Imaging Biomarkers to Predict Outcomes in Patients With Large B-Cell Lymphoma With a Day 28 Partial Response by 18F-FDG PET/CT Imaging Following CAR-T Therapy.

BACKGROUND: CD19 directed CAR-T therapy for Large B-cell lymphoma (LBCL) has shown great therapeutic response in patients with relapsed/refractory disease with response rates of 60-80%. However, in patients with a partial response (PR) on initial day 28 post CAR-T therapy imaging, clinical uncertainty remains as half of these patients will ultimately have relapsed disease.   PATIENTS: In 24 patients receiving CD19 directed CAR-T therapy for relapsed/refractory LBCL achieving a PR on day 28, we utilize imaging biomarkers by 18F-FDG PET/CT imaging at pre CAR-T therapy baseline and day 28 to determine factors that may predict best overall response (B-OR), progression free survival (PFS), and overall survival (OS).   METHODS: Out of 75 patients receiving CAR-T therapy at a single institution, we retrospectively identified and reviewed 25 (33%) as achieving a PR on day 28. PR was defined using the 2014 Lugano classification system. All patients received standard of care CD19 directed CAR-T therapy with axicabtagene ciloleucel. Two independent nuclear medicine physicians measured baseline (pre-CAR-T therapy) and day 28 PET/CT SUVmax, SUVmean and TMV (cm3) of each lesion (node, organ or marrow uptake, if any) using ROVER software. All statistical tests were two-sided and conducted at the 0.05 level of significance. R version 1.3.1099 (R-studio) was used for statistical modeling.   CONCLUSION: We demonstrate that a higher day 28 SUVmax was significantly higher in those with a B-OR of PR and in our modeling, a lower day 28 SUVmax may predict favorable PFS and OS. Additionally, lower TMV, both at baseline and day 28, may also be predictive of longer PFS and OS, while lower TLG at baseline, but not day 28 is significantly associated with a B-OR of CR. While further study is warranted, these imaging biomarkers may allow for early identification of those with a day 28 PR at highest risk for relapse leading to early intervention to improve long term outcomes.

Ocular adverse events associated with chimeric antigen receptor T-cell therapy: a case series and review.

BACKGROUND/AIMS: Chimeric antigen receptor T-cell (CAR T) therapy has been shown to improve the remission rate and survival for patients with refractory haematological malignancies. The aim of this study is to describe ocular adverse effects associated with CAR T therapy in patients with haematological malignancies. METHODS: This is a retrospective, single-institution, case series. Patients aged 18 years or older who received standard of care CAR T therapy for relapsed/refractory large B-cell lymphoma with a documented ophthalmic evaluation were included. The primary outcome was clinician ophthalmic examination findings. RESULTS: A total of 66 patients received CAR T-cell therapy from February 2018 to October 2019 with 11 receiving an ophthalmic examination. Eleven patients (n=22 eyes) who received CAR T-cell therapy were included in review. The median time from CAR T-cell infusion date to ocular examination was 57.5 days. The median patient age at the time of examination was 60.5 years. Ten patients had subjective symptoms prompting ophthalmic examination. Two patients reported floaters and photopsias. One patient had worsening ocular graft-versus-host disease. Two patients were identified with possible reactivation of viral infections, including herpes zoster ophthalmicus and regressing acute retinal necrosis. CONCLUSIONS: The increasing use of CAR T therapy for malignancies underscores the importance of ophthalmologists and oncologists understanding the potential toxicities associated with its use, particularly ocular toxicities and when to refer for an ophthalmic examination.

Hypoxia-inducible lentiviral gene expression in engineered human macrophages.

BACKGROUND: Human immune cells, including monocyte-derived macrophages, can be engineered to deliver proinflammatory cytokines, bispecific antibodies, and chimeric antigen receptors to support immune responses in different disease settings. When gene expression is regulated by constitutively active promoters, lentiviral payload gene expression is unregulated, and can result in potentially toxic quantities of proteins. Regulated delivery of lentivirally encoded proteins may allow localized or conditional therapeutic protein expression to support safe delivery of adoptively transferred, genetically modified cells with reduced capacity for systemic toxicities. METHODS: In this study, we engineered human macrophages to express genes regulated by hypoxia responsive elements included in the lentiviral promoter region to drive conditional lentiviral gene expression only under hypoxic conditions. We tested transduced macrophages cultured in hypoxic conditions for the transient induced expression of reporter genes and the secreted cytokine, interleukin-12. Expression of hypoxia-regulated genes was investigated both transcriptionally and translationally, and in the presence of human tumor cells in a slice culture system. Finally, hypoxia-regulated gene expression was evaluated in a subcutaneous humanized-mouse cancer model. RESULTS: Engineered macrophages were shown to conditionally and tranisently express lentivirally encoded gene protein products, including IL-12 in hypoxic conditions in vitro. On return to normoxic conditions, lentiviral payload expression returned to basal levels. Reporter genes under the control of hypoxia response elements were upregulated under hypoxic conditions in the presence of human colorectal carcinoma cells and in the hypoxic xenograft model of glioblastoma, suggesting utility for systemic engineered cell delivery capable of localized gene delivery in cancer. CONCLUSIONS: Macrophages engineered to express hypoxia-regulated payloads have the potential to be administered systemically and conditionally express proteins in tissues with hypoxic conditions. In contrast to immune cells that function or survive poorly in hypoxic conditions, macrophages maintain a proinflammatory phenotype that may support continued gene and protein expression when regulated by conditional hypoxia responsive elements and naturally traffic to hypoxic microenvironments, making them ideal vehicles for therapeutic payloads to hypoxic tissues, such as solid tumors. With the ability to fine-tune delivery of potent proteins in response to endogenous microenvironments, macrophage-based cellular therapies may therefore be designed for different disease settings.

The impact of bridging therapy prior to CD19-directed chimeric antigen receptor T-cell therapy in patients with large B-cell lymphoma.

In the relapsed/refractory setting for treatment of large B-cell lymphoma (LBCL), chimeric antigen receptor T-cell (CAR-T) therapy has emerged as an effective treatment modality. Patients often have aggressive disease that requires prompt treatment in the form of bridging therapy (BT) for disease stabilisation while CAR-T cells are manufactured. Patients (n = 75) undergoing CAR-T therapy infusion for LBCL at our institution were identified. A total of 52 (69·3%) received BT and 23 (30·7%) received no BT (NBT). BT modalities included systemic BT (SBT) in 28 patients, radiation BT (RBT) in 14, and high-dose steroid BT (HDS) in 10. There was no difference in incidence of cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome between BT and NBT (P = 0·18 and P = 0·53 respectively). Prolonged cytopenias at Day 180 were more common in BT than NBT (50% vs. 13·3%, P = 0·04). The SBT and RBT subgroups had more cytopenias at Day 180 compared to the HDS and NBT subgroups (58·3% and 57·1% vs. 20% and 13·3% respectively, P = 0·04). Disease response at last follow-up, progression-free survival and overall survival were similar between BT, NBT, and BT subgroups. In summary, BT can be safely considered in patients undergoing CAR-T therapy. However, those undergoing BT with SBT or RBT are at higher risk of prolonged cytopenias after CAR-T therapy.

Genetically engineered macrophages persist in solid tumors and locally deliver therapeutic proteins to activate immune responses.

BACKGROUND: Though currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain resistant to these modes of treatment. In solid tumors, the development of effective antitumor immune responses is hampered by restricted immune cell infiltration and an immunosuppressive tumor microenvironment (TME). An immunotherapy that infiltrates and persists in the solid TME, while providing local, stable levels of therapeutic to activate or reinvigorate antitumor immunity could overcome these challenges faced by current immunotherapies. METHODS: Using lentivirus-driven engineering, we programmed human and murine macrophages to express therapeutic payloads, including Interleukin (IL)-12. In vitro coculture studies were used to evaluate the effect of genetically engineered macrophages (GEMs) secreting IL-12 on T cells and on the GEMs themselves. The effects of IL-12 GEMs on gene expression profiles within the TME and tumor burden were evaluated in syngeneic mouse models of glioblastoma and melanoma and in human tumor slices isolated from patients with advanced gastrointestinal malignancies. RESULTS: Here, we present a cellular immunotherapy platform using lentivirus-driven genetic engineering of human and mouse macrophages to constitutively express proteins, including secreted cytokines and full-length checkpoint antibodies, as well as cytoplasmic and surface proteins that overcomes these barriers. GEMs traffic to, persist in, and express lentiviral payloads in xenograft mouse models of glioblastoma, and express a non-signaling truncated CD19 surface protein for elimination. IL-12-secreting GEMs activated T cells and induced interferon-gamma (IFNγ) in vitro and slowed tumor growth resulting in extended survival in vivo. In a syngeneic glioblastoma model, IFNγ signaling cascades were also observed in mice treated with mouse bone-marrow-derived GEMs secreting murine IL-12. These findings were reproduced in ex vivo tumor slices comprised of intact MEs. In this setting, IL-12 GEMs induced tumor cell death, chemokines and IFNγ-stimulated genes and proteins. CONCLUSIONS: Our data demonstrate that GEMs can precisely deliver titratable doses of therapeutic proteins to the TME to improve safety, tissue penetrance, targeted delivery and pharmacokinetics.

Human macrophages engineered to secrete a bispecific T cell engager support antigen-dependent T cell responses to glioblastoma.

BACKGROUND: Targeted and effective treatment options are needed for solid tumors, including glioblastoma (GBM), where survival rates with standard treatments are typically less than 2 years from diagnosis. Solid tumors pose many barriers to immunotherapies, including therapy half-life and persistence, tumor penetrance, and targeting. Therapeutics delivered systemically may not traffic to the tumor site. If cellular therapies or drugs are able to access the tumor site, or can be delivered directly within the tumor, treatments may not persist for the duration necessary to reduce or eliminate tumor burden. An approach that allows durable and titratable local therapeutic protein delivery could improve antitumor efficacy while minimizing toxicities or unwanted on-target, off-tissue effects. METHODS: In this study, human monocyte-derived macrophages were genetically engineered to secrete a bispecific T cell engager (BiTE) specific to the mutated epidermal growth factor variant III (EGFRvIII) expressed by some GBM tumors. We investigated the ability of lentivirally modified macrophages to secrete a functional BiTE that can bind target tumor antigen and activate T cells. Secreted BiTE protein was assayed in a range of T cell functional assays in vitro and in subcutaneous and intracranial GBM xenograft models. Finally, we tested genetically engineered macrophages (GEMs) secreting BiTE and the proinflammatory cytokine interleukin (IL)-12 to amplify T cell responses in vitro and in vivo. RESULTS: Transduced human macrophages secreted a lentivirally encoded functional EGFRvIII-targeted BiTE protein capable of inducing T cell activation, proliferation, degranulation, and killing of antigen-specific tumor cells. Furthermore, BiTE secreting macrophages reduced early tumor burden in both subcutaneous and intracranial mouse models of GBM, a response which was enhanced using macrophages that were dual transduced to secrete both the BiTE protein and single chain IL-12, preventing tumor growth in an aggressive GBM model. CONCLUSIONS: The ability of macrophages to infiltrate and persist in solid tumor tissue could overcome many of the obstacles associated with systemic delivery of immunotherapies. We have found that human GEMs can locally and constitutively express one or more therapeutic proteins, which may help recruit T cells and transform the immunosuppressive tumor microenvironment to better support antitumor immunity.

HPV type 16 E6 and NFX1-123 augment JNK signaling to mediate keratinocyte differentiation and L1 expression.

The HPV life cycle is differentiation-dependent, with cellular differentiation driving initiation of the late, productive stage of the viral life cycle. Here, we identify a role for the protein NFX1-123 in regulating keratinocyte differentiation and events of the late HPV life cycle. NFX1-123 itself increased with differentiation of epithelial cells. Greater NFX1-123 augmented differentiation marker expression and JNK phosphorylation in differentiating 16E6-expressing human foreskin keratinocytes (16E6 HFKs). This was associated with altered expression of MKK4 and MKK7, upstream kinase regulators of JNK phosphorylation. Modulating levels of NFX1-123 in HPV16-positive W12E cells recapitulated the effects on differentiation markers, JNK phosphorylation, and MKK4/7 seen in 16E6 HFKs. Crucially, levels of NFX1-123 also correlated with expression of L1, the capsid protein of HPV. Altogether, these studies define a role for NFX1-123 in mediating epithelial differentiation through the JNK signaling pathway, potentially linking expression of cellular genes and HPV genes during differentiation.

NFX1-123 is highly expressed in cervical cancer and increases growth and telomerase activity in HPV 16E6 expressing cells.

A significant contributor to women's cancer mortality worldwide is cervical cancer, which is caused by high-risk human papillomavirus (HR HPV). The two viral oncoproteins of HR HPV, E6 and E7, partner with host cell proteins to target oncogenic proteins and pathways. Previously, we have shown HR HPV type 16 E6 (16E6) interacts with the host protein NFX1-123 to target telomerase and cellular immortalization, requiring NFX1-123 to fully upregulate telomerase activity. We now report that NFX1-123 is highly expressed in primary cervical cancers. In vitro, cells expressing 16E6 and overexpressing NFX1-123 have extended active growth, decreased senescence marker staining, and more rapid cell cycling compared to 16E6 expressing cells with endogenous amounts of NFX1-123. These findings were associated with increased telomerase activity and augmented expression of its catalytic subunit, hTERT. In complement, HPV 16 positive cervical cancer cell lines with knocked down NFX1-123 had slowed growth and reduced hTERT over time. In cells that express HR HPV E6, greater expression of NFX1-123 can modify active cellular growth and augment hTERT expression and telomerase activity over time, potentially supporting the initiation and progression of HPV-associated cancers.

Quantitative RNAseq analysis of Ugandan KS tumors reveals KSHV gene expression dominated by transcription from the LTd downstream latency promoter.

KSHV is endemic in Uganda and the HIV epidemic has dramatically increased the incidence of Kaposi sarcoma (KS). To investigate the role of KSHV in the development of KS, we obtained KS biopsies from ART-naïve, HIV-positive individuals in Uganda and analyzed the tumors using RNAseq to globally characterize the KSHV transcriptome. Phylogenetic analysis of ORF75 sequences from 23 tumors revealed 6 distinct genetic clusters with KSHV strains exhibiting M, N or P alleles. RNA reads mapping to specific unique coding sequence (UCDS) features were quantitated using a gene feature file previously developed to globally analyze and quantitate KSHV transcription in infected endothelial cells. A pattern of high level expression was detected in the KSHV latency region that was common to all KS tumors. The clear majority of transcription was derived from the downstream latency transcript promoter P3(LTd) flanking ORF72, with little evidence of transcription from the P1(LTc) latency promoter, which is constitutive in KSHV-infected lymphomas and tissue-culture cells. RNAseq data provided evidence of alternate P3(LTd) transcript editing, splicing and termination resulting in multiple gene products, with 90% of the P3(LTd) transcripts spliced to release the intronic source of the microRNAs K1-9 and 11. The spliced transcripts encode a regulatory uORF upstream of Kaposin A with alterations in intervening repeat sequences yielding novel or deleted Kaposin B/C-like sequences. Hierarchical clustering and PCA analysis of KSHV transcripts revealed three clusters of tumors with different latent and lytic gene expression profiles. Paradoxically, tumors with a latent phenotype had high levels of total KSHV transcription, while tumors with a lytic phenotype had low levels of total KSHV transcription. Morphologically distinct KS tumors from the same individual showed similar KSHV gene expression profiles suggesting that the tumor microenvironment and host response play important roles in the activation level of KSHV within the infected tumor cells.