The efficacy of chimeric antigen receptor (CAR) T cell therapy in solid tumors is limited by immunosuppression and antigen heterogeneity. To overcome these barriers, “armored” CAR T cells, which ...secrete proinflammatory cytokines, have been developed. However, their clinical application has been limited due to toxicities related to peripheral expression of the armoring transgene. For instance, studies that have utilized the NFAT-inducible promoter system, which links transgene expression to T cell activation, have indicated detectable levels of transgene product in the periphery. Here, we developed a novel CRISPR knock-in strategy that leverages the regulatory mechanisms of endogenous genes to drive transgene expression in a tumor-localized manner.
CRISPR knock-in was performed in both murine and human CAR T cells targeting the Her2 or Lewis Y antigens with high efficiency (>70%). Approaches were tested in syngeneic and xenograft models of CAR T cell therapy and using patient derived CAR T cells. Efficacy was determined through analysis of tumor burden and analysis of host anti-tumor immune responses (e.g. TCR-Seq).
Through a genome-wide screen for genes with increased transcriptional activity in the tumor relative to the spleen, we identify NR4A2 and RGS16 as promising promoters to support tumor-localized delivery of proinflammatory factors. Thus, expression of IL-12 or IL-2 via the NR4A2 or RGS16 promoters, respectively, led to robust therapeutic responses and tumor eradcation in 3 syngeneic models, including in "cold" tumors that are refractory to standard CAR T. This was associated with robust activation of host anti-tumor immunity. Importantly, this approach demonstrated no toxicity in contrast to NFAT-IL-12 engineered T cells. Additionally, we validate the clinical applicability of our approach in T cells derived from patients undergoing CAR T cell therapy.
Use of CRISPR/HDR to regulate transgene expression to the tumor site was demonstrated to achieve a superior therapeutic and safety profile to analogous technologies. This technology has broad scope given that it can be applied to any transgene of interest, enabling the development of armored CAR T cells that can simultaneously overcome multiple challenges posed by solid tumors. This work represents a major advance to the field as the ability to restrict transgene expression to the tumor site has been a thus far insurmountable challenge.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Allogeneic hematopoietic cell transplantation (allo-HCT) is performed with curative intent for high- risk blood cancers and bone marrow failure syndromes; yet the development of acute and chronic ...graft-vs.-host disease (GVHD) remain preeminent causes of death and morbidity. The IL-12 family of cytokines is comprised of IL-12, IL-23, IL-27, IL-35, and IL-39. This family of cytokines is biologically distinct in that they are composed of functional heterodimers, which bind to cognate heterodimeric receptor chains expressed on T cells. Of these, IL-12 and IL-23 share a common β cytokine subunit, p40, as well as a receptor chain: IL-12Rβ1. IL-12 and IL-23 have been documented as proinflammatory mediators of GVHD, responsible for T helper 1 (Th1) differentiation and T helper 17 (Th17) stabilization, respectively. The role of IL-27 is less defined, seemingly immune suppressive via IL-10 secretion by Type 1 regulatory (Tr1) cells yet promoting inflammation through impairing CD4
T regulatory (Treg) development and/or enhancing Th1 differentiation. More recently, IL-35 was described as a potent anti-inflammatory agent produced by regulatory B and T cells. The role of the newest member, IL-39, has been implicated in proinflammatory B cell responses but has not been explored in the context of allo-HCT. This review is directed at discussing the current literature relevant to each IL-12-family cytokine and cognate receptor engagement, as well as the consequential downstream signaling implications, during GVHD pathogenesis. Additionally, we will provide an overview of translational strategies targeting the IL-12 family cytokines, their receptors, and subsequent signal transduction to control GVHD.
Psoriasis is a chronic skin condition with immune-mediated inflammatory etiology. Many studies have indicated that Interleukin-12 (IL-12) plays an essential role in the severity of psoriasis.So to ...elucidate IL-12 role in regulating psoriasis promotion, the sera levels of IL-12 are detected in psoriasis patients and healthy people. The study contains 45 individuals with psoriasis, and the same number of healthy persons are included as controls .The PASI (Psoriasis Area and Severity Index) was used to assess the severity of psoriasis. Patients were divided into three groups depending on severity: mild psoriatic patients (13), moderate psoriatic patients(15), and severe psoriatic patients (17).Five ml of serum was obtained from each individual in per groups to determine IL-12 levels. The data demonstrated that the age range (36-45) has the highest prevalence of psoriatic patients (51.11 %). In addition, the female to male ratio in patients with psoriasis was 1.2:1.IL-12 levels in psoriasis patients were significantly higher than in healthy persons (3.55 pg/ml and 2.79 pg/ml). Furthermore, there was a significant association between blood IL-12 levels and PASI for psoriatic patient groups. The findings suggest that IL-12 has a key role in the psoriasis pathogenesis and that it might be utilized to evaluate psoriasis and as a follow-up indicator for those with it.
Natural Killer (NK) cells are cytotoxic innate lymphoid cells serving at the front line against infection and cancer. In inflammatory microenvironments, multiple soluble and contact-dependent signals ...modulate NK cell responsiveness. Besides their innate cytotoxic and immunostimulatory activity, it has been uncovered in recent years that NK cells constitute a heterogeneous and versatile cell subset. Persistent memory-like NK populations that mount a robust recall response were reported during viral infection, contact hypersensitivity reactions, and after stimulation by pro-inflammatory cytokines or activating receptor pathways. In this review, we highlight recent findings on the generation, functionality, and clinical applicability of memory-like NK cells and describe common features in comparison to other recent concepts of memory NK cells. Understanding of these features will facilitate the conception and design of novel NK cell-based immunotherapies.
Interleukin-12 (IL-12) is a potent, pro-inflammatory type 1 cytokine that has long been studied as a potential immunotherapy for cancer. Unfortunately, IL-12's remarkable antitumor efficacy in ...preclinical models has yet to be replicated in humans. Early clinical trials in the mid-1990's showed that systemic delivery of IL-12 incurred dose-limiting toxicities. Nevertheless, IL-12's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. The development of strategies which maximize IL-12 delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. Diverse IL-12 delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. Several localized IL-12 delivery approaches have recently reached the clinical stage with several more at the precipice of translation. Taken together, localized delivery systems are supporting an IL-12 renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. This review begins with a brief historical account of cytokine monotherapies and describes how IL-12 went from promising new cure to ostracized black sheep following multiple on-study deaths. The bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for IL-12-based cancer immunotherapies. Advantages and limitations of different delivery technologies are highlighted. Finally, perspectives on how IL-12-based immunotherapies may be utilized for widespread clinical application in the very near future are offered.
Cancer cells are adept at evading cell death, but the underlying mechanisms are poorly understood. IL-12 plays a critical role in the early inflammatory response to infection and in the generation of ...T-helper type 1 cells, favoring cell-mediated immunity. IL-12 is composed of two different subunits, p40 and p35. This study underlines the importance of IL-12 p40 monomer (p40) in helping cancer cells to escape cell death. We found that different mouse and human cancer cells produced greater levels of p40 than p40 homodimer (p40₂), IL-12, or IL-23. Similarly, the serum level of p40 was much greater in patients with prostate cancer than in healthy control subjects. Selective neutralization of p40, but not p40₂, by mAb stimulated death in different cancer cells in vitro and in vivo in a tumor model. Interestingly, p40 was involved in the arrest of IL-12 receptor (IL-12R) IL-12Rβ1, but not IL-12Rβ2, in the membrane, and that p40 neutralization induced the internalization of IL-12Rβ1 via caveolin and caused cancer cell death via the IL-12–IFN-γ pathway. These studies identify a role of p40 monomer in helping cancer cells to escape cell death via suppression of IL-12Rβ1 internalization.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Interleukin-12 (IL-12) is a proinflammatory cytokine that links innate and adaptive immune responses against tumor cells. Single Nucleotide Polymorphisms (SNPs) in IL-12 genes have been associated ...with cancer risk. However, limited studies have assessed the role of IL-12 in breast cancer (BC) risk comprehensively, and these were done in European and Asian populations. Here, we evaluated the association of the IL-12 signaling pathway and BC risk in Puerto Rican women. A genetic association study was completed with 461 BC cases and 463 non-BC controls. By logistic regression, IL-12 signaling SNPs were associated with an increased BC risk, including rs2243123 (
), rs3761041, rs401502 and rs404733 (
), rs7849191 (
), rs280500 (
) and rs4274624 (
). Conversely, other SNPs were associated with reduced BC risk including rs438421 (
), rs6693065 (
), rs10974947, and rs2274471 (
), rs10168266 and rs925847 (
), and rs2069718 (
). Analyses based in hormone receptors such as estrogen (ER) and progesterone (PR) receptors also revealed protective (for SNPs rs3212227-
; rs3024896 and rs3821236-
) and predisposing (for rs2069705-
SNP) BC associations. Haplotype analysis showed a decreased BC risk for
and
SNPs, whereas increased risk for
. This study suggests a role of the IL-12 signaling axis and BC risk. SNPs in this pathway may alter IL-12 induced anti-tumor responses and modulate BC predisposition in a population-specific context. Functional studies will be necessary to confirm these findings, which potentially may benefit IL-12 related immunotherapeutic approaches towards BC.
Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or ...packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two “scaffold” proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.
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Extracellular vesicles (EVs) are natural mediators of cell-cell communication and thus potential vehicles for drug delivery. Dooley et al. describe an EV engineering platform based on two scaffold proteins, PTGFRN and BASP1, which enable high-density EV surface display and luminal loading of therapeutic cargo, leading to potent, pharmacodynamic activity in vitro and in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We aimed in the current study to determine the LC50 of glyphosate in common carp fish (Cyprinus carpio L.) within 24 hours in addition to studying the pathological lesions of sublethal concentration ...of glyphosate (25 and 50% of LC50) in kidney and liver tissues. A total of 100 fish were used to determine the LC50 of glyphosate using the Trevan method (Curve protocol) within 24 hours, in addition to 75 fish used to study the histopathological lesions caused by 25 and 50% of LC50 in common carp after 5, 15 and 25 days of exposure. The current study showed that the LC50 of glyphosate in common carp was 26.151 mg/L within 24 hours, by this result the 25 and 50% of LC50 was 6.54 and 13.08 mg/L, respectively. The histopathological lesions revealed that both the kidney and liver showed degenerative and necrotic changes associated with infiltration of inflammatory cells and interstitial hemorrhages, also the liver sections showed hyperplasia of bile cuniculi and collagen fiber deposition within the portal area, generally, the histopathological lesions were more intensive and advanced in the group exposed to 50% of glyphosate LC50 more than that recorded in the group treated with 25% of glyphosate LC50. In conclusion, common carp fish (Cyprinus carpio L.) showed severe histopathological lesions after exposure to sublethal concentrations of glyphosate, these lesions are life-threatening and can cause high mortality in higher concentrations.
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