scholarly journals 721 Intratumoral immunotherapy with aluminum hydroxide-tethered IL-12 induces potent local and systemic immunity with minimal toxicity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A750-A750
Author(s):  
Michael Schmidt ◽  
Gregory Papastoitsis ◽  
Howard Kaufman ◽  
Darrell Irvine ◽  
K Wittrup
Keyword(s):  
Aluminum Hydroxide ◽  
Systemic Exposure ◽  
Therapeutic Window ◽  
Interleukin 12 ◽  
Hydroxyl Groups ◽  
Single Chain ◽  
Tumor Models ◽  
Syngeneic Tumor ◽  
Tumor Retention

BackgroundInterleukin-12 (IL-12) is a potent pro-inflammatory cytokine that promotes Th1 skewing, IFNγ expression, T- and NK-cell activation, and antigen presentation. In animal models, IL-12 can elicit robust anti-tumor responses through activation of both innate and adaptive immunity. However, clinical translation of IL-12 has been hindered by significant immune-related toxicity when delivered systemically, necessitating low doses that are often insufficient for efficacy. Intratumoral (IT) administration can expand the therapeutic window of IL-12 by increasing the local tumor concentration relative to systemic exposure but is in turn limited by rapid vascular and lymphatic clearance of injected drug from the tumor and corresponding systemic accumulation. Here we describe an approach to locally retain intratumorally administered IL-12 by complexing it to the common vaccine adjuvant aluminum hydroxide (alum) through a novel phosphopeptide linkage.MethodsSingle-chain murine IL-12 (mIL12) was genetically fused at its c-terminus to a short alum-binding peptide (ABP) that is specifically phosphorylated on multiple serines when co-expressed with the kinase Fam20C. Phosphorylated mIL12-ABP proteins were complexed with a 10x mass excess of aluminum hydroxide through a naturally occurring ligand exchange reaction between the phosphoserines in the ABP and surface hydroxyl groups on alum. mIL12-ABP/alum complexes were characterized for in vitro potency and in vivo efficacy in multiple syngeneic tumor models including MC38, CT26, A20, 4T1, and B16F10 following IT administration. Immune analyses and re-challenge experiments are in progress.Results mIL12-ABP is phosphorylated on multiple sites when co-expressed with Fam20C and is stably retained on aluminum hydroxide in vitro under elution conditions containing phosphate and serum. Alum-bound mIL12-ABP remains active in cellular assays with a 3–4 fold increase in EC50 compared to free protein. Following intratumoral administration, the mIL12-ABP/alum complexes have significantly extended tumor retention compared to unmodified mIL12, leading to potent local immune activation for >1 week. One or two doses of IT administered mIL12-ABP/alum is sufficient to induce robust monotherapy efficacy in diverse syngeneic tumor models including cold tumors resistant to checkpoint blockade and other immunotherapies. Locally administered mIL12-ABP/alum is further able to prime a systemic immune response leading to efficacy against non-injected tumors and spontaneous metastases. Doses required for optimal efficacy are well tolerated in mice with no significant weight loss or other evidence of systemic toxicity.ConclusionsAnkyra's platform is a differentiated approach to expand the therapeutic window of IL-12 and other cytokine drugs by enhancing tumor retention following IT administration.

H60/TNT-3 Fusion Protein Activates NK Cells In Vitro and Improves Immunotherapeutic Outcome in Murine Syngeneic Tumor Models

2006 ◽  
Vol 29 (3) ◽  
pp. 274-283 ◽  
Author(s):  
Meg L. Flanagan ◽  
Leslie A. Khawli ◽  
Peisheng Hu ◽  
Alan L. Epstein
Keyword(s):  
Fusion Protein ◽  
Nk Cells ◽  
Tumor Models ◽  
Syngeneic Tumor

Molecular modification of a recombinant anti-CD3ε-directed immunotoxin by inducing terminal cysteine bridging enhances anti-GVHD efficacy and reduces organ toxicity in a lethal murine model

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 1157-1165 ◽  
Author(s):  
Daniel A. Vallera ◽  
David W. Kuroki ◽  
Angela Panoskaltsis-Mortari ◽  
Donald J. Buchsbaum ◽  
Buck E. Rogers ◽  
...  
Keyword(s):  
Therapeutic Index ◽  
Maximum Tolerated Dose ◽  
Cysteine Residue ◽  
Genetically Engineered ◽  
Therapeutic Window ◽  
Single Chain ◽  
Molecular Modification ◽  
Organ Toxicity

Abstract Immunotoxin (IT) therapy shows potential for selectively eliminating GVHD-causing T cells in vivo, but the field has been hampered by toxicity. Previously, we showed that a genetically engineered IT consisting of a single-chain protein, including the anti-CD3sFv spliced to a portion of diphtheria-toxin (DT390) has anti-GVHD effects, but pronounced organ toxicity common to this class of agent. A recombinant DT390 anti-CD3sFv protein previously shown to have anti-GVHD activity was modified to reduce its filtration into kidney by genetically inserting a cysteine residue downstream of the sFv moiety at the c-terminus of the protein. This modification produced an intermolecular disulfide bridge, resulting in a bivalent, rather than a monovalent IT, termed SS2, that selectively inhibited T-cell proliferation in vitro. Although monomer and SS2 were similar in in vitro activity, SS2 had a superior therapeutic index in vivo with at least 8-fold more being tolerated with reduced kidney toxicity. Most importantly, in a lethal model of GVHD, 40 μg SS2 given for 1 day, protected 100% of the mice from lethal GVHD for 3 months, whereas the maximum tolerated dose (MTD) of monomer protected only 33%. To our knowledge, this is the first time disulfide bonded ITs have been created in this way and this simple molecular modification may address several problems in the IT field because it (1) markedly increased efficacy curing mice of GVHD after a single daily treatment, (2) markedly decreased organ toxicity, (3) increased the tolerated dosage, and (4) created a therapeutic window where none existed before.

Molecular modification of a recombinant anti-CD3ε-directed immunotoxin by inducing terminal cysteine bridging enhances anti-GVHD efficacy and reduces organ toxicity in a lethal murine model

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 1157-1165 ◽  
Author(s):  
Daniel A. Vallera ◽  
David W. Kuroki ◽  
Angela Panoskaltsis-Mortari ◽  
Donald J. Buchsbaum ◽  
Buck E. Rogers ◽  
...  
Keyword(s):  
Therapeutic Index ◽  
Maximum Tolerated Dose ◽  
Cysteine Residue ◽  
Genetically Engineered ◽  
Therapeutic Window ◽  
Single Chain ◽  
Molecular Modification ◽  
Organ Toxicity

Immunotoxin (IT) therapy shows potential for selectively eliminating GVHD-causing T cells in vivo, but the field has been hampered by toxicity. Previously, we showed that a genetically engineered IT consisting of a single-chain protein, including the anti-CD3sFv spliced to a portion of diphtheria-toxin (DT390) has anti-GVHD effects, but pronounced organ toxicity common to this class of agent. A recombinant DT390 anti-CD3sFv protein previously shown to have anti-GVHD activity was modified to reduce its filtration into kidney by genetically inserting a cysteine residue downstream of the sFv moiety at the c-terminus of the protein. This modification produced an intermolecular disulfide bridge, resulting in a bivalent, rather than a monovalent IT, termed SS2, that selectively inhibited T-cell proliferation in vitro. Although monomer and SS2 were similar in in vitro activity, SS2 had a superior therapeutic index in vivo with at least 8-fold more being tolerated with reduced kidney toxicity. Most importantly, in a lethal model of GVHD, 40 μg SS2 given for 1 day, protected 100% of the mice from lethal GVHD for 3 months, whereas the maximum tolerated dose (MTD) of monomer protected only 33%. To our knowledge, this is the first time disulfide bonded ITs have been created in this way and this simple molecular modification may address several problems in the IT field because it (1) markedly increased efficacy curing mice of GVHD after a single daily treatment, (2) markedly decreased organ toxicity, (3) increased the tolerated dosage, and (4) created a therapeutic window where none existed before.

Anti-Tumor Study of H6, a 4-Substituted Coumarins Derivative, Loaded Biodegradable Self-Assembly Nano-Micelles In Vitro and In Vivo

2019 ◽  
Vol 15 (7) ◽  
pp. 1515-1531 ◽  
Author(s):  
Zejiang Zhu ◽  
Zhengying Su ◽  
Jianhong Yang ◽  
Huili Liu ◽  
Minghai Tang ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Polymeric Micelles ◽  
Drug Loading ◽  
Therapeutic Window ◽  
Tumor Growth Inhibition ◽  
Mechanism Study ◽  
Tumor Models ◽  
Substituted Coumarins

In our previous study, we identified a class of 4-substituted coumarins as a powerful microtubule inhibitors binding to the colchicine site of β-tubulin. H6 showed potent anti-proliferative ability with IC50 values from 7 to 47 nM, and remarkable ability to reduce tumor growth in several xenograft models including taxol resistant tumor models. However, the extremely hydrophobicity limited its clinical application. In this study, to improve the anticancer activity and reduce the toxicity of H6, we successfully prepared MPEG-PCL with different proportions and H6-loaded polymeric micelles (H6/MPEG2kPCL2k micelles) by a simple thin-film hydration method. The prepared H6/MPEG-PCL micelles had a drug loading of 3.79 ± 0.001%, an encapsulation efficiency of 98.00 ± 0.41%, a mean particle size of 30.45 ± 0.18nm and a polydispersity index (PDI) of 0.096 ± 0.009. Computer simulation results revealed a good compatibility of H6 and MPEG2k-PCL2k copolymer. In in vitro release study and pharmacokinetic study showed H6 micelles can release H6 over an extended period. Furthermore, H6 micelles possessed comparative effect as free H6 in inhibiting cell growth, preventing cell migration, and inducing apoptosis. Mechanism study identified that H6 is a novel reversible microtubule inhibitor. In in vivo studies, H6 micelles exhibited tumor growth inhibition on two pulmonary metastatic tumor models (B16/F10 and 4T1). Importantly, H6 micelles significantly improved the solubility, reduced the toxicity, extended the half-life of drugs, and augmented the therapeutic window. All these results imply that H6 micelles have great potential for suppression of tumor metastasis.

564 Potency-reduced and extended half-life IL12 heterodimeric Fc-fusions exhibit strong anti-tumor activity with potentially improved therapeutic index compared to native IL12 agents

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A598-A598
Author(s):  
Matthew Bernett ◽  
Rajat Varma ◽  
Ke Liu ◽  
Christine Bonzon ◽  
Rumana Rashid ◽  
...  
Keyword(s):  
Nk Cells ◽  
Half Life ◽  
Single Agent ◽  
Therapeutic Index ◽  
Immune Checkpoints ◽  
Therapeutic Window ◽  
Interleukin 12 ◽  
Severe Toxicity ◽  
Anti Tumor Activity

BackgroundInterleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 may help to establish an inflammatory tumor microenvironment critical for anti-tumor responses. Preclinical studies in mice revealed that native IL12 can dramatically shrink syngeneic tumors, however clinical studies in humans resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and extend half-life.MethodsIL12 is a heterodimeric protein consisting of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity was assessed by engrafting MCF-7 cells into PBMC engrafted NSG MHC class I and II double-knockout mice and by measuring tumor volume, lymphocyte activation/proliferation, and IFNγ production over time.ResultsIL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to native IL12-Fc. Anti-tumor activity in the huPBMC-MCF7 model was achieved with potency-reduced IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells, as evidenced by upregulation of CD25. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels.ConclusionsCombined, these data indicate that potency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to small therapeutic index associated with recombinant native IL12 or IL12-Fc agents.

scholarly journals Construction of a Single-Chain Interleukin-12-Expressing Retroviral Vector and Its Application in Cytokine Gene Therapy against Experimental Coccidioidomycosis

1999 ◽  
Vol 67 (6) ◽  
pp. 2996-3001 ◽  
Author(s):  
Chengyong Jiang ◽  
D. Mitchell Magee ◽  
Rebecca A. Cox
Keyword(s):  
Gene Therapy ◽  
Retroviral Vector ◽  
Biologically Active ◽  
Interleukin 12 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Spleen Cells ◽  
J774 Cells ◽  
Ifn Γ

ABSTRACT T-cell-mediated immunity is an important determinant in protection against primary infection with Coccidioides immitis, a dimorphic fungal pathogen that causes the disease coccidioidomycosis. To determine if interleukin-12 (IL-12) gene therapy could potentiate host response against C. immitis, we constructed a single-chain cDNA encoding the p40 and p35 subunits linked by a polylinker and, using a retroviral vector, transfected J774 macrophages with the construct. The transduced J774 cells expressed IL-12 in vitro, with a mean concentration of 28,440 pg from 106 cells in 48 h as measured by an IL-12 (p75)-specific enzyme-linked immunosorbent assay. The secreted IL-12 was biologically active, as judged by its ability to induce the production of gamma interferon (IFN-γ) by spleen cells from BALB/c mice. Treatment of the highly susceptible BALB/c mouse strain with the IL-12-transduced J774 cells inhibitedC. immitis growth in tissues from mice challenged by a pulmonary route, as evidenced by 1.37-, 2.59-, and 1.22-log reductions in the number of CFU in the lungs, spleens, and livers, respectively, compared to the fungal load in mice given vector-transduced J774 cells. The protective effect of IL-12 gene therapy was accompanied by increased levels of IFN-γ in the lungs and sera of mice treated with IL-12-transduced J774 cells and the constitutive production of IFN-γ by their spleen cells cultured in vitro. These results suggest that IL-12 gene therapy could be used as adjunct therapy for coccidioidomycosis.

scholarly journals 186 Development of KSQ-001, an engineered TIL (eTIL) therapy for solid tumors through CRISPR/Cas9-mediated editing of SOCS1

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A198-A198
Author(s):  
Karrie Wong ◽  
Sharon Lin ◽  
Christopher Wrocklage ◽  
Katri Sofjan ◽  
Leila Williams ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Solid Tumor ◽  
Memory Formation ◽  
Tumor Models ◽  
Syngeneic Tumor ◽  
Genome Wide

BackgroundAdoptive cell therapy with ex vivo expanded tumor infiltrating lymphocytes (TIL) offers a potentially curative treatment for cancer. However, the immunosuppressive tumor microenvironment limits the effectiveness of TIL therapy. To address this medical need, we used our Immune-CRISPRomics® Platform to perform a series of genome-wide CRISPR/Cas9 screens to identify targets enhancing the ability of T cells to infiltrate and kill solid tumors in an in vivo setting. These screens identified SOCS1 as a top target that restrains T cell anti-tumor immunity. Based on these findings, we developed KSQ-001, an engineered TIL (eTIL) therapy created via CRISPR/Cas9-mediated editing of SOCS1 for the treatment of solid tumors.MethodsGenome-wide CRISPR/Cas9 screens were conducted in in vitro primary human T cells and TIL cultures and in in vivo primary mouse OT1 and PMEL-TCR-Tg T cells in syngeneic tumor models. The efficacy of surrogate murine KSQ-001 (mKSQ-001), in which the SOCS1 gene is inactivated by CRISPR/Cas9 in OT1 or PMEL-TCR-Tg T cells, was evaluated in both the B16-Ova and CRC-gp100 syngeneic tumor models, with memory formation and efficacy evaluated both in the presence and absence of cyclophosphamide-mediated lymphodepletion. KSQ-001 was manufactured from human TIL using SOCS1-targeting sgRNAs selected for therapeutic use based on potency and selectivity, with KSQ-001 characterized for in vitro function and in vivo anti-tumor efficacy.ResultsUpon adoptive transfer of a single dose into solid tumor-bearing hosts, mKSQ-001 was found to robustly enhance anti-tumor efficacy and eradicate tumors in 7/10 mice in the PD1-sensitive OT1/B16-Ova model and to drive responses in the PD-1 refractory PMEL/CRC-gp100 syngeneic tumor model. mKSQ-001 also showed a ten-fold increase in anti-tumor potency in vivo compared to unengineered T-cell product and established durable anti-tumor memory by persisting in the form of T central memory cells detectable at high frequency in the peripheral blood of complete responder mice. In the setting of lymphodepletion, mKSQ-001 also displayed heightened anti-tumor potency, accumulation, and memory formation in comparison to inactivation of PD-1. Importantly, human KSQ-001 displayed a transcriptional signature indicative of increased anti-tumor function, produced increased amounts of pro-inflammatory cytokines, exhibited a hypersensitivity to IL-12 signaling, and demonstrated increased anti-tumor function both in vitro and in vivo solid tumor models.ConclusionsBased on insights from our Immune-CRISPRomics® platform and demonstrated efficacy across multiple preclinical tumor models, we have developed KSQ-001, a novel eTIL therapy. These preclinical data support clinical testing of KSQ-001 in a variety of solid tumor indications.

scholarly journals 709 Exosome surface display of IL-12 results in tumor-retained pharmacology with superior potency and limited systemic exposure

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A751-A751
Author(s):  
Nuruddeen Lewis ◽  
Chang Ling Sia ◽  
Katherine Kirwin ◽  
Sonya Haupt ◽  
Gauri Mahimkar ◽  
...  
Keyword(s):  
T Cells ◽  
Antitumor Activity ◽  
Cd8 T Cells ◽  
Systemic Exposure ◽  
Intratumoral Injection ◽  
Therapeutic Window ◽  
Murine Splenocytes ◽  
Animal Care

BackgroundThe promise of Interleukin-12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL-12™, a novel, engineered-exosome therapeutic that displays functional IL-12 on the surface of an exosome.MethodsIL-12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN. Potency was assessed in vitro using human PBMCs or murine splenocytes and in vivo using mouse subcutaneous tumor models. Local versus systemic pharmacology was determined with intratumoral injection in mice and subcutaneous injection in monkeys. All studies were benchmarked against recombinant IL-12 (rIL-12).ResultsExosomes engineered to express either murine or human IL-12 had equivalent potency in vitro to rIL-12 as demonstrated by IFNγ production. Following intratumoral injection, exoIL-12 exhibited prolonged tumor retention and greater antitumor activity than rIL-12. Moreover, exoIL-12 was 100-fold more potent than rIL-12 in tumor growth inhibition. In the MC38 tumor model, complete responses were observed in 63% of mice treated with exoIL-12; in contrast, rIL-12 resulted in 0% complete responses at an equivalent IL-12 dose. This correlated with dose-dependent increases in tumor antigen-specific CD8+ T cells. Re-challenge studies of exoIL-12 in complete responder mice showed no tumor regrowth. Moreover, depletion of CD8+ T cells completely abrogated the antitumor activity of exoIL-12. Following intratumoral administration, exoIL-12 exhibited 10-fold higher intratumoral exposure than rIL-12 and prolonged IFNγ production up to 48 hr. Retained, local pharmacology of exoIL-12 was further confirmed using subcutaneous injections in non-human primates.ConclusionsThis work demonstrates that tumor-restricted pharmacology of exoIL-12 results in superior in vivo efficacy and immune memory without systemic IL-12 exposure and related toxicity. exoIL-12 is a novel cancer therapeutic candidate that has the potential to overcome key limitations of rIL-12 and thereby create a therapeutic window for this potent cytokine.Ethics ApprovalAll animals were maintained and treated at the animal care facility of Codiak Biosciences in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee (CB2017-001).

scholarly journals In Vitro and In Vivo Bioactivity of Single-Chain Interleukin-12

1999 ◽  
Vol 50 (6) ◽  
pp. 596-604 ◽  
Author(s):  
Foss ◽  
Moody ◽  
Murphy Jr ◽  
Pazmany ◽  
Zilliox ◽  
...  
Keyword(s):  
Interleukin 12 ◽  
Single Chain

scholarly journals 707 IL12 Fc-fusions engineered for reduced potency and extended half-life exhibit strong anti-tumor activity and improved therapeutic index compared to wild-type IL12 agents

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A736-A736
Author(s):  
Matthew Bernett ◽  
Ke Liu ◽  
Christine Bonzon ◽  
Michael Hackett ◽  
Katrina Bykova ◽  
...  
Keyword(s):  
Nk Cells ◽  
Therapeutic Index ◽  
Immune Checkpoints ◽  
Therapeutic Window ◽  
Interleukin 12 ◽  
Severe Toxicity ◽  
Wild Type ◽  
Anti Tumor Activity

BackgroundInterleukin-12 (IL12) is a proinflammatory cytokine that induces differentiation of Th1 cells and increased cytotoxicity of T and NK cells. Stimulation by IL12 leads to production of IFNγ and an inflammatory tumor microenvironment critical for anti-tumor responses. Studies in mice revealed IL12 can dramatically shrink syngeneic tumors, however human clinical studies resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior therapeutic index (TI) in non-human primates (NHP) by reducing receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve TI.MethodsIL12 is a heterodimer of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and IL12p40 to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity of human IL12-Fc was assessed in huPBMC-NSG-DKO and huCD34+ MCF7 xenograft models. Surrogate mouse potency-reduced IL12-Fc were evaluated in syngeneic tumor models. Tolerability and pharmacodynamic activity were assessed in NHP.ResultsAn IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling and IFNγ production in an MLR assay compared to wild-type IL12-Fc. Anti-tumor activity was achieved with potency-reduced IL12-Fc as single-agents and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Surrogate potency-reduced IL12-Fc had improved tolerability and greater selectivity of IFNγ production in tumors compared to spleen and less production of IL10 compared to wild-type IL12-Fc. In NHP, potency-reduced IL12-Fc had superior exposure with slower, more sustained accumulation of IFNγ and IP10, and a more gradual dose-dependent peak response, as well as more sustained margination of T and NK cells compared to wild-type IL12-Fc.ConclusionsPotency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to narrow TI associated with wild-type IL12 or IL12-Fc agents.

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