26952084
Driving CAR T-cells forward
The engineered expression of chimeric antigen receptors (CARs) on the surface of T cells enables the redirection of T-cell specificity. Early clinical trials using CAR T cells for the treatment of patients with cancer showed modest results, but the impressive outcomes of several trials of CD19-targeted CAR T cells in the treatment of patients with B-cell malignancies have generated an increased enthusiasm for this approach. Important lessons have been derived from clinical trials of CD19-specific CAR T cells, and ongoing clinical trials are testing CAR designs directed at novel targets involved in haematological and solid malignancies. In this Review, we discuss these trials and present strategies that can increase the antitumour efficacy and safety of CAR T-cell therapy. Given the fast-moving nature of this field, we only discuss studies with direct translational application currently or soon-to-be tested in the clinical setting.
206681995
CD19-Targeted T Cells Rapidly Induce Molecular Remissions in Adults with Chemotherapy-Refractory Acute Lymphoblastic Leukemia
Five adults with chemotherapy-refractory B-ALL were induced into molecular remissions after treatment with CD19 CAR-targeted T cells. CARving a Niche for Cancer Immunotherapy Acute lymphoblastic leukemia (ALL) is a cancer of the white blood cells that fend off infection. It’s most common in children but—as with many diseases that primarily affect children—has a much worse prognosis when it affects adults. Adults with relapsed disease have a very low chance of survival, and new therapies are desperately needed. Now, Brentjens et al. test T cells engineered to target CD19, which is expressed on both healthy B lymphocytes and B-ALL cells, in five chemotherapy-refractory adult B-ALL patients. Here, the authors treat patients with the patients’ own T cells altered to express not only CD19 but also a fusion of the costimulatory molecule CD28 with CD3ζ chain—so-called “second-generation chimeric antigen receptor (CAR) T cells.” All patients treated with these cells achieved tumor eradication and complete remission. These CAR T cells were well tolerated, although there was substantial cytokine release in some patients that correlated to tumor burden. These patients were treated with steroid therapy. Long-term follow-up in four of these patients was not possible because the CAR T cell therapy allowed these patients to be eligible for subsequent hematopoietic stem cell transplant (HSCT), which resulted in restored hematopoiesis. The remaining patient experienced a relapse of CD19+ cells that coincided with the lack of persistence of the CAR T cells from circulation. These data suggest that subsequent transfusions should be considered for patients unable to undergo HSCT. Adults with relapsed B cell acute lymphoblastic leukemia (B-ALL) have a dismal prognosis. Only those patients able to achieve a second remission with no minimal residual disease (MRD) have a hope for long-term survival in the context of a subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have treated five relapsed B-ALL subjects with autologous T cells expressing a CD19-specific CD28/CD3ζ second-generation dual-signaling chimeric antigen receptor (CAR) termed 19-28z. All patients with persistent morphological disease or MRD+ disease upon T cell infusion demonstrated rapid tumor eradication and achieved MRD− complete remissions as assessed by deep sequencing polymerase chain reaction. Therapy was well tolerated, although significant cytokine elevations, specifically observed in those patients with morphologic evidence of disease at the time of treatment, required lymphotoxic steroid therapy to ameliorate cytokine-mediated toxicities. Indeed, cytokine elevations directly correlated to tumor burden at the time of CAR-modified T cell infusions. Tumor cells from one patient with relapsed disease after CAR-modified T cell therapy, who was ineligible for additional allo-HSCT or T cell therapy, exhibited persistent expression of CD19 and sensitivity to autologous 19-28z T cell–mediated cytotoxicity, which suggests potential clinical benefit of additional CAR-modified T cell infusions. These results demonstrate the marked antitumor efficacy of 19-28z CAR-modified T cells in patients with relapsed/refractory B-ALL and the reliability of this therapy to induce profound molecular remissions, forming a highly effective bridge to potentially curative therapy with subsequent allo-HSCT.
6359940
T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial
BACKGROUND
Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells.


METHODS
This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 10(6) CAR-transduced T cells per kg (dose 1), 3 × 10(6) CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT01593696.


FINDINGS
Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 10(6) CD19-CAR T cells per kg. All toxicities were fully reversible, with the most severe being grade 4 cytokine release syndrome that occurred in three (14%) of 21 patients (95% CI 3·0-36·3). The most common non-haematological grade 3 adverse events were fever (nine [43%] of 21 patients), hypokalaemia (nine [43%] of 21 patients), fever and neutropenia (eight [38%] of 21 patients), and cytokine release syndrome (three [14%) of 21 patients).


INTERPRETATION
CD19-CAR T cell therapy is feasible, safe, and mediates potent anti-leukaemic activity in children and young adults with chemotherapy-resistant B-precursor acute lymphoblastic leukaemia. All toxicities were reversible and prolonged B-cell aplasia did not occur.


FUNDING
National Institutes of Health Intramural funds and St Baldrick's Foundation.
8474298
Chimeric antigen receptor T cells for sustained remissions in leukemia.
BACKGROUND
Relapsed acute lymphoblastic leukemia (ALL) is difficult to treat despite the availability of aggressive therapies. Chimeric antigen receptor-modified T cells targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease.


METHODS
We infused autologous T cells transduced with a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector in patients with relapsed or refractory ALL at doses of 0.76×10(6) to 20.6×10(6) CTL019 cells per kilogram of body weight. Patients were monitored for a response, toxic effects, and the expansion and persistence of circulating CTL019 T cells.


RESULTS
A total of 30 children and adults received CTL019. Complete remission was achieved in 27 patients (90%), including 2 patients with blinatumomab-refractory disease and 15 who had undergone stem-cell transplantation. CTL019 cells proliferated in vivo and were detectable in the blood, bone marrow, and cerebrospinal fluid of patients who had a response. Sustained remission was achieved with a 6-month event-free survival rate of 67% (95% confidence interval [CI], 51 to 88) and an overall survival rate of 78% (95% CI, 65 to 95). At 6 months, the probability that a patient would have persistence of CTL019 was 68% (95% CI, 50 to 92) and the probability that a patient would have relapse-free B-cell aplasia was 73% (95% CI, 57 to 94). All the patients had the cytokine-release syndrome. Severe cytokine-release syndrome, which developed in 27% of the patients, was associated with a higher disease burden before infusion and was effectively treated with the anti-interleukin-6 receptor antibody tocilizumab.


CONCLUSIONS
Chimeric antigen receptor-modified T-cell therapy against CD19 was effective in treating relapsed and refractory ALL. CTL019 was associated with a high remission rate, even among patients for whom stem-cell transplantation had failed, and durable remissions up to 24 months were observed. (Funded by Novartis and others; CART19 ClinicalTrials.gov numbers, NCT01626495 and NCT01029366.).
22220330
Antitransgene rejection responses contribute to attenuated persistence of adoptively transferred CD20/CD19-specific chimeric antigen receptor redirected T cells in humans.
Immunotherapeutic ablation of lymphoma is a conceptually attractive treatment strategy that is the subject of intense translational research. Cytotoxic T lymphocytes (CTLs) that are genetically modified to express CD19- or CD20-specific, single-chain antibody-derived chimeric antigen receptors (CARs) display HLA-independent antigen-specific recognition/killing of lymphoma targets. Here, we describe our initial experience in applying CAR-redirected autologous CTL adoptive therapy to patients with recurrent lymphoma. Using plasmid vector electrotransfer/drug selection systems, cloned and polyclonal CAR(+) CTLs were generated from autologous peripheral blood mononuclear cells and expanded in vitro to cell numbers sufficient for clinical use. In 2 FDA-authorized trials, patients with recurrent diffuse large cell lymphoma were treated with cloned CD8(+) CTLs expressing a CD20-specific CAR (along with NeoR) after autologous hematopoietic stem cell transplantation, and patients with refractory follicular lymphoma were treated with polyclonal T cell preparations expressing a CD19-specific CAR (along with HyTK, a fusion of hygromycin resistance and HSV-1 thymidine kinase suicide genes) and low-dose s.c. recombinant human interleukin-2. A total of 15 infusions were administered (5 at 10(8)cells/m(2), 7 at 10(9)cells/m(2), and 3 at 2 x 10(9)cells/m(2)) to 4 patients. Overt toxicities attributable to CTL administration were not observed; however, detection of transferred CTLs in the circulation, as measured by quantitative polymerase chain reaction, was short (24 hours to 7 days), and cellular antitransgene immune rejection responses were noted in 2 patients. These studies reveal the primary barrier to therapeutic efficacy is limited persistence, and provide the rationale to prospectively define T cell populations intrinsically programmed for survival after adoptive transfer and to modulate the immune status of recipients to prevent/delay antitransgene rejection responses.
18770177
Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells.
Adoptive immunotherapy with T cells expressing a tumor-specific chimeric T-cell receptor is a promising approach to cancer therapy that has not previously been explored for the treatment of lymphoma in human subjects. We report the results of a proof-of-concept clinical trial in which patients with relapsed or refractory indolent B-cell lymphoma or mantle cell lymphoma were treated with autologous T cells genetically modified by electroporation with a vector plasmid encoding a CD20-specific chimeric T-cell receptor and neomycin resistance gene. Transfected cells were immunophenotypically similar to CD8(+) effector cells and showed CD20-specific cytotoxicity in vitro. Seven patients received a total of 20 T-cell infusions, with minimal toxicities. Modified T cells persisted in vivo 1 to 3 weeks in the first 3 patients, who received T cells produced by limiting dilution methods, but persisted 5 to 9 weeks in the next 4 patients who received T cells produced in bulk cultures followed by 14 days of low-dose subcutaneous interleukin-2 (IL-2) injections. Of the 7 treated patients, 2 maintained a previous complete response, 1 achieved a partial response, and 4 had stable disease. These results show the safety, feasibility, and potential antitumor activity of adoptive T-cell therapy using this approach. This trial was registered at www.clinicaltrials.gov as #NCT00012207.
33417415
CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients.
Targeted T cell immunotherapies using engineered T lymphocytes expressing tumor-directed chimeric antigen receptors (CARs) are designed to benefit patients with cancer. Although incorporation of costimulatory endodomains within these CARs increases the proliferation of CAR-redirected T lymphocytes, it has proven difficult to draw definitive conclusions about the specific effects of costimulatory endodomains on the expansion, persistence, and antitumor effectiveness of CAR-redirected T cells in human subjects, owing to the lack of side-by-side comparisons with T cells bearing only a single signaling domain. We therefore designed a study that allowed us to directly measure the consequences of adding a costimulatory endodomain to CAR-redirected T cells. Patients with B cell lymphomas were simultaneously infused with 2 autologous T cell products expressing CARs with the same specificity for the CD19 antigen, present on most B cell malignancies. One CAR encoded both the costimulatory CD28 and the ζ-endodomains, while the other encoded only the ζ-endodomain. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced expansion and persistence compared with CAR+ T cells lacking this endodomain. These results demonstrate the superiority of CARs with dual signal domains and confirm a method of comparing CAR-modified T cells within individual patients, thereby avoiding patient-to-patient variability and accelerating the development of optimal T cell immunotherapies.
206679854
Decade-Long Safety and Function of Retroviral-Modified Chimeric Antigen Receptor T Cells
Adoptively transferred chimeric antigen receptor T cells have stable stem cell–like persistence for at least a decade and more than 500 years of patient safety. Standing the Test of Time Retroviral vectors were once the mainstay of gene transfer because they could stably integrate into the host genome. However, some patients in early trials developed leukemia because of insertional mutagenesis. Now, Scholler et al. report that retroviral vector–mediated gene transfer in T cells may not have the same safety concerns, and that these cells may persist over a decade in patients. The authors followed patients from three clinical trials who received T cells transduced with gammaretroviruses carrying a chimeric antigen receptor. They found that these cells were present in recipients over a decade after infusion at levels higher than those induced by standard vaccines. These cells were still functional, had stable levels of engraftment, and did not require host immunosuppression before transplant. Moreover, the authors found no evidence of integration-induced immortalization, with no observable enrichment of integration sites near genes involved in growth control or transformation. Thus, the safety of retroviral vectors may be cell type–specific, opening up engineered T cells as a delivery platform for therapeutics. The success of adoptive T cell gene transfer for treatment of cancer and HIV is predicated on generating a response that is both durable and safe. We report long-term results from three clinical trials to evaluate gammaretroviral vector–engineered T cells for HIV. The vector encoded a chimeric antigen receptor (CAR) composed of CD4 linked to the CD3ζ signaling chain (CD4ζ). CAR T cells were detected in 98% of samples tested for at least 11 years after infusion at frequencies that exceeded average T cell levels after most vaccine approaches. The CD4ζ transgene retained expression and function. There was no evidence of vector-induced immortalization of cells; integration site distributions showed no evidence of persistent clonal expansion or enrichment for integration sites near genes implicated in growth control or transformation. The CD4ζ T cells had stable levels of engraftment, with decay half-lives that exceeded 16 years, in marked contrast to previous trials testing engineered T cells. These findings indicate that host immunosuppression before T cell transfer is not required to achieve long-term persistence of gene-modified T cells. Further, our results emphasize the safety of T cells modified by retroviral gene transfer in clinical application, as measured in >500 patient-years of follow-up. Thus, previous safety issues with integrating viral vectors are hematopoietic stem cell or transgene intrinsic, and not a general feature of retroviral vectors. Engineered T cells are a promising form of synthetic biology for long-term delivery of protein-based therapeutics. These results provide a framework to guide the therapy of a wide spectrum of human diseases.
6359940
T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial
BACKGROUND
Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells.


METHODS
This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 10(6) CAR-transduced T cells per kg (dose 1), 3 × 10(6) CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT01593696.


FINDINGS
Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 10(6) CD19-CAR T cells per kg. All toxicities were fully reversible, with the most severe being grade 4 cytokine release syndrome that occurred in three (14%) of 21 patients (95% CI 3·0-36·3). The most common non-haematological grade 3 adverse events were fever (nine [43%] of 21 patients), hypokalaemia (nine [43%] of 21 patients), fever and neutropenia (eight [38%] of 21 patients), and cytokine release syndrome (three [14%) of 21 patients).


INTERPRETATION
CD19-CAR T cell therapy is feasible, safe, and mediates potent anti-leukaemic activity in children and young adults with chemotherapy-resistant B-precursor acute lymphoblastic leukaemia. All toxicities were reversible and prolonged B-cell aplasia did not occur.


FUNDING
National Institutes of Health Intramural funds and St Baldrick's Foundation.
36897995
Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains
Mesothelin is a cell-surface molecule over-expressed on a large fraction of carcinomas, and thus is an attractive target of immunotherapy. A molecularly targeted therapy for these cancers was created by engineering T cells to express a chimeric receptor with high affinity for human mesothelin. Lentiviral vectors were used to express a single-chain variable fragment that binds mesothelin and that is fused to signaling domains derived from T-cell receptor zeta, CD28, and CD137 (4–1BB). When stimulated by mesothelin, lentivirally transduced T cells were induced to proliferate, express the antiapoptotic gene Bcl-XL, and secrete multiple cytokines, all features characteristic of central memory T cells. When transferred intratumorally or intravenously into NOD/scid/IL2rγ−/− mice engrafted with large pre-established tumors, the engineered T cells reduced the tumor burden, and in some cases resulted in complete eradication of the tumors at low effector-to-target ratios. Incorporation of the CD137 signaling domain specifically reprogrammed cells for multifunctional cytokine secretion and enhanced persistence of T cells. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of poorly immunogenic tumors. Genetically redirected T cells have promise of targeting T lymphocytes to tumor antigens, confer resistance to the tumor microenvironment, and providing immunosurveillance.
6864319
Chimeric antigen receptors combining 4-1BB and CD28 signaling domains augment PI3kinase/AKT/Bcl-XL activation and CD8+ T cell-mediated tumor eradication.
To enhance the strength of activation afforded by tumor antigen-specific receptors, we investigated the effect of adding combined CD28 and 4-1BB costimulatory signaling domains to a chimeric antigen receptor (CAR) specific for prostate-specific membrane antigen (PSMA). Having transferred receptors encompassing the CD28, 4-1BB, and/or CD3zeta cytoplasmic domains in primary human CD8(+) T cells, we find that the P28BBz receptor, which includes all three signaling domains, is superior to receptors that only include one or two of these domains in promoting cytokine release, in vivo T-cell survival and tumor elimination following intravenous T-cell administration to tumor-bearing severe combined immunodeficient (SCID)/beige mice. Upon in vitro exposure to PSMA, the P28BBZ receptor-induced the strongest PI(3)Kinase/Akt activation and Bcl-X(L) expression, and the least apoptosis in transduced peripheral blood CD8(+) T cells. These findings further support the concept of integrating optimized costimulatory properties into recombinant antigen receptors to augment the survival and function of genetically targeted T cells within the tumor microenvironment.
5132358
Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.
Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.
10064535
Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo
Adoptive T-cell therapy with gene-modified T cells expressing a tumor-reactive T-cell receptor or chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and has shown success in the treatment of B-cell malignancies and solid tumors. In all reported trials, patients have received T-cell products comprising random compositions of CD4+ and CD8+ naive and memory T cells, meaning that each patient received a different therapeutic agent. This variation may have influenced the efficacy of T-cell therapy, and complicates comparison of outcomes between different patients and across trials. We analyzed CD19 CAR-expressing effector T cells derived from different subsets (CD4+/CD8+ naive, central memory, effector memory). T cells derived from each of the subsets were efficiently transduced and expanded, but showed clear differences in effector function and proliferation in vitro and in vivo. Combining the most potent CD4+ and CD8+ CAR-expressing subsets, resulted in synergistic antitumor effects in vivo. We show that CAR-T-cell products generated from defined T-cell subsets can provide uniform potency compared with products derived from unselected T cells that vary in phenotypic composition. These findings have important implications for the formulation of T-cell products for adoptive therapies.
78158186
Immunotherapy with CD19-specific chimeric antigen receptor (CAR)-modified T cells of defined subset composition.
3006 Background: Genetically modified T cells derived from distinct T cell subsets differ in the capacity to persist after adoptive transfer. We are conducting the first phase I/II clinical trial in which patients (pts) with CD19+ B cell malignancies receive T cells comprised of a defined composition of CD8+ TCM and CD4+ T cells engineered to express a CD19 CAR. Methods: CD8+ TCM and CD4+ T cells were separately enriched from each patient, transduced with a CD19 CAR lentivirus and expanded in vitro. The cell product for infusion was formulated in a 1:1 ratio of CD8+:CD4+ CAR+ T cells and infused at one of three dose levels (2x105 – 2x107 CAR-T cells/kg) after lymphodepleting chemotherapy. Results: Thirty-seven pts with ALL (n = 20), NHL (n = 14) or CLL (n = 3) have been treated and 33/37 received a product that conformed to a prescribed CD8+:CD4+ composition. There was no serious acute infusional toxicity. Severe cytokine release syndrome (sCRS) consisting of fever, hypotension, coagulopathy and neurotoxi...
2932855
Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias.
We report the findings from the first 10 patients with chemotherapy-refractory chronic lymphocytic leukemia (CLL) or relapsed B-cell acute lymphoblastic leukemia (ALL) we have enrolled for treatment with autologous T cells modified to express 19-28z, a second-generation chimeric antigen (Ag) receptor specific to the B-cell lineage Ag CD19. Eight of the 9 treated patients tolerated 19-28z(+) T-cell infusions well. Three of 4 evaluable patients with bulky CLL who received prior conditioning with cyclophosphamide exhibited either a significant reduction or a mixed response in lymphadenopathy without concomitant development of B-cell aplasia. In contrast, one patient with relapsed ALL who was treated in remission with a similar T-cell dose developed a predicted B-cell aplasia. The short-term persistence of infused T cells was enhanced by prior cyclophosphamide administration and inversely proportional to the peripheral blood tumor burden. Further analyses showed rapid trafficking of modified T cells to tumor and retained ex vivo cytotoxic potential of CD19-targeted T cells retrieved 8 days after infusion. We conclude that this adoptive T-cell approach is promising and more likely to show clinical benefit in the setting of prior conditioning chemotherapy and low tumor burden or minimal residual disease. These studies are registered at www.clinicaltrials.org as #NCT00466531 (CLL protocol) and #NCT01044069 (B-ALL protocol).
2710513
B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells.
We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19(+) B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti-CD19-CAR-transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti-CD19-CAR-transduced T-cell infusions. Anti-CD19-CAR-transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19(+) cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR-transduced T-cell infusions.
206688370
Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia
CAR T cells persist and sustain remissions in advanced chronic lymphocytic leukemia. CAR T cells for the long haul Immunotherapy is one of the most promising avenues of cancer therapy, with the potential to induce sustained remissions in patients with refractory disease. Studies with chimeric antigen receptor (CAR)–modified T cells have paved the way in patients with relapsed and refractory chronic lymphocytic leukemia. Porter et al. now report the mature results from their initial CAR T cell trial. CAR T cell persistence correlated with clinical responses, and these cells were functional up to 4 years after treatment. No patient who achieved complete remission relapsed, and no minimal residual disease was detected, suggesting that in a subset of patients, CAR T cells may drive disease eradication. Patients with multiply relapsed or refractory chronic lymphocytic leukemia (CLL) have a poor prognosis. Chimeric antigen receptor (CAR)–modified T cells targeting CD19 have the potential to improve on the low complete response rates with conventional therapies by inducing sustained remissions in patients with refractory B cell malignancies. We previously reported preliminary results on three patients with refractory CLL. We report the mature results from our initial trial using CAR-modified T cells to treat 14 patients with relapsed and refractory CLL. Autologous T cells transduced with a CD19-directed CAR (CTL019) lentiviral vector were infused into patients with relapsed/refractory CLL at doses of 0.14 × 108 to 11 × 108 CTL019 cells (median, 1.6 × 108 cells). Patients were monitored for toxicity, response, expansion, and persistence of circulating CTL019 T cells. The overall response rate in these heavily pretreated CLL patients was 8 of 14 (57%), with 4 complete remissions (CR) and 4 partial remissions (PR). The in vivo expansion of the CAR T cells correlated with clinical responses, and the CAR T cells persisted and remained functional beyond 4 years in the first two patients achieving CR. No patient in CR has relapsed. All responding patients developed B cell aplasia and experienced cytokine release syndrome, coincident with T cell proliferation. Minimal residual disease was not detectable in patients who achieved CR, suggesting that disease eradication may be possible in some patients with advanced CLL.
54160556
Anti-CD19 CAR T Cells Administered after Low-Dose Chemotherapy Can Induce Remissions of Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma
We have treated a total of 30 patients with autologous T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19; 22 of 27 evaluable patients obtained either complete remissions (CR) or partial remissions (PR). Ten patients remain in ongoing CRs of 1 to 37 months duration. The CAR was encoded by a gammaretroviral vector and included the variable regions of an anti-CD19 antibody along with CD28 and CD3-zeta moieties. The first 21 patients treated on this protocol have been reported (Kochenderfer et al. Blood 2010, Blood 2012, and Journal of Clinical Oncology 2014). To enhance the activity of the transferred CAR T cells, T-cell infusions in the previously reported patients were preceded by a chemotherapy regimen of high-dose cyclophosphamide (60-120 mg/kg) plus fludarabine. In an attempt to reduce the overall toxicity of our anti-CD19 CAR treatment protocol, we substantially reduced the doses of chemotherapy administered before CAR T-cell infusions. This abstract communicates results from 9 patients with B-cell lymphoma who received a single infusion of 1x106 anti-CD19-CAR-expressing T cells/kg bodyweight preceded by a low-dose chemotherapy regimen consisting of cyclophosphamide 300 mg/m2 and fludarabine 30 mg/m2 (Table). Each chemotherapy agent was administered daily for 3 days. Eight of the 9 treated patients had DLBCL (diffuse large B-cell lymphoma) that was refractory to chemotherapy (chemo-refractory) or that had relapsed less than 1 year after autologous stem cell transplantation (ASCT). Both of these clinical situations carry a grim prognosis, with median overall survivals of only a few months. Despite the very poor prognoses of our patients, one patient with DLBCL obtained a CR and 4 DLBCL patients obtained PRs. In some patients, PRs included resolution of large lymphoma masses. Compared to our previous experience with anti-CD19 CAR T cells preceded by high-dose chemotherapy, toxicity was reduced when CAR T cells were infused after low-dose chemotherapy. None of the 9 patients treated with low-dose chemotherapy and CAR T cells required vasopressor drugs or mechanical ventilation, although some patients did have short-term neurological toxicity. Cytopenias were mild with a mean of only 1.4 days of blood neutrophils<500/microliter. Blood anti-CD19 CAR T-cell levels were assessed in 6 patients with a quantitative PCR assay; we detected CAR+ cells in the blood of all 6 patients. The mean peak absolute number of blood CAR+ T cells was 73 cells/microliter. Six months after infusion, persisting CAR+ T cells were detected in a lymphoma-involved lymph node by flow cytometry. These results demonstrate that anti-CD19 CAR T cells administered after low-dose chemotherapy have significant activity against chemo-refractory DLBCL and could potentially become a standard treatment for aggressive lymphoma.

| Patient | Age/Gender | Malignancy | Number of Prior Therapies | Clinical Situation | Response (Duration in Months) |
||
| 1 | 66/M | DLBCL | 3 | Post ASCT relapse | PR (7) |
| 2[*][1] | 63/F | DLBCL | 2 | Chemo-refractory | PR (7+) |
| 3 | 63/M | FL | 7 | Not chemo-refractory | PR (6+) |
| 4[*][1] | 22/M | DLBCL | 6 | Chemo-refractory | Progression |
| 5 | 65/M | DLBCL | 4 | Post ASCT relapse | PR (5+) |
| 6 | 47/M | DLBCL | 2 | Chemo-refractory | PR (1) |
| 7 | 28/M | DLBCL | 7 | Chemo-refractory | Progression |
| 8 | 62/M | DLBCL | 7 | Post ASCT relapse | CR (1+) |
| 9 | 54/M | DLBCL | 3 | Chemo-refractory | Progression |

* [↵][2]* Compassionate exemption was obtained from regulatory agencies to enroll these patients because their poor performance status precluded standard enrollment; M = male; F = female; FL = follicular lymphoma; + indicates ongoing response

Table 



Disclosures Rosenberg: Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding.

 [1]: #fn-2
 [2]: #xref-fn-2-1
74129392
Phase IIa trial of chimeric antigen receptor modified T cells directed against CD19 (CTL019) in patients with relapsed or refractory CD19+ lymphomas.
8516 Background: Autologous T cells expressing a chimeric antigen receptor with an external anti-CD19 single chain antibody domain and CD3ζ and 4-1BB signaling domains (CTL019 cells) mediate anti-t...
21175903
Efficacy and Toxicity Management of 19-28z CAR T Cell Therapy in B Cell Acute Lymphoblastic Leukemia
CD19 CAR T cell therapy induces complete remissions in 88% of 16 adult patients with relapsed or refractory acute lymphoblastic leukemia. CARving Out a Niche for CAR T Cell Immunotherapy Relapsed or refractory B acute lymphoblastic leukemia (B-ALL) in adults has a poor prognosis, with an expected median survival of less than 6 months. An emerging therapy for adult B-ALL is through T cells that target tumor cells with chimeric antigen receptors (CARs). Davila et al. now report the results of a phase 1 clinical trial of CAR T cells in 16 relapsed or refractory adult patients. The CD19-targeting CAR T cell therapy resulted in an 88% complete response rate, which allowed most of the patients to transition to allogeneic hematopoietic stem cell transplantation—the current standard of care. Moreover, the authors carefully characterized cytokine release syndrome (CRS), which is a series of toxicities associated with CAR T cell therapy. They found that serum C-reactive protein (CRP) associated with the severity of CRS, which should allow for identification of the subset of patients who will likely require therapeutic intervention with corticosteroids or interleukin-6 receptor blockade to curb the CRS. This is especially important because treatment for CRS may limit the efficacy of the CAR T cell therapy. These data support the need for further multicenter trials for CAR T cell therapy. We report on 16 patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL) that we treated with autologous T cells expressing the 19-28z chimeric antigen receptor (CAR) specific to the CD19 antigen. The overall complete response rate was 88%, which allowed us to transition most of these patients to a standard-of-care allogeneic hematopoietic stem cell transplant (allo-SCT). This therapy was as effective in high-risk patients with Philadelphia chromosome–positive (Ph+) disease as in those with relapsed disease after previous allo-SCT. Through systematic analysis of clinical data and serum cytokine levels over the first 21 days after T cell infusion, we have defined diagnostic criteria for a severe cytokine release syndrome (sCRS), with the goal of better identifying the subset of patients who will likely require therapeutic intervention with corticosteroids or interleukin-6 receptor blockade to curb the sCRS. Additionally, we found that serum C-reactive protein, a readily available laboratory study, can serve as a reliable indicator for the severity of the CRS. Together, our data provide strong support for conducting a multicenter phase 2 study to further evaluate 19-28z CAR T cells in B-ALL and a road map for patient management at centers now contemplating the use of CAR T cell therapy.
27768464
Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.
We designed a lentiviral vector expressing a chimeric antigen receptor with specificity for the B-cell antigen CD19, coupled with CD137 (a costimulatory receptor in T cells [4-1BB]) and CD3-zeta (a signal-transduction component of the T-cell antigen receptor) signaling domains. A low dose (approximately 1.5×10(5) cells per kilogram of body weight) of autologous chimeric antigen receptor-modified T cells reinfused into a patient with refractory chronic lymphocytic leukemia (CLL) expanded to a level that was more than 1000 times as high as the initial engraftment level in vivo, with delayed development of the tumor lysis syndrome and with complete remission. Apart from the tumor lysis syndrome, the only other grade 3/4 toxic effect related to chimeric antigen receptor T cells was lymphopenia. Engineered cells persisted at high levels for 6 months in the blood and bone marrow and continued to express the chimeric antigen receptor. A specific immune response was detected in the bone marrow, accompanied by loss of normal B cells and leukemia cells that express CD19. Remission was ongoing 10 months after treatment. Hypogammaglobulinemia was an expected chronic toxic effect.
205098044
Chimeric Antigen Receptor T Cells against CD19 for Multiple Myeloma.
A patient with refractory multiple myeloma received an infusion of CTL019 cells, a cellular therapy consisting of autologous T cells transduced with an anti-CD19 chimeric antigen receptor, after myeloablative chemotherapy (melphalan, 140 mg per square meter of body-surface area) and autologous stem-cell transplantation. Four years earlier, autologous transplantation with a higher melphalan dose (200 mg per square meter) had induced only a partial, transient response. Autologous transplantation followed by treatment with CTL019 cells led to a complete response with no evidence of progression and no measurable serum or urine monoclonal protein at the most recent evaluation, 12 months after treatment. This response was achieved despite the absence of CD19 expression in 99.95% of the patient's neoplastic plasma cells. (Funded by Novartis and others; ClinicalTrials.gov number, NCT02135406.).
5005560
Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.
UNLABELLED
The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.


SIGNIFICANCE
CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.
23718478
Design and development of therapies using chimeric antigen receptor‐expressing T cells
Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non‐protein antigens) with the persistence, trafficking, and effector functions of a T cell. This article describes how the past two decades have seen a crescendo of research which has now begun to translate these potential benefits into effective treatments for patients with cancer. We describe the basic design of CARs, describe how antigenic targets are selected, and the initial clinical experience with CAR‐T cells. Our review then describes our own and other investigators' work aimed at improving the function of CARs and reviews the clinical studies in hematological and solid malignancies that are beginning to exploit these approaches. Finally, we show the value of adding additional engineering features to CAR‐T cells, irrespective of their target, to render them better suited to function in the tumor environment, and discuss how the safety of these heavily modified cells may be maintained.
17202601
Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.
Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.
11387511
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.
25487356
Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma treated by CD20-directed chimeric antigen receptor-modified T cells.
We conducted a trial testing a CD20-specific CAR coupled with CD137 and the CD3ζ moiety in patients with chemotherapy refractory advanced diffuse large B cell lymphomas (DLBCL). Seven patients were enrolled. One of the two patients with no bulky tumor obtained a 14-month durable and ongoing complete remission by cell infusion only, and another attained a 6-month tumor regression. Four of five patients with bulky tumor burden were evaluable for clinical efficacy, three of which attained 3- to 6-month tumor regression. Delayed toxicities related to cell infusion are directly correlated to tumor burden and tumor-harboring sites, and mainly included cytokine release symptoms, tumor lysis symptoms, massive hemorrhage of the alimentary tract and aggressive intrapulmonary inflammation surrounding extranodal lesions. These results show firstly that anti-CD20 CART cells can cause prolonged tumor regression in combination with debulking conditioning regimens for advanced DLBCL. This study is registered at www.clinicaltrials.gov as NCT01735604.
20727074
The onco-embryonic antigen ROR1 is expressed by a variety of human cancers.
ROR1 is an orphan-receptor tyrosine-kinase-like surface antigen that is expressed by many tissues during embryogenesis, some B-cell malignancies, and various cancer cell lines but not by virtually all normal adult tissues. Here, we report that large proportions of many different human cancers also express ROR1, particularly those cancers that have high-grade histology. Primary cancers that expressed ROR1 more commonly expressed high levels of phosphorylated AKT (p-AKT) and phosphorylated cAMP response element binding-factor (p-CREB) than similar cancers that lacked expression of ROR1. Induced expression of ROR1 could enhance basal p-AKT and p-CREB levels and could promote the growth of a cancer cell line, MEC1. Conversely, silencing ROR1 resulted in lower levels of p-AKT and p-CREB, which was associated with impaired tumor cell growth. In summary, this study found that many different human cancers express ROR1 and that ROR1 may play a functional role in promoting tumor cell growth, suggesting that this orphan-receptor tyrosine-kinase-like protein may be a potential target for therapy directed against a variety of human cancers.
35238280
Expression of the Ror1 and Ror2 receptor tyrosine kinase genes during mouse development
Abstract. Ror1 and Ror2 are orphan receptor tyrosine kinases that are most closely related to MuSK and the Trk family of neurotrophin receptors. We report the results of an extensive in situ hybridisation survey of the expression of these genes during mouse development. Expression of Ror1 and Ror2 differs markedly at early stages (E8.5–E9.5). At these times, Ror2 is expressed much more widely than Ror1, expression of which is largely restricted to head mesenchyme. At later stages of development (E12.5–E14.5), Ror1 expression expands and Ror2 expression becomes more restricted than at earlier times, although expression of Ror1 continues to be more restricted than that of Ror2. These changes result in overlapping expression domains but with major differences remaining. In many cases Ror1 is expressed in a sub-set of Ror2-expressing tissues; in others, there is complementary expression of Ror1 and Ror2. Ror1 and Ror2 are both expressed in derivatives of all three germ layers and in most organ systems, including the nervous, circulatory, respiratory, digestive, urogenital and skeletal systems. Conspicuous themes are the expression in major sense organs, and in neural crest and its derivatives.
28832765
The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor.
Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells.
38974635
CD30 is a signal-transducing molecule that defines a subset of human activated CD45RO+ T cells.
CD30 has been extensively studied as a cell surface marker expressed by Reed-Sternberg cells of Hodgkin's disease and other hematologic malignancies, although little is known about its expression by normal lymphoid cells. We therefore characterized the requirements for the induction of CD30 expression and identified the subsets of T cells that express CD30. CD30 is inducible on approximately 15% of normal PBMC stimulated with any of a variety of nonspecific T cell activators, including PHA, Con A, anti-T11(2) + T11(3), and anti-CD3; ionomycin alone induced lower percentages of CD30+ T cells (3 +/- 2%) compared to other stimuli. Maximal numbers of CD30+ cells were observed at 48 to 72 h of activation and the addition of rIL-2 did not affect these kinetics. However, CD30 expression was enhanced by the addition of exogenous rIL-2 to any of the stimuli tested, although rIL-2 alone did not lead to CD30 expression. The induction of CD30 during anti-CD3 mitogenesis was completely inhibitable by anti-IL-2 antibodies and partially inhibitable by rIL-4, indicating a requirement for both TCR triggering and IL-2 for its expression. Dual immunofluorescence analysis revealed that CD30+ cells were confined to CD3+ T cells that coexpressed higher levels of the p55 IL-2 receptor (CD25) than the CD30- population. Furthermore, CD30 expression was restricted to a subset of cells derived from CD45RO+ T cell precursors. Cell cycle analysis showed that CD30+ expression was not cell cycle dependent. Cross-linking of membrane CD30 induced Ca2+ in TCR+, but not TCR- Jurkat T cells. These results demonstrate that CD30 can serve as a T cell signal-transducing molecule and expressed by a unique subset of activated CD45RO+ T cells.
42526490
An anti-CD30 chimeric receptor that mediates CD3-zeta-independent T-cell activation against Hodgkin's lymphoma cells in the presence of soluble CD30.
Hodgkin's lymphoma patients fail to establish an efficient cellular response against CD30+ Hodgkin/Reed-Sternberg cells. An impaired T-cell receptor/CD3-zeta-mediated activation of T cells is thought to be involved in this situation. We here present a chimeric anti-CD30 receptor that mediates MHC and T-cell receptor/CD3-zeta-independent T-cell activation against CD30+ lymphoma cells even in the presence of soluble CD30. The receptor consists of the binding domain of the monoclonal antibody HRS3 and the signaling unit of the Fc epsilonRI-receptor gamma-chain. After expression in MD45 T cells, receptor cross-linking with immobilized anti-idiotypic monoclonal antibody and CD30+ cells, respectively, results in increased interleukin 2 secretion and specific cytolysis of CD30+ Hodgkin's lymphoma cells. Soluble CD30 in concentrations up to 6000 units/ml did not interfere with cellular activation induced by membrane-bound antigen. This demonstrates the feasibility of the chimeric anti-CD30-scFv-gamma receptor in CD30+ lymphoma cell targeting, even in the presence of as high concentrations of soluble CD30 as are found in patients during progression of the disease.
72639968
CD138-directed adoptive immunotherapy of chimeric antigen receptor (CAR)-modified T cells for multiple myeloma
Abstract Adoptive immunotherapy with T cells expressing a tumor-associated chimeric antigen receptor (CAR) provides a promising approach for tumor therapy. We designed a clinical trial for multiple myeloma (MM) treatment with CAR-modified T cells recognizing CD138 (CART-138). Five patients diagnosed with chemotherapy-refractory MM were enrolled into this trial, although one later advanced to plasma cell leukemia. By intravenous infusions, these patients received CD3+ CART-138 cells in an escalating dose. No intolerable toxicity was observed during this process. CART-138 cells were expanded to a level 1000 times higher than the initial engraftment level and were maintained in the peripheral blood. In addition, increased CART-138 cells were also detected in the bone marrow. Four of the five patients had stable disease (SD) longer than three months, and one patient with advanced plasma cell leukemia had a reduction of the myeloma cells in her peripheral blood (from 10.5% to
25414211
B-cell Maturation Antigen Is a Promising Target for Adoptive T-cell Therapy of Multiple Myeloma
Purpose: Multiple myeloma is a usually incurable malignancy of plasma cells. New therapies are urgently needed for multiple myeloma. Adoptive transfer of chimeric antigen receptor (CAR)–expressing T cells is a promising new therapy for hematologic malignancies, but an ideal target antigen for CAR-expressing T-cell therapies for multiple myeloma has not been identified. B-cell maturation antigen (BCMA) is a protein that has been reported to be selectively expressed by B-lineage cells including multiple myeloma cells. Our goal was to determine if BCMA is a suitable target for CAR-expressing T cells. Experimental Design: We conducted an assessment of BCMA expression in normal human tissues and multiple myeloma cells by flow cytometry, quantitative PCR, and immunohistochemistry. We designed and tested novel anti-BCMA CARs. Results: BCMA had a restricted RNA expression pattern. Except for expression in plasma cells, BCMA protein was not detected in normal human tissues. BCMA was not detected on primary human CD34+ hematopoietic cells. We detected uniform BCMA cell-surface expression on primary multiple myeloma cells from five of five patients. We designed the first anti-BCMA CARs to be reported and we transduced T cells with lentiviral vectors encoding these CARs. The CARs gave T cells the ability to specifically recognize BCMA. The anti-BCMA-CAR–transduced T cells exhibited BCMA-specific functions including cytokine production, proliferation, cytotoxicity, and in vivo tumor eradication. Importantly, anti-BCMA-CAR–transduced T cells recognized and killed primary multiple myeloma cells. Conclusions: BCMA is a suitable target for CAR-expressing T cells, and adoptive transfer of anti-BCMA-CAR–expressing T cells is a promising new strategy for treating multiple myeloma. Clin Cancer Res; 19(8); 2048–60. ©2013 AACR.
19759913
Treatment of relapsed or refractory acute myeloid leukemia with humanized anti-CD33 monoclonal antibody HuM195
HuM195 is a humanized, unconjugated, anti-CD33 monoclonal antibody. Fifty adult patients with relapsed or refractory AML were randomized to receive HuM195 at a dose of 12 or 36 mg/m2 by intravenous infusion over 4 h on days 1–4 and 15–18. Patients with stable or responding disease received two additional cycles on days 29–32 and 43–46. HuM195 was given as first salvage therapy in 24 patients and as second or subsequent salvage therapy in 26 patients. Pretreatment blast percentage in the marrow was between 5 and 30% in 20 patients with the others having blast counts greater than 30%. The median age of patients was 62 years (range 26–86) and CD33 was detected in 95% of patients for whom immunophenotyping was available. Of 49 evaluable patients, two complete and one partial remission were observed. All three responses were in patients treated at the 12 mg/m2 dose level and all had baseline blast percentages less than 30%. Decreases in blast counts ranging from 30 to 74% were seen in nine additional patients. Infusion-related events of fever and chills occurred in the majority of patients and were generally mild and primarily related to the first dose of antibody. No hepatic, renal or cardiac toxicities were observed and other adverse events such as nausea, vomiting, mucositis and diarrhea were uncommon or felt to be unrelated to HuM195. In addition, anti-HuM195 responses were not detected. HuM195 as a single agent has minimal, but observable, anti-leukemic activity in patients with relapsed or refractory AML and activity is confined to patients with low burden disease. No significant differences in clinical efficacy or toxicity were seen between the two dose levels of antibody. HuM195 was well tolerated with infusion-related fevers and chills the predominant toxicities seen. Meaningful clinical efficacy of this unconjugated monoclonal antibody may be realized only in patients with minimal residual disease, or in combination with chemotherapy.
206917728
T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.
Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in select patients and highlights the susceptibility of AML to donor-derived immunotherapy. The interleukin-3 receptor α chain (CD123) has been identified as a potential immunotherapeutic target because it is overexpressed in AML compared with normal hematopoietic stem cells. Therefore, we developed 2 chimeric antigen receptors (CARs) containing a CD123-specific single-chain variable fragment, in combination with a CD28 costimulatory domain and CD3-ζ signaling domain, targeting different epitopes on CD123. CD123-CAR-redirected T cells mediated potent effector activity against CD123+ cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited antileukemic activity in vivo against a xenogeneic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high-risk AML.
31491887
Identification of CD123+ myeloid dendritic cells as an early-stage immature subset with strong tumoristatic potential.
CD123 has been identified as a specific surface marker for plasmacytoid dendritic cells (PDCs). However, CD123 has recently been shown to be expressed on freshly isolated or in vitro generated myeloid dendritic cells (MDCs). In this article, we investigated whether the expression of CD123 on monocyte-derived MDCs was related to their function, especially to tumor-inhibiting potential. MDCs were induced from cord blood CD14+ monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days, and then CD123+ cells were isolated by positive immunomagnetic cell selection. We observed that CD123+ cells lost monocyte CD14 expression, acquired immature myeloid dendritic cell phenotype and morphology. They exerted more significant endocytosis and less antigen-presenting function than CD123(-)MDCs which are often referred to as typical MDCs. Meanwhile, CD123+ MDCs exhibited more significant tumor-inhibiting activity toward hematological tumor cell lines of U937 and Jurkat even at a low effector:target ratio. CD123+ MDCs expressed higher level of cytoplasmic TNF-alpha-related apoptosis-inducing ligand (TRAIL), but no detectable surface TRAIL and very little soluble TRAIL. Pretreatment with recombinant human TRAIL receptor 2:Fc fusion protein significantly reduced the tumor-inhibiting effect of CD123+ MDCs, but not of CD123(-) MDCs. Overall, our data demonstrated that CD123+ MDCs were an early-stage immature DC subset, with a significant tumor-inhibiting activity partially via involvement of enhanced cytoplasmic TRAIL.
20084806
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.
23974912
Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukaemia in first relapse
Gemtuzumab ozogamicin (CMA-676, Mylotarg®), an antibody-targeted chemotherapy agent, was recently approved by the FDA for the treatment of patients with CD33+ acute myeloid leukaemia (AML) in first relapse who are 60 years of age or older and who are not considered candidates for other types of cytotoxic chemotherapy. In combined Phase II studies of 142 patients with CD33+ AML in first relapse, gemtuzumab ozogamicin monotherapy was associated with a 30% overall response rate. While treated patients had relatively high incidences of myelosuppression, grade 3 or grade 4 hyperbilirubinaemia (23%) and elevated hepatic transaminases (17%), the incidences of grade 3 or grade 4 mucositis (4%) and infections (28%) were low compared with what might be expected in association with conventional chemotherapeutic treatment. In contrast with the usual in-patient administration of cytarabine and anthracycline-containing induction regimens, a large number of patients were treated with gemtuzumab ozogamicin as outpatients (38% and 41% for the first and second doses, respectively). Two prognostic factors for patients with AML in first relapse, age and duration of complete remission, had relatively little effect on response rates to gemtuzumab ozogamicin. Preliminary data in pediatric patients also suggest the immunoconjugate to be reasonably well tolerated. Studies of gemtuzumab ozogamicin in combination with anthracycline and cytarabine are underway. Gemtuzumab ozogamicin, administered to patients with CD33+ AML in first relapse, has shown overall response rates comparable to conventional agents and a safety profile that appears to be favourable.
8378475
Treatment of CD33-directed chimeric antigen receptor-modified T cells in one patient with relapsed and refractory acute myeloid leukemia.
We conducted a clinical trial to assess the feasibility and efficacy of CD33-directed chimeric antigen receptor-modified T cells (CART-33) for the treatment of refractory acute myeloid leukemia (AML). A 41-year-old male patient with AML was enrolled and received a total of 1.12 × 10(9) autologous CART-33 cells, of which ~38% were transduced with CAR. The CART-33 infusion alone induced rigorous chills and fevers; drastic fluctuations of his preexisting pancytopenia; elevated serum cytokine levels, including interleukin (IL)-6, IL-8, tumor necrosis factor-α, and interferon-γ; slight transient hyperbilirubinemia within 2 weeks; a subsequent intermittent moderate fever; and reversed fluctuation of the pancytopenia. A marked decrease of blasts in the bone marrow was observed on examination 2 weeks after therapy, and there was a gradual increase until florid disease progression occurred at 9 weeks after the cell infusion. These observations warrant further research on CART-33 treatment in refractory AML and may spur efforts to extend the CART-33-induced tumor burden to the preparation of other intensive strategies, such as hematopoietic stem cell transplantation. This study is registered at www.ClinicalTrials.gov as NCT01864902.
36145684
Lewis blood group-related antigen expression in normal gastric epithelium, intestinal metaplasia, gastric adenoma, and gastric carcinoma.
The expression of blood group-related antigens of the Lewis system in normal gastric mucosa, intestinal metaplasia, gastric adenoma, and gastric carcinoma was examined. Ninety-five percent of normal foveolar epithelial samples stained positive for Lewis(b) antigen, whereas only 10.0% expressed Lewis(a) antigen. In contrast, intestinal metaplasia specimens had increased Lewis(a) antigen expression and slightly decreased Lewis(b) antigen expression. A similar pattern of Lewis(a) and Lewis(b) expression was observed in gastric adenomas and intestinal type adenocarcinomas. Lewis(x) and Lewis(y) were detected in all normal deep glands, but were not expressed in the majority of intestinal metaplasia specimens. In addition, only 20-40% of gastric adenomas and gastric carcinomas expressed Lewis(x) and Lewis(y) antigens. These changes in Lewis antigen expression in intestinal metaplasia, adenomas, and intestinal type adenocarcinomas suggest that altered expression of Lewis blood group-related antigens may correlate with cell transformation processes.
6875533
Gene-modified T cells as immunotherapy for multiple myeloma and acute myeloid leukemia expressing the Lewis Y antigen
We have evaluated the carbohydrate antigen LewisY (LeY) as a potential target for T-cell immunotherapy of hematological neoplasias. Analysis of 81 primary bone marrow samples revealed moderate LeY expression on plasma cells of myeloma patients and myeloblasts of patients with acute myeloid leukemia (AML) (52 and 46% of cases, respectively). We developed a retroviral vector construct encoding a chimeric T-cell receptor that recognizes the LeY antigen in a major histocompatibility complex-independent manner and delivers co-stimulatory signals to achieve T-cell activation. We have shown efficient transduction of peripheral blood-derived T cells with this construct, resulting in antigen-restricted interferon-γ secretion and cell lysis of LeY-expressing tumor cells. In vivo activity of gene-modified T cells was demonstrated in the delayed growth of myeloma xenografts in NOD/SCID mice, which prolonged survival. Therefore, targeting LeY-positive malignant cells with T cells expressing a chimeric receptor recognizing LeY was effective both in vitro and in a myeloma mouse model. Consequently, we plan to use T cells manufactured under Good Manufacturing Practice conditions in a phase I immunotherapy study for patients with LeY-positive myeloma or AML.
42891748
Blood group and blood‐group‐related antigens in normal pancreas and pancreas cancer: Enhanced expression of precursor type 1, Tn and Sialyl‐Tn in pancreas cancer
Expression of blood‐group antigens A, B, Lea, sialyl‐Lea (sLea), Leb, Lex, Ley, precursor type 1, Tn and sialyl‐Tn (sTn) was examined in non‐neoplastic pancreas (n = 37) and pancreas cancer (n = 21) using mouse monoclonal antibodies (MAbs). Immunohistochemical assays were performed on sections of paraffin‐embedded tissues using the avidin‐biotin complex method. In normal pancreas, antibodies detecting Lea, sLea and Tn reacted with ductal epithelium, and antibodies detecting A and B reacted with acini and ducts, independently of secretor status. Lex was weakly expressed in ducts and acini, and sTn could not be detected in normal pancreas. Expression of Leb, Ley and precursor type 1 was regulated by secretor status: Leb and Ley were expressed in ducts of secretor and Lea‐b− individuals, but not in ducts of non‐secretors; precursor type I was weakly expressed in acini and ducts of non‐secretors and Lea‐b− individuals, and was absent in acini and ducts of secretors. The following alterations in the expression of blood‐group antigens were observed in pancreas cancer: (1) enhanced expression of Lex, Tn and sTn; (2) enhanced expression of precursor type I independently of secretor status; (3) loss of regulation of Leb by the secretor gene; (4) decreased expression of Ley. The weak expression of precursor type 1, Tn and sTn in non‐neoplastic pancreas, and their stronger expression in pancreas cancer, suggests that up‐regulation of their expression is associated with malignant transformation of pancreatic duct cells.
205207215
Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia.
In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T-cell therapy of acute myeloid leukemia (AML), we examined the safety and postinfusion persistence of adoptively transferred T cells. Following fludarabine-containing preconditioning, four patients received up to 1.3 × 109 total T cells, of which 14-38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission whereas another with active leukemia had a reduction in peripheral blood (PB) blasts and a third showed a protracted remission. Using an aliquot of In111-labeled CAR T cells, we demonstrated trafficking to the bone marrow (BM) in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR T cells infiltrated proven sites of disease. Serial PCR of PB and BM for the LeY transgene demonstrated that infused CAR T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR-T-cell therapy in high-risk AML, and demonstrates durable in vivo persistence.
87298682
Overview of Prostate-specific Membrane Antigen
In recent years,many domestic and overseas scholars pay close attention to prostate-specific membrane antigen which is a specific prostate cancermarker. This article review focuses on the structure and properties of prostate-specific membrane antigen as well as the role of PSMA in the treatment of prostate cancer.
23182888
Prostate-specific membrane antigen expression in the neovasculature of gastric and colorectal cancers.
Prostate-specific membrane antigen (PSMA), a type II transmembrane metallo-peptidase highly overexpressed in prostate cancer cells, has been studied as a targeting molecule in prostate cancer. Recently, PSMA has also been found to be expressed in the neovasculature of multiple nonprostatic solid tumors. Because of its unique expression pattern limited to tumor-associated endothelial cells, PSMA may also be an interesting molecule for vascular targeting. In this study, PSMA expression was determined by immunohistochemistry in 119 cases of primary gastric adenocarcinoma, 130 cases of primary colorectal adenocarcinoma, and 24 metastasis of colorectal adenocarcinoma. Expression data were correlated with clinicopathologic information. PSMA expression was detected in tumor-associated neovasculature of 79 (66%) of 119 gastric and 110 (85%) of 130 colorectal carcinomas. Furthermore, the neovasculatures of 16 (84%) of 19 liver and 4 (80%) of 5 nodal metastases from colorectal carcinomas were prostate-specific membrane antigen positive. There was a trend for high-grade tumors to higher PSMA expression (Spearman r = 0.18, P = .046) in colorectal cancers. No association between PSMA expression and overall- or disease-free survival was observed in gastric or colorectal cancers. This study provides the first in-depth look at PSMA expression in gastric and colorectal cancer. Because of its highly tumor-restricted expression and its accessibility to targeted therapy, PSMA represents a promising therapeutic and diagnostic target in colorectal and gastric cancer.
20302096
Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRζ /CD28 receptor
Artificial receptors provide a promising approach to target T lymphocytes to tumor antigens. However, the receptors described thus far produce either an activation or a co-stimulatory signal alone, thus limiting the spectrum of functions accomplished by the genetically modified cells. Here we show that human primary T lymphocytes expressing fusion receptors directed to prostate-specific membrane antigen (PSMA) and containing combined T-cell receptor-ζ (TCRζ), and CD28 signaling elements, effectively lyse tumor cells expressing PSMA. When stimulated by cell-surface PSMA, retrovirally transduced lymphocytes undergo robust proliferation, expanding by more than 2 logs in three weeks, and produce large amounts of interleukin-2 (IL-2). Importantly, the amplified cell populations retain their antigen-specific cytolytic activity. These data demonstrate that fusion receptors containing both TCR and CD28 signaling moieties are potent molecules able to redirect and amplify human T-cell responses. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of tumor cells that fail to express major histocompatibility complex antigens and co-stimulatory molecules.
79162494
Targeting castration resistant prostate cancer (CRPC) with autologous PSMA-directed CAR+ T cells.
TPS4700 Background: Based on our preclinical animal models, we initiated a phase I dose-escalating study to assess safety, dose requirement and targeting efficiency of genetically directed autologous human T cells targeted to Prostate Specific Membrane Antigen (PSMA). Our approach is based on the infusion of autologous PSMA-targeted T cells utilizing the P28z second generation chimeric antigen receptor (CAR) in patients (pts) with metastatic CRPC, following iv cyclophosphamide (Cy) (trial NCT01140373). For safety, the herpes simplex virus-1 thymidine kinase (hsvtk) gene is co-expressed with the P28z receptor, and renders T cells sensitive to ganciclovir for immediate T cell elimination if needed. The expression of hsvtk enables PET imaging using radiolabeled FIAU to localize adoptively transferred T cells. The aims of the trial are to assess: (1) safety of PSMA-targeted T cells; (2) biologic and anti-tumor effects; (3) T cell persistence at tumor site; and (4) immune response. Methods: Autologous T cells ...
72746580
Abstract C13: Phase I trial of anti-PSMA designer T cells in advanced prostate cancer
Introduction: We created chimeric antigen receptors (CAR) specific for prostate specific membrane antigen (PSMA). When expressed in patient T cells, these “designer T cells” (dTc) specifically kill prostate cancer cells in vitro and in vivo in animal models (Ma et al. Prostate 2004:61:12-25). A Phase I clinical trial was approved by the FDA under BB-IND 12084. With the recent publicity surrounding use of this technology to suppress and potentially cure CLL [Porter et al. NEJM 2011:365:725-33], the current aim to apply dTc in metastatic HRPC gains a heightened impetus and significance.

Methods: Patient T cells are retrovirally transduced and expanded ex vivo to span the dose range of 10⁁9 to 10⁁11 T cells. Patients undergo prior non-myeloablative (NMA) conditioning to create a “hematologic space” into which dTc are infused for stable engraftment and improved in vivo efficacy. Patients are co-administered continuous infusion IL2. Patients are monitored for safety and response. Outcomes include Phase Ia goals of safety and toxicity and Phase Ib goals of establishing an optimal biologic dose in terms of dTc engraftment and tumor response.

Results: To date five subjects have been treated, three at the 10⁁9 cell dose and two at the 10⁁10 cell dose. Excellent T cell modifications of 30-60% were obtained. After NMA conditioning, T cells were infused and stable engraft—ments of 1-20% were observed one-month post recovery, thus affirming one of the study end-points, even at the lowest 10⁁9 dose level, reflecting near 100-fold expansions of dTc in vivo. Two of five patients (40%) had PSA reductions of 50 and 70% in the two months following treatment, whereas four patients (80%) had delays in PSA progression of 2-5 months. Patients experienced neutropenia and lymphopenia after conditioning, but no designer T cell related toxicities. Patient treatments will continue under the dose escalation plan.

Conclusion: A new approach to adoptive immune therapy in metastatic prostate cancer has been devised with exciting early results. We postulate that the greater potency of higher dTc doses under adequate IL2 support will induce PSA reductions of 100%, potentially with durable remissions of metastatic prostate cancer that is refractory to all other treatments. Patients are being actively recruited. For referrals, contact by phone: (401) 456-2507 or email: RDavies{at}rwmc.org. This clinical trial received partial funding from US Army/DOD. Preclinical work was supported by the Prostate Cancer Foundation.

Note: This abstract was not presented at the conference because the presenter was unable to attend.

Citation Format: Richard P. Junghans, Qiangzhong Ma, Ritesh Rathore, Robin Davies, Anthony Bais, Erica Gomes, Ryan Harvey, Steven I. Cohen. Phase I trial of anti-PSMA designer T cells in advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C13.
19194467
Mesothelin targeted cancer immunotherapy.
Mesothelin is a tumour differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. It is, however, highly expressed in several human cancers including malignant mesothelioma, pancreatic, ovarian and lung adenocarcinoma. The normal biologic function of mesothelin is unknown but recent studies have shown that it binds to CA-125 and may play a role in the peritoneal spread of ovarian cancer. The limited mesothelin expression in normal tissues and high expression in many cancers makes it an attractive candidate for cancer therapy. Three mesothelin targeted agents are in various stages of clinical evaluation in patients. These include SS1P (CAT-5001) a recombinant immunotoxin targeting mesothelin, MORAb-009 a chimeric anti-mesothelin monoclonal antibody and CRS-207 a live-attenuated Listeria monocytogenes vector encoding human mesothelin. These ongoing clinical trials will help define the utility of mesothelin as a target for cancer therapy.
17117070
Mesothelin-Specific Chimeric Antigen Receptor mRNA-Engineered T Cells Induce Antitumor Activity in Solid Malignancies
Beatty, Haas, and colleagues report antitumor activity in two patients treated with autologous T cells transfected with mRNA encoding a chimeric antigen receptor that recognizes mesothelin and contains the CD3-ζ and 4-1BB costimulatory domains (CARTmeso). The short-lived CARTmeso cells induced novel antiself antibodies and a broadly directed epitope spreading. Off-target toxicity due to the expression of target antigens in normal tissue represents a major obstacle to the use of chimeric antigen receptor (CAR)-engineered T cells for treatment of solid malignancies. To circumvent this issue, we established a clinical platform for engineering T cells with transient CAR expression by using in vitro transcribed mRNA encoding a CAR that includes both the CD3-ζ and 4-1BB costimulatory domains. We present two case reports from ongoing trials indicating that adoptive transfer of mRNA CAR T cells that target mesothelin (CARTmeso cells) is feasible and safe without overt evidence of off-tumor on-target toxicity against normal tissues. CARTmeso cells persisted transiently within the peripheral blood after intravenous administration and migrated to primary and metastatic tumor sites. Clinical and laboratory evidence of antitumor activity was shown in both patients, and the CARTmeso cells elicited an antitumor immune response revealed by the development of novel antiself antibodies. These data show the potential of using mRNA-engineered T cells to evaluate, in a controlled manner, potential off-tumor on-target toxicities and show that short-lived CAR T cells can induce epitope spreading and mediate antitumor activity in patients with advanced cancer. Thus, these findings support the development of mRNA CAR-based strategies for carcinoma and other solid tumors. Cancer Immunol Res; 2(2); 112–20. ©2013 AACR.
5951078
T Cells Expressing Chimeric Antigen Receptors Can Cause Anaphylaxis in Humans
Chimeric antigen receptor–expressing T cells (CAR-T cells) represent a promising, novel form of adoptive immunotherapy to overcome tolerance to cancer. Using an intermittent dosing schedule of autologous CAR-T cells electroporated with mRNA encoding a hybrid single chain molecule comprising the extracellular domain of murine monoclonal antibody against human mesothelin and the human transmembrane and cytoplasmic T-cell signaling domains, Maus and colleagues characterized and reported a serious adverse event that occurred in one of four patients receiving repeated modified T-cell infusions, with proposed modifications to address and minimize future adverse occurrence. T cells can be redirected to overcome tolerance to cancer by engineering with integrating vectors to express a chimeric antigen receptor (CAR). In preclinical models, we have previously shown that transfection of T cells with mRNA coding for a CAR is an alternative strategy that has antitumor efficacy and the potential to evaluate the on-target off-tumor toxicity of new CAR targets safely due to transient mRNA CAR expression. Here, we report the safety observed in four patients treated with autologous T cells that had been electroporated with mRNA coding for a CAR derived from a murine antibody to human mesothelin. Because of the transient nature of CAR expression on the T cells, subjects in the clinical study were given repeated infusions of the CAR-T cells to assess their safety. One subject developed anaphylaxis and cardiac arrest within minutes of completing the third infusion. Although human anti-mouse immunoglobulin (Ig)G antibodies have been known to develop with CAR-transduced T cells, they have been thought to have no adverse clinical consequences. This is the first description of clinical anaphylaxis resulting from CAR-modified T cells, most likely through IgE antibodies specific to the CAR. These results indicate that the potential immunogenicity of CARs derived from murine antibodies may be a safety issue for mRNA CARs, especially when administered using an intermittent dosing schedule. Cancer Immunol Res; 1(1); 26–31. ©2013 AACR.
41152805
The gene for fibroblast activation protein alpha (FAP), a putative cell surface-bound serine protease expressed in cancer stroma and wound healing, maps to chromosome band 2q23.
The human fibroblast activation protein {alpha} (FAP{alpha}) is an inducible cell surface glycoprotein of M{sub r} 95,000 recognized by a number of monoclonal antibodies (mAbs), including the prototype mAb F19. Immunohistochemical studies have shown that FAP{alpha} expression in vivo is tightly regulated, with transient expression in some fetal mesenchymal tissues but absence of expression in most normal adult tissues. Reexpression of FAP{alpha} is observed in the reactive stromal fibroblasts of several common types of epithelial cancers, including >90% of breast, colorectal, and lung carcinomas and healing wounds. Cloning and sequence analysis of an FAP{alpha}-specific cDNA has revealed that the molecule is encoded by a novel gene, FAP, which shows sequence similarity to members of the serine protease family of integral membrane proteins, namely dipeptidyl peptidase IV (DPPIV, also known as lymphocyte activation antigen, CD26, or adenosine dearoinase binding protein) and DPPX, a DPPIV-related molecule of unknown function. 15 refs., 1 fig.
10008199
Re-directed T cells for the treatment of fibroblast activation protein (FAP)-positive malignant pleural mesothelioma (FAPME-1)
BackgroundAsbestos is the main cause of MPM in industrialized countries. Even since asbestos is banned in most developed countries, the peak wave of MPM incidence is anticipated for the next years due to the long latency of asbestos induced MPM. MPM patients not eligible for surgical procedures like decortication or pleuro-pneumectomie have a median survival of 12 months with palliative chemotherapy. Therefore, new therapeutic approaches are of crucial need in this clinical situation.Methods/designThis is a phase I trial for patients with malignant pleural mesothelioma with pleural effusion testing the safety of a fixed single dose of 1x106 adoptively transferred FAP-specific re-directed T cells given directly in the pleural effusion. Lymphocytes will be taken 21 days before transfer from peripheral blood. CD8 positive T cells will be isolated and re-programmed by retroviral transfer of a chimeric antigen receptor recognizing FAP which serves as target structure in MPM. At day 0 of the protocol, re-directed T cells will be injected in the pleural effusion and patients will be monitored for 48h under intermediate care conditions. AE, SAE, SADR and SUSAR will be monitored for 35 days and evaluated by an independent safety board to define any dose limiting toxicity (DLT). No further patient can be treated before the previous patient passed day 14 after T cell transfer. The protocol will be judged as save when no DLT occurred in the first 3 patients, or 1 DLT in 6 patients. Secondary objectives are feasibility and immune monitoring.DiscussionAdoptive T cell transfer is a new and rapidly expanding branch of immunotherapies focusing on cancer treatment. Recently, objective responses could be observed in patients with chronic lymphatic leukemia treated with adoptively transferred CD19-specific re-directed T cells. The choice of the target antigen determines the possible on-target off-tissue toxicity of such approaches. There are reports of severe toxicity in patients who received T cells intravenously due to unexpected expression of the target antigen (on-target) in other tissues than the tumor (off-tissue). To minimize the risk of on-target off-tissue toxicity and to maximize the on-target anti-tumor effect we propose a clinical protocol with loco-regional administration of re-directed T cells. FAP-specific T cells will be directly injected in the pleural effusion of patients with MPM.Trial registrationClinicalTrials.gov (NCT01722149)
14227694
Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific re-directed T cells
IntroductionMalignant pleural mesothelioma (MPM) is an incurable malignant disease, which results from chronic exposition to asbestos in at least 70% of the cases. Fibroblast activation protein (FAP) is predominantly expressed on the surface of reactive tumor-associated fibroblasts as well as on particular cancer types. Because of its expression on the cell surface, FAP is an attractive target for adoptive T cell therapy. T cells can be re-directed by retroviral transfer of chimeric antigen receptors (CAR) against tumor-associated antigens (TAA) and therefore represent a therapeutic strategy of adoptive immunotherapy.MethodsTo evaluate FAP expression immunohistochemistry was performed in tumor tissue from MPM patients. CD8+ human T cells were retrovirally transduced with an anti-FAP-F19-∆CD28/CD3ζ-CAR. T cell function was evaluated in vitro by cytokine release and cytotoxicity assays. In vivo function was tested with an intraperitoneal xenograft tumor model in immunodeficient mice.ResultsFAP was found to be expressed in all subtypes of MPM. Additionally, FAP expression was evaluated in healthy adult tissue samples and was only detected in specific areas in the pancreas, the placenta and very weakly for cervix and uterus. Expression of the anti-FAP-F19-∆CD28/CD3ζ-CAR in CD8+ T cells resulted in antigen-specific IFNγ release. Additionally, FAP-specific re-directed T cells lysed FAP positive mesothelioma cells and inflammatory fibroblasts in an antigen-specific manner in vitro. Furthermore, FAP-specific re-directed T cells inhibited the growth of FAP positive human tumor cells in the peritoneal cavity of mice and significantly prolonged survival of mice.ConclusionFAP re-directed CD8+ T cells showed antigen-specific functionality in vitro and in vivo. Furthermore, FAP expression was verified in all MPM histotypes. Therefore, our data support performing a phase I clinical trial in which MPM patients are treated with adoptively transferred FAP-specific re-directed T cells.
178655
Epidermal growth factor receptor dependence in human tumors: more than just expression?
The epidermal growth factor receptor (EGFR) is a rational target for antitumor strategies. EGFR signaling causes increased proliferation, decreased apoptosis, and enhanced tumor cell motility and neo-angiogenesis. The EGFR is expressed or highly expressed in a variety of human tumors of epithelial origin. ZD1839 (Iressa) is an orally active, selective EGFR tyrosine kinase inhibitor, which blocks signal transduction pathways implicated in proliferation and survival of cancer cells. The lack of a consistent method of evaluating levels of EGFR has caused a disparity in reports of the EGFR as a prognostic factor; however, for some tumors, EGFR is a strong prognostic indicator associated with more aggressive disease and reduced survival. So far, no clear association between EGFR levels and response to EGFR-targeted agents has been found. Preclinical studies with ZD1839 have noted a relationship between the two in some cases, but not others. EGFR signaling may be increased by a number of mechanisms in addition to high expression levels of EGFR, including receptor mutations, heterodimerization with other members of this receptor family such as HER2 (erbB2), increased expression of (autocrine/ paracrine) ligands, and alterations in molecules that control receptor signaling output. Each of these components could be assessed to give an indication of the magnitude of EGFR signal amplification. Evaluation of signaling components downstream from EGFR should provide information on the activation of the EGFR pathway. Until EGFR-based assays predictive of a response to receptor-targeted therapies are available, there is no clear justification for stratifying patients by EGFR status or excluding patients with low EGFR levels from trials with ZD1839 or other EGFR inhibitors.
25579906
Cetuximab: adverse event profile and recommendations for toxicity management.
Cetuximab (Erbitux, IMC-C225, ImClone Systems Incorporated, New York, NY) is a monoclonal antibody targeted to the epidermal growth factor receptor. Expression of the epidermal growth factor receptor is associated with disease progression, poor survival, poor response to therapy, and the development of resistance to therapy in many solid tumors. Cetuximab blocks the binding of natural ligands to the epidermal growth factor receptor, thus inhibiting oncogenic processes associated with its activation. Infusion reactions, acneform skin rash, and nail disorder are the most clinically relevant adverse events observed. Because infusion reactions can be life threatening when severe, nurses must administer prophylactic treatment with an H1 antagonist prior to infusion and actively manage cetuximab-related infusion reactions when they occur. Management of infusion reactions typically includes vigilant patient monitoring, appropriate medical supervision, readily available resources for the treatment of infusion reactions, and initiation of institution- or practice-specific protocols when necessary. Acneform skin rash is the most common adverse event, but severe (grade 3 or 4) rash requiring interruption of treatment is not common. Topical and systemic antibiotic therapies may be administered to reduce symptoms. Nail disorder typically is mild to moderate and is observed infrequently; this also may be treated with systemic and topical antibiotics. Overall, the safety profile of cetuximab is favorable compared to that typically seen with chemotherapeutic agents. The acneform skin rash and nail disorder, which may affect quality of life, rarely threaten the general well-being of patients and typically are manageable.
8519139
Rational development and characterization of humanized anti–EGFR variant III chimeric antigen receptor T cells for glioblastoma
A chimeric antigen receptor redirects T cells to treat glioblastoma. CAR T cells drive glioblastoma therapy Immunotherapy with chimeric antigen receptor (CAR) T cells can successfully treat B cell malignancies, but expansion into solid tumors has been limited by the lack of availability of tumor-specific antigens. Now, Johnson et al. target CAR T cells to a variant III mutation of the epidermal growth factor receptor (EGFRvIII), which is thought to be enriched in glioblastoma stem cells. They found that a low-affinity single-chain variable fragment was specific for EGFRvIII over wild-type EGFR and that CAR T cells transduced with this fragment were able to target antigen-expressing cells in vitro and in vivo in multiple mouse xenograft models of human glioblastoma. These cells are currently being moved into the clinic in a phase 1 clinical trial. Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv–based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII+ glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).
7670230
Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.
No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.
32523011
The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues.
The human CEA family has been fully characterized. It comprises 29 genes of which 18 are expressed; 7 belonging to the CEA subgroup and 11 to the pregnancy specific glycoprotein subgroup. CEA is an important tumor marker for colorectal and some other carcinomas. The CEA subgroup members are cell membrane associated and show a complex expression pattern in normal and cancerous tissues with notably CEA showing a selective epithelial expression. Several CEA subgroup members possess cell adhesion properties and the primordial member, biliary glycoprotein, seems to function in signal transduction or regulation of signal transduction possibly in association with other CEA sub-family members. A modified ITAM/ITIM motif is identified in the cytoplasmatic domain of BGP. A role of CEA in innate immunity is envisioned.
10752873
T Cells Targeting Carcinoembryonic Antigen Can Mediate Regression of Metastatic Colorectal Cancer but Induce Severe Transient Colitis.
Autologous T lymphocytes genetically engineered to express a murine T cell receptor (TCR) against human carcinoembryonic antigen (CEA) were administered to three patients with metastatic colorectal cancer refractory to standard treatments. All patients experienced profound decreases in serum CEA levels (74-99%), and one patient had an objective regression of cancer metastatic to the lung and liver. However, a severe transient inflammatory colitis that represented a dose limiting toxicity was induced in all three patients. This report represents the first example of objective regression of metastatic colorectal cancer mediated by adoptive T cell transfer and illustrates the successful use of a TCR, raised in human leukocyte antigen (HLA) transgenic mice, against a human tumor associated antigen. It also emphasizes the destructive power of small numbers of highly avid T cells and the limitations of using CEA as a target for cancer immunotherapy.
24478939
Phase I Hepatic Immunotherapy for Metastases Study of Intra-Arterial Chimeric Antigen Receptor–Modified T-cell Therapy for CEA+ Liver Metastases
Purpose: Chimeric antigen receptor–modified T cells (CAR-T) have demonstrated encouraging results in early-phase clinical trials. Successful adaptation of CAR-T technology for CEA-expressing adenocarcinoma liver metastases, a major cause of death in patients with gastrointestinal cancers, has yet to be achieved. We sought to test intrahepatic delivery of anti-CEA CAR-T through percutaneous hepatic artery infusions (HAIs). Experimental Design: We conducted a phase I trial to test HAI of CAR-T in patients with CEA+ liver metastases. Six patients completed the protocol, and 3 received anti-CEA CAR-T HAIs alone in dose-escalation fashion (108, 109, and 1010 cells). We treated an additional 3 patients with the maximum planned CAR-T HAI dose (1010 cells × 3) along with systemic IL2 support. Results: Four patients had more than 10 liver metastases, and patients received a mean of 2.5 lines of conventional systemic therapy before enrollment. No patient suffered a grade 3 or 4 adverse event related to the CAR-T HAIs. One patient remains alive with stable disease at 23 months following CAR-T HAI, and 5 patients died of progressive disease. Among the patients in the cohort that received systemic IL2 support, CEA levels decreased 37% (range, 19%–48%) from baseline. Biopsies demonstrated an increase in liver metastasis necrosis or fibrosis in 4 of 6 patients. Elevated serum IFNγ levels correlated with IL2 administration and CEA decreases. Conclusions: We have demonstrated the safety of anti-CEA CAR-T HAIs with encouraging signals of clinical activity in a heavily pretreated population with large tumor burdens. Further clinical testing of CAR-T HAIs for liver metastases is warranted. Clin Cancer Res; 21(14); 3149–59. ©2015 AACR.
7011852
Expression profile analysis in multiple human tumors identifies L1 (CD171) as a molecular marker for differential diagnosis and targeted therapy.
L1 cell adhesion molecule (CD171) represents a strongly unfavorable prognostic biomarker for ovarian and endometrial carcinomas. Here we carried out an immunohistochemical survey of L1 expression in normal adults and in a broad range of benign and malignant tumors using monoclonal antibody L1-11A and the novel monoclonal antibody L1-14.10. In normal tissues, L1 was expressed in the collecting tubules of adult tissues and pediatric kidney and in peripheral nerve bundles. In tumors of the female genital tract, L1 was detected in adenocarcinomas of the cervix and fallopian tubes, in addition to ovarian and endometrial carcinomas. Nongynecological tumors expressing L1 comprised malignant melanoma, colon adenocarcinoma positive to chromogranin, clear-cell adenocarcinoma of the urinary bladder, pheochromocytoma, small cell lung carcinoma, and tumors of the nervous system. L1 was absent in breast carcinoma, gastrointestinal tract carcinomas, gastrointestinal carcinoids, renal clear-cell carcinomas, prostate adenocarcinomas, and mesotheliomas. Surprisingly, L1 expression in established breast and renal carcinoma cell lines was not a predictor for its presence in these human tumors in vivo. Our results suggest that L1 expression in tumors is not ubiquitous but restricted to certain subtypes and may be a helpful molecular marker for differential diagnosis and target for antibody-based therapy.
27990505
Anti‐neuroblastoma antibody chCE7 binds to an isoform of L1‐CAM present in renal carcinoma cells
Immunoprecipitation after cell surface labeling of human neuroblastoma cells showed that the anti‐neuroblastoma monoclonal antibody (mAb) chCE7 binds to a 200,000 Mr cell surface protein. The protein was partially purified by immuno‐affinity chromatography from a human renal carcinoma and a human neuroblastoma cell line, which both showed high levels of binding of MAb chCE7. NH2‐terminal sequences of 18 and 15 amino acid residues were determined. Both sequences isolated from the renal carcinoma and the neuroblastoma cells showed strong homology to human cell adhesion molecule L1 (L1‐CAM), and both were characterized by the NH2‐terminal deletion of 5 amino acids, comprising exon 2 of L1‐CAM. Reverse trancription‐polymerase chain reaction (RT‐PCR) analysis of the regions spanning exon 2 and exon 27 of L1‐CAM indicated that in neuroblastoma cells both transcripts for the full‐length and exon‐deleted forms are present, whereas in the renal carcinoma cell lines only the exon‐deleted L1‐CAM isoform were detected. Western blot analysis showed that 6 of 7 tested renal carcinoma cell lines and 5 of 15 renal carcinoma tissues expressed L1‐CAM. In normal adult kidney tissue, very low levels of protein expression were found. Northern blot analysis confirmed that in renal carcinoma and neuroblastoma cell lines L1‐CAM mRNA levels are correlated with protein expression. Int. J. Cancer 83: 401–408, 1999 © 1999 Wiley‐Liss, Inc.
205553197
Diverse Solid Tumors Expressing a Restricted Epitope of L1-CAM Can Be Targeted by Chimeric Antigen Receptor Redirected T Lymphocytes
Adhesion molecule L1-CAM (CD171) was originally reported to be overexpressed on neuroblastoma and to play an important role during tumor progression. More recently, it has been shown to be overexpressed on many other solid tumors such as melanoma and carcinomas of the cervix, ovary, bladder, and others. Thus, there has been a growing interest in using this cell-surface molecule as a target for both antibody-based and cellular-based therapy—our group has previously examined the clinical utility of chimeric antigen receptor (CAR)–redirected cytolytic T cells that specifically target the CE7 epitope of L1-CAM on neuroblastoma patients. Here, we sought to determine whether this CE7 epitope is present on other recently identified L1-CAM+ tumors and whether it too can be targeted by CAR T cells. Our studies demonstrate that a diverse array of human tumor cell lines and primary solid tumors (ovarian, lung, and renal carcinoma, glioblastoma and neuroblastoma) do express the CE7 epitope and can efficiently stimulate CE7-specific CAR-redirected (CE7R+) T-cell lytic activity and secretion of proinflamatory cytokines. L1-CAM was also detected on a limited number of normal tissues; however, L1-CAM expressed on normal human monocytes was not bound by the CE7 mAb nor was it targeted by CE7R+ T cells, suggesting that the CE7 epitope is more tumor restricted and not expressed on all L1-CAM+ tissues. Overall, the CE7 epitope of L1-CAM on a variety of tumors may be amenable to targeting by CE7R+ T cells, making it a promising target for adoptive immunotherapy.
38534953
Disialoganglioside GD2 as a therapeutic target for human diseases
Introduction: Ganglioside GD2 is found in vertebrates and invertebrates, overexpressed among pediatric and adult solid tumors, including neuroblastoma, glioma, retinoblastoma, Ewing’s family of tumors, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, small cell lung cancer and melanoma. It is also found on stem cells, neurons, some nerve fibers and basal layer of the skin. Areas covered: GD2 provides a promising clinical target for radiolabeled antibodies, bispecific antibodies, chimeric antigen receptor (CAR)-modified T cells, drug conjugates, nanoparticles and vaccines. Here, we review its biochemistry, normal physiology, role in tumorigenesis, important characteristics as a target, as well as anti-GD2-targeted strategies. Expert opinion: Bridging the knowledge gaps in understanding the interactions of GD2 with signaling molecules within the glycosynapses, and the regulation of its cellular expression should improve therapeutic strategies targeting this ganglioside. In addition to anti-GD2 IgG mAbs, their drug conjugates, radiolabeled forms especially when genetically engineered to improve therapeutic index and novel bispecific forms or CARs to retarget T-cells are promising candidates for treating metastatic cancers.
22303340
Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides
Understanding the distribution of tumor‐associated antigens on cancers and normal tissues is essential for selection of targets for cancer immunotherapy. Seven carbohydrate antigens, potential targets for immunotherapy, were studied using a panel of well‐characterized MAbs by immunohistochemistry on cryostat‐cut tissue sections of 13 types of cancers and 18 normal tissues. GD2 and GD3 were present on most cancers of neuroectodermal origin and GD2 was also present on B cell lymphomas. 9‐O‐acetyl‐GD3 was detected only on melanoma while fucosyl GM1 was detected only on small cell lung cancers (SCLC). Surprisingly, GM2 was strongly expressed on all tested tumors, including cancers of neuroectodermal origin and cancers of epithelial origin. Polysialic acid was primarily expressed on SCLC and neuroblastomas. Globo H was present on most cancers of epithelial origin. These antigens were also identified in normal tissues. Fucosyl GM1 was not expressed significantly on any of the normal tissues analyzed. GD3, GD2, GM2 and polysialic acid were detected in normal brain to varying degrees. GM2 and Globo H were expressed on the luminal surface of epithelia of a variety of organs. The unexpected expression of GM2 on a broad range of cancers and normal epithelial tissues was confirmed by loss after methanol fixation and by immune thin layer chromatography. Int. J. Cancer 73:42–49, 1997. © 1997 Wiley‐Liss, Inc.
471912
Different cytokine and stimulation conditions influence the expansion and immune phenotype of third-generation chimeric antigen receptor T cells specific for tumor antigen GD2.
BACKGROUND AIMS
Chimeric antigen receptor (CAR) T cells are a novel immunotherapy for cancer. To achieve anti-tumor efficacy, these cells must survive, expand, and persist after infusion into patients, functions that are reportedly best achieved by cells with a stem or central-memory rather than effector-memory phenotype. We have developed third-generation CAR T cells specific for the tumor-associated antigen GD2 for use in a phase I clinical trial. We investigated the optimal cell culture conditions for CAR T-cell production, and here we describe the relative effects of 3 activation and cytokine conditions on CAR T-cell expansion, effector function and phenotype.


METHODS
Peripheral blood mononuclear cells were activated by anti-CD3 and anti-CD28 or anti-CD3 and Retronectin. Activated cells were transduced with the CAR-encoding retroviral vector and expanded in either interleukin (IL)-2 or IL-7 and IL-15. Immune phenotype and expansion were tracked throughout the culture, and transduction efficiency, and subsequent GD2-specific effector functions were evaluated by flow cytometry and cytotoxic T lymphocytes assay.


RESULTS
CD3/Retronectin stimulation with IL-2 resulted in poorer activation, expansion and Th1 cytokine secretion of CAR T cells than CD3/CD28 stimulation with either IL-2 or IL-7 and IL-15. However, CAR T cells cultured in CD3/CD28/IL7/IL-15 and CD3/Retronectin/IL-2 had superior cytotoxic T lymphocyte activity and a more stem-like phenotype.


DISCUSSION
The combination of CD3 and CD28 with IL-7 and IL-15 gave the best balance of CAR T-cell expansion and potent GD2-specific effector functions while retaining a stem/memory phenotype, and these growth conditions will therefore be used to manufacture CAR T cells for our phase I clinical trial.
29774126
Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues.
The HER-2/neu proto-oncogene is homologous with, but distinct from, the epidermal growth factor receptor. Current evidence indicates that this gene is frequently amplified and/or overexpressed in some human breast and ovarian cancers and that these alterations may be clinically important; however, little is known about the expression pattern of the gene in normal tissues. Using immunohistochemistry and northern blot analyses to identify the HER-2/neu protein and transcript respectively, we have evaluated a variety of normal adult and fetal tissues for HER-2/neu expression. HER-2/neu protein was identified on cell membranes of epithelial cells in the gastro-intestinal, respiratory, reproductive, and urinary tract as well as in the skin, breast and placenta. Northern hybridization confirmed the presence of the 4.5 kb transcript encoding the protein in these tissues. The amount of HER-2/neu message and protein was generally higher in fetal tissues than in the corresponding normal adult tissues. HER-2/neu expression levels in these normal tissues were similar to the levels found in non-amplified, non-overexpressing breast cancers and breast cancer cell lines. Southern hybridization of extracted DNA showed that none of the normal tissues expressing HER-2/neu had amplification of the gene. These results confirm that HER-2/neu is normally a membrane constituent of a variety of epithelial cell types.
39909239
Human Epidermal Growth Factor Receptor 2 (HER2) -Specific Chimeric Antigen Receptor-Modified T Cells for the Immunotherapy of HER2-Positive Sarcoma.
PURPOSE
The outcome for patients with metastatic or recurrent sarcoma remains poor. Adoptive therapy with tumor-directed T cells is an attractive therapeutic option but has never been evaluated in sarcoma.


PATIENTS AND METHODS
We conducted a phase I/II clinical study in which patients with recurrent/refractory human epidermal growth factor receptor 2 (HER2) -positive sarcoma received escalating doses (1 × 10(4)/m(2) to 1 × 10(8)/m(2)) of T cells expressing an HER2-specific chimeric antigen receptor with a CD28.ζ signaling domain (HER2-CAR T cells).


RESULTS
We enrolled 19 patients with HER2-positive tumors (16 osteosarcomas, one Ewing sarcoma, one primitive neuroectodermal tumor, and one desmoplastic small round cell tumor). HER2-CAR T-cell infusions were well tolerated with no dose-limiting toxicity. At dose level 3 (1 × 10(5)/m(2)) and above, we detected HER2-CAR T cells 3 hours after infusion by quantitative polymerase chain reaction in 14 of 16 patients. HER2-CAR T cells persisted for at least 6 weeks in seven of the nine evaluable patients who received greater than 1 × 10(6)/m(2) HER2-CAR T cells (P = .005). HER2-CAR T cells were detected at tumor sites of two of two patients examined. Of 17 evaluable patients, four had stable disease for 12 weeks to 14 months. Three of these patients had their tumor removed, with one showing ≥ 90% necrosis. The median overall survival of all 19 infused patients was 10.3 months (range, 5.1 to 29.1 months).


CONCLUSION
This first evaluation of the safety and efficacy of HER2-CAR T cells in patients with cancer shows the cells can persist for 6 weeks without evident toxicities, setting the stage for studies that combine HER2-CAR T cells with other immunomodulatory approaches to enhance their expansion and persistence.
8358407
A Herceptin-Based Chimeric Antigen Receptor with Modified Signaling Domains Leads to Enhanced Survival of Transduced T Lymphocytes and Antitumor Activity1
To generate chimeric Ag receptors (CARs) for the adoptive immunotherapy of cancer patients with ErbB2-expressing tumors, a single-chain Ab derived from the humanized mAb 4D5 Herceptin (trastuzumab) was initially linked to T cell signaling domains derived from CD28 and the CD3ζ to generate a CAR against ErbB2. Human PBLs expressing the 4D5 CAR demonstrated Ag-specific activities against ErbB2+ tumors. However, a gradual loss of transgene expression was noted for PBLs transduced with this 4D5 CAR. When the CD3ζ signaling domain of the CAR was truncated or mutated, loss of CAR expression was not observed, suggesting that the CD3ζ signaling caused the transgene decrease, which was supported by the finding that T cells expressing 4D5 CARs with CD3ζ ITAM mutations were less prone to apoptosis. By adding 4-1BB cytoplasmic domains to the CD28-CD3ζ signaling moieties, we found increased transgene persistence in 4D5 CAR-transduced PBLs. Furthermore, constructs with 4-1BB sequences demonstrated increased cytokine secretion and lytic activity in 4D5 CAR-transduced T cells. More importantly, PBLs expressing this new version of the 4D5 CAR could not only efficiently lyse the autologous fresh tumor digests, but they could strongly suppress tumor growth in a xenogenic mouse model.
15506190
Receptor for interleukin 13 is a marker and therapeutic target for human high-grade gliomas.
Glioblastoma multiforme (GBM) is an incurable brain tumor. Due to the striking heterogeneity that characterizes GBM, there is no known tumor-specific antigen or receptor that is expressed by a majority of GBM patients. We found that virtually all studied human GBM specimens (23 samples) abundantly expressed a receptor for interleukin (IL)-13 in situ, whereas normal human brain had few, if any, IL-13-binding sites. The GBM-associated IL-13 receptor was both quantitatively and qualitatively different from and, thus, more restrictive than the shared signaling receptor of normal tissue: it was IL-4 independent. The receptor for IL-13 was overexpressed by a majority of cancer cells in situ. Furthermore, cytotoxins targeted to this more restrictive IL-13R produced cures in animals bearing xenografts of human high-grade gliomas. Thus, unexpectedly, the receptor for an immune regulatory cytokine may be a long sought marker and, concomitantly, a unique imaging site and therapeutic target for GBM, the most malignant and the most heterogeneous of brain tumors.
207661395
Bioactivity and Safety of IL13Rα2-Redirected Chimeric Antigen Receptor CD8+ T Cells in Patients with Recurrent Glioblastoma
Purpose: A first-in-human pilot safety and feasibility trial evaluating chimeric antigen receptor (CAR)–engineered, autologous primary human CD8+ cytotoxic T lymphocytes (CTL) targeting IL13Rα2 for the treatment of recurrent glioblastoma (GBM). Experimental Design: Three patients with recurrent GBM were treated with IL13(E13Y)-zetakine CD8+ CTL targeting IL13Rα2. Patients received up to 12 local infusions at a maximum dose of 108 CAR-engineered T cells via a catheter/reservoir system. Results: We demonstrate the feasibility of manufacturing sufficient numbers of autologous CTL clones expressing an IL13(E13Y)-zetakine CAR for redirected HLA-independent IL13Rα2-specific effector function for a cohort of patients diagnosed with GBM. Intracranial delivery of the IL13-zetakine+ CTL clones into the resection cavity of 3 patients with recurrent disease was well-tolerated, with manageable temporary brain inflammation. Following infusion of IL13-zetakine+ CTLs, evidence for transient anti-glioma responses was observed in 2 of the patients. Analysis of tumor tissue from 1 patient before and after T-cell therapy suggested reduced overall IL13Rα2 expression within the tumor following treatment. MRI analysis of another patient indicated an increase in tumor necrotic volume at the site of IL13-zetakine+ T-cell administration. Conclusions: These findings provide promising first-in-human clinical experience for intracranial administration of IL13Rα2-specific CAR T cells for the treatment of GBM, establishing a foundation on which future refinements of adoptive CAR T-cell therapies can be applied. Clin Cancer Res; 21(18); 4062–72. ©2015 AACR.
43122775
Local Delivery of lnterleukin-12 Using T Cells Targeting VEGF Receptor-2 Eradicates Multiple Vascularized Tumors in Mice
Purpose: We investigated the feasibility of delivering the proinflammatory cytokine interleukin (IL)-12 into tumor using T cells genetically engineered to express a chimeric antigen receptor (CAR) against the VEGF receptor-2 (VEGFR-2). Experimental Design: Two different strains of mice bearing five different established subcutaneous tumors were treated with syngeneic T cells cotransduced with an anti–VEGFR-2 CAR and a constitutively expressed single-chain murine IL-12 or an inducible IL-12 gene after host lymphodepletion. Tumor regression, survival of mice, and persistence of the transferred cells were evaluated. Results: Adoptive transfer of syngeneic T cells cotransduced with an anti–VEGFR-2 CAR and a constitutively expressing single-chain IL-12 resulted in the regression of five different established tumors of different histologies without the need for IL-2 administration. T cells transduced with either anti–VEGFR-2 CAR or single-chain IL-12 alone did not alter the tumor growth indicating that both of them had to be expressed in the same cell to mediate tumor regression. Anti–VEGFR-2 CAR and IL-12–cotransduced T cells infiltrated the tumors, expanded, and persisted for prolonged periods. The antitumor effect did not require the presence of host T and B cells but was dependent on host IL-12R–expressing cells. The anti–VEGFR-2 CAR changed the immunosuppressive tumor environment by altering/reducing both the systemic and the intratumoral CD11b+Gr1+ myeloid suppressor cell subsets that expressed VEGFR-2. Conclusions: These results suggest that targeted delivery of IL-12 into the tumor environment with T cells redirected against VEGFR-2 is a promising approach for treating patients with a variety of solid tumor types. Clin Cancer Res; 18(6); 1672–83. ©2012 AACR.
207553569
IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression.
During malignant progression cancer cells tend to lose cell surface expression of MHC and other immune antigens, making them invisible to cytotoxic T cells and therefore inaccessible to tumor antigen-directed immunotherapy. Moreover, cancer cell variants that have lost antigen expression frequently contribute to deadly tumor relapses that occur following treatments that had been initially effective. In an effort to destroy antigen-loss cancer cells in tumors, we created a strategy that combines a chimeric antigen receptor (CAR)-redirected T-cell attack with an engineered local release of the cytokine interleukin 12 (IL-12), which recruits and reinforces macrophage function. Cytotoxic T cells were engineered to release inducible IL-12 upon CAR engagement in the tumor lesion, resulting in destruction of antigen-loss cancer cells that would normally escape. Importantly, elimination of the antigen-loss cancer cells was accompanied by an accumulation of activated macrophages that was critical to the antitumor response, because removing the macrophages abolished the response and restoring them reengaged it. Neutralizing TNF-α also abrogated the elimination of antigen-loss cancer cells, implying this proinflammatory factor in the process. Taken together, our results show how IL-12 supplementation by CAR T cells can target otherwise inaccessible tumor lesions, in a manner associated with reduced systemic toxicity, by recruiting and activating innate immune cells for a proinflammatory response.
4936924
IL-12 triggers a programmatic change in dysfunctional myeloid-derived cells within mouse tumors.
Solid tumors are complex masses with a local microenvironment, or stroma, that supports tumor growth and progression. Among the diverse tumor-supporting stromal cells is a heterogeneous population of myeloid-derived cells. These cells are alternatively activated and contribute to the immunosuppressive environment of the tumor; overcoming their immunosuppressive effects may improve the efficacy of cancer immunotherapies. We recently found that engineering tumor-specific CD8(+) T cells to secrete the inflammatory cytokine IL-12 improved their therapeutic efficacy in the B16 mouse model of established melanoma. Here, we report the mechanism underlying this finding. Surprisingly, direct binding of IL-12 to receptors on lymphocytes or NK cells was not required. Instead, IL-12 sensitized bone marrow-derived tumor stromal cells, including CD11b(+)F4/80(hi) macrophages, CD11b(+)MHCII(hi)CD11c(hi) dendritic cells, and CD11b(+)Gr-1(hi) myeloid-derived suppressor cells, causing them to enhance the effects of adoptively transferred CD8(+) T cells. This reprogramming of myeloid-derived cells occurred partly through IFN-γ. Surprisingly, direct presentation of antigen to the transferred CD8(+) T cells by tumor was not necessary; however, MHCI expression on host cells was essential for IL-12-mediated antitumor enhancements. These results are consistent with a model in which IL-12 enhances the ability of CD8(+) T cells to collapse large vascularized tumors by triggering programmatic changes in otherwise suppressive antigen-presenting cells within tumors and support the use of IL-12 as part of immunotherapy for the treatment of solid tumors.
206908004
Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning.
Adoptive cell therapy with tumor-targeted T cells is a promising approach to cancer therapy. Enhanced clinical outcome using this approach requires conditioning regimens with total body irradiation, lymphodepleting chemotherapy, and/or additional cytokine support. However, the need for prior conditioning precludes optimal application of this approach to a significant number of cancer patients intolerant to these regimens. Herein, we present preclinical studies demonstrating that treatment with CD19-specific, chimeric antigen receptor (CAR)-modified T cells that are further modified to constitutively secrete IL-12 are able to safely eradicate established disease in the absence of prior conditioning. We demonstrate in a novel syngeneic tumor model that tumor elimination requires both CD4(+) and CD8(+) T-cell subsets, autocrine IL-12 stimulation, and subsequent IFNγ secretion by the CAR(+) T cells. Importantly, IL-12-secreting, tumor-targeted T cells acquire intrinsic resistance to T regulatory cell-mediated inhibition. Based on these preclinical data, we anticipate that adoptive therapy using CAR-targeted T cells modified to secrete IL-12 will obviate or reduce the need for potentially hazardous conditioning regimens to achieve optimal antitumor responses in cancer patients.
3191225
Tumor-Infiltrating Lymphocytes Genetically Engineered with an Inducible Gene Encoding Interleukin-12 for the Immunotherapy of Metastatic Melanoma
Purpose: Infusion of interleukin-12 (IL12) can mediate antitumor immunity in animal models, yet its systemic administration to patients with cancer results in minimal efficacy and severe toxicity. Here, we evaluated the antitumor activity of adoptively transferred human tumor-infiltrating lymphocytes (TILs) genetically engineered to secrete single-chain IL12 selectively at the tumor site. Experimental Design: Thirty-three patients with metastatic melanoma were treated in a cell dose–escalation trial of autologous TILs transduced with a gene encoding a single-chain IL12 driven by a nuclear factor of the activated T cells promoter (NFAT.IL12). No IL2 was administered. Results: The administration of 0.001 to 0.1 × 109 NFAT.IL12–transduced TILs to 17 patients resulted in a single, objective response (5.9%). However, at doses between 0.3 and 3 × 109 cells, 10 of 16 patients (63%) exhibited objective clinical responses. The responses tended to be short, and the administered IL12-producing cells rarely persisted at 1 month. Increasing cell doses were associated with high serum levels of IL12 and IFNγ as well as clinical toxicities, including liver dysfunction, high fevers, and sporadic life-threatening hemodynamic instability. Conclusions: In this first-in-man trial, administration of TILs transduced with an inducible IL12 gene mediated tumor responses in the absence of IL2 administration using cell doses 10- to 100-fold lower than conventional TILs. However, due to toxicities, likely attributable to the secreted IL12, further refinement will be necessary before this approach can be safely used in the treatment of cancer patients. Clin Cancer Res; 21(10); 2278–88. ©2015 AACR.
2049187
Successful Eradication of Established Peritoneal Ovarian Tumors in SCID-Beige Mice following Adoptive Transfer of T Cells Genetically Targeted to the MUC16 Antigen
Purpose: Most patients diagnosed with ovarian cancer will ultimately die from their disease. For this reason, novel approaches to the treatment of this malignancy are needed. Adoptive transfer of a patient's own T cells, genetically modified ex vivo through the introduction of a gene encoding a chimeric antigen receptor (CAR) targeted to a tumor-associated antigen, is a novel approach to the treatment of ovarian cancer. Experimental Design: We have generated several CARs targeted to the retained extracellular domain of MUC16, termed MUC-CD, an antigen expressed on most ovarian carcinomas. We investigate the in vitro biology of human T cells retrovirally transduced to express these CARs by coculture assays on artificial antigen-presenting cells as well as by cytotoxicity and cytokine release assays using the human MUC-CD+ ovarian tumor cell lines and primary patient tumor cells. Further, we assess the in vivo antitumor efficacy of MUC-CD–targeted T cells in SCID-Beige mice bearing peritoneal human MUC-CD+ tumor cell lines. Results: CAR-modified, MUC-CD–targeted T cells exhibited efficient MUC-CD–specific cytolytic activity against both human ovarian cell and primary ovarian carcinoma cells in vitro. Furthermore, expanded MUC-CD–targeted T cells infused through either i.p. injection or i.v. infusion into SCID-Beige mice bearing orthotopic human MUC-CD+ ovarian carcinoma tumors either delayed progression or fully eradicated disease. Conclusion: These promising preclinical studies justify further investigation of MUC-CD–targeted T cells as a potential therapeutic approach for patients with high-risk MUC16+ ovarian carcinomas. Clin Cancer Res; 16(14); 3594–606. ©2010 AACR.
33663578
Molecular Cloning of the CA125 Ovarian Cancer Antigen
CA125 is an ovarian cancer antigen that is the basis for a widely used serum assay for the monitoring of patients with ovarian cancer; however, detailed information on its biochemical and molecular nature is lacking. We now report the isolation of a long, but partial, cDNA that corresponds to the CA125 antigen. A rabbit polyclonal antibody produced to purified CA125 antigen was used to screen a λZAP cDNA library from OVCAR-3 cells inEscherichia coli. The longest insert from the 54 positive isolated clones had a 5797-base pair sequence containing a stop codon and a poly(A) sequence but no clear 5′ initiation sequence. The deduced amino acid sequence has many of the attributes of a mucin molecule and was designated CA125/MUC16 (gene MUC16). These features include a high serine, threonine, and proline content in an N-terminal region of nine partially conserved tandem repeats (156 amino acids each) and a C-terminal region non-tandem repeat sequence containing a possible transmembrane region and a potential tyrosine phosphorylation site. Northern blotting showed that the level of MUC16 mRNA correlated with the expression of CA125 in a panel of cell lines. The molecular cloning of the CA125 antigen will lead to a better understanding of its role in ovarian cancer.
36901139
Selective Expansion of Chimeric Antigen Receptor-targeted T-cells with Potent Effector Function using Interleukin-4*
Polyclonal T-cells can be directed against cancer using transmembrane fusion molecules known as chimeric antigen receptors (CARs). Although preclinical studies have provided encouragement, pioneering clinical trials using CAR-based immunotherapy have been disappointing. Key obstacles are the need for robust expansion ex vivo followed by sustained survival of infused T-cells in patients. To address this, we have developed a system to achieve selective proliferation of CAR+ T-cells using IL-4, a cytokine with several pathophysiologic and therapeutic links to cancer. A chimeric cytokine receptor (4αβ) was engineered by fusion of the IL-4 receptor α (IL-4Rα) ectodomain to the βc subunit, used by IL-2 and IL-15. Addition of IL-4 to T-cells that express 4αβ resulted in STAT3/STAT5/ERK phosphorylation and exponential proliferation, mimicking the actions of IL-2. Using receptor-selective IL-4 muteins, partnering of 4αβ with γc was implicated in signal delivery. Next, human T-cells were engineered to co-express 4αβ with a CAR specific for tumor-associated MUC1. These T-cells exhibited an unprecedented capacity to elicit repeated destruction of MUC1-expressing tumor cultures and expanded through several logs in vitro. Despite prolonged culture in IL-4, T-cells retained specificity for target antigen, type 1 polarity, and cytokine dependence. Similar findings were observed using CARs directed against two additional tumor-associated targets, demonstrating generality of application. Furthermore, this system allows rapid ex vivo expansion and enrichment of engineered T-cells from small blood volumes, under GMP-compliant conditions. Together, these findings provide proof of principle for the development of IL-4-enhanced T-cell immunotherapy of cancer.
22991841
Flexible Targeting of ErbB Dimers That Drive Tumorigenesis by Using Genetically Engineered T Cells
Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z+ T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28z+ T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain.
38608401
NKG2D CAR T‐cell therapy inhibits the growth of NKG2D ligand heterogeneous tumors
Tumor heterogeneity presents a substantial barrier to increasing clinical responses mediated by targeted therapies. Broadening the immune response elicited by treatments that target a single antigen is necessary for the elimination of tumor variants that fail to express the targeted antigen. In this study, it is shown that adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) inhibited the growth of target‐expressing and ‐deficient tumor cells within ovarian and lymphoma tumors. Mice bearing the ID8 ovarian or RMA lymphoma tumors were treated with T cells transduced with a NKG2D‐based CAR (chNKG2D). NKG2D CAR T‐cell therapy protected mice from heterogeneous RMA tumors. Moreover, adoptive transfer of chNKG2D T cells mediated tumor protection against highly heterogeneous ovarian tumors in which 50, 20 or only 7% of tumor cells expressed significant amounts of NKG2D ligands. CAR T cells did not mediate an in vivo response against tumor cells that did not express sufficient amounts of NKG2D ligands, and the number of ligand‐expressing tumor cells correlated with therapeutic efficacy. In addition, tumor‐free surviving mice were protected against a tumor re‐challenge with NKG2D ligand‐negative ovarian tumor cells. These data indicate that NKG2D CAR T‐cell treatment can be an effective therapy against heterogeneous tumors and induce tumor‐specific immunity against ligand‐deficient tumor cells.
5242474
NKG2D CARs as Cell Therapy for Cancer
AbstractThe NKG2D cell receptor and its ligands have attracted considerable interest as a potential strategy to attack tumor cells. NKG2D ligands are expressed on most types of tumors, and they demonstrate relative selectivity of ligand expression on tumor cells compared to healthy cells. Several different variants of NKG2D-based chimeric antigen receptors (CARs) have been developed, and extensive in vivo mechanistic studies performed demonstrated that cytotoxicity and cytokines are important for the efficacy NKG2D CAR adoptive T-cell therapy. NKG2D CARs target tumor cells, and they also target immunosuppressive cells within the tumor microenvironment. Under certain conditions, NKG2D ligand expression can be found on nontumor tissue, so potential off-tumor toxicity remains. In this article, we review the use of NKG2D as a basis for CAR targeting of tumors.
21498300
Chimeric NKG2D modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways.
In this study, the efficacy and mechanisms of chimeric NKG2D receptor (chNKG2D)-modified T cells in eliminating NKG2D ligand-positive RMA/Rae1 lymphoma cells were evaluated. Intravenous injection of RMA/Rae1 cells led to significant tumor formation in spleens and lymph nodes within 2 weeks. Adoptive transfer of chNKG2D-modified T cells after tumor injection significantly reduced tumor burdens in both spleens and lymph nodes, and prolonged the survival of tumor-bearing mice. Multiple treatments with chNKG2D T cells resulted in long-term tumor-free survival. Moreover, these long-term survivors were resistant to rechallenge with RMA tumor cells (NKG2D ligand-negative), and their spleen and lymph node cells produced IFN-gamma in response to RMA but not to other tumors in vitro, indicating immunity against RMA tumor antigens. ChNKG2D T cell-derived IFN-gamma and granulocyte-macrophage colony-stimulating factor, but not perforin (Pfp), tumor necrosis factor-related apoptosis-inducing ligand, or Fas ligand (FasL) alone were critical for in vivo efficacy. T cells deficient in both Pfp and FasL did not kill NKG2D ligand-positive RMA cells in vitro. Adoptive transfer of Pfp(-/-)FasL(-/-) chNKG2D T cells had reduced in vivo efficacy, indicating that chNKG2D T cells used both mechanisms to attack RMA/Rae1 cells. Taken together, these results indicate that chNKG2D T-cell-mediated therapeutic effects are mediated by both cytokine-dependent and cytotoxic mechanisms in vivo.
5087652
Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer
Immune checkpoint inhibitors, which unleash a patient’s own T cells to kill tumors, are revolutionizing cancer treatment. To unravel the genomic determinants of response to this therapy, we used whole-exome sequencing of non–small cell lung cancers treated with pembrolizumab, an antibody targeting programmed cell death-1 (PD-1). In two independent cohorts, higher nonsynonymous mutation burden in tumors was associated with improved objective response, durable clinical benefit, and progression-free survival. Efficacy also correlated with the molecular smoking signature, higher neoantigen burden, and DNA repair pathway mutations; each factor was also associated with mutation burden. In one responder, neoantigen-specific CD8+ T cell responses paralleled tumor regression, suggesting that anti–PD-1 therapy enhances neoantigen-specific T cell reactivity. Our results suggest that the genomic landscape of lung cancers shapes response to anti–PD-1 therapy. An anticancer drug is more effective against tumors that carry more mutations. More mutations predict better efficacy Despite the remarkable success of cancer immunotherapies, many patients do not respond to treatment. Rizvi et al. studied the tumors of patients with non–small-cell lung cancer undergoing immunotherapy. In two independent cohorts, treatment efficacy was associated with a higher number of mutations in the tumors. In one patient, a tumor-specific T cell response paralleled tumor regression. Science, this issue p. 124
41000029
Pembrolizumab versus Ipilimumab in Advanced Melanoma.
BACKGROUND
The immune checkpoint inhibitor ipilimumab is the standard-of-care treatment for patients with advanced melanoma. Pembrolizumab inhibits the programmed cell death 1 (PD-1) immune checkpoint and has antitumor activity in patients with advanced melanoma.


METHODS
In this randomized, controlled, phase 3 study, we assigned 834 patients with advanced melanoma in a 1:1:1 ratio to receive pembrolizumab (at a dose of 10 mg per kilogram of body weight) every 2 weeks or every 3 weeks or four doses of ipilimumab (at 3 mg per kilogram) every 3 weeks. Primary end points were progression-free and overall survival.


RESULTS
The estimated 6-month progression-free-survival rates were 47.3% for pembrolizumab every 2 weeks, 46.4% for pembrolizumab every 3 weeks, and 26.5% for ipilimumab (hazard ratio for disease progression, 0.58; P<0.001 for both pembrolizumab regimens versus ipilimumab; 95% confidence intervals [CIs], 0.46 to 0.72 and 0.47 to 0.72, respectively). Estimated 12-month survival rates were 74.1%, 68.4%, and 58.2%, respectively (hazard ratio for death for pembrolizumab every 2 weeks, 0.63; 95% CI, 0.47 to 0.83; P=0.0005; hazard ratio for pembrolizumab every 3 weeks, 0.69; 95% CI, 0.52 to 0.90; P=0.0036). The response rate was improved with pembrolizumab administered every 2 weeks (33.7%) and every 3 weeks (32.9%), as compared with ipilimumab (11.9%) (P<0.001 for both comparisons). Responses were ongoing in 89.4%, 96.7%, and 87.9% of patients, respectively, after a median follow-up of 7.9 months. Efficacy was similar in the two pembrolizumab groups. Rates of treatment-related adverse events of grade 3 to 5 severity were lower in the pembrolizumab groups (13.3% and 10.1%) than in the ipilimumab group (19.9%).


CONCLUSIONS
The anti-PD-1 antibody pembrolizumab prolonged progression-free survival and overall survival and had less high-grade toxicity than did ipilimumab in patients with advanced melanoma. (Funded by Merck Sharp & Dohme; KEYNOTE-006 ClinicalTrials.gov number, NCT01866319.).
489805
Genomic correlates of response to CTLA-4 blockade in metastatic melanoma
Is cancer immunotherapy a private affair? Immune checkpoint blockade, a relatively new cancer treatment, substantially extends the survival of a subset of patients. Previous work has shown that patients whose tumors harbor the largest number of mutations—and thus produce a large number of “neoantigens” recognized as foreign by the immune system—are most likely to benefit. Expanding on these earlier studies, Van Allen et al. studied over 100 patients with melanoma and found a similar correlation (see the Perspective by Gubin and Schreiber). There was no evidence, however, that specific neoantigen sequences were shared by patients who responded. Science, this issue p. 207, see also p. 158 Melanoma patients who respond to immunotherapy do not appear to share common tumor neoantigens. [Also see Perspective by Gubin and Schreiber ] Monoclonal antibodies directed against cytotoxic T lymphocyte–associated antigen-4 (CTLA-4), such as ipilimumab, yield considerable clinical benefit for patients with metastatic melanoma by inhibiting immune checkpoint activity, but clinical predictors of response to these therapies remain incompletely characterized. To investigate the roles of tumor-specific neoantigens and alterations in the tumor microenvironment in the response to ipilimumab, we analyzed whole exomes from pretreatment melanoma tumor biopsies and matching germline tissue samples from 110 patients. For 40 of these patients, we also obtained and analyzed transcriptome data from the pretreatment tumor samples. Overall mutational load, neoantigen load, and expression of cytolytic markers in the immune microenvironment were significantly associated with clinical benefit. However, no recurrent neoantigen peptide sequences predicted responder patient populations. Thus, detailed integrated molecular characterization of large patient cohorts may be needed to identify robust determinants of response and resistance to immune checkpoint inhibitors.
11419286
Anti-PD-1 Antibody Therapy Potently Enhances the Eradication of Established Tumors By Gene-Modified T Cells
Purpose: To determine the antitumor efficacy and toxicity of a novel combination approach involving adoptive T-cell immunotherapy using chimeric antigen receptor (CAR) T cells with an immunomodulatory reagent for blocking immunosuppression. Experimental Design: We examined whether administration of a PD-1 blocking antibody could increase the therapeutic activity of CAR T cells against two different Her-2+ tumors. The use of a self-antigen mouse model enabled investigation into the efficacy, mechanism, and toxicity of this combination approach. Results: In this study, we first showed a significant increase in the level of PD-1 expressed on transduced anti-Her-2 CD8+ T cells following antigen-specific stimulation with PD-L1+ tumor cells and that markers of activation and proliferation were increased in anti-Her-2 T cells in the presence of anti-PD-1 antibody. In adoptive transfer studies in Her-2 transgenic recipient mice, we showed a significant improvement in growth inhibition of two different Her-2+ tumors treated with anti-Her-2 T cells in combination with anti-PD-1 antibody. The therapeutic effects observed correlated with increased function of anti-Her-2 T cells following PD-1 blockade. Strikingly, a significant decrease in the percentage of Gr1+ CD11b+ myeloid-derived suppressor cells (MDSC) was observed in the tumor microenvironment of mice treated with the combination therapy. Importantly, increased antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen. Conclusion: This study shows that specifically blocking PD-1 immunosuppression can potently enhance CAR T-cell therapy that has significant implications for potentially improving therapeutic outcomes of this approach in patients with cancer. Clin Cancer Res; 19(20); 5636–46. ©2013 AACR.
24242642
Gene therapy using genetically modified lymphocytes targeting VEGFR-2 inhibits the growth of vascularized syngenic tumors in mice.
Immunotherapies based on adoptive cell transfer are highly effective in the treatment of metastatic melanoma, but the use of this approach in other cancer histologies has been hampered by the identification of appropriate target molecules. Immunologic approaches targeting tumor vasculature provide a means for the therapy of multiple solid tumor types. We developed a method to target tumor vasculature, using genetically redirected syngeneic or autologous T cells. Mouse and human T cells were engineered to express a chimeric antigen receptor (CAR) targeted against VEGFR-2, which is overexpressed in tumor vasculature and is responsible for VEGF-mediated tumor progression and metastasis. Mouse and human T cells expressing the relevant VEGFR-2 CARs mediated specific immune responses against VEGFR-2 protein as well as VEGFR-2-expressing cells in vitro. A single dose of VEGFR-2 CAR-engineered mouse T cells plus exogenous IL-2 significantly inhibited the growth of 5 different types of established, vascularized syngeneic tumors in 2 different strains of mice and prolonged the survival of mice. T cells transduced with VEGFR-2 CAR showed durable and increased tumor infiltration, correlating with their antitumor effect. This approach provides a potential method for the gene therapy of a variety of human cancers.
207037416
Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor.
PURPOSE
T cells can be genetically modified to express an anti-CD19 chimeric antigen receptor (CAR). We assessed the safety and efficacy of administering autologous anti-CD19 CAR T cells to patients with advanced CD19(+) B-cell malignancies.


PATIENTS AND METHODS
We treated 15 patients with advanced B-cell malignancies. Nine patients had diffuse large B-cell lymphoma (DLBCL), two had indolent lymphomas, and four had chronic lymphocytic leukemia. Patients received a conditioning chemotherapy regimen of cyclophosphamide and fludarabine followed by a single infusion of anti-CD19 CAR T cells.


RESULTS
Of 15 patients, eight achieved complete remissions (CRs), four achieved partial remissions, one had stable lymphoma, and two were not evaluable for response. CRs were obtained by four of seven evaluable patients with chemotherapy-refractory DLBCL; three of these four CRs are ongoing, with durations ranging from 9 to 22 months. Acute toxicities including fever, hypotension, delirium, and other neurologic toxicities occurred in some patients after infusion of anti-CD19 CAR T cells; these toxicities resolved within 3 weeks after cell infusion. One patient died suddenly as a result of an unknown cause 16 days after cell infusion. CAR T cells were detected in the blood of patients at peak levels, ranging from nine to 777 CAR-positive T cells/μL.


CONCLUSION
This is the first report to our knowledge of successful treatment of DLBCL with anti-CD19 CAR T cells. These results demonstrate the feasibility and effectiveness of treating chemotherapy-refractory B-cell malignancies with anti-CD19 CAR T cells. The numerous remissions obtained provide strong support for further development of this approach.
205093443
Inducible apoptosis as a safety switch for adoptive cell therapy.
BACKGROUND
Cellular therapies could play a role in cancer treatment and regenerative medicine if it were possible to quickly eliminate the infused cells in case of adverse events. We devised an inducible T-cell safety switch that is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iCasp9) becomes activated and leads to the rapid death of cells expressing this construct.


METHODS
We tested the activity of our safety switch by introducing the gene into donor T cells given to enhance immune reconstitution in recipients of haploidentical stem-cell transplants. Patients received AP1903, an otherwise bioinert small-molecule dimerizing drug, if graft-versus-host disease (GVHD) developed. We measured the effects of AP1903 on GVHD and on the function and persistence of the cells containing the iCasp9 safety switch.


RESULTS
Five patients between the ages of 3 and 17 years who had undergone stem-cell transplantation for relapsed acute leukemia were treated with the genetically modified T cells. The cells were detected in peripheral blood from all five patients and increased in number over time, despite their constitutive transgene expression. A single dose of dimerizing drug, given to four patients in whom GVHD developed, eliminated more than 90% of the modified T cells within 30 minutes after administration and ended the GVHD without recurrence.


CONCLUSIONS
The iCasp9 cell-suicide system may increase the safety of cellular therapies and expand their clinical applications. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00710892.).
12629497
A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells.
An unmet need in cell engineering is the availability of a single transgene encoded, functionally inert, human polypeptide that can serve multiple purposes, including ex vivo cell selection, in vivo cell tracking, and as a target for in vivo cell ablation. Here we describe a truncated human EGFR polypeptide (huEGFRt) that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity but retains the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux). After lentiviral transduction of human T cells with vectors that coordinately express tumor-specific chimeric antigen receptors and huEGFRt, we show that huEGFRt serves as a highly efficient selection epitope for chimeric antigen receptor(+) T cells using biotinylated cetuximab in conjunction with current good manufacturing practices (cGMP)-grade anti-biotin immunomagnetic microbeads. Moreover, huEGFRt provides a cell surface marker for in vivo tracking of adoptively transferred T cells using both flow cytometry and immunohistochemistry, and a target for cetuximab-mediated antibody-dependent cellular cytotoxicity and in vivo elimination. The versatility of huEGFRt and the availability of pharmaceutical-grade reagents for its clinical application denote huEGFRt as a significant new tool for cellular engineering.