Next-generation chimeric antigen receptors for T- and natural killer-cell therapies against cancer.

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cells has led to a paradigm shift in the treatment of various hematologic malignancies. However, the broad application of this approach for myeloid malignancies and solid cancers has been limited by the paucity and heterogeneity of target antigen expression, and lack of bona fide tumor-specific antigens that can be targeted without cross-reactivity against normal tissues. This may lead to unwanted on-target off-tumor toxicities that could undermine the desired antitumor effect. Recent advances in synthetic biology and genetic engineering have enabled reprogramming of immune effector cells to enhance their selectivity toward tumors, thus mitigating on-target off-tumor adverse effects. In this review, we outline the current strategies being explored to improve CAR selectivity toward tumor cells with a focus on natural killer (NK) cells, and the progress made in translating these strategies to the clinic.

Safety and long-term survival results of the addition of inotuzumab ozogamicin to the conditioning regimen of allogeneic stem cell transplantation: A single-center phase 1,2 trial.

Here we report on the first prospective study evaluating the safety and long-term survival when an escalating dose of inotuzumab ozogamicin (INO) (0.6, 1.2, or 1.8 mg/m2 on day 13) was added to one alkylator-containing conditioning regimen in patients with relapsed CD22 (+) lymphoid malignancies who were candidates for hematopoietic stem cell transplantation (HSCT). Twenty-six patients were enrolled. Six (23%) of these patients entered the phase 1 study: four were treated at an INO dose of 0.6 mg/m2 and two at dose of 1.2 mg/m2. None of these patients experienced dose-limiting toxicities. The remaining 20 (77%) patients entered the phase 2 part of the study at the maximum dose of 1.8 mg/m2. One patient developed VOD; this patient had received nivolumab immediately before HSCT while simultaneously experiencing hyperacute graft-vs-host disease (GVHD). Treatment-related mortality (TRM) at 5 years was 12%. With a median follow-up of 48.7 months, the 5-year overall survival (OS) and progression-free survival (PFS) rates were 84% and 80%, respectively. Compared with a historical cohort who received same conditioning for HSCT but without INO (n = 56), the INO group showed no significant differences in incidence of liver toxicity, engraftment time, TRM, or risk of acute GVHD. Patients with lymphoma who received INO had a trend for a better 5-year OS (93% versus 68%) and PFS (93% versus 58%) than those in the control group. In conclusion, our results showed that INO is safe with no increased risk of VOD when combined with one alkylator-containing regimen of HSCT.

Microbiome-intestine cross talk during acute graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) offers cure for a variety of conditions, in particular, but not limited to, hematologic malignancies. However, it can be associated with life-threatening complications, including graft-versus-host disease (GVHD) and infections, which are factors limiting its widespread use. Technical advances in the field of microbiome research have allowed for a better understanding of the microbial flora of the human intestine, as well as dissection of their interactions with the host immune system in allo-SCT and posttransplant complications. There is growing evidence that the commensal microbiome is frequently dysregulated following allo-SCT and that this dysbiosis can predispose to adverse clinical outcomes, especially including acute intestinal GVHD and reduced overall survival. In this review, we discuss the interactions between the microbiome and the components of the immune system that play a major role in the pathways leading to the inflammatory state of acute intestinal GVHD. We also discuss the microbiome-centered strategies that have been devised or are actively being investigated to improve the outcomes of allo-SCT patients in regard to acute intestinal GVHD.

Chimeric antigen receptor (CAR) natural killer (NK)-cell therapy: leveraging the power of innate immunity.

Chimeric antigen receptor (CAR) T cells are a rapidly emerging form of cancer treatment, and have resulted in remarkable responses in refractory lymphoid malignancies. However, their widespread clinical use is limited by toxicity related to cytokine release syndrome and neurotoxicity, the logistic complexity of their manufacturing, cost and time-to-treatment for autologous CAR-T cells, and the risk of graft-versus-host disease (GvHD) associated with allogeneic CAR-T cells. Natural killer (NK) cells have emerged as a promising source of cells for CAR-based therapies due to their ready availability and safety profile. NK cells are part of the innate immune system, providing the first line of defence against pathogens and cancer cells. They produce cytokines and mediate cytotoxicity without the need for prior sensitisation and have the ability to interact with, and activate other immune cells. NK cells for immunotherapy can be generated from multiple sources, such as expanded autologous or allogeneic peripheral blood, umbilical cord blood, haematopoietic stem cells, induced pluripotent stem cells, as well as cell lines. Genetic engineering of NK cells to express a CAR has shown impressive preclinical results and is currently being explored in multiple clinical trials. In the present review, we discuss both the preclinical and clinical trial progress made in the field of CAR NK-cell therapy, and the strategies to overcome the challenges encountered.

Landscape of Immunotherapy in Genitourinary Malignancies.

The past decade has witnessed a revolution in the development of immune checkpoint inhibitors for the treatment of multiple tumor types, including genitourinary cancers. Immune checkpoint inhibitors have notably improved the treatment outcomes of patients with metastatic renal cell carcinoma and metastatic urothelial carcinoma. In prostate cancer, the role of immunotherapy with checkpoint inhibitors is not yet established except for microsatellite instability high (MSI-H) tumors. Other immunotherapeutic approaches that have been explored in these malignancies include cytokines, vaccines, and cellular therapy. Ongoing studies are exploring the use of immunotherapy combinations as well as combination with chemotherapy and targeted therapy in these types of tumors. The use of immunotherapy beyond the metastatic setting is an active area of research. Moreover, there is great interest in biomarker development to predict response to immunotherapy and risk of toxicity. This book chapter is a comprehensive review of immunotherapeutic approaches, both approved and investigational, for the treatment of renal cell carcinoma, urothelial carcinoma, and prostate cancer.

Targeted therapy paves the way for the cure of acute lymphoblastic leukaemia.

The past decade has witnessed tremendous progress in the treatment of acute lymphoblastic leukaemia (ALL), primarily due to the development of targeted therapies, including tyrosine kinase inhibitors targeting BCR-ABL1 tyrosine kinase, monoclonal antibodies targeting cell surface antigens (CD19, CD20 and CD22), bispecific antibodies and chimeric antigen receptor T- cell therapy. A number of new therapies have been approved by the US Food and Drug Administration in the past 5 years, including blinatumomab in 2014, inotuzumab ozagamicin in 2017 and tisagenlecleucel in 2017 for relapsed/refractory ALL. This has led to tremendous improvement in long-term survival, of more than 50% in patients with precursor B-ALL [50-70% in patients with Philadelphia chromosome (Ph)-positive ALL)], 50-60% in T-ALL and 80% in mature B-ALL. Research is ongoing to optimize the benefit of targeted therapeutics with the goal of decreasing the use of cytotoxic therapies.

Current Landscape of Immunotherapy in Genitourinary Malignancies.

The past decade has witnessed a revolution of immune checkpoint inhibitors in the treatment of multiple tumor types, including genitourinary cancers. Immune checkpoint inhibitors improved the treatment outcomes of patients with metastatic renal cell carcinoma and metastatic urothelial carcinoma. In prostate cancer, the role of immunotherapy with checkpoint inhibitors is not yet established, but clinical trials investigating their use are ongoing. Other immunotherapeutic approaches that have been explored in these malignancies include cytokines, vaccines, and cellular therapy. Ongoing studies are exploring the use of immunotherapy combinations as well as combination with chemotherapy and targeted therapy in these types of tumors. The use of immunotherapy beyond the metastatic setting is an active area of research. Moreover, there is a great interest in biomarker development to predict response to immunotherapy and risk of toxicity. This chapter is a comprehensive review of the immunotherapeutic approaches, both approved and investigational, for the treatment of renal cell carcinoma, urothelial carcinoma, and prostate cancer.

Targeting advanced urothelial carcinoma-developing strategies.

PURPOSE OF REVIEW: Advanced urothelial carcinoma is a heterogeneous disease with high burden of morbidity, mortality, and cost. Significant progress has been made in understanding the biology of the disease and the development of immunotherapies and targeted therapies. In this review, we summarize the current and future therapeutic approaches in the management of urothelial carcinoma. RECENT FINDINGS: Advances in immune checkpoint inhibitors resulted in the Food and Drug Administration (FDA) approvals of atezolizumab in 2016, and pembrolizumab, avelumab, durvalumab, and nivolumab in 2017 for the treatment of advanced urothelial carcinoma. More recently, development of inhibitors targeting the fibroblast growth factor receptor genetic alterations and antibody-drug conjugates targeting specific cell surface antigens (trop2, nectin4, and SLITRK6) resulted in several FDA breakthrough designations for urothelial carcinoma. CONCLUSION: The development of novel therapies targeting the immune and molecular pathways of advanced urothelial carcinoma is promising for the improvement of outcomes in this lethal disease. Ongoing efforts are poised to optimize therapeutic options in the post-chemotherapy arena. In the era of precision medicine, the future of urothelial carcinoma lies in using less cytotoxic chemotherapy, more targeted therapy and immunotherapy, and possibly a combination of these therapeutic approaches.

Allogeneic Hematopoietic Stem Cell Transplantation for GATA2 Deficiency Using a Busulfan-Based Regimen.

Allogeneic hematopoietic stem cell transplantation (HSCT) reverses the bone marrow failure syndrome due to GATA2 deficiency. The intensity of conditioning required to achieve reliable engraftment and prevent relapse remains unclear. Here, we describe the results of a prospective study of HSCT in 22 patients with GATA2 deficiency using a busulfan-based conditioning regimen. The study included 2 matched related donor (MRD) recipients, 13 matched unrelated donor (URD) recipients, and 7 haploidentical related donor (HRD) recipients. MRD and URD recipients received 4 days of busulfan and 4 days of fludarabine. HRD recipients received low-dose cyclophosphamide for 2 days, fludarabine for 5 days, 2 to 3 days of busulfan depending on cytogenetics, and 200 cGy total body irradiation. MRD and URD recipients received tacrolimus and short-course methotrexate for graft-versus-host disease (GVHD) prophylaxis. HRD recipients received high-dose post-transplant cyclophosphamide (PTCy) followed by tacrolimus and mycophenolate mofetil. At a median follow-up of 24 months (range, 9 to 50), 19 of 22 patients were alive with reversal of the disease phenotype and correction of the myelodysplastic syndrome, including eradication of cytogenetic abnormalities. Three patients died: 1 from refractory acute myelogenous leukemia, 1 from GVHD, and 1 from sepsis. There was a 26% incidence of grades III to IV acute GVHD in the MRD and URD groups and no grades III to IV acute GVHD in the HRD cohort. Similarly, there was a 46% incidence of chronic GVHD in the MRD and URD cohorts, whereas only 28% of HRD recipients developed chronic GVHD. Despite excellent overall disease-free survival (86%), GVHD remains a limitation using standard prophylaxis for GVHD. We are currently extending the use of PTCy to the MRD and URD cohorts to reduce GVHD.

DA-EPOCH-R for post-transplant lymphoproliferative disorders.

BACKGROUND: Post-transplant lymphoproliferative disorders (PTLD) are a potentially fatal group of neoplasms arising in an immunodeficient environment. Although the cornerstone of treatment is reduced immunosuppression (RI), advanced cases often warrant treatment with chemoimmunotherapy. The chemoimmunotherapy regimen of dose-adjusted (DA)-EPOCH-R is superior to R-CHOP in HIV associated aggressive lymphomas, suggesting that it might also be favorable in the setting of PTLD. METHODS: We performed a retrospective analysis of patients with advanced monomorphic PTLD treated with first line DA-EPOCH-R in addition to RI at our institution from 2003-2016. RESULTS: Seven patients were included. Mean age was 51 and mean time from transplant to diagnosis was 71 months. Six of the seven patients received a kidney transplant, six had stage III or IV disease, six had tumors that were EBV positive, and six completed therapy. All six patients who completed therapy achieved a complete response. Mean PFS and OS were 46.6 and 52.6 months, respectively. Treatment was well-tolerated with no significant treatment related morbidity or mortality. CONCLUSIONS: Our findings support several observations in the literature that DA-EPOCH-R is efficacious and well-tolerated for the treatment of advanced, monomorphic PTLD.

Perspective of Lebanese oncologists on the symptom burden among adult cancer patients.

Background: Proper approach to symptom control in cancer patients requires a good understanding of the prevalence of the various symptoms these patients have. Aim: This study aims at assessing the Lebanese oncologists' point of view concerning the symptom burden among cancer patients of Lebanon and comparing their opinions to the real complaints of patients themselves. Methods: A cross-sectional study was conducted among a representative sample of the Lebanese medical oncologists. Thirty-six physicians filled out a questionnaire regarding their demographics as well as the symptom profile of their patients. Those results were compared to the ones obtained from our previous study about symptom profile as reported by patients. Results: Fatigue was the symptom most our patients suffered from according to their physicians (64.167%). Also, a good percentage of physicians agreed that patients suffer from appetite loss, pain, weight loss, and nausea. When compared to the patients' reports of their own symptoms, a statistically significant difference existed between the two profiles for the majority of symptoms (14 out of 19). Also, for the majority of symptoms, physicians were found to underestimate the percentage of patients suffering from each symptom. Conclusion: This study shows that there is a statistically significant difference between the physicians' and the patients' perspectives on most of the common distressing symptoms. This entails more detailed questioning of the cancer patients about their distressing symptoms.

Role of baseline echocardiography prior to initiation of anthracycline-based chemotherapy in breast cancer patients.

BACKGROUND: Anthracycline adjuvant therapy has taken a particular role in the treatment of early stage breast cancer with an associated decrease in rates of both relapse and death. Their success however has been limited by their myelosuppression and their well-established risk of cardiac dysfunction. Guidelines have emerged that would limit the maximum lifetime dose of anthracyclines and make a baseline assessment and periodic monitoring of cardiac function part of the routine practice, which could be cumbersome, and may condemn the patient to an unwarranted modification of his/her regimen. Our study aimed at assessing the incidence of abnormal baseline echocardiography in asymptomatic women with breast cancer prior to anthracycline therapy and establishing risk criteria associated with abnormal echocardiograms at baseline. METHODS: 220 Patients seen at AUBMC (American University of Beirut Medical Center) who had non- metastatic breast cancer, and had an echocardiography performed before starting anthracycline chemotherapy were chosen. Data about demographic characteristics, tumor characteristics, baseline echocardiography results, and change in clinical decision was collected. Patients with suboptimal (less than 50%) ejection fraction (EF) on baseline echocardiography were analyzed for the prevalence of cardiac risk factors. Results were compared to those among the overall study group using Fisher's Exact test. A p- value of = < 0.05 was used as reference for statistical significance. RESULTS: All 220 of our patients had received a baseline echo prior to initiation of anthracycline therapy. 6.7% of these patients had already some abnormality in wall motion but only 2.7% had a suboptimal ejection fraction. 1.3% had a change in chemotherapy regimen based on ejection fraction. The patients with depressed EF had higher rates of CAD (coronary artery disease), diabetes, hypertension and dyslipidemia than the overall study group but without statistical significance. CONCLUSIONS: Our study, as well as the previous contingent studies raise the question about routine echocardiography prior to anthracycline therapy and might eventually lead to a modification of current practice guidelines.

Elucidating immune-related gene transcriptional programs via factorization of large-scale RNA-profiles.

Recent developments in immunotherapy, including immune checkpoint blockade (ICB) and adoptive cell therapy (ACT), have encountered challenges such as immune-related adverse events and resistance, especially in solid tumors. To advance the field, a deeper understanding of the molecular mechanisms behind treatment responses and resistance is essential. However, the lack of functionally characterized immune-related gene sets has limited data-driven immunological research. To address this gap, we adopted non-negative matrix factorization on 83 human bulk RNA sequencing (RNA-seq) datasets and constructed 28 immune-specific gene sets. After rigorous immunologist-led manual annotations and orthogonal validations across immunological contexts and functional omics data, we demonstrated that these gene sets can be applied to refine pan-cancer immune subtypes, improve ICB response prediction and functionally annotate spatial transcriptomic data. These functional gene sets, informing diverse immune states, will advance our understanding of immunology and cancer research.

Elucidating immune-related gene transcriptional programs via factorization of large-scale RNA-profiles.

Recent developments in immunotherapy, including immune checkpoint blockade (ICB) and adoptive cell therapy, have encountered challenges such as immune-related adverse events and resistance, especially in solid tumors. To advance the field, a deeper understanding of the molecular mechanisms behind treatment responses and resistance is essential. However, the lack of functionally characterized immune-related gene sets has limited data-driven immunological research. To address this gap, we adopted non-negative matrix factorization on 83 human bulk RNA-seq datasets and constructed 28 immune-specific gene sets. After rigorous immunologist-led manual annotations and orthogonal validations across immunological contexts and functional omics data, we demonstrated that these gene sets can be applied to refine pan-cancer immune subtypes, improve ICB response prediction and functionally annotate spatial transcriptomic data. These functional gene sets, informing diverse immune states, will advance our understanding of immunology and cancer research.

Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19+ B cell tumors: a phase 1/2 trial.

There is a pressing need for allogeneic chimeric antigen receptor (CAR)-immune cell therapies that are safe, effective and affordable. We conducted a phase 1/2 trial of cord blood-derived natural killer (NK) cells expressing anti-CD19 chimeric antigen receptor and interleukin-15 (CAR19/IL-15) in 37 patients with CD19+ B cell malignancies. The primary objectives were safety and efficacy, defined as day 30 overall response (OR). Secondary objectives included day 100 response, progression-free survival, overall survival and CAR19/IL-15 NK cell persistence. No notable toxicities such as cytokine release syndrome, neurotoxicity or graft-versus-host disease were observed. The day 30 and day 100 OR rates were 48.6% for both. The 1-year overall survival and progression-free survival were 68% and 32%, respectively. Patients who achieved OR had higher levels and longer persistence of CAR-NK cells. Receiving CAR-NK cells from a cord blood unit (CBU) with nucleated red blood cells ≤ 8 × 107 and a collection-to-cryopreservation time ≤ 24 h was the most significant predictor for superior outcome. NK cells from these optimal CBUs were highly functional and enriched in effector-related genes. In contrast, NK cells from suboptimal CBUs had upregulation of inflammation, hypoxia and cellular stress programs. Finally, using multiple mouse models, we confirmed the superior antitumor activity of CAR/IL-15 NK cells from optimal CBUs in vivo. These findings uncover new features of CAR-NK cell biology and underscore the importance of donor selection for allogeneic cell therapies. ClinicalTrials.gov identifier: NCT03056339 .

CD28 costimulation augments CAR signaling in NK cells via the LCK/CD3Z/ZAP70 signaling axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3Z creates a platform that recruits critical kinases, such as LCK and ZAP70, initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy.

Umbilical Cord Blood Transplantation: Connecting Its Origin to Its Future.

Transplantation of umbilical cord blood (UCB) is an attractive alternative source of hematopoietic stem cells (HSCs). The unique properties of cord blood and its distinct immune tolerance and engraftment kinetics compared to bone marrow (BM) and peripheral blood progenitor cells, permit a wider disparity in human leukocyte antigen levels between a cord blood donor and recipient after an unrelated umbilical cord blood transplant (UCBT). In addition, it is readily available and has a lowered risk of graft-versus-host disease (GvHD), with similar long-term clinical outcomes, compared to BM transplants. However, the relatively low number of cells administered by UCB units, as well as the associated delayed engraftment and immune reconstitution, pose limitations to the wide application of UCBT. Research into several aspects of UCBT has been evaluated, including the ex vivo expansion of cord blood HSCs and the process of fucosylation to enhance engraftment. Additionally, UCB has also been used in the treatment of several neurodegenerative and cardiovascular disorders with varying degrees of success. In this article, we will discuss the biology, clinical indications, and benefits of UCBT in pediatric and adult populations. We will also discuss future directions for the use of cord blood.

Loss of metabolic fitness drives tumor resistance after CAR-NK cell therapy and can be overcome by cytokine engineering.

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells is promising, with early-phase clinical studies showing encouraging responses. However, the transcriptional signatures that control the fate of CAR-NK cells after infusion and factors that influence tumor control remain poorly understood. We performed single-cell RNA sequencing and mass cytometry to study the heterogeneity of CAR-NK cells and their in vivo evolution after adoptive transfer, from the phase of tumor control to relapse. Using a preclinical model of noncurative lymphoma and samples from a responder and a nonresponder patient treated with CAR19/IL-15 NK cells, we observed the emergence of NK cell clusters with distinct patterns of activation, function, and metabolic signature associated with different phases of in vivo evolution and tumor control. Interaction with the highly metabolically active tumor resulted in loss of metabolic fitness in NK cells that could be partly overcome by incorporation of IL-15 in the CAR construct.

Engineered cord blood megakaryocytes evade killing by allogeneic T-cells for refractory thrombocytopenia.

The current global platelet supply is often insufficient to meet all the transfusion needs of patients, in particular for those with alloimmune thrombocytopenia. To address this issue, we have developed a strategy employing a combination of approaches to achieve more efficient production of functional megakaryocytes (MKs) and platelets collected from cord blood (CB)-derived CD34+ hematopoietic cells. This strategy is based on ex-vivo expansion and differentiation of MKs in the presence of bone marrow niche-mimicking mesenchymal stem cells (MSCs), together with two other key components: (1) To enhance MK polyploidization, we used the potent pharmacological Rho-associated coiled-coil kinase (ROCK) inhibitor, KD045, resulting in liberation of increased numbers of functional platelets both in-vitro and in-vivo; (2) To evade HLA class I T-cell-driven killing of these expanded MKs, we employed CRISPR-Cas9-mediated ß-2 microglobulin (ß2M) gene knockout (KO). We found that coculturing with MSCs and MK-lineage-specific cytokines significantly increased MK expansion. This was further increased by ROCK inhibition, which induced MK polyploidization and platelet production. Additionally, ex-vivo treatment of MKs with KD045 resulted in significantly higher levels of engraftment and donor chimerism in a mouse model of thrombocytopenia. Finally, ß2M KO allowed MKs to evade killing by allogeneic T-cells. Overall, our approaches offer a novel, readily translatable roadmap for producing adult donor-independent platelet products for a variety of clinical indications.

KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape.

Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKTROG+) and NK cell hyporesponsiveness. This phenomenon could be offset by a dual-CAR system incorporating both an activating CAR against the cognate tumor antigen and an NK self-recognizing inhibitory CAR that transferred a 'don't kill me' signal to NK cells upon engagement with their TROG+ siblings. This system prevented trogocytic antigen-mediated fratricide, while sparing activating CAR signaling against the tumor antigen, and resulted in enhanced CAR-NK cell activity.