Prognostic discrimination for early chronic phase chronic myeloid leukemia in imatinib era: comparison of Sokal,Euro, and EUTOS scores in Korean population

Adoptive transfer of tumor-reactive T cells into cancer patients with the intent of inducing a cytotoxic anti-tumor effector response and durable immunity has long been proposed as a novel therapy for a broad range of malignancies; however, local and systemic tolerance mechanisms have hindered the generation of effective T cell therapies and limited the clinical efficacy of this approach in cancer patients. Chimeric antigen receptors (CARs) are recombinant receptors that comprise an extracellular antigen-targeting domain in conjunction with one or more intracellular T cell signaling domains that can be introduced into T cells by genetic modification to redirect their specificity to the CAR-targeted antigen. Administration of CD19-specific CAR-modified T cells that target B cell non-Hodgkin lymphomas and leukemia has been remarkably effective in recent clinical trials, energizing the field and stimulating new efforts to identify the critical parameters of CAR design and T cell engineering that are necessary for effective cancer therapy.     

Chimeric antigen receptors for the adoptive T cell therapy of hematologic malignancies

The genetic modification of autologous T cells with chimeric antigen receptors (CARs) represents a breakthrough for gene engineering as a cancer therapy for hematologic malignancies. By targeting the CD19 antigen, we have demonstrated robust and rapid anti-leukemia activity in patients with heavily pre-treated and chemotherapy-refractory B cell acute lymphoblastic leukemia (B-ALL). We demonstrated rapid induction of deep molecular remissions in adults, which has been recently confirmed in a case report involving a child with B-ALL. In contrast to the results when treating B-ALL, outcomes have been more modest in patients with chronic lymphocytic leukemia (CLL) or other non-hodgkin’s lymphoma (NHL). We review the clinical trial experience targeting B-ALL and CLL and speculate on the possible reasons for the different outcomes and propose potential optimization to CAR T cell therapy when targeting CLL or other indolent NHL. Lastly, we discuss the pre-clinical development and potential for clinical translation for using CAR T cells against multiple myeloma and acute myeloid leukemia. We highlight the potential risks and benefits by targeting these poor outcome hematologic malignancies.     

Advances in CAR design

Chimeric antigen receptor (CAR) T cells have revolutionized the management of B cell malignancies. These synthetic molecules are composed of peptide fragments from several distinct immune cell proteins and link highly-specific antigen recognition with potent T cell activation. Despite impressive results in many, less than half of patients treated will achieve durable remission after CAR therapy. Recent studies have identified the central role that each structural component of the CAR molecule plays in regulating T cell function. Significant effort has been dedicated to exploring strategies to improve the design of CARs themselves or integrate their activity with other regulatory circuits to enable more precise function. In this review, we will summarize recent pre-clinical and clinical studies that have evaluated novel CAR design formats.     

T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells

There has been interest in generating T cells expressing chimeric artificial receptors (CARs) targeting CD19/CD20 antigens to treat B-cell lymphomas. If successful, however, this approach would likely impair humoral immunity because T cells may persist long-term. Most low-grade lymphoma and chronic lymphocytic leukemia (B-CLL) cells express monoclonal immunoglobulins carrying either kappa or lambda light chains. We, therefore, explored whether T lymphocytes could be genetically modified to target the tumor-associated light chain, sparing B lymphocytes expressing the reciprocal light chain, and consequently reduce impairment of humoral immunity. We found that T lymphocytes expressing the anti-kappa light chain CAR showed cytotoxic activity against Igkappa(+) tumor cell lines and B-CLL cells both in vitro and in vivo. We also found that the incorporation of the CD28 endodomain within the CAR enhanced the in vitro and in vivo expansion of transgenic T cells after tumor-associated antigen stimulation. Free Igkappa(+) did not compromise the ability of redirected T lymphocytes to eliminate Igkappa(+) tumors because these free immunoglobulins served to sustain proliferation of CAR-CD28 transgenic T cells. Thus, adoptive transfer of T lymphocytes targeting the appropriate light chain could be a useful immunotherapy approach to treat B-lymphocyte malignancies that clonally express immunoglobulin without entirely compromising humoral immunity.     

T cells redirected against CD70 for the immunotherapy of CD70-positive malignancies

T-cell therapy with genetically modified T cells targeting CD19 or CD20 holds promise for the immunotherapy of hematologic malignancies. These targets, however, are only present on B cell-derived malignancies, and because they are broadly expressed in the hematopoietic system, their targeting may have unwanted consequences. To expand T-cell therapies to hematologic malignancies that are not B cell-derived, we determined whether T cells can be redirected to CD70, an antigen expressed by limited subsets of normal lymphocytes and dendritic cells, but aberrantly expressed by a broad range of hematologic malignancies and some solid tumors. To generate CD70-specific T cells, we constructed a chimeric antigen receptor (CAR) consisting of the CD70 receptor (CD27) fused to the CD3-ζ chain. Stimulation of T cells expressing CD70-specific CARs resulted in CD27 costimulation and recognition of CD70-positive tumor cell lines and primary tumor cells, as shown by IFN-γ and IL-2 secretion and by tumor cell killing. Adoptively transferred CD70-specific T cells induced sustained regression of established murine xenografts. Therefore, CD70-specific T cells may be a promising immunotherapeutic approach for CD70-positive malignancies.     

CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results

Cellular immune responses have the potential to elicit dramatic and sustained clinical remissions in lymphoma patients. Recent clinical trial data demonstrate that modification of T cells with chimeric antigen receptors (CARs) is a promising strategy. T cells containing CARs with costimulatory domains exhibit improved activity against tumors. We conducted a pilot clinical trial testing a "third-generation" CD20-specific CAR with CD28 and 4-1BB costimulatory domains in patients with relapsed indolent B-cell and mantle cell lymphomas. Four patients were enrolled, and 3 received T-cell infusions after cyclophosphamide lymphodepletion. Treatment was well tolerated, although one patient developed transient infusional symptoms. Two patients without evaluable disease remained progression-free for 12 and 24 months. The third patient had an objective partial remission and relapsed at 12 months after infusions. Modified T cells were detected by quantitative PCR at tumor sites and up to 1 year in peripheral blood, albeit at low levels. No evidence of host immune responses against infused cells was detected. In conclusion, adoptive immunotherapy with CD20-specific T cells was well tolerated and was associated with antitumor activity. We will pursue alternative gene transfer technologies and culture conditions in future studies to improve CAR expression and cell production efficiency.     

Chimeric antigen receptor modified T cells that target chemokine receptor CCR4 as a therapeutic modality for T‐cell malignancies

With the emerging success of treating CD19 expressing B cell malignancies with ex vivo modified, autologous T cells that express CD19‐directed chimeric antigen receptors (CAR), there is intense interest in expanding this evolving technology to develop effective modalities to treat other malignancies including solid tumors. Exploiting this approach to develop a therapeutic modality for T cell malignancies for which the available regimens are neither curative, nor confer long term survival we generated a lentivirus‐based CAR gene transfer system to target the chemokine receptor CCR4 that is over‐expressed in a spectrum of T cell malignancies as well as in CD4⁺CD25⁺Foxp3⁺T regulatory cells that accumulate in the tumor microenvironment constituting a barrier against anti‐tumor immunity. Ex vivo modified, donor‐derived T cells that expressed CCR4 directed CAR displayed antigen‐dependent potent cytotoxicity against patient‐derived cell lines representing ATL, CTCL, ALCL and a subset of HDL. Furthermore, these CAR T cells also eradicated leukemia in a mouse xenograft model of ATL illustrating the potential utility of this modality in the treatment of a wide spectrum of T cell malignancies.     

Chimeric antigen receptor T-cells: Properties,production, and quality control

Chimeric antigen receptor (CAR) T-cell therapy is a novel adoptive T-cell immunotherapy for haematological malignancies. First introduced into clinical practice in 2017, CAR T-cell therapy is now finding its place in the management of lymphoid malignancies, primarily of B-cell lineage, including lymphoblastic leukaemia, non-Hodgkin lymphoma and plasma cell myeloma, with remarkable therapeutic outcomes. CAR T-cells are a customised therapeutic product for each patient. Manufacture commences with collection of autologous T-cells, which are then genetically engineered ex vivo to express transmembrane CARs. These chimeric proteins consist of an antibody-like extracellular antigen-binding domain, to recognise specific antigens on the surface of tumour cells (e.g. CD19), linked to the intracellular co-stimulatory signalling domains of a T-cell receptor (e.g. CD137). The latter is required for in vivo CAR T-cell proliferation, survival, and durable efficacy. Following reinfusion, CAR T-cells harness the cytotoxic capacity of a patient's immune system. They overcome major mechanisms of tumour immuno-evasion and have potential to generate robust cytotoxic anti-tumour responses. This review discusses the background to CAR T-cell therapies, including their molecular design, mechanisms of action, methods of production, clinical applications and established and emerging technologies for CAR T-cell evaluation. It highlights the need for standardisation, quality control and monitoring of CAR T-cell therapies, to ensure their safety and efficacy in clinical management.     

Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma

We generated MHC-independent chimeric antigen receptors (CARs) directed to the GD2 antigen expressed by neuroblastoma tumor cells and treated patients with this disease. Two distinguishable forms of this CAR were expressed in EBV-specific cytotoxic T lymphocytes (EBV-CTLs) and activated T cells (ATCs). We have previously shown that EBV-CTLs expressing GD2-CARs (CAR-CTLs) circulated at higher levels than GD2-CAR ATCs (CAR-ATCs) early after infusion, but by 6 weeks, both subsets became low or undetectable. We now report the long-term clinical and immunologic consequences of infusions in 19 patients with high-risk neuroblastoma: 8 in remission at infusion and 11 with active disease. Three of 11 patients with active disease achieved complete remission, and persistence of either CAR-ATCs or CAR-CTLs beyond 6 weeks was associated with superior clinical outcome. We observed persistence for up to 192 weeks for CAR-ATCs and 96 weeks for CAR-CTLs, and duration of persistence was highly concordant with the percentage of CD4(+) cells and central memory cells (CD45RO(+)CD62L(+)) in the infused product. In conclusion, GD2-CAR T cells can induce complete tumor responses in patients with active neuroblastoma; these CAR T cells may have extended, low-level persistence in patients, and such persistence was associated with longer survival. This study is registered at www.clinialtrials.gov as #NCT00085930.     

New targets for CAR T therapy in hematologic malignancies

As we expand our acumen of the intricacies of hematological malignancies at a genetic and cellular level, we have paved the way in advancing novel targeted therapeutic avenues such as chimeric antigen receptor T-cell therapies (CAR T). Engineering cells to target a specific antigen has led to dramatic remission rates in cases of relapsed/refractory non-Hodgkin lymphoma, acute lymphoblastic leukemia as well as multiple myeloma thus far with trials in place to further advance targeted therapies in other hematological malignancies. Most currently available CAR T therapies target CD19 antigen. Studies are underway exploring novel CAR T products aimed at other tumor-specific antigens with potential to improve the efficacy and reduce the toxicities. Early studies have confirmed safety and efficacy of CD22 and BCMA targeted CAR T therapies. Moreover, various other targets including CD20, CD30, CD123, kappa, and lambda light chains among others are under clinical investigation as potential avenues of targeted therapy. This review highlights the shift in the treatment paradigm in pursuing diverse antigen targets while addressing the challenges in terms of the efficacy and toxicity of current CAR T-cell therapies.     

Targeting of acute myeloid leukaemia by cytokine‐induced killer cells redirected with a novel CD123‐specific chimeric antigen receptor

Current therapeutic regimens for acute myeloid leukaemia (AML) are still associated with high rates of relapse. Immunotherapy with T‐cells genetically modified to express chimeric antigen receptors (CARs) represents an innovative approach. Here we investigated the targeting of the interleukin three receptor alpha (IL3RA; CD123) molecule, which is overexpressed on AML bulk population, CD34⁺ leukaemia progenitors, and leukaemia stem cells (LSC) compared to normal haematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine‐induced killer (CIK) cells were transduced with SFG‐retroviral‐vector encoding an anti‐CD123 CAR. Transduced cells were able to strongly kill CD123⁺ cell lines, as well as primary AML blasts. Interestingly, secondary colony experiments demonstrated that anti‐CD123.CAR preserved in vitro HSPCs, in contrast to a previously generated anti‐CD33.CAR, while keeping an identical cytotoxicity profile towards AML. Furthermore, limited killing of normal monocytes and CD123‐low‐expressing endothelial cells was noted, thus indicating a low toxicity profile of the anti‐CD123.CAR. Taken together, our results indicate that CD123‐specific CARs strongly enhance anti‐AML CIK functions, while sparing HSPCs and normal low‐expressing antigen cells, paving the way to develop novel immunotherapy approaches for AML treatment.     

An introduction to chimeric antigen receptor (CAR) T-cell immunotherapy for human cancer

Chimeric antigen receptor (CAR) T-cell therapy represents a major advancement in personalized cancer treatment. In this strategy, a patient's own T cells are genetically engineered to express a synthetic receptor that binds a tumor antigen. CAR T cells are then expanded for clinical use and infused back into the patient's body to attack and destroy chemotherapy-resistant cancer. Dramatic clinical responses and high rates of complete remission have been observed in the setting of CAR T-cell therapy of B-cell malignancies. This resulted in two recent FDA approvals of CAR T cells directed against the CD19 protein for treatment of acute lymphoblastic leukemia and diffuse large B-cell lymphoma. Thus, CAR T cells are arguably one of the first successful examples of synthetic biology and personalized cellular cancer therapy to become commercially available. In this review, we introduce the concept of using CAR T cells to break immunological tolerance to tumors, highlight several challenges in the field, discuss the utility of biomarkers in the context of predicting clinical responses, and offer prospects for developing next-generation CAR T cell-based approaches that will improve outcome.     

In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric antigen receptor

Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19(+)CD5(+)CD20(dim) B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR(+) T cells showed specific cytotoxic activity against CD23(+) tumor cell lines (average lysis 42%) and primary CD23(+) CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR(+) T cells released inflammatory cytokines (1445-fold more TNF-β, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR(+) T cells. Redirected T cells were also effective in vivo in a CLL Rag2(-/-)γ(c)(-/-) xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR(+) T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR(+) T cells represent a selective immunotherapy for the elimination of CD23(+) leukemic cells in patients with CLL.     

Toxicities associated with adoptive cellular therapies

Chimeric antigen receptor (CAR) T cell therapy is an effective strategy for the treatment of relapsed/refractory hematologic malignancies leading to the Food and Drug Administration (FDA) approval of five CAR T cell products. Despite encouraging efficacy, the widespread utilization of CAR T cell therapy is limited by unique immune mediated toxicities, primarily cytokine release syndrome (CRS) and neurologic toxicity. Data regarding late effects and long-term toxicities of CAR T cell therapy is evolving and includes prolonged cytopenias, hypogammaglobulinemia, infections and secondary malignancies. In this review, we will describe the clinical presentation, diagnosis, mechanisms and management of short- and long-term toxicities of CAR T cell therapy.     

A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR

Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR(+) T cells to eliminate expression of the endogenous αβ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR(+)TCR(neg) T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.     

Improved Activity against Acute Myeloid Leukemia with Chimeric Antigen Receptor (CAR)-NK-92 Cells Designed to Target CD123

Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123⁺ AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123⁺ AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.     

CD19 chimeric antigen receptor T cell therapy for haematological malignancies

T cells can be redirected to recognize tumour antigens by genetic modification to express a chimeric antigen receptor (CAR). These consist of antibody‐derived antigen‐binding regions linked to T cell signalling elements. CD19 is an ideal target because it is expressed on most B cell malignancies as well as normal B cells but not on other cell types, restricting any ‘on target, off tumour’ toxicity to B cell depletion. Recent clinical studies involving CD19 CAR‐directed T cells have shown unprecedented responses in a range of B cell malignancies, even in patients with chemorefractory relapse. Durable responses have been achieved, although the persistence of modified T cells may be limited. This therapy is not without toxicity, however. Cytokine release syndrome and neurotoxicity appear to be frequent but are treatable and reversible. CAR T cell therapy holds the promise of a tailored cellular therapy, which can form memory and be adapted to the tumour microenvironment. This review will provide a perspective on the currently available data, as well as on future developments in the field.     

Epstein Barr virus specific cytotoxic T lymphocytes expressing the anti-CD30zeta artificial chimeric T-cell receptor for immunotherapy of Hodgkin disease

Adoptive transfer of Epstein Barr virus (EBV)-specific cytotoxic T-lymphocytes (EBV-CTLs) has shown that these cells persist in patients with EBV(+) Hodgkin lymphoma (HD) to produce complete tumor responses. Treatment failure, however, occurs if a subpopulation of malignant cells in the tumor lacks or loses expression of EBV antigens. We have therefore determined whether we could prepare EBV-CTLs that retained the antitumor activity conferred by their native receptor while expressing a chimeric antigen receptor (CAR) specific for CD30, a molecule highly and consistently expressed on malignant Hodgkin Reed-Sternberg cells. We made a CD30CAR and were able to express it on 26% (+/- 11%) and 22% (+/- 5%) of EBV-CTLs generated from healthy donors and HD patients, respectively. These CD30CAR(+) CTLs killed both autologous EBV(+) cells through their native receptor and EBV(-)/CD30(+) targets through their major histocompatibility complex (MHC)-unrestricted CAR. A subpopulation of activated T cells also express CD30, but the CD30CAR(+) CTLs did not impair cellular immune responses, probably because normal T cells express lower levels of the target antigen. In a xenograft model, CD30CAR(+) EBV-CTLs could be costimulated by EBV-infected cells and produce antitumor effects even against EBV(-)/CD30(+) tumors. EBV-CTLs expressing both a native and a chimeric antigen receptor may therefore have added value for treatment of HD.     

Chimeric antigen receptor-engineered T-cell therapy for liver cancer

Background

Chimeric antigen receptor-engineered T-cell (CAR-T) therapy is a newly developed immunotherapy used in the treatment of cancers. Because CAR-T therapy has shown great success in treating CD19-positive hematological malignancies, its application has been explored in the treatment of solid tumors, such as liver cancer. In this review, we discuss the immune characteristics of liver cancer, the obstacles encountered during the application of CAR-T therapy, and preclinical and clinical progress in the use of CAR-T therapy in patients with liver cancer.

Chimeric Antigen Receptor (CAR) therapy for multiple myeloma

The introduction of chimeric antigen receptor (CAR)‐modified T cells has revolutionized immunotherapy and cancer treatment as a whole. However, so far, clinical efficacy has only been demonstrated for CD19‐positive B cell lymphomas. For Multiple Myeloma (MM), the second most common haematological malignancy, there are currently no clinical results supporting the usefulness of the adoptive transfer of CAR‐modified T cells. This might be related to the fact that an ideal surface target has not yet been identified or the presence of strong local immunosuppression in the tumour microenvironment, which is a hallmark of MM. In this review, we provide a comprehensive overview of promising target molecules for CAR T cell approaches in MM and we outline a number of ways in which the local immunosuppression in MM can be overcome. By providing a strategy for the design of CAR T cell treatments for MM we hope to transform this new therapeutic approach into a valuable tool within the therapeutic armamentarium for MM.