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What is this summary about? This is a summary of two studies that looked at the safety and effectiveness of a potential new treatment, N-803 (Anktiva), in combination with a standard treatment bacillus Calmette-Guerin (BCG) for people with non-muscle invasive bladder cancer (NMIBC). One study was a Phase 1b study that tested increasing doses of N-803 in combination with the same dose of BCG in people with NMIBC who had never received BCG previously (BCG-naive). The other study is a Phase 2/3 study of N-803 and BCG in people with NMIBC whose cancer wasn't eliminated by BCG alone (BCGunresponsive). What happened in the studies? In the Phase 1b study, the nine participants were split into three groups of 3 participants who received a dose of 100, 200, or 400 μg N-803 along with a standard 50 mg dose of BCG. In the Phase 2/3 study, one group (cohort A) of participants with carcinoma in situ (CIS) disease and another group (cohort B) with papillary disease were treated with 400 μg N-803 plus 50 mg BCG. There was also a cohort C that received only 400 μg N-803. Treatments were delivered directly into the bladder once a week for 6 weeks in a row. What were the key takeaways? N-803 plus BCG eliminated NMIBC in all nine BCG-naive participants and the effects were long-lasting, with participants remaining NMIBC-free for a range of 8.3 to 9.2 years. As reported in 2022, cancer was eliminated in 58 of 82 (71%) participants with BCG-unresponsive CIS disease and the effect was also long-lasting. Importantly, approximately 90% of the successfully treated participants avoided surgical removal of the bladder. In cohort B participants with papillary disease, 40 of 72 (55.4%) were cancer-free 12 months after treatment. N-803 used alone was only effective in 2 of 10 participants. In both studies, the combination of N-803 and BCG was found to be associated with very few adverse events. Based on results from the Phase 2/3 study, the U.S. Food and Drug Association (FDA) approved the use of N-803 plus BCG for the treatment of BCG-unresponsive bladder CIS with or without Ta/T1 papillary disease. Clinical Trial Registration:NCT02138734 (Phase 1b study), NCT03022825 (Phase 2/3 study) Tweetable Abstract Addition of the IL-15 superagonist N-803 to BCG therapy produces a high rate of success in eliminating non-muscle invasive bladder cancer in both BCG-naive and BCG-unresponsive patients, with long-lasting effects that allow patients to avoid surgical removal of the bladder [ABSTRACT FROM AUTHOR]

Systemic agents including immune checkpoint inhibitors, antibody-drug conjugates, and targeted therapies play a critical role in the management of bladder cancer. Novel localized delivery mechanisms for existing systemic agents explore solutions to improve treatment response without compromising safety. Herein, we review the contemporary innovations in modern intravesical agents, hyperthermic drug delivery, reverse-thermal gels, nanocarriers, gene therapy, and subcutaneous therapies.

Background Natural killer (NK) cells are dysfunctional in chronic human immunodeficiency virus (HIV) infection as they are not able to clear virus. We hypothesized that an infusion of NK cells, supported by interleukin 2 (IL-2) or IL-15, could decrease virus-producing cells in the lymphatic tissues. Methods We conducted a phase 1 pilot study in 6 persons with HIV (PWH), where a single infusion of haploidentical related donor NK cells was given plus either IL-2 or N-803 (an IL-15 superagonist). Results The approach was well tolerated with no unexpected adverse events. We did not pretreat recipients with cyclophosphamide or fludarabine to "make immunologic space," reasoning that PWH on stable antiretroviral treatment remain T-cell depleted in lymphatic tissues. We found donor cells remained detectable in blood for up to 8 days (similar to what is seen in cancer pretreatment with lymphodepleting chemotherapy) and in the lymph nodes and rectum up to 28 days. There was a moderate decrease in the frequency of viral RNA-positive cells in lymph nodes. Conclusions There was a moderate decrease in HIV-producing cells in lymph nodes. Further studies are warranted to determine the impact of healthy NK cells on HIV reservoirs and if restoring NK-cell function could be part of an HIV cure strategy. Clinical Trials Registration. NCT03346499 and NCT03899480. [ABSTRACT FROM AUTHOR]

Simple Summary: Bladder cancer is a common disease in urological patients. The approach to treatment depends on the severity of the tumor; in this article, we focus on tumors that do not invade the muscle layer of the bladder. Those tumors are resected during an endoscopic procedure (TURBT), but often reoccur. Treating bladder cancer with drugs instead of surgical removal of the bladder (radical cystectomy) is paramount for patients quality of life and overall well-being. The aim of this paper is to review methods of conservative treatment of tumors unresponsive to the typical treatment of choice (BCG instillations). Bladder cancer is the 10th most popular cancer in the world, and non-muscle-invasive bladder cancer (NMIBC) is diagnosed in ~80% of all cases. Treatments for NMIBC include transurethral resection of the bladder tumor (TURBT) and intravesical instillations of Bacillus Calmette-Guérin (BCG). Treatment of BCG-unresponsive tumors is scarce and usually leads to Radical Cystectomy. In this paper, we review recent advancements in conservative treatment of BCG-unresponsive tumors. The main focus of the paper is FDA-approved medications: Pembrolizumab and Nadofaragene Firadenovec (Adstiladrin). Other, less researched therapeutic possibilities are also included, namely: N-803 immunotherapy, TAR-200 and TAR-210 intravesical delivery systems and combined Cabazitaxel, Gemcitabine and Cisplatin chemotherapy. Conservative treatment and delaying radical cystectomy would greatly benefit patients' quality of life; it is undoubtedly the future of BCG-unresponsive NMIBC. [ABSTRACT FROM AUTHOR]

In spite of the advances in antiretroviral therapy to treat HIV infection, the presence of a latent reservoir of HIV-infected cells represents the largest barrier towards finding a cure. Among the different strategies being pursued to eliminate or reduce this latent reservoir, the γc-cytokine IL-15 or its superagonist N-803 are currently under clinical investigation, either alone or with other interventions. They have been shown to reactivate latent HIV and enhance immune effector function, both of which are potentially required for effective reduction of latent reservoirs. In here, we present a comprehensive literature review of the different in vitro, ex vivo, and in vivo studies conducted to date that are aimed at targeting HIV reservoirs using IL-15 and N-803. [ABSTRACT FROM AUTHOR]

Vaccine strategies aimed at eliciting human immunodeficiency virus (HIV)-specific CD8+ T cells are one major target of interest in HIV functional cure strategies. We hypothesized that CD8+ T cells elicited by therapeutic vaccination during antiretroviral therapy (ART) would be recalled and boosted by treatment with the interleukin 15 (IL-15) superagonist N-803 after ART discontinuation. We intravenously immunized four simian immunodeficiency virus-positive (SIV1) Mauritian cynomolgus macaques receiving ART with vesicular stomatitis virus (VSV), modified vaccinia virus Ankara strain (MVA), and recombinant adenovirus serotype 5 (rAd-5) vectors all expressing SIVmac239 Gag. Immediately after ART cessation, these animals received three doses of N-803. Four control animals received no vaccines or N-803. The vaccine regimen generated a high-magnitude response involving Gag-specific CD8+ T cells that were proliferative and biased toward an effector memory phenotype. We then compared cells elicited by vaccination (Gag specific) to cells elicited by SIV infection and unaffected by vaccination (Nef specific). We found that N-803 treatment enhanced the frequencies of both bulk and proliferating antigen-specific CD8+ T cells elicited by vaccination and the antigen-specific CD8+ T cells elicited by SIV infection. In sum, we demonstrate that a therapeutic heterologous prime-boost-boost (HPBB) vaccine can elicit antigen-specific effector memory CD8+ T cells that are boosted by N-803. IMPORTANCE While antiretroviral therapy (ART) can suppress HIV replication, it is not a cure. It is therefore essential to develop therapeutic strategies to enhance the immune system to better become activated and recognize virus-infected cells. Here, we evaluated a novel therapeutic vaccination strategy delivered to SIV+ Mauritian cynomolgus macaques receiving ART. ART was then discontinued and we delivered an immunotherapeutic agent (N-803) after ART withdrawal with the goal of eliciting and boosting anti-SIV cellular immunity. Immunologic and virologic analysis of peripheral blood and lymph nodes collected from these animals revealed transient boosts in the frequency, activation, proliferation, and memory phenotype of CD4+ and CD8+ T cells following each intervention. Overall, these results are important in educating the field of the transient nature of the immunological responses to this particular therapeutic regimen and the similar effects of N-803 on boosting T cells elicited by vaccination or elicited naturally by infection. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are cytotoxic innate lymphoid cells that are emerging as a cellular immunotherapy for various malignancies. NK cells are particularly dependent on interleukin (IL)-15 for their survival, proliferation, and cytotoxic function. NK cells differentiate into memory-like cells with enhanced effector function after a brief activation with IL-12, IL-15, and IL-18. N-803 is an IL-15 superagonist composed of an IL-15 mutant (IL-15N72D) bound to the sushi domain of IL-15Rα fused to the Fc region of IgG1, which results in physiological trans-presentation of IL-15. Here, we describe the creation of a novel triple-cytokine fusion molecule, 18/12/TxM, using the N-803 scaffold fused to IL-18 via the IL-15N72D domain and linked to a heteromeric single-chain IL-12 p70 by the sushi domain of the IL-15Rα. This molecule displays trispecific cytokine activity through its binding and signaling through the individual cytokine receptors. Compared with activation with the individual cytokines, 18/12/TxM induces similar short-term activation and memory-like differentiation of NK cells on both the transcriptional and protein level and identical in vitro and in vivo anti-tumor activity. Thus, N-803 can be modified as a functional scaffold for the creation of cytokine immunotherapies with multiple receptor specificities to activate NK cells for adoptive cellular therapy.

In spite of the advances in antiretroviral therapy to treat HIV infection, the presence of a latent reservoir of HIV-infected cells represents the largest barrier towards finding a cure. Among the different strategies being pursued to eliminate or reduce this latent reservoir, the γc-cytokine IL-15 or its superagonist N-803 are currently under clinical investigation, either alone or with other interventions. They have been shown to reactivate latent HIV and enhance immune effector function, both of which are potentially required for effective reduction of latent reservoirs. In here, we present a comprehensive literature review of the different in vitro, ex vivo, and in vivo studies conducted to date that are aimed at targeting HIV reservoirs using IL-15 and N-803.

The IL-15 superagonist N-803 has been shown to enhance the function of CD8 T cells and NK cells. We previously found that in a subset of vaccinated, ART-naive, SIV1 rhesus macaques, N-803 treatment led to a rapid but transient decline in plasma viremia that positively correlated with an increase in the frequency of CD8 T cells. Here, we tested the hypothesis that prophylactic vaccination was required for the N-803 mediated suppression of SIV plasma viremia. We either vaccinated rhesus macaques with a DNA prime/Ad5 boost regimen using vectors expressing SIVmac239 gag with or without a plasmid expressing IL-12 or left them unvaccinated. The animals were then intravenously infected with SIVmac239M. 6 months after infection, the animals were treated with N-803. We found no differences in the control of plasma viremia during N-803 treatment between vaccinated and unvaccinated macaques. Interestingly, when we divided the SIV1 animals based on their plasma viral load set-points prior to the N-803 treatment, N-803 increased the frequency of SIV-specific T cells expressing ki-671 and granzyme B1 in animals with low plasma viremia (,104 copies/mL; SIV controllers) compared to animals with high plasma viremia (.104 copies/mL; SIV noncontrollers). In addition, Gag-specific CD8 T cells from the SIV1 controllers had a greater increase in CD81CD107a1 T cells in ex vivo functional assays than did the SIV1 noncontrollers. Overall, our results indicate that N-803 is most effective in SIV1 animals with a preexisting immunological ability to control SIV replication. IMPORTANCE N-803 is a drug that boosts the immune cells involved in combating HIV/SIV infection. Here, we found that in SIV1 rhesus macaques that were not on antiretroviral therapy, N-803 increased the proliferation and potential capacity for killing of the SIV-specific immune cells to a greater degree in animals that spontaneously controlled SIV than in animals that did not control SIV. Understanding the mechanism of how N-803 might function differently in individuals that control HIV/SIV (for example, individuals on antiretroviral therapy or spontaneous controllers) compared to settings where HIV/SIV are not controlled, could impact the efficacy of N-803 utilization in the field of HIV cure. [ABSTRACT FROM AUTHOR]

Multimodal temporal therapy orchestrated to leverage immunotherapy, tumor-targeted chemotherapy, and natural killer (NK) cell therapy may provide an opportunity to induce immunogenic cell death for tumor response and increased survival in patients with recurrent cancer. The interleukin-15 (IL-15) superagonist N-803, an enhancer of NK cells, CD4 + T cells, cytotoxic CD8 + T cells, and memory T-cell activity, combined with off-the-shelf PD-L1-targeted high-affinity NK (PD-L1 t-haNK) cells represent novel immunotherapies designed to overcome an immunosuppressive tumor microenvironment (TME). The epidermal growth factor receptor-targeted antibody-nanocell conjugate E-EDV-D682 provides tumor-targeted chemotherapy in the form of its anthracycline metabolite PNU159682 (nemorubicin) cargo and is currently being studied in combination with immunomodulatory EDVs delivering the adjuvant α-galactosyl ceramide (GC). Here, we report the compassionate use treatment of this combination in a patient with recurrent, metastatic pancreatic cancer (mPC) after 3 lines of therapy. Under the initial single-patient Investigational New Drug (spIND) protocol, the patient received N-803, PD-L1 t-haNK cells, and the albumin doxorubicin conjugate aldoxorubicin for ~27 months. The patient's disease became stable on this regimen, and a transient complete response was observed by ~14 months of therapy. Due to progression, a second spIND protocol was designed whereby the patient received E-EDV-D682 plus EDV-GC for more than 24 months, which resulted in stable disease and the patient's continued survival at the time this report was written. The patient's extended survival despite the dire prognosis associated with recurrent mPC points to the merits of this temporal combination regimen in overcoming immuno-chemo resistance with enhanced immune activity required for tumor response and extended survival., (© The Author(s) 2024. Published by Oxford University Press.)

Maintenance therapy may improve natural killer (NK) cell surveillance after allogeneic donor hematopoietic cell transplant (HCT) for myeloid malignancies and represents a potential approach to improve cure rates. Interleukin-15 (IL-15) enhances lymphocyte proliferation and antitumor activity. In a prior Phase 1 study of an IL-15 superagonist (N-803) in patients with AML who relapsed after HCT, we observed in vivo expansion of NK cells and antitumor responses. The primary objective of this Phase 2 trial was to determine if post-transplant N-803 could reduce relapse. We administered N-803 (n = 20) (dosed 6 mcg/kg subcutaneously [SQ] at day 60 after HCT to patients with myelodysplastic syndrome [MDS] or acute myeloid leukemia [AML] who were in complete remission [CR]). N-803 treatment was planned weekly, biweekly or every 4 weeks in 2 sequential cohorts. The most common adverse events after administration were self-limited injection sites skin rashes (n = 20). One week after an N-803 dose, we observed enhanced NK cell proliferation and improved antitumor cytotoxicity without inducing immune exhaustion. Five patients who developed acute graft versus host disease (aGVHD) after N-803 responded promptly to steroids and 4 patients developed chronic GVHD. Patients receiving >4 doses of N-803 had a 3-fold decrease in relapse at two years (P = .06). These findings support the safety, immune activation, and potential efficacy of N-803 to prevent relapse of AML/MDS after HSCT., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Introduction: Bladder cancer remains a prevalent and often lethal cancer. Fortunately, recent clinical trials using immunotherapeutics demonstrate promise to improve treatment outcomes. Several of these immunotherapies have already established their value in the metastatic space and likely will soon have indications in the non-metastatic space. Areas covered: This review will cover immunotherapies ranging for well-studied BCG administration to more novel medications such as PD-L1/PD-1 inhibitors, CTLA-4 inhibitors, IL-15 super agonists, and immune modulating gene therapies. Specifically, this article will address the mechanisms of actions, clinical evidence supporting their use, and the presence of any FDA regulatory approval for these medications in the treatment of high-risk non-muscle invasive bladder cancer. Expert opinion: With the publication of clinical data supporting the use of immunotherapies, these novel medications are likely to be adopted for treatment of high-grade non-metastatic bladder cancer. While BCG is likely to remain a primary medication for high-grade bladder cancer for the near future, BCG will likely be co-administered with immunomodulatory medications in some patients to enhance the medications effect in the future. Clinical trials are still ongoing and will demonstrate which of these multiple treatment options yield results worthy of a modification in our current treatment paradigm. [ABSTRACT FROM AUTHOR]

Abstract Background Immunotherapy targeting PD-1/PD-L1 fails to induce clinical responses in most patients with solid cancers. N-803, formerly ALT-803, is an IL-15 superagonist mutant and dimeric IL-15RαSushi-Fc fusion protein complex that enhances CD8+ T and NK cell expansion and function and exhibits anti-tumor efficacy in preclinical models. Previous in vitro studies have shown that IL-15 increases PD-L1 expression, a negative regulator of CD8+ T and NK cell function. Most reported preclinical studies administered N-803 intraperitoneally not subcutaneously, the current clinical route of administration. N-803 is now being evaluated clinically in combination with PD-1/PD-L1 inhibitors. However, the mechanism of action has not been fully elucidated. Here, we examined the anti­tumor efficacy and immunomodulatory effects of combining N-803 with an anti-PD-L1 antibody in preclinical models of solid carcinomas refractory to anti-PD-L1 or N-803. Methods Subcutaneous N-803 and an anti-PD-L1 monoclonal antibody were administered as monotherapy or in combination to 4T1 triple negative breast and MC38-CEA colon tumor-bearing mice. Anti-tumor efficacy was evaluated, and a comprehensive analysis of the immune-mediated effects of each therapy was performed on the primary tumor, lung as a site of metastasis, and spleen. Results We demonstrate that N-803 treatment increased PD-L1 expression on immune cells in vivo, supporting the combination of N-803 and anti-PD-L1. N-803 plus anti-PD-L1 was well-tolerated, reduced 4T1 lung metastasis and MC38-CEA tumor burden, and increased survival as compared to N-803 and anti-PD-L1 monotherapies. Efficacy of the combination therapy was dependent on both CD8+ T and NK cells and was associated with increased numbers of these activated immune cells in the lung and spleen. Most alterations to NK and CD8+ T cell phenotype and number were driven by N-803. However, the addition of anti-PD-L1 to N-803 significantly enhanced CD8+ T cell effector function versus N-803 and anti-PD-L1 monotherapies, as indicated by increased Granzyme B and IFNγ production, at the site of metastasis and in the periphery. Increased CD8+ T cell effector function correlated with higher serum IFNγ levels, without related toxicities, and enhanced anti-tumor efficacy of the N-803 plus anti-PD-L1 combination versus either monotherapy. Conclusions We provide novel insight into the mechanism of action of N-803 plus anti-PD-L1 combination and offer preclinical proof of concept supporting clinical use of N-803 in combination with checkpoint inhibitors, including for patients non- and/or minimally responsive to either monotherapy.

Allogeneic natural killer (NK) cell transfer is a potential immunotherapy to eliminate and control cancer. A promising source are CD34 + hematopoietic progenitor cells (HPCs), since large numbers of cytotoxic NK cells can be generated. Effective boosting of NK cell function can be achieved by interleukin (IL)-15. However, its in vivo half-life is short and potent trans-presentation by IL-15 receptor α (IL-15Rα) is absent. Therefore, ImmunityBio developed IL-15 superagonist N-803, which combines IL-15 with an activating mutation, an IL-15Rα sushi domain for trans-presentation, and IgG1-Fc for increased half-life. Here, we investigated whether and how N-803 improves HPC-NK cell functionality in leukemia and ovarian cancer (OC) models in vitro and in vivo in OC-bearing immunodeficient mice. We used flow cytometry-based assays, enzyme-linked immunosorbent assay, microscopy-based serial killing assays, and bioluminescence imaging, for in vitro and in vivo experiments. N-803 increased HPC-NK cell proliferation and interferon (IFN)γ production. On leukemia cells, co-culture with HPC-NK cells and N-803 increased ICAM-1 expression. Furthermore, N-803 improved HPC-NK cell-mediated (serial) leukemia killing. Treating OC spheroids with HPC-NK cells and N-803 increased IFNγ-induced CXCL10 secretion, and target killing after prolonged exposure. In immunodeficient mice bearing human OC, N-803 supported HPC-NK cell persistence in combination with total human immunoglobulins to prevent Fc-mediated HPC-NK cell depletion. Moreover, this combination treatment decreased tumor growth. In conclusion, N-803 is a promising IL-15-based compound that boosts HPC-NK cell expansion and functionality in vitro and in vivo. Adding N-803 to HPC-NK cell therapy could improve cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Introduction: In the phase 2/3 study QUILT-3.032 (NCT03022825), the ability of the IL-15RαFc superagonist N-803 (nogapendekin alfa inbakicept) plus bacillus Calmette-Guérin (BCG) to elicit durable complete responses in patients with BCG-unresponsive nonmuscle-invasive bladder cancer (NMIBC) was demonstrated. As a secondary end point, patient-reported outcomes (PROs) were assessed., Methods: Both cohort A patients with carcinoma in situ with or without Ta/T1 disease and cohort B patients with high-grade Ta/T1 papillary disease who received N-803 plus BCG therapy completed the EORTC (European Organization for Research and Treatment of Cancer) Core 30 and Quality of Life NMIBC-Specific 24 questionnaires at baseline and months 6, 12, 18, and 24 on study. Scores were analyzed using descriptive statistics, and multivariable analyses were performed to identify baseline variables associated with PROs., Results: On study, mean physical function (PF) and global health (GH) scores remained relatively stable from baseline for cohorts A (n = 86) and B (n = 78). At month 6, cohort A patients with a complete response reported higher PF scores than those without ( P = .0659); at month 12, > 3 as compared with ≤ 3 prior transurethral resections of bladder tumor was associated ( P = .0729) with lower GH scores. In cohort B, baseline disease type was associated ( P = .0738) with PF and race was significantly associated ( P = .0478) with GH at month 6. NMIBC-Specific 24 summary scores also remained stable on study for both cohorts., Conclusions: The overall stability of PROs scores, taken together with the efficacy findings, indicates a favorable risk-benefit ratio and quality of life following N-803 plus BCG.

Background: Immunotherapy targeting PD-1/PD-L1 fails to induce clinical responses in most patients with solid cancers. N-803, formerly ALT-803, is an IL-15 superagonist mutant and dimeric IL-15RαSushi-Fc fusion protein complex that enhances CD8+ T and NK cell expansion and function and exhibits anti-tumor efficacy in preclinical models. Previous in vitro studies have shown that IL-15 increases PD-L1 expression, a negative regulator of CD8+ T and NK cell function. Most reported preclinical studies administered N-803 intraperitoneally not subcutaneously, the current clinical route of administration. N-803 is now being evaluated clinically in combination with PD-1/PD-L1 inhibitors. However, the mechanism of action has not been fully elucidated. Here, we examined the antitumor efficacy and immunomodulatory effects of combining N-803 with an anti-PD-L1 antibody in preclinical models of solid carcinomas refractory to anti-PD-L1 or N-803. Methods: Subcutaneous N-803 and an anti-PD-L1 monoclonal antibody were administered as monotherapy or in combination to 4T1 triple negative breast and MC38-CEA colon tumor-bearing mice. Anti-tumor efficacy was evaluated, and a comprehensive analysis of the immune-mediated effects of each therapy was performed on the primary tumor, lung as a site of metastasis, and spleen. Results: We demonstrate that N-803 treatment increased PD-L1 expression on immune cells in vivo, supporting the combination of N-803 and anti-PD-L1. N-803 plus anti-PD-L1 was well-tolerated, reduced 4T1 lung metastasis and MC38-CEA tumor burden, and increased survival as compared to N-803 and anti-PD-L1 monotherapies. Efficacy of the combination therapy was dependent on both CD8+ T and NK cells and was associated with increased numbers of these activated immune cells in the lung and spleen. Most alterations to NK and CD8+ T cell phenotype and number were driven by N-803. However, the addition of anti-PD-L1 to N-803 significantly enhanced CD8+ T cell effector function versus N-803 and anti-PD-L1 monotherapies, as indicated by increased Granzyme B and IFNγ production, at the site of metastasis and in the periphery. Increased CD8+ T cell effector function correlated with higher serum IFNγ levels, without related toxicities, and enhanced anti-tumor efficacy of the N-803 plus anti-PD-L1 combination versus either monotherapy. Conclusions: We provide novel insight into the mechanism of action of N-803 plus anti-PD-L1 combination and offer preclinical proof of concept supporting clinical use of N-803 in combination with checkpoint inhibitors, including for patients non- and/or minimally responsive to either monotherapy. [ABSTRACT FROM AUTHOR]

IL-15, a proinflammatory cytokine critical for the generation, maintenance, and homeostasis of T cell responses, is produced naturally in response to HIV/SIV infection, but has also demonstrated therapeutic potential. IL-15 can boost CD4+ and CD8+ T cell and NK cell proliferation, activation, and function. However, IL-15 treatment may cause aberrant immune activation and accelerated disease progression in certain circumstances. Moreover, the relationship between the timing of IL-15 administration and disease progression remains unclear. The IL-15 superagonist N-803 was developed to expand the therapeutic potential of IL-15 by maximizing its tissue distribution and half-life. N-803 has garnered enthusiasm recently as a way to enhance the innate and cellular immune responses to HIV/SIV by improving CD8+ T cell recognition and killing of virus-infected cells and directing immune cells to mucosal sites and lymph nodes, the primary sites of virus replication. N-803 has also been evaluated in “shock and kill” strategies due to its potential to reverse latency (shock) and enhance antiviral immunity (kill). This review examines the current literature about the effects of IL-15 and N-803 on innate and cellular immunity, viral burden, and latency reversal in the context of HIV/SIV, and their therapeutic potential both alone and combined with additional interventions such as antiretroviral therapy (ART) and vaccination.

Here we describe a novel bifunctional fusion protein, designated N-809. This molecule comprises the IL-15/IL15Rα superagonist complex containing the Fc-domain of IgG1 (N-803, formerly designated as ALT-803) fused to two single chain anti-PD-L1 domains. The fully human IgG1 portion of the N-809 molecule was designed to potentially mediate antibody dependent cellular cytotoxicity (ADCC). The studies reported here show that N-809 has the same ability to bind PD-L1 as an anti-PD-L1 monoclonal antibody. RNAseq studies show the ability of N-809 to alter the expression of an array of genes of both CD4+ and CD8+ human T cells, and to enhance their proliferation; CD8+ T cells exposed to N-809 also have enhanced ability to lyse human tumor cells. An array of genes was differentially expressed in human natural killer (NK) cells following N-809 treatment, and there was increased expression of several surface activating receptors; there was, however, no increase in the expression of inhibitory receptors known to be upregulated in exhausted NK cells. N-809 also increased the cytotoxic potential of NK cells, as shown by increased expression of granzyme B and perforin. The lysis of several tumor cell types was increased when either NK cells or tumor cells were exposed to N-809. Similarly, the highest level of ADCC was seen when both NK cells (from donors or cancer patients) and tumor cells were exposed to N-809. These studies thus demonstrate the multi-functionality of this novel agent.

Introduction: SCLC is a highly aggressive tumor with a 5-year survival rate of less than 6%. A heterogeneous disease, SCLC is classified into four subtypes that include tumors with neuroendocrine and non-neuroendocrine features. Immune checkpoint blockade has been recently added for the frontline treatment of SCLC; however, this therapy has only led to modest clinical improvements. The lack of clinical benefit in a cancer type known to have a high tumor mutational burden has been attributed to poor T-cell infiltration and low expression of MHC-class I in most SCLC tumors. In an attempt to devise a more effective immunotherapeutic regimen, this study investigated an alternate approach on the basis of the use of the clinical-stage interleukin-15 superagonist, N-803., Methods: Preclinical models of SCLC spanning all molecular subtypes were used to evaluate the susceptibility of SCLC to natural killer (NK)-mediated lysis in vitro, including NK cells activated by N-803. Antitumor activity of N-803 was evaluated in vivo with a xenograft model of SCLC., Results: In vitro and in vivo data revealed differences in susceptibility of SCLC subtypes to lysis by NK cells and that NK cells activated by N-803 effectively lyse SCLC tumor cells across all variant subtypes, regardless of their expression of MHC-class I., Conclusions: These findings highlight the potential of a novel immune-based intervention using a cytokine-based therapeutic option for the treatment of SCLC. We hypothesize that N-803 may provide benefit to most patients with SCLC, including those with immunologically cold tumors lacking MHC expression., (Published by Elsevier Inc.)

Mucosal associated invariant T (MAIT) cells are innate T cells that recognize bacterial metabolites and secrete cytokines and cytolytic enzymes to destroy infected target cells. This makes MAIT cells promising targets for immunotherapy to combat bacterial infections. Here, we analyzed the effects of an immunotherapeutic agent, the IL-15 superagonist N-803, on MAIT cell activation, trafficking, and cytolytic function in macaques. We found that N-803 could activate MAIT cells in vitro and increase their ability to produce IFN-γ in response to bacterial stimulation. To expand upon this, we examined the phenotypes and functions of MAIT cells present in samples collected from PBMC, airways (bronchoalveolar lavage [BAL] fluid), and lymph nodes (LN) from rhesus macaques that were treated in vivo with N-803. N-803 treatment led to a transient 6 to 7-fold decrease in the total number of MAIT cells in the peripheral blood, relative to pre N-803 time points. Concurrent with the decrease in cells in the peripheral blood, we observed a rapid decline in the frequency of CXCR3 + CCR6 + MAITs. This corresponded with an increase in the frequency of CCR6 + MAITs in the BAL fluid, and higher frequencies of ki-67 + and granzyme B + MAITs in the blood, LN, and BAL fluid. Finally, N-803 improved the ability of MAIT cells collected from PBMC and airways to produce IFN-γ in response to bacterial stimulation. Overall, N-803 shows the potential to transiently alter the phenotypes and functions of MAIT cells, which could be combined with other strategies to combat bacterial infections.

IL-15, a proinflammatory cytokine critical for the generation, maintenance, and homeostasis of T cell responses, is produced naturally in response to HIV/SIV infection, but has also demonstrated therapeutic potential. IL-15 can boost CD4+ and CD8+ T cell and NK cell proliferation, activation, and function. However, IL-15 treatment may cause aberrant immune activation and accelerated disease progression in certain circumstances. Moreover, the relationship between the timing of IL-15 administration and disease progression remains unclear. The IL-15 superagonist N-803 was developed to expand the therapeutic potential of IL-15 by maximizing its tissue distribution and half-life. N-803 has garnered enthusiasm recently as a way to enhance the innate and cellular immune responses to HIV/SIV by improving CD8+ T cell recognition and killing of virus-infected cells and directing immune cells to mucosal sites and lymph nodes, the primary sites of virus replication. N-803 has also been evaluated in "shock and kill" strategies due to its potential to reverse latency (shock) and enhance antiviral immunity (kill). This review examines the current literature about the effects of IL-15 and N-803 on innate and cellular immunity, viral burden, and latency reversal in the context of HIV/SIV, and their therapeutic potential both alone and combined with additional interventions such as antiretroviral therapy (ART) and vaccination. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are cytotoxic innate lymphoid cells that are emerging as a cellular immunotherapy for various malignancies. NK cells are particularly dependent on interleukin (IL)-15 for their survival, proliferation, and cytotoxic function. NK cells differentiate into memory-like cells with enhanced effector function after a brief activation with IL-12, IL-15, and IL-18. N-803 is an IL-15 superagonist composed of an IL-15 mutant (IL-15N72D) bound to the sushi domain of IL-15Rα fused to the Fc region of IgG1, which results in physiological trans-presentation of IL-15. Here, we describe the creation of a novel triple-cytokine fusion molecule, 18/12/TxM, using the N-803 scaffold fused to IL-18 via the IL-15N72D domain and linked to a heteromeric single-chain IL-12 p70 by the sushi domain of the IL-15Rα. This molecule displays trispecific cytokine activity through its binding and signaling through the individual cytokine receptors. Compared with activation with the individual cytokines, 18/12/TxM induces similar short-term activation and memory-like differentiation of NK cells on both the transcriptional and protein level and identical in vitro and in vivo anti-tumor activity. Thus, N-803 can be modified as a functional scaffold for the creation of cytokine immunotherapies with multiple receptor specificities to activate NK cells for adoptive cellular therapy., Competing Interests: This study was funded in part by ImmunityBio. M.M.B.-E. and T.A.F. consult for Wugen (equity) and are inventors of technology that Washington University has licensed to Wugen. T.A.F. has received research support from ImmunityBio, Compass Therapeutics, HCW Biologics, Wugen and advises Kiadis, Nkarta, Indapta, and Orca Biosystems., (© 2022 The Authors.)

The "shock and kill" strategy predicates that virus reactivation in latently infected cells is required to eliminate the human immunodeficiency virus (HIV) reservoir. In a recent study, we showed robust and persistent induction of plasma viremia in antiretroviral therapy (ART)-treated simian immunodeficiency virus-infected rhesus macaques (RMs) undergoing CD8α depletion and treated with the interleukin-15 (IL-15) superagonist N-803 (J. B. McBrien et al., Nature 578:154-159, 2020, https://doi.org/10.1038/s41586-020-1946-0). Of note, in that study we used an antibody targeting CD8α, thereby depleting NK cells, NKT cells, and γδ T cells, in addition to CD8 + T cells. In the current proof-of-concept study, we tested whether virus reactivation can be induced by administration of N-803 to simian-human chimeric immunodeficiency virus-infected, ART-treated RMs that are selectively depleted of CD8 + T cells via the CD8β-targeting antibody CD8b255R1. CD8β depletion was performed in five SHIV SF162P3 -infected RMs treated with ART for 12 months and with plasma viremia consistently below 3 copies/ml. All animals received four weekly doses of N-803 starting at the time of CD8b255R1 administration. The induction of detectable plasma viremia was observed in three out of five RMs, with the level of virus reactivation seemingly correlated with the frequency of CD8 + T cells following CD8β depletion as well as the level of virus reactivation observed when the same animals underwent CD8α depletion and N-803 administration after 24 weeks of ART. These data indicate that CD8β depletion and N-803 administration can induce virus reactivation in SHIV SF162P3 -infected RMs despite suboptimal depletion of CD8 + T cells and profound ART-induced suppression of virus replication, confirming a critical role for these cells in suppressing virus production and/or reactivation in vivo under ART. IMPORTANCE The "shock and kill" HIV cure strategy attempts to reverse and eliminate the latent viral infection that prevents eradication of the virus. Latency-reversing agents tested in clinical trials to date have failed to affect the HIV viral reservoir. IL-15 superagonist N-803, currently involved in a clinical trial for HIV cure, was recently shown by our laboratory to induce robust and persistent induction of plasma viremia during ART in three in vivo animal models of HIV infection. These results suggest a substantial role for CD8 + lymphocytes in suppressing the latency reversal effect of N-803 by promoting the maintenance of viral latency. In this study, we tested whether the use of a CD8β-targeting antibody, which would specifically deplete CD8 + T cells, would yield similar levels of virus reactivation. We observed the induction of plasma viremia, which correlated with the efficacy of the CD8 depletion strategy., (Copyright © 2020 McBrien et al.)

Here we describe a novel bifunctional fusion protein, designated N-809. This molecule comprises the IL-15/IL15Rα superagonist complex containing the Fc-domain of IgG1 (N-803, formerly designated as ALT-803) fused to two single chain anti-PD-L1 domains. The fully human IgG1 portion of the N-809 molecule was designed to potentially mediate antibody dependent cellular cytotoxicity (ADCC). The studies reported here show that N-809 has the same ability to bind PD-L1 as an anti-PD-L1 monoclonal antibody. RNAseq studies show the ability of N-809 to alter the expression of an array of genes of both CD4+ and CD8+ human T cells, and to enhance their proliferation; CD8+ T cells exposed to N-809 also have enhanced ability to lyse human tumor cells. An array of genes was differentially expressed in human natural killer (NK) cells following N-809 treatment, and there was increased expression of several surface activating receptors; there was, however, no increase in the expression of inhibitory receptors known to be upregulated in exhausted NK cells. N-809 also increased the cytotoxic potential of NK cells, as shown by increased expression of granzyme B and perforin. The lysis of several tumor cell types was increased when either NK cells or tumor cells were exposed to N-809. Similarly, the highest level of ADCC was seen when both NK cells (from donors or cancer patients) and tumor cells were exposed to N-809. These studies thus demonstrate the multi-functionality of this novel agent. [ABSTRACT FROM AUTHOR]

Here we describe a novel bifunctional fusion protein, designated N-809. This molecule comprises the IL-15/IL15Rα superagonist complex containing the Fc-domain of IgG1 (N-803, formerly designated as ALT-803) fused to two single chain anti-PD-L1 domains. The fully human IgG1 portion of the N-809 molecule was designed to potentially mediate antibody dependent cellular cytotoxicity (ADCC). The studies reported here show that N-809 has the same ability to bind PD-L1 as an anti-PD-L1 monoclonal antibody. RNAseq studies show the ability of N-809 to alter the expression of an array of genes of both CD4 + and CD8 + human T cells, and to enhance their proliferation; CD8 + T cells exposed to N-809 also have enhanced ability to lyse human tumor cells. An array of genes was differentially expressed in human natural killer (NK) cells following N-809 treatment, and there was increased expression of several surface activating receptors; there was, however, no increase in the expression of inhibitory receptors known to be upregulated in exhausted NK cells. N-809 also increased the cytotoxic potential of NK cells, as shown by increased expression of granzyme B and perforin. The lysis of several tumor cell types was increased when either NK cells or tumor cells were exposed to N-809. Similarly, the highest level of ADCC was seen when both NK cells (from donors or cancer patients) and tumor cells were exposed to N-809. These studies thus demonstrate the multi-functionality of this novel agent.