Anti-TACI single and dual-targeting CAR T cells overcome BCMA antigen loss in multiple myeloma.

Chimeric Antigen Receptor (CAR) T cells directed to B cell maturation antigen (BCMA) mediate profound responses in patients with multiple myeloma, but most patients do not achieve long-term complete remissions. In addition, recent evidence suggests that high-affinity binding to BCMA can result in on-target, off-tumor activity in the basal ganglia and can lead to fatal Parkinsonian-like disease. Here we develop CAR T cells against multiple myeloma using a binder to targeting transmembrane activator and CAML interactor (TACI) in mono and dual-specific formats with anti-BCMA. These CARs have robust, antigen-specific activity in vitro and in vivo. We also show that TACI RNA expression is limited in the basal ganglia, which may circumvent some of the toxicities recently reported with BCMA CARs. Thus, single-targeting TACI CARs may have a safer toxicity profile, whereas dual-specific BCMA-TACI CAR T cells have potential to avoid the antigen escape that can occur with single-antigen targeting.

Mesenchymal stromal cells expressing a dominant-negative high mobility group A1 transgene exhibit improved function during sepsis.

High mobility group (HMG)A proteins are nonhistone chromatin proteins that bind to the minor groove of DNA, interact with transcriptional machinery, and facilitate DNA-directed nuclear processes. HMGA1 has been shown to regulate genes involved with systemic inflammatory processes. We hypothesized that HMGA1 is important in the function of mesenchymal stromal cells (MSCs), which are known to modulate inflammatory responses due to sepsis. To study this process, we harvested MSCs from transgenic (Tg) mice expressing a dominant-negative (dn) form of HMGA1 in mesenchymal cells. MSCs harvested from Tg mice contained the dnHMGA1 transgene, and transgene expression did not change endogenous HMGA1 levels. Immunophenotyping of the cells, along with trilineage differentiation revealed no striking differences between Tg and wild-type (WT) MSCs. However, Tg MSCs growth was decreased compared with WT MSCs, although Tg MSCs were more resistant to oxidative stress-induced death and expressed less IL-6. Tg MSCs administered after the onset of Escherichia coli-induced sepsis maintained their ability to improve survival when given in a single dose, in contrast with WT MSCs. This survival benefit of Tg MSCs was associated with less tissue cell death, and also a reduction in tissue neutrophil infiltration and expression of neutrophil chemokines. Finally, Tg MSCs promoted bacterial clearance and enhanced neutrophil phagocytosis, in part through their increased expression of stromal cell-derived factor-1 compared with WT MSCs. Taken together, these data demonstrate that expression of dnHMGA1 in MSCs provides a functional advantage of the cells when administered during bacterial sepsis.

Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide.

Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.

A Distinct Transcriptional Program in Human CAR T Cells Bearing the 4-1BB Signaling Domain Revealed by scRNA-Seq.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have produced impressive outcomes for the treatment of B cell malignancies, but different products vary in kinetics, persistence, and toxicity profiles based on the co-stimulatory domains included in the CAR. In this study, we performed transcriptional profiling of bulk CAR T cell populations and single cells to characterize the transcriptional states of human T cells transduced with CD3ζ, 4-1BB-CD3ζ (BBζ), or CD28-CD3ζ (28ζ) co-stimulatory domains at rest and after activation by triggering their CAR or their endogenous T cell receptor (TCR). We identified a transcriptional signature common across CARs with the CD3ζ signaling domain, as well as a distinct program associated with the 4-1BB co-stimulatory domain at rest and after activation. CAR T cells bearing BBζ had increased expression of human leukocyte antigen (HLA) class II genes, ENPP2, and interleukin (IL)-21 axis genes, and decreased PD1 compared to 28ζ CAR T cells. Similar to previous studies, we also found BBζ CAR CD8 T cells to be enriched in a central memory cell phenotype and fatty acid metabolism genes. Our data uncovered transcriptional signatures related to costimulatory domains and demonstrated that signaling domains included in CARs uniquely shape the transcriptional programs of T cells.

CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma.

Despite remarkable success in the treatment of hematological malignancies, CAR T-cell therapies for solid tumors have floundered, in large part due to local immune suppression and the effects of prolonged stimulation leading to T-cell dysfunction and exhaustion. One mechanism by which gliomas and other cancers can hamper CAR T cells is through surface expression of inhibitory ligands such as programmed cell death ligand 1 (PD-L1). Using the CRIPSR-Cas9 system, we created universal CAR T cells resistant to PD-1 inhibition through multiplexed gene disruption of endogenous T-cell receptor (TRAC), beta-2 microglobulin (B2M) and PD-1 (PDCD1). Triple gene-edited CAR T cells demonstrated enhanced activity in preclinical glioma models. Prolonged survival in mice bearing intracranial tumors was achieved after intracerebral, but not intravenous administration. CRISPR-Cas9 gene-editing not only provides a potential source of allogeneic, universal donor cells, but also enables simultaneous disruption of checkpoint signaling that otherwise impedes maximal antitumor functionality.

Rational design of a trimeric APRIL-based CAR-binding domain enables efficient targeting of multiple myeloma.

Chimeric antigen receptor (CAR) T cells (CARTs) have shown tremendous potential for the treatment of certain B-cell malignancies, including patients with relapsed/refractory multiple myeloma (MM). Targeting the B-cell maturation antigen (BCMA) has produced the most promising results for CART therapy of MM to date, but not all remissions are sustained. Emergence of BCMA escape variants has been reported under the selective pressure of monospecific anti-BCMA CART treatment. Thus, there is a clinical need for continuous improvement of CART therapies for MM. Here, we show that a novel trimeric APRIL (a proliferation-inducing ligand)-based CAR efficiently targets both BCMA+ and BCMA- MM. Modeled after the natural ligand-receptor pair, APRIL-based CARs allow for bispecific targeting of the MM-associated antigens BCMA and transmembrane activator and CAML interactor (TACI). However, natural ligands as CAR antigen-binding domains may require further engineering to promote optimal binding and multimerization to adequately trigger T-cell activation. We found that using a trimeric rather than a monomeric APRIL format as the antigen-binding domain enhanced binding to BCMA and TACI and CART activity against MM in vitro and in vivo. Dual-specific, trimeric APRIL-based CAR are a promising therapeutic approach for MM with potential for preventing and treating BCMA escape.

Glycoengineering of chimeric antigen receptor (CAR) T-cells to enforce E-selectin binding.

Tissue colonization (homing) by blood-borne cells critically hinges on the ability of the cells to adhere to vascular endothelium with sufficient strength to overcome prevailing hemodynamic shear stress. These adhesive interactions are most effectively engendered via binding of the endothelial lectin E-selectin (CD62E) to its cognate ligand, sialyl Lewis-X (sLe X ), displayed on circulating cells. Although chimeric antigen receptor (CAR) T-cell immunotherapy holds promise for treatment of various hematologic and non-hematologic malignancies, there is essentially no information regarding the efficiency of CAR T-cell homing. Accordingly, we performed integrated biochemical studies and adhesion assays to examine the capacity of human CAR T-cells to engage E-selectin. Our data indicate that CAR T-cells do not express sLe X and do not bind E-selectin. However, enforced sLe X display can be achieved on human CAR T-cells by surface fucosylation, with resultant robust E-selectin binding under hemodynamic shear. Importantly, following intravascular administration into mice, fucosylated human CAR-T cells infiltrate marrow with 10-fold higher efficiency than do unfucosylated cells. Collectively, these findings indicate that custom installation of sLe X programs tissue colonization of vascularly administered human CAR T-cells, offering a readily translatable strategy to augment tissue delivery, thereby lowering the pertinent cell dosing and attendant cell production burden, for CAR T-cell immunotherapy applications.

Chimeric Antigen Receptor T Cells Targeting CD79b Show Efficacy in Lymphoma with or without Cotargeting CD19.

PURPOSE: T cells engineered to express a chimeric antigen receptor (CAR) against CD19 have recently been FDA approved for the treatment of relapsed or refractory large B-cell lymphoma. Despite the success and curative potential of CD19 CAR T cells, several reports describing disease relapse due to antigen loss are now emerging. EXPERIMENTAL DESIGN: We developed a novel CAR construct directed against CD79b, a critical receptor for successful B-cell development that remains highly expressed in several subtypes of B-cell lymphoma, including mantle cell lymphoma (MCL). We tested CAR T cells directed against CD79b alone or in combination with CD19 targeting in a single construct, against cell line- and patient-derived xenograft models. RESULTS: We demonstrate CAR79b antigen-specific recognition and cytotoxicity against a panel of cell lines and patient-derived xenograft models of MCL. Importantly, we show that downregulation of CD19 does not influence surface expression of CD79b and that anti-CD79b CAR T cells alone or arranged in a dual-targeting format with a CD19 single-chain variable fragment (scFv) are able to recognize and eliminate CD19+, CD19-, and mixed CD19+/CD19-B-cell lymphoma. CONCLUSIONS: Our findings demonstrate that CAR T cells targeting CD79b alone or in combination have promise for treating and preventing CD19 antigen escape in B-cell lymphomas.

CAR-T cells secreting BiTEs circumvent antigen escape without detectable toxicity.

Chimeric antigen receptor (CAR)-T-cell therapy for solid tumors is limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR-T cells directed against single antigens. Here, we developed a bicistronic construct to drive expression of a CAR specific for EGFRvIII, a glioblastoma-specific tumor antigen, and a bispecific T-cell engager (BiTE) against EGFR, an antigen frequently overexpressed in glioblastoma but also expressed in normal tissues. CART.BiTE cells secreted EGFR-specific BiTEs that redirect CAR-T cells and recruit untransduced bystander T cells against wild-type EGFR. EGFRvIII-specific CAR-T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive glioblastoma. However, CART.BiTE cells eliminated heterogenous tumors in mouse models of glioblastoma. BiTE-EGFR was locally effective but was not detected systemically after intracranial delivery of CART.BiTE cells. Unlike EGFR-specific CAR-T cells, CART.BiTE cells did not result in toxicity against human skin grafts in vivo.

Anti-CD37 chimeric antigen receptor T cells are active against B- and T-cell lymphomas.

Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.

Frontline Science: Targeted expression of a dominant-negative high mobility group A1 transgene improves outcome in sepsis.

High mobility group (HMG) proteins are a family of architectural transcription factors, with HMGA1 playing a role in the regulation of genes involved in promoting systemic inflammatory responses. We speculated that blocking HMGA1-mediated pathways might improve outcomes from sepsis. To investigate HMGA1 further, we developed genetically modified mice expressing a dominant negative (dn) form of HMGA1 targeted to the vasculature. In dnHMGA1 transgenic (Tg) mice, endogenous HMGA1 is present, but its function is decreased due to the mutant transgene. These mice allowed us to specifically study the importance of HMGA1 not only during a purely pro-inflammatory insult of endotoxemia, but also during microbial sepsis induced by implantation of a bacterial-laden fibrin clot into the peritoneum. We found that the dnHMGA1 transgene was only present in Tg and not wild-type (WT) littermate mice, and the mutant transgene was able to interact with transcription factors (such as NF-κB), but was not able to bind DNA. Tg mice exhibited a blunted hypotensive response to endotoxemia, and less mortality in microbial sepsis. Moreover, Tg mice had a reduced inflammatory response during sepsis, with decreased macrophage and neutrophil infiltration into tissues, which was associated with reduced expression of monocyte chemotactic protein-1 and macrophage inflammatory protein-2. Collectively, these data suggest that targeted expression of a dnHMGA1 transgene is able to improve outcomes in models of endotoxin exposure and microbial sepsis, in part by modulating the immune response and suggest a novel modifiable pathway to target therapeutics in sepsis.

Enhancing T cell therapy through TCR-signaling-responsive nanoparticle drug delivery.

Adoptive cell therapy (ACT) with antigen-specific T cells has shown remarkable clinical success; however, approaches to safely and effectively augment T cell function, especially in solid tumors, remain of great interest. Here we describe a strategy to 'backpack' large quantities of supporting protein drugs on T cells by using protein nanogels (NGs) that selectively release these cargos in response to T cell receptor activation. We designed cell surface-conjugated NGs that responded to an increase in T cell surface reduction potential after antigen recognition and limited drug release to sites of antigen encounter, such as the tumor microenvironment. By using NGs that carried an interleukin-15 super-agonist complex, we demonstrated that, relative to systemic administration of free cytokines, NG delivery selectively expanded T cells 16-fold in tumors and allowed at least eightfold higher doses of cytokine to be administered without toxicity. The improved therapeutic window enabled substantially increased tumor clearance by mouse T cell and human chimeric antigen receptor (CAR)-T cell therapy in vivo.

CpG oligodeoxynucleotide as immune adjuvant enhances photodynamic therapy response in murine metastatic breast cancer.

Breast cancer is the most common cause of cancer death in women. The side effects and complications following current breast cancer therapy can be devastating. Moreover, the prognosis in late stages of the diseases is usually poor. Photodynamic therapy (PDT) is a promising cancer treatment modality that is capable of both local tumor destruction and immune stimulation. However, treatment with PDT alone is often non-curative due to tumor-induced immune cell dysfunction and immune suppression. This phenomenon has motivated a new approach by combining immunostimulants with PDT to enhance anti-tumor immunity. In the present study, we investigated PDT mediated by verteporfin and 690 nm light delivered 15 min later, in combination with an immunomodulation approach using CpG oligodeoxynucleotide for the treatment of 4T1 metastatic breast cancer in a BALB/c immunocompetent mouse model. In vitro, CpG primed immature dendritic cells (DC) via toll like receptor 9 to phagocytose PDT killed tumor cells leading to DC maturation and activation. Peritumoral injection of CpG after PDT in mice gave improved local tumor control and a survival advantage compared to either treatment alone (p < 0.05). CpG may be a valuable dendritic cell targeted immunoadjuvant to combine with PDT.

MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways.

Scarring of the kidney is a major public health concern, directly promoting loss of kidney function. To understand the role of microRNA (miRNA) in the progression of kidney scarring in response to injury, we investigated changes in miRNA expression in two kidney fibrosis models and identified 24 commonly up-regulated miRNAs. Among them, miR-21 was highly elevated in both animal models and in human transplanted kidneys with nephropathy. Deletion of miR-21 in mice resulted in no overt abnormality. However, miR-21(-/-) mice suffered far less interstitial fibrosis in response to kidney injury, a phenotype duplicated in wild-type mice treated with anti-miR-21 oligonucleotides. Global derepression of miR-21 target mRNAs was readily detectable in miR-21(-/-) kidneys after injury. Analysis of gene expression profiles up-regulated in the absence of miR-21 identified groups of genes involved in metabolic pathways, including the lipid metabolism pathway regulated by peroxisome proliferator-activated receptor-α (Pparα), a direct miR-21 target. Overexpression of Pparα prevented ureteral obstruction-induced injury and fibrosis. Pparα deficiency abrogated the antifibrotic effect of anti-miR-21 oligonucleotides. miR-21 also regulated the redox metabolic pathway. The mitochondrial inhibitor of reactive oxygen species generation Mpv17l was repressed by miR-21, correlating closely with enhanced oxidative kidney damage. These studies demonstrate that miR-21 contributes to fibrogenesis and epithelial injury in the kidney in two mouse models and is a candidate target for antifibrotic therapies.

The melanoma-associated transmembrane glycoprotein Gpnmb controls trafficking of cellular debris for degradation and is essential for tissue repair.

Kidney damage due to injury rarely resolves completely, and there are currently no therapies capable of promoting repair. In addition to understanding mechanisms by which tissues are damaged, illuminating mechanisms of repair and regeneration is also of great importance. Here we show that the melanoma-associated, transmembrane glycoprotein, Gpnmb, is up-regulated 15-fold following ischemic damage in kidney tissue and by more than 10-fold in macrophages and 3-fold in surviving epithelial cells. Gpnmb-expressing macrophages and epithelial cells were found to contain apoptotic bodies at 3 times the rate of nonexpressing cells. Either mutation of Gpnmb or ablation of inflammatory macrophages prevents normal repair of the kidney. Significantly, the kidneys from postischemic Gpnmb mutant mice exhibited a 5-fold increase in apoptotic cellular debris compared to wild-type mice. These mice also experienced an 85% increase in mortality following bilateral ischemic kidney. Finally, we demonstrate that Gpnmb is a phagocytic protein that is necessary for recruitment of the autophagy protein LC3 to the phagosome where these proteins are colocalized and for lysosomal fusion with the phagosome and hence bulk degradation of their content. Therefore, Gpnmb is a novel prorepair gene that is necessary for crosstalk between the macroautophagic degradation pathway and phagocytosis.

Bone marrow Ly6Chigh monocytes are selectively recruited to injured kidney and differentiate into functionally distinct populations.

Roles for monocyte/macrophages (Mphi) in directing the development of tissue fibrosis are increasingly recognized. Macrophages form a heterogeneous group of inflammatory leukocytes, and the mechanisms by which they acquire heterogeneity and its functional significance are unclear. We used the unilateral ureteral obstruction model of progressive kidney fibrosis to explore macrophage heterogeneity and function further. Unilateral ureteral obstruction kidney Mphis form three distinct subpopulations defined by the marker Ly6C, all of which are derived from a single Ly6C(high) bone marrow monocyte population selectively recruited to the kidney. Conditional ablation of these Mphis in vivo in CD11b-DTR mice is potently antifibrotic. The mRNA transcription profile of these populations is consistent with differential functional roles for each subpopulation, with Ly6C(low) macrophages transcribing genes consistent with selective profibrotic or M2-type function. Furthermore, bone marrow chimerism studies indicate that although resident kidney macrophages proliferate markedly to comprise up to 40% of the inflammatory macrophage population, they do not contribute to fibrosis. Our data identify Ly6C as a marker of functionally discrete tissue macrophage subsets and support a model of selective recruitment of Ly6C(high) bone marrow monocytes to the kidney that differentiate into three populations of kidney macrophages, including a profibrotic Ly6C(low) population.

Serum amyloid P inhibits fibrosis through Fc gamma R-dependent monocyte-macrophage regulation in vivo.

New therapies that target chronic inflammation with fibrosis are urgently required. Increasing evidence points to innate activation of inflammatory cells in driving chronic organ fibrosis. Serum amyloid P is a naturally circulating soluble pattern recognition receptor, a member of the family of pentraxin proteins. It links danger-associated molecular pattern recognition to Fc gamma receptor-mediated phagocytosis. Here we show that fibrosis progression in the mouse kidney is significantly inhibited by therapeutic administration of human serum amyloid P, regulated by activating Fc gamma receptors, and dependent on inflammatory monocytes and macrophages, but not fibrocytes. Human serum amyloid P-mediated inhibition of mouse kidney fibrosis correlated with specific binding of human serum amyloid P to cell debris and with subsequent suppression of inflammatory monocytes and kidney macrophages in vitro and in vivo, and was dependent on regulated binding to activating Fc gamma receptors and interleukin-10 expression. These studies uncover previously unidentified roles for Fc gamma receptors in sterile inflammation and highlight serum amyloid P as a potential antifibrotic therapy through local generation of interleukin-10.

Chitosan acetate bandage as a topical antimicrobial dressing for infected burns.

An engineered chitosan acetate bandage preparation (HemCon) is used as a hemostatic dressing, and its chemical structure suggests that it should also be antimicrobial. We previously showed that when a chitosan acetate bandage was applied to full-thickness excisional wounds in mice that had been infected with pathogenic bioluminescent bacteria (Pseudomonas aeruginosa, Proteus mirabilis, and Staphylococcus aureus), it was able to rapidly kill the bacteria and save the mice from developing fatal infections. Wound healing was also stimulated. In the present study, we asked whether a chitosan acetate bandage could act as a topical antimicrobial dressing when it was applied to third-degree burns in mice contaminated with two of these bacterial species (P. aeruginosa and P. mirabilis). Preliminary experiments established the length of burn time and the number of bacteria needed to produce fatal infections in untreated mice and established that the chitosan acetate bandage could adhere to the infected burn for up to 21 days. In the case of P. aeruginosa infections, the survival rate of mice treated with the chitosan acetate bandage was 73.3% (whereas the survival rate of mice treated with a nanocrystalline silver dressing was 27.3% [P = 0.0055] and that of untreated mice was 13.3% [P < 0.0002]). For P. mirabilis infections, the comparable survival rates were 66.7%, 62.5%, and 23.1% respectively. Quantitative bioluminescent signals showed that the chitosan acetate bandage effectively controlled the growth of bacteria in the burn and prevented the development of systemic sepsis, as shown by blood culture. These data suggest that chitosan acetate bandage is efficacious in preventing fatal burn infections.

Effect of chitosan acetate bandage on wound healing in infected and noninfected wounds in mice.

HemCon bandage is an engineered chitosan acetate preparation designed as a hemostatic dressing, and is under investigation as a topical antimicrobial dressing. We studied its effects on healing of excisional wounds that were or were not infected with Staphylococcus aureus, in normal mice or mice previously pretreated with cyclophosphamide (CY). CY significantly suppressed wound healing in both the early and later stages, while S. aureus alone significantly stimulated wound healing in the early stages by preventing the initial wound expansion. CY plus S. aureus showed an advantage in early stages by preventing expansion, but a significant slowing of wound healing in later stages. In order to study the conflicting clamping and stimulating effects of chitosan acetate bandage on normal wounds, we removed the bandage from wounds at times after application ranging from 1 hour to 9 days. Three days application gave the earliest wound closure, and all application times gave a faster healing slope after removal compared with control wounds. Chitosan acetate bandage reduced the number of inflammatory cells in the wound at days 2 and 4, and had an overall beneficial effect on wound healing especially during the early period where its antimicrobial effect is most important.