Chimeric Antigen Receptor T Cells in Refractory B-Cell Lymphomas.

BACKGROUND: Patients with diffuse large B-cell lymphoma or follicular lymphoma that is refractory to or that relapses after immunochemotherapy and transplantation have a poor prognosis. High response rates have been reported with the use of T cells modified by chimeric antigen receptor (CAR) that target CD19 in B-cell cancers, although data regarding B-cell lymphomas are limited. METHODS: We used autologous T cells that express a CD19-directed CAR (CTL019) to treat patients with diffuse large B-cell lymphoma or follicular lymphoma that had relapsed or was refractory to previous treatments. Patients were monitored for response to treatment, toxic effects, the expansion and persistence of CTL019 cells in vivo, and immune recovery. RESULTS: A total of 28 adult patients with lymphoma received CTL019 cells, and 18 of 28 had a response (64%; 95% confidence interval [CI], 44 to 81). Complete remission occurred in 6 of 14 patients with diffuse large B-cell lymphoma (43%; 95% CI, 18 to 71) and 10 of 14 patients with follicular lymphoma (71%; 95% CI, 42 to 92). CTL019 cells proliferated in vivo and were detectable in the blood and bone marrow of patients who had a response and patients who did not have a response. Sustained remissions were achieved, and at a median follow-up of 28.6 months, 86% of patients with diffuse large B-cell lymphoma who had a response (95% CI, 33 to 98) and 89% of patients with follicular lymphoma who had a response (95% CI, 43 to 98) had maintained the response. Severe cytokine-release syndrome occurred in 5 patients (18%). Serious encephalopathy occurred in 3 patients (11%); 2 cases were self-limiting and 1 case was fatal. All patients in complete remission by 6 months remained in remission at 7.7 to 37.9 months (median, 29.3 months) after induction, with a sustained reappearance of B cells in 8 of 16 patients and with improvement in levels of IgG in 4 of 10 patients and of IgM in 6 of 10 patients at 6 months or later and in levels of IgA in 3 of 10 patients at 18 months or later. CONCLUSIONS: CTL019 cells can be effective in the treatment of relapsed or refractory diffuse large B-cell lymphoma and follicular lymphoma. High rates of durable remission were observed, with recovery of B cells and immunoglobulins in some patients. Transient encephalopathy developed in approximately one in three patients and severe cytokine-release syndrome developed in one in five patients. (Funded by Novartis and others; ClinicalTrials.gov number, NCT02030834 .).

Neurotoxicity after CTL019 in a pediatric and young adult cohort.

OBJECTIVE: To characterize the incidence and clinical characteristics of neurotoxicity in the month following CTL019 infusion in children and young adults, to define the relationship between neurotoxicity and cytokine release syndrome (CRS), and to identify predictive biomarkers for development of neurotoxicity following CTL019 infusion. METHODS: We analyzed data on 51 subjects, 4 to 22 years old, who received CTL019, a chimeric antigen receptor-modified T-cell therapy against CD19, between January 1, 2010 and December 1, 2015 through a safety/feasibility clinical trial (NCT01626495) at our institution. We recorded incidence of significant neurotoxicity (encephalopathy, seizures, and focal deficits) and CRS, and compared serum cytokine levels in the first month postinfusion between subjects who did and did not develop neurotoxicity. RESULTS: Neurotoxicity occurred in 23 of 51 subjects (45%, 95% confidence interval = 31-60%) and was positively associated with higher CRS grade (p < 0.0001) but was not associated with demographic characteristics or prior oncologic treatment history. Serum interleukin (IL)-2, IL-15, soluble IL-4, and hepatocyte growth factor concentrations were higher in subjects with neurotoxicity than those with isolated CRS. Differences in peak levels of select cytokines including IL-12 and soluble tumor necrosis factor receptor-1 within the first 3 days were seen in subjects with neurotoxicity. INTERPRETATION: Neurotoxicity is common after CTL019 infusion in children and young adults, and is associated with higher CRS grade. Differences in serum cytokine profiles between subjects with neurotoxicity and those with isolated CRS suggest unique pathophysiological mechanisms. Serum cytokine profiles in the first 3 days postinfusion may help identify children and young adults at risk for neurotoxicity, and may provide a foundation for investigation into potential mitigation strategies. Ann Neurol 2018;84:537-546.

Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy.

The mechanisms underlying the maintenance of long-lasting humoral immunity are not well understood. Studies in mice indicate that plasma cells (PCs) can survive up to a lifetime, even in the absence of regeneration by B cells, implying the presence of long-lived PCs as a mechanism for long-lasting immunity. Evidence from humans treated with anti-CD20, which depletes circulating B cells, also suggests B-cell-independent long-term survival of some PCs. On the other hand, antibody responses may be sustained solely by short-lived PCs with repopulation from clonally related memory B cells. To explore PC longevity and humoral immunity in humans, we investigated the fate of PCs and their antibodies in adult and pediatric patients who received chimeric antigen receptor-based adoptive T-cell immunotherapy targeting CD19 to treat B-cell lineage malignancies (CTL019). Treatment with CTL019 is frequently associated with B-cell aplasia that can persist for years. Serum antibody titers to vaccine-related antigens were measured, and quantitative assessment of B cells and PCs in blood and bone marrow was performed at various time points before and after CTL019 therapy. While total serum immunoglobulin concentrations decline following CTL019-induced B-cell aplasia, several vaccine/pathogen-specific serum immunoglobulin G and A (IgG and IgA) titers remain relatively stable for at least 6 and 12 months posttreatment, respectively. Analysis of bone marrow biopsies after CTL019 revealed 8 patients with persistence of antibody-secreting PCs at least 25 months post-CTL019 infusion despite absence of CD19(+)CD20(+) B cells. These results provide strong evidence for the existence of memory B-cell-independent, long-lived PCs in humans that contribute to long-lasting humoral immunity.

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.).

Graft versus leukemia response without graft-versus-host disease elicited by adoptively transferred multivirus-specific T-cells.

A 12-year-old boy with refractory acute lymphoblastic leukemia received a haploidentical transplant from his mother. As prophylaxis for Epstein-Barr virus (EBV), cytomegalovirus (CMV) and adenovirus, he received ex vivo expanded virus-specific donor T cells 3.5 months after transplant. Four weeks later leukemic blasts bearing the E2A deletion, identified by fluorescent in situ hybridization (FISH), appeared transiently in the blood followed by a FISH-negative hematological remission, which was sustained until a testicular relapse 3.5 months later. Clearance of the circulating leukemic cells coincided with a marked increase in circulating virus-specific T cells. The virus-specific cytotoxic T-cell (CTL) line showed strong polyfunctional reactivity with the patient's leukemic cells but not phytohemagglutinin (PHA) blasts, suggesting that virus-specific CTL lines may have clinically significant antileukemia activity.

KIT with D816 mutations cooperates with CBFB-MYH11 for leukemogenesis in mice.

KIT mutations are the most common secondary mutations in inv(16) acute myeloid leukemia (AML) patients and are associated with poor prognosis. It is therefore important to verify that KIT mutations cooperate with CBFB-MYH11, the fusion gene generated by inv(16), for leukemogenesis. Here, we transduced wild-type and conditional Cbfb-MYH11 knockin (KI) mouse bone marrow (BM) cells with KIT D816V/Y mutations. KIT transduction caused massive BM Lin(-) cell death and fewer colonies in culture that were less severe in the KI cells. D816Y KIT but not wild-type KIT enhanced proliferation in Lin(-) cells and led to more mixed lineage colonies from transduced KI BM cells. Importantly, 60% and 80% of mice transplanted with KI BM cells expressing D816V or D816Y KIT, respectively, died from leukemia within 9 months, whereas no control mice died. Results from limiting dilution transplantations indicate higher frequencies of leukemia-initiating cells in the leukemia expressing mutated KIT. Signaling pathway analysis revealed that p44/42 MAPK and Stat3, but not AKT and Stat5, were strongly phosphorylated in the leukemia cells. Finally, leukemia cells carrying KIT D816 mutations were sensitive to the kinase inhibitor PKC412. Our data provide clear evidence for cooperation between mutated KIT and CBFB-MYH11 during leukemogenesis.

Immune reconstitution in recipients of photodepleted HLA-identical sibling donor stem cell transplantations: T cell subset frequencies predict outcome.

We evaluated an ex vivo photodepletion (PD) technique to selectively deplete graft-versus-host disease (GVHD) alloreacting T cells given to 24 human leukocyte antigen (HLA)-identical sibling stem cell transplantation (SCT) recipients. Donor lymphocytes were activated by 72-hour exposure to irradiated in vitro expanded recipient T lymphocytes and pulsed with a TH9402 photosensitizer. Alloactivated T cells preferentially retaining the photosensitizer were eliminated by light exposure. The PD product showed an inverted CD4(+)/CD8(+) ratio with greatest depletion occurring in the CD4(+) naive and central memory populations. In contrast, the CD8(+) naive and effector cells were relatively conserved, reflecting the differential extrusion of TH9402 by T cell subsets. Cytomegalovirus reactive T cells were reduced in the PD product and in recipient blood 100 days after SCT when compared with contemporaneous HLA-identical sibling donor T cell-depleted SCT recipients. Although PD SCT recipients experienced similar absolute lymphocyte counts during the first 100 days after SCT, they achieved 100% donor T cell chimerism more rapidly and had higher CD8(+) naive T cell counts early after SCT. SCT recipients of PD products with the lowest CD4 central memory content had the highest risk of developing chronic GVHD (cGVHD) (P = .04) and a poorer survival (P = .03). Although the persistence of CD8(+) naive T cells may have contributed to important antileukemia responses resulting in a relatively low relapse rate, our findings emphasize the role of donor memory T cells and CD4 cells in establishing immune competence post-SCT. Although PD is associated with excellent outcomes in the haploidentical setting, the low frequency of alloactivations in HLA-matched pairs makes the PD approach used by our group for allodepletion in HLA-matched sibling transplantations an inefficient technique.

The transfer of adaptive immunity to CMV during hematopoietic stem cell transplantation is dependent on the specificity and phenotype of CMV-specific T cells in the donor.

The successful reconstitution of adaptive immunity to human cytomegalovirus (CMV) in hematopoietic stem cell transplantation (HSCT) recipients is central to the reduction of viral reactivation-related morbidity and mortality. Here, we characterized the magnitude, specificity, phenotype, function, and clonotypic composition of CMV-specific T-cell responses in 18 donor-recipient pairs both before and after HSCT. The principal findings were: (1) the specificity of CMV-specific T-cell responses in the recipient after HSCT mirrors that in the donor; (2) the maintenance of these targeting patterns reflects the transfer of epitope-specific T-cell clonotypes from donor to recipient; (3) less differentiated CD27(+)CD57(-) CMV-specific memory T cells are more likely to persist in the recipient after HSCT compared with more terminally differentiated CD27(-) CD57(+) CMV-specific memory T cells; (4) the presence of greater numbers of less differentiated CD8(+) CMV-specific T cells in the donor appears to confer protection against viral reactivation in the recipient after HSCT; and (5) CMV-specific T cells acquire a more differentiated phenotype and a restricted functional profile after HSCT. Overall, these findings define the immunologic factors that influence the successful adoptive transfer of antigen-specific T-cell immunity during HSCT, which enables the identification of recipients at particular risk of CMV reactivation after HSCT.

The clonal composition of human CD4+CD25+Foxp3+ cells determined by a comprehensive DNA-based multiplex PCR for TCRB gene rearrangements.

The characterization of the T-cell receptor (TCR) repertoire of CD4+ regulatory T cells (T(R)) has been limited due to the RNA degradation that results following permeabilization and fixation as routinely used for intracellular staining of Foxp3. In the present study the clonal composition of human umbilical cord blood (UCB) and adult peripheral blood mononuclear cell (PBMC) CD4+ T(R) and non-T(R) was characterized by a DNA-based multiplex PCR which allowed for the consistent clonotypic characterization of cells that have undergone fixation and permeabilization. To validate this method, CD8+ T cells from two HLA A()0201 individuals were sorted and compared clonotypically based upon their ability either to secrete interferon-gamma in response to a CMV pp65 epitope or to bind to the corresponding pMHC I tetramer. Clonotypes shared between the CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- subsets were observed in all 3 UCB and in one adult PBMCs, suggesting that naïve and memory CD4+ T(R) can share the same clonotypes as CD4+ non-T(R) in humans.

Chimeric Antigen Receptor T Cells: Self-Replicating Drugs for Cancer.

The chimeric antigen receptor (CAR) technology started out as a tool to understand lymphocyte biology but rapidly developed into a T cell therapeutic agent for the treatment of cancers. Here, we describe the technological advances in the field of CARs and highlight critical components of its success. Additionally, we describe how various laboratories have worked toward developing new, safer, and more potent CARs for cancer.

A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma.

We conducted a first-in-human study of intravenous delivery of a single dose of autologous T cells redirected to the epidermal growth factor receptor variant III (EGFRvIII) mutation by a chimeric antigen receptor (CAR). We report our findings on the first 10 recurrent glioblastoma (GBM) patients treated. We found that manufacturing and infusion of CAR-modified T cell (CART)-EGFRvIII cells are feasible and safe, without evidence of off-tumor toxicity or cytokine release syndrome. One patient has had residual stable disease for over 18 months of follow-up. All patients demonstrated detectable transient expansion of CART-EGFRvIII cells in peripheral blood. Seven patients had post-CART-EGFRvIII surgical intervention, which allowed for tissue-specific analysis of CART-EGFRvIII trafficking to the tumor, phenotyping of tumor-infiltrating T cells and the tumor microenvironment in situ, and analysis of post-therapy EGFRvIII target antigen expression. Imaging findings after CART immunotherapy were complex to interpret, further reinforcing the need for pathologic sampling in infused patients. We found trafficking of CART-EGFRvIII cells to regions of active GBM, with antigen decrease in five of these seven patients. In situ evaluation of the tumor environment demonstrated increased and robust expression of inhibitory molecules and infiltration by regulatory T cells after CART-EGFRvIII infusion, compared to pre-CART-EGFRvIII infusion tumor specimens. Our initial experience with CAR T cells in recurrent GBM suggests that although intravenous infusion results in on-target activity in the brain, overcoming the adaptive changes in the local tumor microenvironment and addressing the antigen heterogeneity may improve the efficacy of EGFRvIII-directed strategies in GBM.

Measuring IL-6 and sIL-6R in serum from patients treated with tocilizumab and/or siltuximab following CAR T cell therapy.

T cells expressing a CD19-specific chimeric antigen receptor (CAR19) are demonstrating remarkable efficacy in hematologic malignancies. Treatment is often associated with life-threatening cytokine release syndrome (CRS) which can be effectively treated with cytokine blockade using the antibodies, Siltuximab or Tocilizumab respectively targeting IL-6 or the IL-6 receptor. As IL-6 blockade is moving into the clinic for the treatment of CRS as well as IL-6-driven rheumatologic and malignant diseases, clinicians are utilizing serum cytokine panels more frequently to assess the effects of IL-6 inhibitors. It is paramount to ascertain whether levels obtained are accurate, especially as certain drugs may, in theory, affect quantification. We report the comparative quantification of IL-6 and sIL-6R using Luminex-based immunoassay kits from two vendors. Our results indicate good agreement of the commercial immunoassays in measurement of IL-6 but disagreement in quantitation of sIL-6R. We found that both Siltuximab and Tocilizumab can interfere with the measurement of their respective ligands using reagents from one vendor but not the second. This has significant implications for the analysis of IL-6 and sIL-6R pharmacokinetics analysis in Siltuximab or Tocilizumab-treated patients. We found that high levels of IL-6 can falsely reduce the measured levels of sIL-6R and high levels of sIL-6R can reduce levels of IL-6 when measured with some commercial assays. These data demonstrate the importance of assessing the impact of cytokine-blocking agents on accuracy of clinical biomarker assays in other diseases, as drugs targeting TNF-alpha, IL1B, and IL5 are being used more frequently in a large number of diseases.

Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies.

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies.

Autoreactive, cytotoxic T lymphocytes specific for peptides derived from normal B-cell differentiation antigens in healthy individuals and patients with B-cell malignancies.

PURPOSE: To investigate potential immunotherapeutic strategies in B lymphocytic malignancies we looked for CTLs recognizing CD19 and CD20 epitopes. EXPERIMENTAL DESIGN: Three CD19 and CD20 peptides binding to HLA-A*0201 were identified and used to detect peptide specific CTLs by a quantitative real-time PCR to measure IFN-gamma mRNA expression in 23 healthy individuals and 28 patients (18 chronic lymphocytic leukemia (CLL), 7 follicular lymphoma, 2 acute lymphocytic leukemia, and 1 large cell lymphoma). Peptide-specific CTLs were expanded in culture with CD40-activated B cells to test lytic activity in three patients. RESULTS: In healthy individuals, CD8+ T-cell responses were detected in one to CD19(74-82), in three to CD20(127-135), and three to CD20(188-196). Seven of 27 patients (6 with CLL) had CD8+ T cells recognizing CD19(74-82). Seven patients responded to CD20(127-135) and three to CD20(188-196). All were CLL patients. CD19(74-82)-specific CTLs from three patients were expanded over 4 weeks. These cells were HLA-A*0201 specific and lytic for peptide-loaded antigen-presenting cells but not to malignant or unpulsed B cells. CONCLUSIONS: CTLs that recognize CD19 and CD20 epitopes exist in healthy individuals and may be increased in CLL patients. They are of low avidity and require high doses of peptide for activation. Strategies to increase T-cell avidity would be necessary for T-cell immunotherapeutic approaches using the peptides studied.

Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.

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 × 10(8) to 11 × 10(8) CTL019 cells (median, 1.6 × 10(8) 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.

CMV-specific T cells generated from naïve T cells recognize atypical epitopes and may be protective in vivo.

Adoptive transfer of cytomegalovirus (CMV)-specific T cells derived from adult seropositive donors can effectively restore antiviral immunity after transplantation. However, CMV-seronegative donors lack CMV-specific memory T cells, which restricts the availability of virus-specific T cells for immunoprophylaxis. We demonstrate the feasibility of deriving CMV-specific T cells from naïve cells for T cell therapy. Naïve T cells primed to recognize CMV were restricted to different, atypical epitopes than T cells derived from CMV-seropositive individuals; however, these two cell populations had similar avidities. CMV-seropositive individuals also had T cells recognizing these atypical epitopes, but these cells had a lower avidity than those derived from the seronegative subjects, which suggests that high-avidity T cells to these epitopes may be lost over time. Indeed, recipients of cord blood (CB) grafts who did not develop CMV were found by clonotypic analysis to have T cells recognizing atypical CMVpp65 epitopes. Therefore, we examined unmanipulated CB units and found that T cells with T cell receptors restricted by atypical epitopes were the most common, which may explain why these T cells expanded. When infused to recipients, naïve donor-derived virus-specific T cells that recognized atypical epitopes were associated with prolonged periods of CMV-free survival and complete remission. These data suggest that naïve-derived T cells from seronegative patients may be an additional source of cells for CMV immunoprophylaxis.

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.