Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice.

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

Engineering Tolerance toward Allogeneic CAR-T Cells by Regulation of MHC Surface Expression with Human Herpes Virus-8 Proteins.

Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human T cells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for T cells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic T cells, K3-transduced T cells selectively expanded in vivo. Ectopic K5 expression in MHC class I-, MIC-A+/B+ K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, T cell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products.

Ten-year survival rate of 89% after distal femoral osteotomy surgery for lateral compartment osteoarthritis of the knee.

PURPOSE: The purpose of this study was to assess the accuracy, safety, and survival of distal femoral osteotomy (DFO) surgery for lateral compartment OA of the knee. METHODS: A retrospective cohort study was conducted at a single UK centre, using prospectively collected data over an 8-year period (2009-2017). All patients had pre-operative radiographic analysis and digital planning of their deformity correction in addition to post-operative analysis of the achieved correction and yearly face-to-face follow-up. Complications (defined as an undesirable medical or surgical event as a direct result of the operation), reoperations, and failure (defined as conversion to arthroplasty or revision) were recorded. RESULTS: From a total of 83 patients, 81 patients undergoing 86 primary DFOs were included in this study, with a mean follow-up of 99 months (SD 27 months). The mean pre-operative percentage Mikulicz point was 78.7% (SD 19.1%) and post-operative 35.9% (SD 14.8%). The mean accuracy of correction (intended correction - achieved correction) was an 8.2% overcorrection (SD 13.7%). The complication rate was 4.7%. Using Kaplan-Meier analysis, the mean survival was 113 months (95% CI 106-120) with the probability of surviving 10 years 89%. CONCLUSION: DFO for valgus alignment and lateral compartment arthritis is associated with low complications, long-term joint preservation, and the prevention of arthroplasty surgery. However, the accuracy of correction still requires improvement in intra-operative technique. LEVEL OF EVIDENCE: IV.

Correction to: Paediatric proximal ACL tears managed with direct ACL repair is safe, effective and has excellent short-term outcomes.

Authors would like to update the full author names.

Inducible MyD88/CD40 synergizes with IL-15 to enhance antitumor efficacy of CAR-NK cells.

Natural killer (NK) cells expressing chimeric antigen receptors (CARs) are a promising anticancer immunotherapy, leveraging both innate NK cell antitumor activity and target-specific cytotoxicity. Inducible MyD88/CD40 (iMC) is a potent, rimiducid-regulated protein switch that has been deployed previously as a T-cell activator to enhance proliferation and persistence of CAR-modified T cells. In this study, iMC was extended to CAR-NK cells to enhance their growth and augment cytotoxicity against tumor cells. iMC-activated NK cells substantially increased cytokine and chemokine secretion and displayed higher levels of perforin and granzyme B degranulation. In addition, iMC activation could be coupled with ectopic interleukin-15 (IL-15) to further enhance NK cell proliferation. When coexpressed with a target-specific CAR (CD123 or BCMA), this IL-15/iMC system showed further augmented antitumor activity through enhanced CAR-NK cell expansion and cytolytic activity. To protect against potential toxicity from engineered NK cells, an orthogonal rapamycin-regulated Caspase-9 (iRC9) was included in a 4-gene, dual-switch platform. After infusion of dual-switch NK cells, pharmacologic iRC9 dimerization led to rapid elimination of a majority of expanded transduced NK cells. Thus, CAR-NK cells utilizing dual molecular switches provide an innovative and effective approach to cancer immunotherapy with controlled specificity, efficacy, and safety.

Paediatric proximal ACL tears managed with direct ACL repair is safe, effective and has excellent short-term outcomes.

PURPOSE: Anterior cruciate ligament (ACL) surgery in the paediatric population has long been a challenge. Non-operative treatment will result in persistent instability which can lead to chondral and meniscal injuries. The results of primary open ACL repair are poor. Concerns of growth plate disturbance with transphyseal techniques and issues with relatively small-diameter grafts in Tanner 1 and 2 patients, which are inadequate, have contributed to these challenges. With advancing instrumentation, there is renewed interest in ACL repair. The minimally invasive approach of arthroscopic primary ACL repair retains the native ligament. The objective and subjective outcomes at 2 years are presented. METHODS: Paediatric patients, less than 16 years of age, presenting acutely with complete proximal ACL ruptures underwent direct arthroscopic ACL repair, reinforced by a temporary internal brace, which was subsequently removed after 3 months. Patient-reported outcome measures including the Lysholm, Tegner and KOOS scores were collected at 6 months, 1 year and 2 years post-operatively. RESULTS: Twenty patients (age 6-16) completed data at 2 years post-operatively. There were no failures, no complications and no growth disturbance out to 2 years. The 2-year postoperative outcomes; Lysholm 95 (90-100), Tegner 7 (6-10), KOOS-Child 96.5 (88.9-100) demonstrated statistically significant improvements following surgery (p < 0.001). Objective measurements with an accelerometer did not demonstrate any significant side-to-side difference. CONCLUSION: ACL repair for proximal ACL tears in the paediatric population demonstrates the potential for excellent outcomes at short-term follow-up. This presents an attractive alternative to ACL reconstruction when an adequate ACL remnant permits direct repair. Our results demonstrate that paediatric ACL repair is safe and effective.

Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies.

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting "low" cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

Two-Dimensional Regulation of CAR-T Cell Therapy with Orthogonal Switches.

Use of chimeric antigen receptors (CARs) as the basis of targeted adoptive T cell therapies has enabled dramatic efficacy against multiple hematopoietic malignancies, but potency against bulky and solid tumors has lagged, potentially due to insufficient CAR-T cell expansion and persistence. To improve CAR-T cell efficacy, we utilized a potent activation switch based on rimiducid-inducible MyD88 and CD40 (iMC)-signaling elements. To offset potential toxicity risks by this enhanced CAR, an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch (iRC9) was developed to allow in vivo elimination of CAR-T cells. iMC costimulation induced by systemic rimiducid administration enhanced CAR-T cell proliferation, cytokine secretion, and antitumor efficacy in both in vitro assays and xenograft tumor models. Conversely, rapamycin-mediated iRC9 dimerization rapidly induced apoptosis in a dose-dependent fashion as an approach to mitigate therapy-related toxicity. This novel, regulatable dual-switch system may promote greater CAR-T cell expansion and prolonged persistence in a drug-dependent manner while providing a safety switch to mitigate toxicity concerns.

Regulated Expansion and Survival of Chimeric Antigen Receptor-Modified T Cells Using Small Molecule-Dependent Inducible MyD88/CD40.

Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4+ and CD8+ T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells. Importantly, the concurrent activation of iMC (with rimiducid) and CAR (by antigen recognition) is required for interleukin (IL)-2 production and robust CAR-T cell expansion and may provide a user-controlled mechanism to amplify CAR-T cell levels in vivo and augment anti-tumor efficacy.

Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model.

Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.

In vivo gold nanoparticle delivery of peptide vaccine induces anti-tumor immune response in prophylactic and therapeutic tumor models.

Gold nanoparticles (AuNPs) are promising vehicles for cancer immunotherapy, with demonstrated efficacy in immune delivery and innate cell stimulation. Nevertheless, their potential has yet to be assessed in the in vivo application of peptide cancer vaccines. In this study, it is hypothesized that the immune distribution and adjuvant qualities of AuNPs could be leveraged to facilitate delivery of the ovalbumin (OVA) peptide antigen and the CpG adjuvant and enhance their therapeutic effect in a B16-OVA tumor model. AuNP delivery of OVA (AuNP-OVA) and of CpG (AuNP-CpG) enhanced the efficacy of both agents and induced strong antigen-specific responses. In addition, it is found that AuNP-OVA delivery alone, without CpG, is sufficient to promote significant antigen-specific responses, leading to subsequent anti-tumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This enhanced therapeutic efficacy is likely due to the adjuvant effect of peptide coated AuNPs, as they induce inflammatory cytokine release when cultured with bone marrow dendritic cells. Overall, AuNP-mediated OVA peptide delivery can produce significant therapeutic benefits without the need of adjuvant, indicating that AuNPs are effective peptide vaccine carriers with the potential to permit the use of lower and safer adjuvant doses during vaccination.

Modification of T lymphocytes to express tumor antigens.

Human T cells can be genetically modified to express tumor-associated antigens (TAA) for the induction of tumor-specific immunity, suggesting that T cells may be alternative candidates of effective antigen-presenting cells (TAPC) and may be useful in vivo as cellular cancer vaccines. The effective induction of TAA-specific T cell immune responses requires activation of T cells by CD3/CD28 antibodies and the presence of proinflammatory cytokines such as interleukin-7 (IL-7) and interleukin-12 (IL-12). Here, we describe the technique of preparing activated human TAPC pulsed with TAA peptides for the induction of tumor antigen-specific T cell immunity in vitro.

Ultra low-dose IL-2 for GVHD prophylaxis after allogeneic hematopoietic stem cell transplantation mediates expansion of regulatory T cells without diminishing antiviral and antileukemic activity.

PURPOSE: GVHD after allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with low numbers of circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). Because Tregs express high levels of the interleukin (IL)-2 receptor, they may selectively expand in vivo in response to doses of IL-2 insufficient to stimulate T effector T-cell populations, thereby preventing GVHD. EXPERIMENTAL DESIGN: We prospectively evaluated the effects of ultra low-dose (ULD) IL-2 injections on Treg recovery in pediatric patients after alloSCT and compared this recovery with Treg reconstitution post alloSCT in patients without IL-2. Sixteen recipients of related (n = 12) or unrelated (n = 4) donor grafts received ULD IL-2 post hematopoietic stem cell transplantation (HSCT; 100,000-200,000 IU/m(2) ×3 per week), starting

In vivo immune cell distribution of gold nanoparticles in naïve and tumor bearing mice.

Gold nanoparticles (AuNP) have been widely used for drug delivery and have recently been explored for applications in cancer immunotherapy. Although AuNPs are known to accumulate heavily in the spleen, the particle distribution within immune cells has not been thoroughly studied. Here, cellular distribution of Cy5 labeled 50 nm AuNPs is characterized within the immune populations of the spleen from naïve and tumor bearing mice using flow cytometry. Surprisingly, approximately 30% of the detected AuNPs are taken up by B cells at 24 h, with about 10% in granulocytes, 18% in dendritic cells, and 8% in T cells. In addition, 3% of the particles are detected within myeloid derived suppressor cells, an immune suppressive population that could be targeted for cancer immunotherapy. Furthermore, it is observed that, over time, the particles traveled from the red pulp and marginal zone to the follicles of the spleen. Taking into consideration that the particle cellular distribution does not change at 1, 6 and 24 h, it is highly suggestive that the immune populations carry the particles and migrate through the spleen instead of the particles migrating through the tissue by cell-cell transfer. Finally, no difference is observed in particle distribution between naïve and tumor bearing mice in the spleen, and nanoparticles are detected within 0.7% of dendritic cells of the tumor microenvironment. Overall, these results can help inform and influence future AuNP delivery design criteria including future applications for nanoparticle-mediated immunotherapy.

The miR-223/nuclear factor I-A axis regulates glial precursor proliferation and tumorigenesis in the CNS.

Contemporary views of tumorigenesis regard its inception as a convergence of genetic mutation and developmental context. Glioma is the most common and deadly malignancy in the CNS; therefore, understanding how regulators of glial development contribute to its formation remains a key question. Previously we identified nuclear factor I-A (NFIA) as a key regulator of developmental gliogenesis, while miR-223 has been shown to repress NFIA expression in other systems. Using this relationship as a starting point, we found that miR-223 can suppress glial precursor proliferation via repression of NFIA during chick spinal cord development. This relationship is conserved in glioma, as miR-223 and NFIA expression is negatively correlated in human glioma tumors, and the miR-223/NFIA axis suppresses tumorigenesis in a human glioma cell line. Subsequent analysis of NFIA function revealed that it directly represses p21 and is required for tumorigenesis in a mouse neural stem cell model of glioma. These studies represent the first characterization of miR-223/NFIA axis function in glioma and demonstrate that it is a conserved proliferative mechanism across CNS development and tumorigenesis.

Elimination of metastatic melanoma using gold nanoshell-enabled photothermal therapy and adoptive T cell transfer.

Ablative treatments such as photothermal therapy (PTT) are attractive anticancer strategies because they debulk accessible tumor sites while simultaneously priming antitumor immune responses. However, the immune response following thermal ablation is often insufficient to treat metastatic disease. Here we demonstrate that PTT induces the expression of proinflammatory cytokines and chemokines and promotes the maturation of dendritic cells within tumor-draining lymph nodes, thereby priming antitumor T cell responses. Unexpectedly, however, these immunomodulatory effects were not beneficial to overall antitumor immunity. We found that PTT promoted the infiltration of secondary tumor sites by CD11b(+)Ly-6G/C(+) myeloid-derived suppressor cells, consequently failing to slow the growth of poorly immunogenic B16-F10 tumors and enhancing the growth of distant lung metastases. To exploit the beneficial effects of PTT activity against local tumors and on antitumor immunity whilst avoiding the adverse consequences, we adoptively transferred gp100-specific pmel T cells following PTT. The combination of local control by PTT and systemic antitumor immune reactivity provided by adoptively transferred T cells prevented primary tumor recurrence post-ablation, inhibited tumor growth at distant sites, and abrogated the outgrowth of lung metastases. Hence, the combination of PTT and systemic immunotherapy prevented the adverse effects of PTT on metastatic tumor growth and optimized overall tumor control.

Gold nanoparticle delivery of modified CpG stimulates macrophages and inhibits tumor growth for enhanced immunotherapy.

Gold nanoparticle accumulation in immune cells has commonly been viewed as a side effect for cancer therapeutic delivery; however, this phenomenon can be utilized for developing gold nanoparticle mediated immunotherapy. Here, we conjugated a modified CpG oligodeoxynucleotide immune stimulant to gold nanoparticles using a simple and scalable self-assembled monolayer scheme that enhanced the functionality of CpG in vitro and in vivo. Nanoparticles can attenuate systemic side effects by enhancing CpG delivery passively to innate effector cells. The use of a triethylene glycol (TEG) spacer on top of the traditional poly-thymidine spacer increased CpG macrophage stimulatory effects without sacrificing DNA content on the nanoparticle, which directly correlates to particle uptake. In addition, the immune effects of modified CpG-AuNPs were altered by the core particle size, with smaller 15 nm AuNPs generating maximum immune response. These TEG modified CpG-AuNP complexes induced macrophage and dendritic cell tumor infiltration, significantly inhibited tumor growth, and promoted survival in mice when compared to treatments with free CpG.

High-density sub-100-nm peptide-gold nanoparticle complexes improve vaccine presentation by dendritic cells in vitro.

Nanocarriers have been explored to improve the delivery of tumor antigens to dendritic cells (DCs). Gold nanoparticles are attractive nanocarriers because they are inert, non-toxic, and can be readily endocytosed by DCs. Here, we designed novel gold-based nanovaccines (AuNVs) using a simple self-assembling bottom-up conjugation method to generate high-peptide density delivery and effective immune responses with limited toxicity. AuNVs were synthesized using a self-assembling conjugation method and optimized using DC-to-splenocyte interferon-γ enzyme-linked immunosorbent spot assays. The AuNV design has shown successful peptide conjugation with approximately 90% yield while remaining smaller than 80 nm in diameter. DCs uptake AuNVs with minimal toxicity and are able to process the vaccine peptides on the particles to stimulate cytotoxic T lymphocytes (CTLs). These high-peptide density AuNVs can stimulate CTLs better than free peptides and have great potential as carriers for various vaccine types.