Engineered NKG2C + NK-like T cells exhibit superior antitumor efficacy while mitigating cytokine release syndrome.

Engineered T and NK cell therapies have widely been used to treat hematologic malignancies and solid tumors, with promising clinical results. Current chimeric antigen receptor (CAR) T cell therapeutics have, however, been associated with treatment-related adverse events such as cytokine release syndrome (CRS) and are prone to immunologic exhaustion. CAR-NK therapeutics, while not associated with CRS, have limited in vivo persistence. We now demonstrate that an NK-like TCRαß + CD8 T cell subset, identified and expanded ex vivo through its expression of the activating receptor NKG2C (NKG2C + NK-like T cells), can be transduced to express a second-generation CD19 CAR (1928z), resulting in superior tumor clearance, longer persistence and decreased exhaustion compared to conventional 1928z CAR + CD8 T cells and 1928z CAR+ NK cells. Moreover, CAR-modified NKG2C + NK-like T cells resulted in significantly reduced CRS compared to conventional CAR + CD8 T cells. Similarly, NKG2C + NK-like T cells engineered with a TCR targeting the NY-ESO-1 antigen exhibit robust tumor control and minimal exhaustion compared to TCR-engineered conventional CD8 T cells. These data establish NKG2C + NK-like T cells as a robust platform for cell engineering, and offer a safer, more durable alternative to conventional CAR-T and CAR-NK therapies.

Augmenting CAR T-cell Functions with LIGHT.

Chimeric antigen receptor (CAR) T-cell therapy has resulted in remarkable clinical success in the treatment of B-cell malignancies. However, its clinical efficacy in solid tumors is limited, primarily by target antigen heterogeneity. To overcome antigen heterogeneity, we developed CAR T cells that overexpress LIGHT, a ligand of both lymphotoxin-ß receptor on cancer cells and herpes virus entry mediator on immune cells. LIGHT-expressing CAR T cells displayed both antigen-directed cytotoxicity mediated by the CAR and antigen-independent killing mediated through the interaction of LIGHT with lymphotoxin-ß receptor on cancer cells. Moreover, CAR T cells expressing LIGHT had immunostimulatory properties that improved the cells' proliferation and cytolytic profile. These data indicate that LIGHT-expressing CAR T cells may provide a way to eliminate antigen-negative tumor cells to prevent antigen-negative disease relapse.

Developing a membrane-proximal CD33-targeting CAR T cell.

BACKGROUND: CD33 is a tractable target in acute myeloid leukemia (AML) for chimeric antigen receptor (CAR) T cell therapy, but clinical success is lacking. METHODS: We developed 3P14HLh28Z, a novel CD33-directed CD28/CD3Z-based CAR T cell derived from a high-affinity binder obtained through membrane-proximal fragment immunization in humanized mice. RESULTS: We found that immunization exclusively with the membrane-proximal domain of CD33 is necessary for identification of membrane-proximal binders in humanized mice. Compared with clinically validated lintuzumab-based CAR T cells targeting distal CD33 epitopes, 3P14HLh28Z showed enhanced in vitro functionality as well as superior tumor control and increased overall survival in both low antigen density and clinically relevant patient-derived xenograft models. Increased activation and enhanced polyfunctionality led to enhanced efficacy. CONCLUSIONS: Showing for the first time that a membrane-proximal CAR is superior to a membrane-distal one in the setting of CD33 targeting, our results demonstrate the rationale for targeting membrane-proximal epitopes with high-affinity binders. We also demonstrate the importance of optimizing CAR T cells for functionality in settings of both low antigen density and clinically relevant patient-derived models.

The Effect of Neutropenia and Filgrastim (G-CSF) on Cancer Patients With Coronavirus Disease 2019 (COVID-19) Infection.

BACKGROUND: Neutropenia is commonly encountered in cancer patients. Recombinant human granulocyte colony-stimulating factor (G-CSF, filgrastim), a cytokine that initiates proliferation and differentiation of mature granulocytes, is widely given to oncology patients to counteract neutropenia, reducing susceptibility to infection. However, the clinical impact of neutropenia and G-CSF use in cancer patients with coronavirus disease 2019 (COVID-19) remains unknown. METHODS: An observational cohort of 379 actively treated cancer patients with COVID-19 was assembled to investigate links between concurrent neutropenia and G-CSF administration on COVID-19-associated respiratory failure and death. These factors were encoded as time-dependent predictors in an extended Cox model, controlling for age and underlying cancer diagnosis. To determine whether the degree of granulocyte response to G-CSF affected outcomes, the degree of response to G-CSF, based on rise in absolute neutrophil count (ANC) 24 hours after growth factor administration, was also incorporated into a similar Cox model. RESULTS: In the setting of active COVID-19 infection, outpatient receipt of G-CSF led to an increased number of hospitalizations (hazard ratio [HR]: 3.54, 95% confidence interval [CI]: 1.25-10.0, P value: .017). Furthermore, among inpatients, G-CSF administration was associated with increased need for high levels of oxygen supplementation and death (HR: 3.56, 95% CI: 1.19-10.2, P value: .024). This effect was predominantly seen in patients that exhibited a high response to G-CSF based on their ANC increase post-G-CSF administration (HR: 7.78, 95% CI: 2.05-27.9, P value: .004). CONCLUSIONS: The potential risks versus benefits of G-CSF administration should be considered in neutropenic cancer patients with COVID-19, because G-CSF administration may lead to worsening clinical and respiratory status.

Human cytomegalovirus expands a CD8+ T cell population with loss of BCL11B expression and gain of NK cell identity.

CD8+ T cells not only are critical mediators of adaptive immunity but also may exhibit innate-like properties such as surface expression of NKG2C, an activating receptor typically associated with natural killer (NK) cells. We demonstrate that, similar to NK cells, NKG2C+TCRαß+CD8+ T cells are associated with prior human cytomegalovirus (HCMV) exposure. In addition to expressing several NK cell markers such as CD56 and KIR, NKG2C+CD8+ T cells are oligoclonal and do not up-regulate PD-1 even in response to persistent activation. Furthermore, we found that NKG2C+CD8+ T cells from some individuals exhibited strong effector function against leukemia cells and HCMV-infected fibroblasts, which was dictated by both NKG2C and TCR specificity. Transcriptomic analysis revealed that the transcription factor BCL11B, a regulator of T cell developmental fate, is down-regulated in NKG2C+CD8+ T cells when compared with conventional NKG2C−CD8+ T cells. BCL11B deletion in conventional CD8+ T cells resulted in the emergence of a similar innate-like CD56+CD94+DAP12+NKG2C+CD45RA+CCR7−PD-1−/low T cell population with activity against HLA-E+ targets. On the basis of their intrinsic capacity to recognize diseased cells coupled with lack of PD-1 induction, NKG2C+CD8+ T cells represent a lymphocyte population that resides at the boundary between innate and adaptive immunity, presenting an attractive alternative for cellular therapy, including CAR T cell­based therapies.

Clinical and molecular predictors of response and survival following venetoclax therapy in relapsed/refractory AML.

Azacitidine + venetoclax, decitabine + venetoclax, and low-dose cytarabine + venetoclax are now standard treatments for newly diagnosed older or unfit patients with acute myeloid leukemia (AML). Although these combinations are also commonly used in relapsed or refractory AML (RR-AML), clinical and molecular predictors of response and survival in RR-AML are incompletely understood. We retrospectively analyzed clinical and molecular characteristics and outcomes for 86 patients with RR-AML who were treated with venetoclax combinations. The complete remission (CR) or CR with incomplete hematologic recovery (CRi) rate was 24%, and the overall response rate was 31% with the inclusion of a morphologic leukemia-free state. Azacitidine + venetoclax resulted in higher response rates compared with low-dose cytarabine + venetoclax (49% vs 15%; P = .008). Median overall survival (OS) was 6.1 months, but it was significantly longer with azacitidine + venetoclax compared with low-dose cytarabine + venetoclax (25 vs 3.9 months; P = .003). This survival advantage of azacitidine + venetoclax over low-dose cytarabine + venetoclax persisted when patients were censored for subsequent allogeneic stem cell transplantation (8.1 vs 3.9 months; P = .035). Mutations in NPM1 were associated with higher response rates, whereas adverse cytogenetics and mutations in TP53, KRAS/NRAS, and SF3B1 were associated with worse OS. Relapse was driven by diverse mechanisms, including acquisition of novel mutations and an increase in cytogenetic complexity. Venetoclax combination therapy is effective in many patients with RR-AML, and pretreatment molecular characteristics may predict outcomes. Trials that evaluate novel agents in combination with venetoclax therapy in patients with RR-AML that have adverse risk genomic features are warranted.

Oncologic immunomodulatory agents in patients with cancer and COVID-19.

Corticosteroids, anti-CD20 agents, immunotherapies, and cytotoxic chemotherapy are commonly used in the treatment of patients with cancer. It is unclear how these agents affect patients with cancer who are infected with SARS-CoV-2. We retrospectively investigated associations between SARS-CoV-2-associated respiratory failure or death with receipt of the aforementioned medications and with pre-COVID-19 neutropenia. The study included all cancer patients diagnosed with SARS-CoV-2 at Memorial Sloan Kettering Cancer Center until June 2, 2020 (N = 820). We controlled for cancer-related characteristics known to predispose to worse COVID-19 as well as level of respiratory support during corticosteroid administration. Corticosteroid administration was associated with worse outcomes prior to use of supplemental oxygen; no statistically significant difference was observed in sicker cohorts. In patients with metastatic thoracic cancer, 9 of 25 (36%) and 10 of 31 (32%) had respiratory failure or death among those who did and did not receive immunotherapy, respectively. Seven of 23 (30%) and 52 of 187 (28%) patients with hematologic cancer had respiratory failure or death among those who did and did not receive anti-CD20 therapy, respectively. Chemotherapy itself was not associated with worse outcomes, but pre-COVID-19 neutropenia was associated with worse COVID-19 course. Relative prevalence of chemotherapy-associated neutropenia in previous studies may account for different conclusions regarding the risks of chemotherapy in patients with COVID-19. In the absence of prospective studies and evidence-based guidelines, our data may aid providers looking to assess the risks and benefits of these agents in caring for cancer patients in the COVID-19 era.

Cytokine IL-36γ improves CAR T-cell functionality and induces endogenous antitumor response.

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable responses in B-cell malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR T cells and immune escape. These clinical challenges have compromised the long-term efficacy of CAR T-cell therapy and call for the development of novel CAR designs. We demonstrated that CAR T cells secreting a cytokine interleukin-36γ (IL-36γ) showed significantly improved CAR T-cell expansion and persistence, and resulted in superior tumor eradication compared with conventional CAR T cells. The enhanced cellular function by IL-36γ was mediated through an autocrine manner. In addition, activation of endogenous antigen-presenting cells (APCs) and T cells by IL-36γ aided the formation of a secondary antitumor response, which delayed the progression of antigen-negative tumor challenge. Together, our data provide preclinical evidence to support the translation of this design for an improved CAR T-cell-mediated antitumor response.

CD103+ cDC1 and endogenous CD8+ T cells are necessary for improved CD40L-overexpressing CAR T cell antitumor function.

While effective in specific settings, adoptive chimeric antigen receptor (CAR) T cell therapy for cancer requires further improvement and optimization. Our previous results show that CD40L-overexpressing CAR T cells mobilize endogenous immune effectors, resulting in improved antitumor immunity. However, the cell populations required for this protective effect remain to be identified. Here we show, by analyzing Batf3-/- mice lacking the CD103+ conventional dendritic cell type 1 (cDC1) subpopulation important for antigen cross-presentation, that CD40L-overexpressing CAR T cells elicit an impaired antitumor response in the absence of cDC1s. We further find that CD40L-overexpressing CAR T cells stimulate tumor-resident CD11b-CD103- double-negative (DN) cDCs to proliferate and differentiate into cDC1s in wild-type mice. Finally, re-challenge experiments show that endogenous CD8+ T cells are required for protective antitumor memory in this setting. Our findings thus demonstrate the stimulatory effect of CD40L-overexpressing CAR T cells on innate and adaptive immune cells, and provide a rationale for using CD40L-overexpressing CAR T cells to improve immunotherapy responses.

Functional impact of cancer patient-associated Bcl-xL mutations.

Bcl-xL, an antiapoptotic protein, is frequently overexpressed in cancer to promote survival of tumor cells. However, we have previously shown that Bcl-xL promotes migration, invasion, and metastasis independent of its antiapoptotic function in mitochondria. The pro-metastatic function of Bcl-xL may require its translocation into the nucleus. Besides overexpression, patient-associated mutations of Bcl-xL have been identified in large-scale cancer genomics projects. Understanding the functions of these mutations will guide the development of precision medicine. Here, we selected four patient-associated Bcl-xL mutations, R132W, N136K, R165W, and A201T, to investigate their impacts on antiapoptosis, migration, and nuclear translocation. We found that all four mutation proteins could be detected in both the nucleus and cytosol. Although all four mutations disrupted the antiapoptosis function, one of these mutants, N136K, significantly improved the ability to promote cell migration. These data suggest the importance of developing novel Bcl-xL inhibitors to ablate both antiapoptotic and pro-metastatic functions of Bcl-xL in cancer.

BCMA-Targeted CAR T-cell Therapy plus Radiotherapy for the Treatment of Refractory Myeloma Reveals Potential Synergy.

We present a case of a patient with multiply relapsed, refractory myeloma whose clinical course showed evidence of a synergistic abscopal-like response to chimeric antigen receptor (CAR) T-cell therapy and localized radiotherapy (XRT). Shortly after receiving B-cell maturation antigen (BCMA)-targeted CAR T-cell therapy, the patient required urgent high-dose steroids and XRT for spinal cord compression. Despite the steroids, the patient had a durable systemic response that could not be attributed to XRT alone. Post-XRT findings included a second wave of fever and increased CRP and IL6, beginning 21 days after CAR T cells, which is late for cytokine-release syndrome from CAR T-cell therapy alone on this trial. Given this response, which resembled cytokine-release syndrome, immediately following XRT, we investigated changes in the patient's T-cell receptor (TCR) repertoire over 10 serial time points. Comparing T-cell diversity via Morisita's overlap indices (CD ), we discovered that, although the diversity was initially stable after CAR T-cell therapy compared with baseline (CD = 0.89-0.97, baseline vs. 4 time points after CAR T cells), T-cell diversity changed after the conclusion of XRT, with >30% newly expanded TCRs (CD = 0.56-0.69, baseline vs. 4 time points after XRT). These findings suggest potential synergy between radiation and CAR T-cell therapies resulting in an abscopal-like response.

CD40 Ligand-Modified Chimeric Antigen Receptor T Cells Enhance Antitumor Function by Eliciting an Endogenous Antitumor Response.

Chimeric antigen receptor (CAR) T cells provide great efficacy in B cell malignancies. However, improved CAR T cell therapies are still needed. Here, we engineered tumor-targeted CAR T cells to constitutively express the immune-stimulatory molecule CD40 ligand (CD40L) and explored efficacy in different mouse leukemia/lymphoma models. We observed that CD40L+ CAR T cells circumvent tumor immune escape via antigen loss through CD40/CD40L-mediated cytotoxicity and induction of a sustained, endogenous immune response. After adoptive cell transfer, the CD40L+ CAR T cells displayed superior antitumor efficacy, licensed antigen-presenting cells, enhanced recruitment of immune effectors, and mobilized endogenous tumor-recognizing T cells. These effects were absent in Cd40-/- mice and provide a rationale for the use of CD40L+ CAR T cells in cancer treatment.

CARs of the future.

CAR T cells have revolutionized the treatment of relapsed and refractory CD19-positive leukemia and lymphoma. Unfortunately, the majority of patients treated will not achieve durable remissions. Reasons for these suboptimal clinical outcomes can be tied back to intrinsic CAR T cell design and manufacturing processes, factors that are highly amenable to modification and improvement. As CAR T cell therapy is being deployed in spaces outside of CD19-positive disease, these limitations, complications, and setbacks need to be overcome, allowing for the full potential of this novel therapy to be realized. Preclinical work has begun tackling these major roadblocks, paving the way for potentially off-the-shelf products that are safer and more potent. In time, a number of these advances will be translated to the clinic and usher in an era of CARs of the future.

Engineered Tumor-Targeted T Cells Mediate Enhanced Anti-Tumor Efficacy Both Directly and through Activation of the Endogenous Immune System.

Chimeric antigen receptor (CAR) T cell therapy has proven clinically beneficial against B cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma. However, suboptimal clinical outcomes have been associated with decreased expansion and persistence of adoptively transferred CAR T cells, antigen-negative relapses, and impairment by an immunosuppressive tumor microenvironment. Improvements in CAR T cell design are required to enhance clinical efficacy, as well as broaden the applicability of this technology. Here, we demonstrate that interleukin-18 (IL-18)-secreting CAR T cells exhibit enhanced in vivo expansion and persistence and significantly increase long-term survival in syngeneic mouse models of both hematological and solid malignancies. In addition, we demonstrate that IL-18-secreting CAR T cells are capable of modulating the tumor microenvironment, as well as enhancing an effective endogenous anti-tumor immune response. IL-18-secreting CAR T cells represent a promising strategy to enhance the clinical outcomes of adoptive T cell therapy.

Cytokine release syndrome after radiation therapy: case report and review of the literature.

BACKGROUND: Cytokine release syndrome (CRS) has been reported after immunologic manipulations, most often through therapeutic monoclonal antibodies. To our knowledge, CRS after radiation therapy (RT) for cancer has not been reported before. The development of unusual clinical signs and symptoms after RT led us to investigate the possibility of CRS after RT and review the medical literature on this topic. CASE PRESENTATION: A 65 year-old man with untreated chronic lymphocytic leukemia and recurrent, metastatic Merkel cell carcinoma undergoing anti-programmed death 1 (PD1) immunotherapy was referred for palliative RT to sites of progressing metastases. Within hours of each weekly dose of RT, he experienced fever, tachycardia, hypotension, rash, dyspnea, and rigors. Based on clinical suspicion for CRS, blood cytokine measurements were performed 1 h after the second and third dose of RT and demonstrated tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) levels approximately ten-fold higher than normal. These were near normal immediately prior to the third dose of RT, and resolved to normal levels 3 weeks after RT. He experienced rapid regression of irradiated tumors, with development of new sites of metastases soon thereafter. A literature review revealed no clinical cases of CRS after RT for cancer. CONCLUSIONS: RT during anti-PD1 immunotherapy in a patient with underlying immune dysfunction appeared to be the putative mediator of an immune process which yielded significant increases in pro-inflammatory cytokines, and produced the clinical symptoms meeting the definition of grade 3 CRS. This case demonstrates the capability of RT to elicit immune-related adverse events.

At the Bench: Chimeric antigen receptor (CAR) T cell therapy for the treatment of B cell malignancies.

The chimeric antigen receptor (CAR) represents the epitome of cellular engineering and is one of the best examples of rational biologic design of a synthetic molecule. The CAR is a single polypeptide with modular domains, consisting of an antibody-derived targeting moiety, fused in line with T cell-derived signaling domains, allowing for T cell activation upon ligand binding. T cells expressing a CAR are able to eradicate selectively antigen-expressing tumor cells in a MHC-independent fashion. CD19, a tumor-associated antigen (TAA) present on normal B cells, as well as most B cell-derived malignancies, was an early target of this technology. Through years of experimental refinement and preclinical optimization, autologously derived CD19-targeting CAR T cells have been successfully, clinically deployed, resulting in dramatic and durable antitumor responses but not without therapy-associated toxicity. As CD19-targeted CAR T cells continue to show clinical success, work at the bench continues to be undertaken to increase further the efficacy of this therapy, while simultaneously minimizing the risk for treatment-related morbidities. In this review, we cover the history and evolution of CAR technology and its adaptation to targeting CD19. Furthermore, we discuss the future of CAR T cell therapy and the need to ask, as well as answer, critical questions as this treatment modality is being translated to the clinic.