05), IL-2 (p < 0.01), IL-4 (p < 0.01), IL-6 (p < 0.01), and IFN-γ (p < 0.05) were also significantly increased by supplementation of AF or GF. These results indicate that AF has immune enhancing effects by activation of adaptive immunity via increase of expression of immunoglobulins and cytokines such as IgM, IgG, IL-2, IL-4, IL-6 and thereby proliferating the weight of thymus. Our findings provide a pharmacological rationale for AF-enriched natural products such as ginseng and red ginseng that can possibly have immune-enhancement potential and should be further evaluated.

Phase II study of E7777 in Japanese patients with relapsed/refractory peripheral and cutaneous T-cell lymphoma.

E7777 is a recombinant cytotoxic fusion protein composed of the diphtheria toxin fragments A and B and human interleukin-2. It shares an amino acid sequence with denileukin diftitox, but has improved purity and an increased percentage of active monomer. We undertook a multicenter, single-arm phase II study of E7777 in patients with relapsed or refractory peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL) to evaluate its efficacy, safety, pharmacokinetics, and immunogenicity. A total of 37 patients were enrolled, of which 17 and 19 patients had PTCL and CTCL, respectively, and one patient with another type of lymphoma (extranodal natural killer/T-cell lymphoma, nasal type), diagnosed by the Central Pathological Diagnosis Committee. Among the 36 patients with PTCL and CTCL, objective response rate based on the independent review was 36% (41% and 31%, respectively). The median progression-free survival was 3.1 months (2.1 months in PTCL and 4.2 months in CTCL). The common adverse events (AEs) observed were increased aspartate aminotransferase (AST) / alanine aminotransferase (ALT), hypoalbuminemia, lymphopenia, and pyrexia. Our results indicated that a 9 µg/kg/d dose of E7777 shows efficacy and a manageable safety profile in Japanese patients with relapsed or refractory PTCL and CTCL, with clinical activity observed across the range of CD25 expression. The common AEs were manageable, but increase in ALT / AST, hypoalbuminemia, and capillary leak syndrome should be carefully managed during the treatment.

An Improved Synthesis of N-(4-[18F]Fluorobenzoyl)-Interleukin-2 for the Preclinical PET Imaging of Tumour-Infiltrating T-cells in CT26 and MC38 Colon Cancer Models.

Positron emission tomography (PET) imaging of activated T-cells with N-(4-[18F]fluorobenzoyl)-interleukin-2 ([18F]FB-IL-2) may be a promising tool for patient management to aid in the assessment of clinical responses to immune therapeutics. Unfortunately, existing radiosynthetic methods are very low yielding due to complex and time-consuming chemical processes. Herein, we report an improved method for the synthesis of [18F]FB-IL-2, which reduces synthesis time and improves radiochemical yield. With this optimized approach, [18F]FB-IL-2 was prepared with a non-decay-corrected radiochemical yield of 3.8 ± 0.7% from [18F]fluoride, 3.8 times higher than previously reported methods. In vitro experiments showed that the radiotracer was stable with good radiochemical purity (>95%), confirmed its identity and showed preferential binding to activated mouse peripheral blood mononuclear cells. Dynamic PET imaging and ex vivo biodistribution studies in naïve Balb/c mice showed organ distribution and kinetics comparable to earlier published data on [18F]FB-IL-2. Significant improvements in the radiochemical manufacture of [18F]FB-IL-2 facilitates access to this promising PET imaging radiopharmaceutical, which may, in turn, provide useful insights into different tumour phenotypes and a greater understanding of the cellular nature and differential immune microenvironments that are critical to understand and develop new treatments for cancers.

Long-Acting Human Interleukin 2 Bioconjugate Modified with Fatty Acids by Sortase A.

Human Interleukin 2 (IL-2) has already achieved impressive results as a therapeutic agent for cancer and autoimmune diseases. However, one of the limitations associated with the clinical application of IL-2 is its short half-life owing to rapid clearance by the kidneys. Modification with fatty acids, as an albumin noncovalent ligand with the advantage of deep penetration into tissues and high activity-to-mass ratio, is a commonly used approach to improve the half-life of native peptides and proteins. In this investigation, we attempted to extend the half-life of IL-2 through conjugation with a fatty acid using sortase A (srtA). We initially designed and optimized three IL-2 analogues with different peptide linkers between the C-terminus of IL-2 and srtA recognition sequence (LPETG). Among these, analogue A3 was validated as the optimal IL-2 analogue for further modification. Next, six fatty acid moieties with the same fatty acid and different hydrophilic spacers were conjugated to A3 through srtA. The six bioconjugates generated were screened for in vitro biological activity, among which bioconjugate B6 was identified as near-optimal to IL-2. Additionally, B6 could effectively bind albumin through the conjugated fatty acid, which contributed to a significant improvement in its pharmacokinetic properties in vivo. In summary, we have developed a novel IL-2 bioconjugate, B6, modified with fatty acids using srtA, which may effectively serve as a new-generation long-acting IL-2 immunotherapeutic agent.

Computational study for suppression of CD25/IL-2 interaction.

Cancer recurrence presents a huge challenge in cancer patient management. Immune escape is a key mechanism of cancer progression and metastatic dissemination. CD25 is expressed in regulatory T (Treg) cells including tumor-infiltrating Treg cells (TI-Tregs). These cells specially activate and reinforce immune escape mechanism of cancers. The suppression of CD25/IL-2 interaction would be useful against Treg cells activation and ultimately immune escape of cancer. Here, software, web servers and databases were used, at which in silico designed small interfering RNAs (siRNAs), de novo designed peptides and virtual screened small molecules against CD25 were introduced for the prospect of eliminating cancer immune escape and obtaining successful treatment. We obtained siRNAs with low off-target effects. Further, small molecules based on the binding homology search in ligand and receptor similarity were introduced. Finally, the critical amino acids on CD25 were targeted by a de novo designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further in vitro and in vivo studies.

Predicting cryptic ligand binding sites based on normal modes guided conformational sampling.

To greatly expand the druggable genome, fast and accurate predictions of cryptic sites for small molecules binding in target proteins are in high demand. In this study, we have developed a fast and simple conformational sampling scheme guided by normal modes solved from the coarse-grained elastic models followed by atomistic backbone refinement and side-chain repacking. Despite the observations of complex and diverse conformational changes associated with ligand binding, we found that simply sampling along each of the lowest 30 modes is near optimal for adequately restructuring cryptic sites so they can be detected by existing pocket finding programs like fpocket and concavity. We further trained machine-learning protocols to optimize the combination of the sampling-enhanced pocket scores with other dynamic and conservation scores, which only slightly improved the performance. As assessed based on a training set of 84 known cryptic sites and a test set of 14 proteins, our method achieved high accuracy of prediction (with area under the receiver operating characteristic curve >0.8) comparable to the CryptoSite server. Compared with CryptoSite and other methods based on extensive molecular dynamics simulation, our method is much faster (1-2 hours for an average-size protein) and simpler (using only pocket scores), so it is suitable for high-throughput processing of large datasets of protein structures at the genome scale.

Treatment of mice with S4B6 IL-2 complex prevents lethal toxoplasmosis via IL-12- and IL-18-dependent interferon-gamma production by non-CD4 immune cells.

Toxoplasmic encephalitis is an AIDS-defining condition. The decline of IFN-γ-producing CD4+ T cells in AIDS is a major contributing factor in reactivation of quiescent Toxoplasma gondii to an actively replicating stage of infection. Hence, it is important to characterize CD4-independent mechanisms that constrain acute T. gondii infection. We investigated the in vivo regulation of IFN-γ production by CD8+ T cells, DN T cells and NK cells in response to acute T. gondii infection. Our data show that processing of IFN-γ by these non-CD4 cells is dependent on both IL-12 and IL-18 and the secretion of bioactive IL-18 in response to T. gondii requires the sensing of viable parasites by multiple redundant inflammasome sensors in multiple hematopoietic cell types. Importantly, our results show that expansion of CD8+ T cells, DN T cells and NK cell by S4B6 IL-2 complex pre-treatment increases survival rates of mice infected with T. gondii and this is dependent on IL-12, IL-18 and IFN-γ. Increased survival is accompanied by reduced pathology but is independent of expansion of TReg cells or parasite burden. This provides evidence for a protective role of IL2C-mediated expansion of non-CD4 cells and may represent a promising lead to adjunct therapy for acute toxoplasmosis.

A simple bacterial expression system for human ppGalNAc-T and used for the synthesis of O-GalNAc glycosylated interleukin 2.

Mucin-type O-glycosylation (hereafter referred to as O-GalNAc glycosylation) is one of the most abundant glycosylation on proteins. It is initiated by the members of polypeptide N-acetyl-α-galactosaminyltransferases (ppGalNAc-Ts) family. The ppGalNAc-Ts could be used as tool enzymes to modify target proteins including therapeutic glycoprotein drugs with O-GalNAc glycosylation at specific glycosylated sites in vitro. Obtaining a large amount of ppGalNAc-T can greatly increase the yield of therapeutic O-glycoprotein and reduce the culture costs. In this study, we reported a simple Escherichia coli (E. coli) expression system capable of producing human ppGalNAc-Ts. By co-expressing human PDI, we could simply obtain active ppGalNAc-Ts with high efficiency. Using the E. coli expressed ppGalNAc-T2, we site-specifically synthesized O-glycosylated IL-2 at the native glycosylated site Thr23 residue. These results reveal the E. coli system we constructed is suitable to produce active ppGalNAc-Ts and thus has the potential value for basic research and production of therapeutic O-glycoproteins in vitro.

The advent of de novo proteins for cancer immunotherapy.

Engineered proteins are revolutionizing immunotherapy, but advances are still needed to harness their full potential. Traditional protein engineering methods use naturally existing proteins as a starting point, and therefore, are intrinsically limited to small alterations of a protein's natural structure and function. Conversely, computational de novo protein design is free of such limitation, and can produce a virtually infinite number of novel protein sequences, folds, and functions. Recently, we used de novo protein engineering to create Neoleukin-2/15 (Neo-2/15), a protein mimetic of the function of both interleukin-2 (IL-2) and interleukin-15 (IL-15). To our knowledge, Neo-2/15 is the first de novo protein with immunotherapeutic activity, and in murine cancer models, it has demonstrated enhanced therapeutic potency and reduced toxicity compared to IL-2. De novo protein design is already showcasing its tremendous potential for driving the next wave of protein-based therapeutics that are explicitly engineered to treat disease.

Expression and purification of soluble and functional fusion protein DAB389 IL-2 into the E. coli strain Rosetta-gami (DE3).

DAB389 IL-2 (Denileukin diftitox) is considered an immunotoxin, and it is the first immunotoxin approved by Food and Drug Administration. It is used for the treatment of a cutaneous form of T-cell lymphoma. This fusion protein has two disulfide bonds in its structure that play an essential role in toxicity and functionality of the immunotoxin. Escherichia coli (E. coli) strain BL21 (DE3) is not capable of making disulfide bonds in its reductive cytoplasm, but the E. coli strain Rosetta-gami (DE3) is a proper strain for the correct expression of the protein due to mutations in glutaredoxin reductase and thioredoxin reductase. In this study, a pET21a vector with the His6-tag fused at the N-terminus of DAB389 IL-2 was used to express the soluble immunotoxin in E. coli Rosetta-gami (DE3). After the purification of the soluble protein by two-step column chromatographies, the structure of DAB389 IL-2 was analyzed using the Native-PAGE and circular dichroism methods. In the following, the nuclease activity of soluble DAB389 IL-2 and its cytotoxicity activity were determined. It is concluded that the soluble recombinant protein expressed in the E. coli Rosetta-gami (DE3) has an intact structure and also functional; hence, this form of immunotoxin could be competitive with its commercial counterparts.

Structural Basis for Signaling Through Shared Common γ Chain Cytokines.

The common γ chain (γc) family of hematopoietic cytokines consists of six distinct four α-helix bundle soluble ligands that signal through receptors which include the shared γc subunit to coordinate a wide range of physiological processes, in particular, those related to innate and adaptive immune function. Since the first crystallographic structure of a γc family cytokine/receptor signaling complex (the active Interleukin-2 [IL-2] quaternary complex) was determined in 2005 [1], tremendous progress has been made in the structural characterization of this protein family, transforming our understanding of the molecular mechanisms underlying immune activity. Although many conserved features of γc family cytokine complex architecture have emerged, distinguishing details have been observed for individual cytokine complexes that rationalize their unique functional properties. Much work remains to be done in the molecular characterization of γc family signaling, particularly with regard to intracellular activation events, and looking forward, new technologies in structural biophysics will offer further insight into the biology of cytokine signaling to inform the design of targeted therapeutics for treatment of immune-linked diseases such as cancer, infection, and autoimmune disorders.

Interleukin-2 (IL-2) in flounder (Paralichthys olivaceus): Molecular cloning, characterization and bioactivity analysis.

Interleukin-2 (IL-2) is mainly produced by CD4+ T helper lymphocytes, which is an important immunomodulatory cytokine that primarily promotes activation, proliferation and differentiation of T cells. In the present study, flounder (Paralichthys olivaceus) interleukin 2 homologue (poIL-2) was identified for the first time, and its expression patterns were characterized in healthy, virus- or bacteria-infected flounder. The full-length cDNA sequences of poIL-2 was 989 bp with an open reading frame of 423 bp coding a polypeptide of 140 amino acids (aa). The deduced aa sequences shared low similarities (<53%) with other known fish IL-2s. Multiple alignment of aa sequences revealed that poIL-2 own the classical IL-2 family signature of "C-X(3)-EL-X(2)-(T/V)-(V/M/L)-(K/T/R)-X-EC" and "DS-X-(F/L)Y(A/T/S)P". In healthy flounder, IL-2 mRNA was highly expressed in PBLs, spleen and hindgut, and moderately expressed in gill, trunk kidney and stomach. PHA, LPS and Con-A could effectively induce poIL-2 expression in primary cultured peripheral blood leukocytes in vitro. poIL-2 transcripts were significantly up-regulated in spleen, kidney, gill and hindgut post infections with Edwardsiella tarda and Hirame novirhabdovirus (HIRRV). The eukaryotic expression vector encoding poIL-2 (pcIL-2) was constructed and intramuscularly injected, which could be successfully expressed in flounders and induced significantly higher expressions of six immune related genes including poIL-2, ß-defensin, CD4-1, CD8α, IFN-γ and TNF-α compared with the injection with control plasmid. Moreover, pretreatment with pcIL-2 could markedly increase the survival rate of flounder challenged with HIRRV. Our results demonstrated that poIL-2 plays an important role in the induction of immune responses and immune defense against bacterial and virus infection, which indicated its potential use as an immunopotentiator to prevent diseases in flounder.

Structure and Function of an Inflammatory Cytokine, Interleukin-2, Analyzed Using the Bioinformatic Approach.

The inflammatory cytokine, interleukin-2 (IL-2), is an important regulator of cellular functions. This relatively less studied member of the interleukin protein family is responsible for multiple immuno-modulatory and immuno-stimulatory tasks, like T cell activation, triggering of natural killer cells, inflammation, as well as proliferation and progression of autoimmune diseases and cancers. In this communication we report the temporally variant structural aspects of the IL-2 ligand and its receptor interfaces, based on the available crystal structures. The intended goal of this effort is to generate simulated results that could potentially aid the designs of novel structure based therapeutics.

Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti-PD-1 in melanoma.

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.