A "Prime and Expand" strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy.

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming ("Prime" step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 ("Expand" step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting "Prime and Expand" ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the "Prime and Expand" strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

Immunotherapeutic HCW9218 augments anti-tumor activity of chemotherapy via NK cell-mediated reduction of therapy-induced senescent cells.

Therapy induced senescence (TIS) in tumors and TIS cancer cells secrete proinflammatory senescence-associated secretory phenotype (SASP) factors. SASP factors promote TIS cancer cells to re-enter the growth cycle with stemness characteristics, resulting in chemo-resistance and disease relapse. Herein, we show that the immunotherapeutic HCW9218, comprising transforming growth factor-ß (TGF-ß) receptor II and interleukin (IL)-15/IL-15 receptor α domains, enhances metabolic and cytotoxic activities of immune cells and reduces TIS tumor cells in vivo to improve the efficacy of docetaxel and gemcitabine plus nab-paclitaxel against B16F10 melanoma and SW1990 pancreatic tumors, respectively. Mechanistically, HCW9218 treatment reduces the immunosuppressive tumor microenvironment and enhances immune cell infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immuno-depletion analysis suggests that HCW9218-activated natural killer cells play a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhances efficacy of tumor antigen-specific and anti-programmed death-ligand 1 (PD-L1) antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment decreases TIS cells and lowers SASP factors in off-target tissues caused by chemotherapy of tumor-bearing mice. Collectively, HCW9218 has the potential to significantly enhance anti-tumor efficacy of chemotherapy, therapeutic antibodies, and checkpoint blockade by eliminating TIS cancer cells while reducing TIS-mediated proinflammatory side effects in normal tissues.

The Lower Limit of Regulatory CD4+ Foxp3+ TCRß Repertoire Diversity Required To Control Autoimmunity.

The TCR repertoire of regulatory T cells (Tregs) is highly diverse. The relevance of this diversity to maintain self-tolerance remains unknown. We established a model where the TCR repertoire of normal polyclonal Tregs was limited by serial transfers into IL-2Rß-/- mice, which lack functional Tregs. After a primary transfer, the donor Treg TCR repertoire was substantially narrowed, yet the recipients remained autoimmune-free. Importantly, upon purification and transfer of donor-derived Tregs from an individual primary recipient into neonatal IL-2Rß-/- mice, the secondary recipients developed autoimmunity. In this study, the Treg TCRß repertoire was reshaped and further narrowed. In contrast, secondary IL-2Rß recipients showed fewer symptoms of autoimmunity when they received donor Tregs that were premixed from several primary recipients to increase their TCRß repertoire diversity. About 8-11% of the Treg TCRß repertoire was estimated to be the minimum required to establish and maintain tolerance in primary IL-2Rß-/- recipients. Collectively, these data quantify where limitations imposed on the Treg TCRß repertoire results in a population of Tregs that cannot fully suppress polyclonal autoreactive T cells. Our data favor a model where the high diversity of the Treg TCR provides a mechanism for Tregs to actively adapt and effectively suppress autoreactive T cells, which are not fixed, but are evolving as they encounter self-antigens.

Developmental Progression and Interrelationship of Central and Effector Regulatory T Cell Subsets.

Resting central Tregs (cTregs) and activated effector Tregs (eTregs) are required for self-tolerance, but the heterogeneity and relationships within and between phenotypically distinct subsets of cTregs and eTregs are poorly understood. By extensive immune profiling and deep sequencing of TCR-ß V regions, two subsets of cTregs, based on expression of Ly-6C, and three subsets of eTregs, based on distinctive expression of CD62L, CD69, and CD103, were identified. Ly-6C(+) cTregs exhibited lower basal activation, expressed on average lower affinity TCRs, and less efficiently developed into eTregs when compared with Ly-6C(-) cTregs. The dominant TCR Vßs of Ly-6C(+) cTregs were shared by eTregs at a low frequency. A single TCR clonotype was also identified that was largely restricted to Ly-6C(+) cTregs, even under conditions that promoted the development of eTregs. Collectively, these findings indicate that some Ly-6C(+) cTregs may persist as a lymphoid-specific subset, with minimal potential to develop into highly activated eTregs, whereas other cTregs readily develop into eTregs. In contrast, subsets of CD62L(lo) eTregs showed higher clonal expansion and were more highly interrelated than cTreg subsets based on their TCR-ß repertoires, but exhibited varied immune profiles. The CD62L(lo) CD69(-) CD103(-) eTreg subset displayed properties of a transitional intermediate between cTregs and more activated eTreg subsets. Thus, eTreg subsets appear to exhibit substantial flexibility, most likely in response to environmental cues, to adopt defined immune profiles that are expected to optimize suppression of autoreactive T cells.

IL-2Rß-dependent signaling and CD103 functionally cooperate to maintain tolerance in the gut mucosa.

A network of mechanisms operates to maintain tolerance in the gut mucosa. Although CD103 marks many lymphoid cells within the gut, its direct functional role in intestinal tolerance is poorly understood. CD103 may be part of a redundant pathway, as CD103(-/-) mice do not exhibit autoimmunity. To reduce such redundancy, CD103(-/-) mice were crossed to mice (designated Y3) whose T cells expressed a mutant IL-2Rß-chain that lowers IL-2R signaling. Unlike overtly healthy Y3 mice, all Y3/CD103(-/-) mice rapidly developed severe colitis. The large intestine of these mice contained an increase in CD4(+) Th1 and Th17 effector cells and a reduced ratio of regulatory T cells (Tregs). Importantly, colitis was effectively prevented by the transfer of wild-type Tregs into Y3/CD103(-/-) mice. Impaired intestinal tolerance was not attributed to an obvious lack of CD103-dependent gene regulation or intestinal homing/retention by Tregs nor a lack of functional activities typically associated with CD103(+) dendritic cells, such as peripherally induced Treg development or imprinting CCR9 and α4ß7 homing molecules on Tregs and T effector cells. Transcriptome analysis of Tregs was consistent with altered homeostasis due to impaired IL-2Rß-dependent signaling with minimal dysregulation added by the absence of CD103. Rather, the absence of CD103 functioned to alter the localization of the cells within the gut microenvironment that may alter Treg homeostasis. Thus, IL-2Rß-dependent signaling and CD103 normally cooperate through distinctive processes to promote Treg homeostasis and immune tolerance.

Heat shock protein vaccination and directed IL-2 therapy amplify tumor immunity rapidly following bone marrow transplantation in mice.

Tumor relapse is the primary cause of mortality in patients with hematologic cancers following autologous hematopoietic stem cell transplantation (HSCT). Vaccination early after HSCT can exploit both the state of lymphopenia and minimal residual disease for generating antitumor immunity. Here, multiple vaccinations using lymphoma cells engineered to secrete heat shock protein fusion gp96-Ig within 2 weeks of T cell-replete syngeneic HSCT led to cross-presentation and increased survival of lymphoma-bearing mice. To enhance vaccine efficacy, interleukin (IL)-2 was directed to predominantly memory phenotype CD8(+) T lymphocytes and natural killer (NK) cells via administration bound to anti-IL-2 monoclonal antibody clone S4B6 (IL-2S4B6). Combination therapy with gp96-Ig vaccination and coordinated infusions of IL-2S4B6 resulted in marked prolongation of survival, which directly correlated with ~500% increase in effector CD8(+) T-cell numbers. Notably, this dual regimen elicited large increases in both donor CD8(+) T and NK cells, but not CD4(+) T lymphocytes; the former 2 populations are essential for both vaccine efficacy and protection against opportunistic infections after HSCT. Indeed, IL-2S4B6-treated HSCT recipients infected with Listeria monocytogenes exhibited decreased bacterial levels. These preclinical studies validate a new strategy particularly well suited to the post-HSCT environment, which may augment adaptive and innate immune function in patients with malignant disease receiving autologous HSCT.

IL-2R signaling is essential for functional maturation of regulatory T cells during thymic development.

CD4(+) Foxp3(+) regulatory T cells (Tregs) are an independent cell lineage, and their developmental progression during thymic development depends on IL-2R signaling. However, the role of IL-2R signaling during thymic Treg development remains only partially understood. The current study assessed the contribution of IL-2 to the expansion and functional programming of developing Tregs. In the absence of IL-2Rß signaling, predominantly CD4(+) CD25(-) Foxp3(lo) T cells were found, and these cells exhibited somewhat lower expression of the proliferative marker Ki67. These immature Tregs, which represent products of failed development, were also found in normal mice and were characterized by markedly lower expression of several Treg functional molecules. Therefore, IL-2R is required for the progression, functional programming, and expansion of Tregs during thymic development. An IL-2R-signaling mutant that lowers STAT5 activation readily supported Treg functional programming, but Treg proliferation remained somewhat impaired. The requirement for IL-2 during thymic Treg expansion was best illustrated in mixed chimeras where the Tregs with mutant IL-2Rs were forced to compete with wild-type Tregs during their development. Tregs with impaired IL-2R signaling were more prevalent in the thymus than spleen in these competitive experiments. The general effectiveness of mutant IL-2Rs to support thymic Treg development is partially accounted for by a heightened capacity of thymic Tregs to respond to IL-2. Overall, our data support a model in which limiting IL-2R signaling is amplified by thymic Tregs to readily support their development and functional programming, whereas these same conditions are not sufficient to support peripheral Treg homeostasis.

Transient enhanced IL-2R signaling early during priming rapidly amplifies development of functional CD8+ T effector-memory cells.

Much is known concerning the cellular and molecular basis for CD8(+) T memory immune responses. Nevertheless, conditions that selectively support memory generation have remained elusive. In this study, we show that an immunization regimen that delivers TCR signals through a defined antigenic peptide, inflammatory signals through LPS, and growth and differentiation signals through the IL-2R initially favors Ag-specific CD8(+) T cells to develop rapidly and substantially into T effector-memory cells by TCR transgenic OVA-specific OT-I CD8(+) T cells. Amplified CD8(+) T memory development depends upon a critical frequency of Ag-specific T cells and direct responsiveness to IL-2. A homologous prime-boost immunization protocol with transiently enhanced IL-2R signaling in normal mice led to persistent polyclonal Ag-specific CD8(+) T cells that supported protective immunity to Listeria monocytogenes. These results identify a general approach for amplified T memory development that may be useful to optimize vaccines aimed at generating robust cell-mediated immunity.

The basis of distinctive IL-2- and IL-15-dependent signaling: weak CD122-dependent signaling favors CD8+ T central-memory cell survival but not T effector-memory cell development.

Recent work suggests that IL-2 and IL-15 induce distinctive levels of signaling through common receptor subunits and that such varied signaling directs the fate of Ag-activated CD8(+) T cells. In this study, we directly examined proximal signaling by IL-2 and IL-15 and CD8(+) T cell primary and memory responses as a consequence of varied CD122-dependent signaling. Initially, IL-2 and IL-15 induced similar p-STAT5 and p-S6 activation, but these activities were only sustained by IL-2. Transient IL-15-dependent signaling is due to limited expression of IL-15Rα. To investigate the outcome of varied CD122 signaling for CD8(+) T cell responses in vivo, OT-I T cells were used from mouse models where CD122 signals were attenuated by mutations within the cytoplasmic tail of CD122 or intrinsic survival function was provided in the absence of CD122 expression by transgenic Bcl-2. In the absence of CD122 signaling, generally normal primary response occurred, but the primed CD8(+) T cells were not maintained. In marked contrast, weak CD122 signaling supported development and survival of T central-memory (T(CM)) but not T effector-memory (T(EM)) cells. Transgenic expression of Bcl-2 in CD122(-/-) CD8(+) T cells also supported the survival and persistence of T(CM) cells but did not rescue T(EM) development. These data indicate that weak CD122 signals readily support T(CM) development largely through providing survival signals. However, stronger signals, independent of Bcl-2, are required for T(EM) development. Our findings are consistent with a model whereby low, intermediate, and high CD122 signaling support T(CM) memory survival, T(EM) programming, and terminal T effector cell differentiation, respectively.

Immunotherapeutic approach to reduce senescent cells and alleviate senescence-associated secretory phenotype in mice.

Accumulation of senescent cells (SNCs) with a senescence-associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age-related pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF-ß and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet ß cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro-inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single-nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell-cycle, and senescence-associated pathways in naturally aged mice. Long-term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence-associated diseases.

A Fusion Protein Complex that Combines IL-12, IL-15, and IL-18 Signaling to Induce Memory-Like NK Cells for Cancer Immunotherapy.

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)-grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.