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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.

Background Waldenström macroglobulinemia (WM) is an incurable disease and, while treatable, can develop resistance to available therapies and be fatal. Chimeric antigen receptor (CAR) T cell therapy directed against the CD19 antigen has demonstrated efficacy in relapsed or refractory B lymphoid malignancies, and is now approved for B cell acute lymphoblastic leukemia and certain B cell lymphomas. However, CAR T therapy has not been evaluated for use in WM.Methods and results We performed preclinical studies demonstrating CAR T cell activity against WM cells in vitro, and developed an in vivo murine model of WM which demonstrated prolonged survival with use of CAR T therapy. We then report the first three patients with multiply relapsed and refractory WM treated for their disease with CD19-directed CAR T cells on clinical trials. Treatment was well tolerated, and observed toxicities were consistent with those seen in CAR T treatment for other diseases, and no grade 3 or higher cytokine release syndrome or neurotoxicity events occurred. All three patients attained at least a clinical response to treatment, including one minimal residual disease-negative complete response, though all three eventually developed recurrent disease between 3 and 26 months after initial treatment.Conclusions This report summarizes preclinical and clinical activity of CD19-directed CAR T therapy in WM, demonstrating early tolerability and efficacy in patients with WM, and representing a possible treatment option in patients with heavily pretreated and relapsed or refractory WM. Larger studies evaluating CAR T therapy in WM are warranted, along with further evaluation into mechanisms of resistance to CAR T therapy.

Acute leukemia is a constellation of rapidly progressing diseases that affect a wide range of patients regardless of age or gender. Traditional treatment options for patients with acute leukemia include chemotherapy and hematopoietic cell transplantation. The advent of cancer immunotherapy has had a significant impact on acute leukemia treatment. Novel immunotherapeutic agents including antibody-drug conjugates, bispecific T cell engagers, and chimeric antigen receptor T cell therapies have efficacy and have recently been approved by the US Food and Drug Administration (FDA) for the treatment of patients with acute leukemia. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to develop a clinical practice guideline composed of consensus recommendations on immunotherapy for the treatment of acute lymphoblastic leukemia and acute myeloid leukemia.

T cells can be genetically modified to target tumors through the expression of a chimeric antigen receptor (CAR). Most notably, CAR T cells have demonstrated clinical efficacy in hematologic malignancies with more modest responses when targeting solid tumors. However, CAR T cells also have the capacity to elicit expected and unexpected toxicities including: cytokine release syndrome, neurologic toxicity, “on target/off tumor” recognition, and anaphylaxis. Theoretical toxicities including clonal expansion secondary to insertional oncogenesis, graft versus host disease, and off-target antigen recognition have not been clinically evident. Abrogating toxicity has become a critical step in the successful application of this emerging technology. To this end, we review the reported and theoretical toxicities of CAR T cells and their management.

Xenograft mouse models represent helpful tools for preclinical studies on human tumors. For modeling the complexity of the human disease, primary tumor cells are by far superior to established cell lines. As qualified exemplary model, patients' acute lymphoblastic leukemia cells reliably engraft in mice inducing orthotopic disseminated leukemia closely resembling the disease in men. Unfortunately, disease monitoring of acute lymphoblastic leukemia in mice is hampered by lack of a suitable readout parameter.Patients' acute lymphoblastic leukemia cells were lentivirally transduced to express the membrane-bound form of Gaussia luciferase. In vivo imaging was established in individual patients' leukemias and extensively validated.Bioluminescence in vivo imaging enabled reliable and continuous follow-up of individual mice. Light emission strictly correlated to post mortem quantification of leukemic burden and revealed a logarithmic, time and cell number dependent growth pattern. Imaging conveniently quantified frequencies of leukemia initiating cells in limiting dilution transplantation assays. Upon detecting a single leukemia cell within more than 10,000 bone marrow cells, imaging enabled monitoring minimal residual disease, time to tumor re-growth and relapse. Imaging quantified therapy effects precisely and with low variances, discriminating treatment failure from partial and complete responses.For the first time, we characterized in detail how in vivo imaging reforms preclinical studies on patient-derived tumors upon increasing monitoring resolution. In the future, in vivo imaging will enable performing precise preclinical studies on a broad range of highly demanding clinical challenges, such as treatment failure, resistance in leukemia initiating cells, minimal residual disease and relapse.

Despite therapeutic efficacy observed with immune checkpoint blockade in advanced melanoma, many tumors do not respond to treatment, representing a need for new therapies. Here, we have generated chimeric antigen receptor (CAR) T cells targeting TYRP1, a melanoma differentiation antigen expressed on the surface of melanomas, including rare acral and uveal melanomas. TYRP1-targeted CAR T cells demonstrate antigen-specific activation and cytotoxic activity in vitro and in vivo against human melanomas independent of the MHC alleles and expression. In addition, the toxicity to pigmented normal tissues observed with T lymphocytes expressing TYRP1-targeted TCRs was not observed with TYRP1-targeted CAR T cells. Anti-TYRP1 CAR T cells provide a novel means to target advanced melanomas, serving as a platform for the development of similar novel therapeutic agents and as a tool to interrogate the immunobiology of melanomas.

With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin’s lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.

CD40L-expressing chimeric antigen receptor (CAR) T cells show enhanced anti-tumor immunity, but the cellular mechanisms are still unclear. Here we show, by analyzing mice deficient of conventional dendritic cell type 1 (cDC1) that cDC1s are induced by CD40L+ CAR T cells to prime endogenous CD8 T cells for a stronger anti-tumor immune response.

Immunotherapy for ovarian cancer is an area of intense investigation since the majority of women with relapsed disease develop resistance to conventional cytotoxic therapy. The paucity of safe and validated target antigens has limited the development of clinically relevant antibody-based immunotherapeutics for this disease. Although MUC16 expression is almost universal in High Grade Serous Ovarian Cancers, engagement of the shed circulating MUC16 antigen (CA-125) presents a theoretical risk of systemic activation and toxicity. We designed and evaluated a series of bispecific tandem single-chain variable fragments specific to the retained portion of human MUC16 ectodomain (MUC16ecto) and human CD3. These MUC16ecto- BiTEDs retain binding in the presence of soluble MUC16 (CA-125) and show cytotoxicity against a panel of ovarian cancer cells in vitro. MUC16ecto- BiTEDs delay tumor progression in vivo and significantly prolong survival in a xenograft model of ovarian peritoneal carcinomatosis. This effect was significantly enhanced by antiangiogenic (anti-VEGF) therapy and immune checkpoint inhibition (anti-PD1). However, the combination of BiTEDs with anti-VEGF was superior to combination with anti-PD1, based on findings of decreased peritoneal tumor burden and ascites with the former. This study shows the feasibility and efficacy of MUC16ecto- specific BiTEDs and provides a basis for the combination with anti-VEGF therapy for ovarian cancer.

Background: Adoptive immunotherapy using T cells expressing chimeric antigen receptors (CARs) targeting CD19 has produced remarkable clinical outcomes. However, much of the mechanisms of action, such as the development of memory responses and sources of immune cytokines, remain elusive largely due to the challenge of characterizing human CAR T cell function in vivo. The lack of a suitable in vivo model also hinders the development of new CAR T cell therapies. Methods: We established a humanized mouse (hu-mouse) model with a functional human immune system and genetically-matched (autologous) primary acute B-lymphoblastic leukemia (B-ALL) that permits modeling of CD19-targeted CAR T cell therapy in immunocompetent hosts without allogeneic or xenogeneic immune responses. Findings: Anti-CD19 CAR T cells were detected in blood of leukemic hu-mice with kinetics and levels similar to those seen in patients receiving CAR T cell therapy. The levels of CAR T cells were correlated inversely with the burden of leukemia cells and positively with the survival times in anti-CD19 CAR T cell-treated leukemic hu-mice. Infusion of anti-CD19 CAR T cells also resulted in rapid production of T cell- and monocyte/macrophage-derived cytokines and an increase in frequency of regulatory T cells as reported in clinical studies. Interpretation: These results provide a proof-of-principle that this novel preclinical model has the potential to be used to model human CAR T cell therapy and facilitate the design of new CARs with improved antitumor activity.

Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of vector products used in the clinical trials were generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral vector product generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value. Keywords: retrovirus, safety testing, replicating virus, lentivirus

Patients with small cell lung cancer (SCLC) generally have a poor prognosis and a median overall survival of only about 13 months, indicating the urgent need for novel therapies. Delta-like protein 3 (DLL3) has been identified as a tumor-specific cell surface marker on neuroendocrine cancers, including SCLC. In this study, we developed a chimeric antigen receptor (CAR) against DLL3 that displays antitumor efficacy in xenograft and murine SCLC models. CAR T cell expression of the proinflammatory cytokine IL-18 greatly enhanced the potency of DLL3-targeting CAR T cell therapy. In a murine metastatic SCLC model, IL-18 production increased the activation of both CAR T cells and endogenous tumor-infiltrating lymphocytes. We also observed an increased infiltration, repolarization, and activation of antigen-presenting cells. Additionally, human IL-18–secreting anti-DLL3 CAR T cells showed an increased memory phenotype, less exhaustion, and induced durable responses in multiple SCLC models, an effect that could be further enhanced with anti–PD-1 blockade. All together, these results define DLL3-targeting CAR T cells that produce IL-18 as a potentially promising novel strategy against DLL3-expressing solid tumors.

Ovarian cancer cells often metastasize to the peritoneal cavity, forming spheroid-like structures and promoting a highly immunosuppressive tumor microenvironment. Here, the authors show that the ubiquitin ligase UBR5 is required for ovarian cancer growth and metastasis, sustaining spheroid formation and the infiltration of immunosuppressive tumor associated macrophages.

Abstract Chimeric Antigen Receptor (CAR) T cell therapies – adoptive T cell therapies that have been genetically engineered for a new antigen-specificity - have displayed significant success in treating patients with hematologic malignancies, leading to three recent US Food and Drug Administration approvals. Based on the promise generated from these successes, the field is rapidly evolving to include new disease indications and CAR designs, while simultaneously reviewing and optimizing toxicity and management protocols. As such, this review provides expert perspective on the significance and clinical considerations of CAR T cell therapies in order to provide timely information to clinicians about this revolutionary new therapeutic class.

Abstract: A National Cancer Institute consensus study on prioritization of cancer antigens ranked the Wilms tumor 1 (WT1) protein as the top immunotherapy target in cancer. We previously reported a pilot study of a multivalent WT1 peptide vaccine (galinpepimut-S) in acute myeloid leukemia (AML) patients. We have now conducted a phase 2 study investigating this vaccine in adults with AML in first complete remission (CR1). Patients received 6 vaccinations administered over 10 weeks with the potential to receive 6 additional monthly doses if they remained in CR1. Immune responses (IRs) were evaluated after the 6th and 12th vaccinations by CD4+ T-cell proliferation, CD8+ T-cell interferon-γ secretion (enzyme-linked immunospot), or the CD8-relevant WT1 peptide major histocompatibility complex tetramer assay (HLA-A*02 patients only). Twenty-two patients (7 males; median age, 64 years) were treated. Fourteen patients (64%) completed ≥6 vaccinations, and 9 (41%) received all 12 vaccine doses. Fifteen patients (68%) relapsed, and 10 (46%) died. The vaccine was well tolerated, with the most common toxicities being grade 1/2 injection site reactions (46%), fatigue (32%), and skin induration (32%). Median disease-free survival from CR1 was 16.9 months, whereas the overall survival from diagnosis has not yet been reached but is estimated to be ≥67.6 months. Nine of 14 tested patients (64%) had an IR in ≥1 assay (CD4 or CD8). These results indicated that the WT1 vaccine was well tolerated, stimulated a specific IR, and was associated with survival in excess of 5 years in this cohort of patients. This trial was registered at www.clinicaltrials.gov as #NCT01266083.

During recent years, it has become increasingly evident that donor leukemia following allogeneic transplant may be more common then realized in the past. We identified five cases of potential donor leukemia cases during past five years. The precise mechanism of the origin of such leukemias, however, remains poorly defined. In this short communication, we report a well documented case of donor-derived de novo acute myeloid leukemia (AML) that developed fourteen years after allogeneic stem cell transplantation for treatment induced AML for his primary malignancy Immunoblastic lymphoma. This case allows us to postulate a possible mechanism of the origin of donor leukemia. The de novo AML clone contained a distinct cytogenetic abnormality, trisomy 11, which was simultaneously detected in preserved peripheral blood obtained at the time of transplantation as well as in the current bone marrow from an otherwise clinically and phenotypically normal donor. The findings from this unique case, provides insight into the process of leukemogenesis, and suggests that the sequence of events leading to leukemogenesis in this patient involved the senescence/apoptosis of normal donor hematopoietic cells due to telomere shortening resulting in the selective proliferation and transformation of this clone with MLL (mixed-lineage leukemia) gene amplification.