Permeability Characteristics of Various Intestinal Regions of Rabbit, Dog, and Monkey

The in vitro permeability of a series of both hydrophilic and lipophilic compounds, as defined by the octanol/water partition coefficient, was measured in four segments of rabbit, monkey, and dog intestine using a side-by-side diffusion cell. A linear relationship was established for tissue resistance to hydrophilic compound diffusion in jejunum and colon among rabbit, monkey, and dog. The results suggest that rabbit jejunum is twice as permeable as monkey and dog jejunum. The colonic tissues of monkey, rabbit, and dog demonstrate similar permeabilities. Measuring the permeabilities of different tissues with compounds of similar physicochemical properties allows comparison of tissue restriction to transport. Thus, in vitro permeability measurements may be used to investigate physiological differences of various intestinal tissue segments that influence tissue permeability. Investigating the permeability of different intestinal segments from various species could allow the identification of an appropriate in vitro intestinal permeability model that will lead to the prediction of intestinal absorption in humans, eliminating the need for extensive and often misleading in vivo animal testing.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41567/1/11095_2004_Article_305334.pd

Comparison of the Permeability Characteristics of a Human Colonic Epithelial (Caco-2) Cell Line to Colon of Rabbit, Monkey, and Dog Intestine and Human Drug Absorption

The in vitro permeabilities of Caco-2 monolayers and permeabilities in tissue sections from colon of monkey, rabbit, and dog were compared using a series of compounds. The selected compounds differed in their physicochemical properties, such as octanol/water partition coefficient, water solubility, and molecular weight. Their structure included steroids, carboxylic acids, xanthins, alcohols, and polyethylene glycols. A linear permeability relationship was established between Caco-2 and colon tissue from both rabbit and monkey. The results suggest that Caco-2 is twice as permeable as rabbit and five times as permeable as monkey colon. However, no clear relationship could be established between Caco-2 monolayers and dog colon permeability. A relationship between permeability in Caco-2 monolayers and human absorption was found. The results suggest that within certain limits, permeability of Caco-2 monolayers may be used as a predictive tool to estimate human drug absorption.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41425/1/11095_2004_Article_304739.pd

451 Combining Bempegaldesleukin (CD122-preferential IL-2 pathway agonist) and NKTR-262 (TLR7/8 agonist) pairs local innate activation with systemic CD8+ T cell expansion to enhance anti-tumor immunity

Background Previously, we demonstrated that radiation therapy (RT) combined with Bempegaldesleukin (BEMPEG;NKTR-214), a first-in-class CD122-preferential IL-2 pathway agonist, led to enhanced anti-tumor efficacy through a T cell-dependent mechanism. However, we observed only modest systemic responses to BEMPEG/RT across several murine tumor models. Therefore, we explored alternative approaches to improve systemic tumor-specific immunity. We evaluated whether intratumoral NKTR-262, a polymer-modified toll-like receptor (TLR) 7/8 agonist, combined with systemic BEMPEG treatment resulted in improved tumor-specific immunity and survival compared to BEMPEG combined with RT. We hypothesized that BEMPEG/NKTR-262 immunotherapy would promote synergistic activation of local immunostimulatory innate immune responses followed by systemic adaptive immunity to significantly improve tumor regression and overall survival. Methods Tumor-bearing mice (CT26; EMT6) received BEMPEG (0.8 mg/kg; iv), RT (12 Gy x 1), and/or intratumoral NKTR-262 (0.5 mg/kg). Flow cytometry was used to evaluate CD4+ and CD8+ T cell activation status in the blood and/or tumor (7 days post-treatment) and NK cell activity in the tumor (1, 3 days post-treatment). The contribution of specific immune subsets was determined by depletion of CD4+, CD8+, or NK cells. CD8+ T cell activity was determined in vitro by tracking apoptosis in an Incucyte assay. Data are representative of 1–2 independent experiments (n=5–14/group) and statistical significance was determined by 1-way ANOVA (p-value cut-off of 0.05). Results BEMPEG/NKTR-262 resulted in significantly improved survival compared to BEMPEG/RT. BEMPEG/NKTR-262 efficacy was NK and CD8+ T cell-dependent, while BEMPEG/RT primarily relied on CD8+ T cells. Response to BEMPEG/NKTR-262 was characterized by a significant expansion of activated CD8+ T cells (GzmA+; Ki-67+; ICOS+; PD-1+) in the blood, which correlated with reduced tumor size (p\u3c0.05). In the tumor, NKTR-262/BEMPEG induced higher frequencies of GzmA+ CD8+ T cells exhibiting reduced expression of suppressive molecules (PD-1+, TIM-3+), compared to BEMPEG/RT. Indeed, CD8+ T cells isolated from BEMPEG/NKTR-262-treated tumors had greater cytolytic capacity than those from BEMPEG/RT-treated mice. CD8+ T cell expansion (blood) and activity (tumor) depended upon the initial NK response, as neither occurred in the absence of NK cells. BEMPEG/NKTR-262 uniquely induced the expansion of early and high effector NK cells. Conclusions Combining BEMPEG with NKTR-262 lead to an early and robust NK cell expansion not observed in the BEMPEG/RT combination. The improved tumor regression and survival was dependent on the NKTR-262 driven expansion of NK cells. A clinical trial of BEMPEG/NKTR-262 for patients with metastatic solid tumors is in progress (NCT03435640). http://dx.doi.org/10.1136/jitc-2020-SITC2020.045

Combining bempegaldesleukin (CD122-preferential IL-2 pathway agonist) and NKTR-262 (TLR7/8 agonist) improves systemic antitumor CD8

Background: Tumor cell death caused by radiation therapy (RT) triggers antitumor immunity in part because dying cells release adjuvant factors that amplify and sustain dendritic cell and T cell responses. We previously demonstrated that bempegaldesleukin (BEMPEG: NKTR-214, an immunostimulatory IL-2 cytokine prodrug) significantly enhanced the antitumor efficacy of RT through a T cell-dependent mechanism. Because RT can induce either immunogenic or tolerogenic cell death, depending on various factors (radiation dose, cell cycle phase), we hypothesized that providing a specific immunogenic adjuvant, like intratumoral therapy with a novel toll-like receptor (TLR) 7/8 agonist, NKTR-262, would improve systemic tumor-specific responses through the activation of local innate immunity. Therefore, we evaluated whether intratumoral NKTR-262 combined with systemic BEMPEG treatment would elicit improved tumor-specific immunity and survival compared with RT combined with BEMPEG. Methods: Tumor-bearing mice (CT26; EMT6) received BEMPEG (0.8 mg/kg; intravenously), RT (12 Gy × 1), and/or intratumoral NKTR-262 (0.5 mg/kg). Flow cytometry was used to evaluate CD4+ and CD8+ T cell responses in the blood and tumor 7 days post-treatment. The contribution of specific immune subsets was determined by depletion of CD4+, CD8+, or NK cells. CD8+ T cell cytolytic activity was determined by an in vitro CTL assay. Data are representative of 1-2 independent experiments (n=5-14/group) and statistical significance was determined by 1-way analysis of variance (ANOVA) or repeated measures ANOVA (p value cut-off of 0.05). Results: BEMPEG+NKTR-262 significantly improved survival compared with BEMPEG+RT in a CD8+ T cell-dependent manner. Response to BEMPEG+NKTR-262 was characterized by a significant expansion of activated CD8+ T cells (GzmA+; Ki-67+; ICOS+; PD-1+) in the blood, which correlated with reduced tumor size (p\u3c0.05). In the tumor, BEMPEG+NKTR-262 induced higher frequencies of GzmA+ CD8+ T cells exhibiting reduced expression of suppressive molecules (PD-1+), compared with BEMPEG+RT (p\u3c0.05). Further, BEMPEG+NKTR-262 treatment induced greater tumor-specific CD8+ T cell cytolytic function than BEMPEG+RT. Conclusions: BEMPEG+NKTR-262 therapy elicited more robust expansion of activated CD8+ T cells compared with BEMPEG+RT, suggesting that intratumoral TLR stimulation provides superior antigen presentation and costimulatory activity compared with RT. A clinical trial of BEMPEG+NKTR-262 for patients with metastatic solid tumors is in progress (NCT03435640). Keywords: Adjuvants, Immunologic; Cytokines; Immunotherapy; Lymphocytes, Tumor-Infiltrating; Radiotherapy

596 Combining Bempegaldesleukin (CD122-preferential IL-2 pathway agonist) and NKTR-262 (TLR7/8 agonist) pairs local innate activation with systemic CD8+ T cell expansion to enhance anti-tumor immunity

Background Tumor cell death caused by radiation therapy (RT) can trigger anti-tumor immune responses in part because dying cells release adjuvant factors that amplify and sustain DC and T cell responses. We previously demonstrated that bempegaldesleukin (BEMPEG:NKTR-214, a first-in-class CD122-preferential IL-2 pathway agonist), significantly enhanced the anti-tumor efficacy of RT through a T cell-dependent mechanism. Because RT can induce either immunogenic or tolerogenic cell death, depending on a multitude of factors (radiation dose, cell cycle phase, and tumor microenvironment), we hypothesized that providing a specific immunogenic adjuvant, like intratumoral NKTR-262, a novel toll-like receptor (TLR) 7/8 agonist, to the tumor site would further improve systemic tumor-specific immunity by promoting synergistic activation of local immunostimulatory innate immune responses. Therefore, we evaluated whether intratumoral NKTR-262, combined with systemic BEMPEG treatment would result in improved tumor-specific immunity and survival compared to BEMPEG combined with RT. Methods Tumor-bearing mice (CT26; EMT6) received BEMPEG (0.8 mg/kg; iv), RT (16 Gy x 1), and/or intratumoral NKTR-262 (0.5 mg/kg). Flow cytometry was used to evaluate CD4+ and CD8+ T cell activation status in the blood and tumor (7 days post-treatment). The contribution of specific immune subsets was determined by depletion of CD4+, CD8+, or NK cells. CD8+ T cell cytolytic activity was determined in vitro with an Incucyte assay. Data are representative of 1–2 independent experiments (n=5–14/group) and statistical significance was determined by 1-way ANOVA (p-value cut-off of 0.05). Results BEMPEG/NKTR-262 resulted in significantly improved survival compared to BEMPEG/RT. Both combination therapies were CD8+ T cell dependent. However, response to BEMPEG/NKTR-262 was characterized by a significant expansion of activated CD8+ T cells (GzmA+; Ki-67+; ICOS+; PD-1+) in the blood, which correlated with reduced tumor size (p\u3c0.05). In the tumor, BEMPEG/NKTR-262 induced higher frequencies of GzmA+ CD8+ T cells exhibiting reduced expression of suppressive molecules (PD-1+, TIM-3+), compared to BEMPEG/RT. Additionally, CD8+ T cells isolated from BEMPEG/NKTR-262-treated tumors had greater cytolytic capacity than those from BEMPEG/RT-treated mice. Conclusions Combining BEMPEG with NKTR-262 lead to a more robust expansion of activated CD8+ T cells compared to the BEMPEG/RT combination. Enhancement of the activated CD8+ T cell response in mice treated with NKTR-262 in combination with BEMPEG suggests that intratumoral TLR stimulation provides superior antigen presentation and costimulatory activity compared to RT. A clinical trial of BEMPEG/NKTR-262 for patients with metastatic solid tumors is in progress (NCT03435640). http://dx.doi.org/10.1136/jitc-2021-SITC2021.59

Modeling the receptor pharmacology, pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy.

Cytokines are potent immune modulating agents but are not ideal medicines in their natural form due to their short half-life and pleiotropic systemic effects. NKTR-214 is a clinical-stage biologic that comprises interleukin-2 (IL2) protein bound by multiple releasable polyethylene glycol (PEG) chains. In this highly PEG-bound form, the IL2 is inactive; therefore, NKTR-214 is a biologic prodrug. When administered in vivo, the PEG chains slowly release, creating a cascade of increasingly active IL2 protein conjugates bound by fewer PEG chains. The 1-PEG-IL2 and 2-PEG-IL2 species derived from NKTR-214 are the most active conjugated-IL2 species. Free-IL2 protein is undetectable in vivo as it is eliminated faster than formed. The PEG chains on NKTR-214 are located at the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex, IL2Rαβγ, reducing its ability to bind and activate the heterotrimer. The IL2Rαβγ complex is constitutively expressed on regulatory T cells (Tregs). Therefore, without the use of mutations, PEGylation reduces the affinity for IL2Rαβγ to a greater extent than for IL2Rβγ, the receptor complex predominant on CD8 T cells. NKTR-214 treatment in vivo favors activation of CD8 T cells over Tregs in the tumor microenvironment to provide anti-tumor efficacy in multiple syngeneic models. Mechanistic modeling based on in vitro and in vivo kinetic data provides insight into the mechanism of NKTR-214 pharmacology. The model reveals that conjugated-IL2 protein derived from NKTR-214 occupy IL-2Rβγ to a greater extent compared to free-IL2 protein. The model accurately describes the sustained in vivo signaling observed after a single dose of NKTR-214 and explains how the properties of NKTR-214 impart a unique kinetically-controlled immunological mechanism of action