CD19 Chimeric Antigen Receptor Therapy Research Articles

Cytopenias After CD19 Chimeric Antigen Receptor T-Cells (CAR-T) Therapy for Diffuse Large B-Cell Lymphomas or Transformed Follicular Lymphoma: A Single Institution Experience.

IntroductionPatients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) have poor outcomes. Treatment with CD19 chimeric antigen receptor (CAR-T) cells, tisagenlecleucel and axicabtagene ciloleucel, has been associated with improved outcomes. Cytopenias were observed in clinical trials with both products; however, little is known regarding the patterns and outcomes of these cytopenias.Subjects and MethodsWe reviewed DLBCL patients (n=32) receiving either product between January and September 2018 at our institution.ResultsMedian duration of leukopenia, neutropenia, lymphopenia, anemia, and thrombocytopenia was 49, 9, 117.5, 125, and 95.5 days after CAR-T infusion, respectively. Filgrastim was used in 63% of patients, and 50% of patients received red cell or platelet transfusions. With the exception of neutropenia, increase in the duration of cytopenia of any lineage was associated with improvement in progression-free survival, and in overall survival in case of anemia. There was no association between the duration of cytopenias with either cytokine release syndrome or neurotoxicity.DiscussionOur data suggest a correlation between cytopenias and survival outcomes after CD19 CAR-T therapy. If validated, cytopenia may be proven useful as a biomarker of response and survival after CAR-T therapy.

Modulation of CD22 Antigen Density Improves Efficacy of CD22 Chimeric Antigen Receptor (CAR) T Cells Against CD22lo B-Lineage Leukemia and Lymphoma

While overall survival for acute lymphoblastic leukemia (ALL) in children treated with risk-adapted, multi-agent chemotherapeutic regimens is greater than 85%, relapsed ALL remains a major challenge. Chimeric antigen receptor (CAR) T cell therapy targeting CD19 provides these patients with a remission induction rate of 70-90% across multiple clinical trials, but loss of the targeted epitope results in relapse following CD19 CAR T cells. In the setting of these relapses, our group conducted a CD22 CAR T cell trial, which induces remission in approximately 70% percent of patients with relapsed/refractory CD22-positive pre-B cell ALL, including those relapsing after CD19 CAR therapy (NCT02315612). However, relapse associated with reduced CD22 site density has shortened remission duration in some patients. To test the impact of low CD22 site density on CD22 CAR T cell functionality, we modified Nalm6 ALL cell lines using CRISPR/Cas9 to eliminate the CD22 gene, followed by lentiviral transduction of full-length CD22 and single-cell cloning based on CD22 expression levels. CD22 site density was assessed with the Quantibrite-PE Assay Kit and four cell lines were identified for a spectrum of CD22 expression: CD22 negative (CD22neg); CD22 low (CD22lo) - site density comparable to that seen in ALL patients relapsing following CD22 CAR therapy; CD22 normal (Nalm6) - site density comparable to ALL CD22 expression; and CD22 high (CD22hi) - site density comparable to CD19 levels. As CD22 site density decreases, CD22 CAR T cells produce incrementally less interferon-gamma and do not produce IL-2 against CD22lo ALL. CD22 CAR delayed, but was unable to prevent, progression of CD22lo ALL in murine xenografts. We next evaluated two potential approaches to overcoming low site density antigen escape: improving CAR activity or increasing tumor sensitivity. To improve CAR activity, we generated a high-binding affinity CD22 CAR (CD22 CARH1) using an affinity-enhanced version of the scFv from the CD22 CAR construct in our ongoing trial. Despite higher affinity, the CD22 CARH1 T cell did not improve in vitro killing or in vivo clearance of CD22lo ALL. To increase tumor sensitivity, we sought to increase CD22 site density using the PKC inhibitor Bryostatin1, a drug previously shown to increase CD22 expression in CLL. Bryostatin1 increases CD22 site density on a panel of pre-B cell ALL and diffuse large B cell lymphoma (DLBCL) cell lines. Interestingly, the CD22 increase is durable for at least 1 week after exposure to Bryostatin1. Bryostatin1-mediated CD22 increased site density improves cytokine production by CD22 CAR T cells. To evaluate the mechanism of Bryostatin1-mediated upregulation of CD22 expression, RNAseq was performed on Bryostatin1-exposed ALL. Preliminary analysis demonstrates that Bryostatin1 does not directly affect CD22 mRNA expression levels, but modulates early transcription factors within the B-cell receptor pathway. Based on these results, we are further evaluating pathways affected by Bryostatin1 through protein expression analysis and assessing in vivo function of Bryostatin1 in the setting of a patient-derived CD22 CAR-resistant xenograft model. In conclusion, these results demonstrate a clear threshold antigen site density, below which the CD22 CAR is unable to clear CD22-expressing ALL. We further demonstrate that increased CD22 CAR affinity does not seem to improve CD22 CAR activity against CD22lo ALL. Bryostatin1-mediated CD22 modulation can improve CD22 CAR response against leukemia and lymphoma providing a potential rational combination to rescue CD22 CAR activity and remission durability. DisclosuresNo relevant conflicts of interest to declare.

Clinical Factors Associated with Improved Survival Following Allogeneic HSCT after CD19 CAR Therapy in Adult Patients with Relapsed B-ALL

Introduction: Autologous chimeric antigen receptor (CAR) T cell therapy has shown to be effective in CD19+ relapsed or refractory (R/R) B-ALL patients (pts). However, relapses are common and the role of post-CAR allogeneic hematopoietic stem cell transplant (alloHSCT) remains unclear, particularly in adults with relapsed B-ALL. We previously reported that there was no survival benefit with post-CAR alloHSCT in overall adult ALL population (Park J et al. NEJM 2018), but it is unclear whether there is a subset of pts who may benefit the most from post-CAR alloHSCT. Therefore, we sought to identify clinical features that are associated with a better long-term survival in adult B-ALL pts treated with CD19 CAR T cells followed by alloHSCT. Methods: We performed a retrospective chart review of adult pts with R/R ALL treated with 19-28z CAR T therapy at MSKCC (NCT01044069), with a particular focus on pts who achieved minimal residual disease (MRD) negative CR to the CAR therapy and subsequently proceeded to alloHSCT. Clinical data was updated with the data cutoff date of July 15, 2019. We examined the association between overall survival and the following clinical factors: impact of age, prior HSCT status, presence of Philadelphia chromosome (Ph), disease status at the time of CAR therapy (MRD vs. morphologic), severity of cytokine release syndrome (CRS) and neurotoxicity (NTX) experienced during CAR therapy. Survival was calculated from the time of CAR T cell infusion. Continuous variables were analyzed univariately using cox PH models. Categorical variables were analyzed using Kaplan Meier methodology and log rank tests. Results: Of the 53 pts who received 1928z CAR T cells, 16 pts proceeded to alloHSCT after achieving MRD negative CR to the CAR therapy. The median age of these pts was 38 (range, 23-66), and baseline disease and pt characteristics as well as response and toxicity to CAR T therapy are listed in Table 1. Of the 16 pts, 6 pts remain alive in remission; 4 pts died of relapse (median time to relapse: 104.5 days, 57-194), and 6 pts died of transplant-related mortality (TRM) in remission (n=4, infection; n=1, pulmonary VOD; n=1, respiratory failure of unclear etiology). Detailed disease and treatment characteristics by the outcome categories are listed in Table 2. For those pts who are alive in remission, the median survival is 47.5 months (range, 39.7-92.4). Among the examined clinical variables, we found younger age and no severe NTX (i.e. grade 0-2) after CAR therapy to be significantly associated with improved overall survival (p=0.014 and 0.05, respectively) but prior lines of therapy, time to HSCT, prior HSCT, Ph chromosome, disease status at the time of T cell therapy and severity of CRS did not impact the survival after alloHSCT. Conclusions: In this retrospective analysis, we found that younger age and no severe NTX to CD19 CAR therapy were associated with improved overall survival following alloHSCT. Number of relapses following alloHSCT (4 of 16 pts) appears to be lower compared to the previously reported number of relapse in the adult ALL cohort without alloHSCT (17 of 26 pts). However, the occurrence of TRM due to infection is high, likely a reflection of heavily pretreated pt population and possibly further immunosuppression from CAR + alloHSCT. The link between severe NTX and worse survival is unclear but could be related to prolonged corticosteroid use and high disease burden. While no definitive conclusion can be drawn due to a small sample size, our data suggests a certain subset of adult pts with R/R B-ALL may benefit more from alloHSCT as a consolidation therapy following CD19 CAR T cells, and acute and late infectious complications following alloHSCT should be carefully monitored. These findings should be validated prospectively and compared between different CAR constructs. Disclosures Park: Allogene: Consultancy; Amgen: Consultancy; AstraZeneca: Consultancy; Autolus: Consultancy; GSK: Consultancy; Incyte: Consultancy; Kite Pharma: Consultancy; Novartis: Consultancy; Takeda: Consultancy. Riviere:Fate Therapeutics: Consultancy; Juno Therapeutics: Consultancy, Equity Ownership, Research Funding; Memorial Sloan Kettering Cancer Center: Employment. Sadelain:Memorial Sloan Kettering Cancer Center: Employment; Fate Therapeutics: Consultancy, Patents & Royalties; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding. Brentjens:JUNO Therapeutics: Consultancy, Patents & Royalties, Research Funding; Celgene: Consultancy.

Long Term Follow-up after SCRI-CAR19v1 Reveals Late Recurrences As Well As a Survival Advantage to Consolidation with HCT after CAR T Cell Induced Remission

Background: CD19 chimeric antigen receptor (CAR) T cell therapy has demonstrated robust responses in refractory/relapsed subjects with CD19+ acute lymphoblastic leukemia (ALL). Our Phase 1 clinical trial demonstrated a minimal residual disease (MRD) negative complete remission (CR) rate of 93% at 21 days following SCRI-CAR19v1 (a CD19 specific CAR T cell product) infusion (PMID: 29171004). Though remission is frequently attained, approximately half of patients recur. Controversy exists regarding the benefit of hematopoietic cell transplant (HCT) following CD19 CAR T cell therapy. Although not mandated by the study, our current institutional recommendation for relapsed/refractory patients without a history of allogeneic HCT is to undergo a HCT once in remission following SCRI-CAR19v1. Additionally, we have recommended HCT to those who have a short duration of persistence of SCRI-CAR19v1 in vivo regardless of prior HCT status. We report here the leukemia free survival (LFS) of subjects who proceeded to HCT following remission after SCRI-CAR19v1 infusion.Methods/Results: We analyzed the first 64 subjects on our Phase 1/2 trial, PLAT-02 (NCT02028455), with follow-up of ≥1 year to evaluate the potential benefit of consolidative HCT. We excluded subjects that did not respond (n=5) or relapsed prior to day 63 (n=9). Thirty-two of the evaluated subjects were treated on the Phase I dose finding portion of the study and 18 were treated on the Phase 2 portion at dose level of 1x106cells/kg. Of the 50 evaluable subjects, 33 had a history of at least one prior HCT, whereas 17 had no history of HCT. Figure A demonstrates the LFS for patients that did and did not undergo HCT following SCRI-CAR19v1.There is a trend towards improved LFS (p-value 0.057; Figure B) in patients who underwent first HCT following SCRI-CAR19v1. Of the 17 patients without a history of HCT, 3 did not pursue HCT following CAR therapy. Of those 3 subjects, only 1 remains in remission at 28 months. The other 2 subjects relapsed at 6 and 7 months with CD19+ and CD19- disease, respectively. Of the 14 patients that underwent first HCT after SCRI-CAR19v1, 2 relapsed following HCT. One had evidence of MRD by flow at the time of HCT and the other subject relapsed with CD19- disease. The role of second HCT following CAR therapy remains unclear. Of the 33 subjects with a history of HCT, 10 underwent a second HCT following SCRI-CAR19v1 infusion, and 5 are alive and remain in remission. The recurrences included 2 lineage switches and 2 CD19+ (1 was MRD positive by deep sequence prior to HCT), and one transplant related mortality (TRM). Of the 23 subjects that did not undergo a second HCT, 8 remain in remission (p-value not significant (NS); Figure C).We previously reported that subjects with a short duration of B cell aplasia (BCA) ≤63 days following SCRI-CAR19v1 have an increased risk of relapse. Here we show that subjects with short BCA who attained a CR and did not relapse prior to day 63 demonstrate a clear benefit of consolidative HCT (p-value 0.007; Figure D). Of the 15 subjects with short BCA (regardless of HCT history), 6 did not pursue HCT and all recurred (5 CD19+, 1 CD19-). Of the short BCA subjects that underwent HCT, 1 subject died of TRM (2ndHCT for this subject) and 2 subjects relapsed following HCT (1 was MRD positive prior to HCT).All events in the consolidative HCT group have occurred by 20 months following SCRI-CAR19v1. However, we continue to see late relapses following CAR T cell therapy in the group who did not proceed with HCT. Three subjects have relapsed beyond 2 years: 1 with CD19+ disease at 27months, 1 with CD19- disease at 41 months, and 1 patient with lineage switch AML at 38 months. Further analysis is needed to understand the continued long-term risk of relapse following CD19 CAR T cell therapy and the potential role and timing for consolidative HCT in patients with a previous HCT.Conclusions: We demonstrate a trend towards improved LFS for subjects without a history of HCT who undergo a consolidative HCT following CD19 CAR T cell therapy on PLAT-02. In addition, HCT appears to benefit subjects who attain a CR but are at increased risk of relapse with short-term BCA. Currently, the benefit of second HCT following CD19 CAR T cell therapy is unclear and may be restricted to those that have short functional persistence of SCRI-CAR19v1. Late relapses after SCRI-CAR19v1 have only occurred in those without consolidative HCT, but longer follow up is needed to confirm these findings. DisclosuresJensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Early Clinical Experience of CD19 x CD22 Dual Specific CAR T Cells for Enhanced Anti-Leukemic Targeting of Acute Lymphoblastic Leukemia

Introduction:Advances in chimeric antigen receptor (CAR) T cell therapy have yielded complete remission (CR) rates in relapsed/refractory B-ALL (rrB-ALL) of 70-95%. However, disease recurrence after CD19 or CD22 CAR therapy is greater than 50% at 1 year, and approximately half of recurrences are due to antigen escape. To reduce antigen escape and optimize the durability of remission, we sought to design a CAR T cell product with dual specificity that is capable of simultaneously targeting both CD19 and CD22. Preclinical testing of our bi-specific CAR showed a preference for signaling through CD22 over the CD19 CAR. In contrast, dual transduced T cells signaled through both the CD19 and CD22 CAR with lytic activity and cytokine production similar to single transduced CAR T cells of the same specificity. Therefore, we opted to move forward with dual transduced T cells for clinical use. We are currently testing SCRI-CAR19x22v1 in PLAT-05 (NCT03330691), a phase 1 clinical trial for pediatric and young adult patients with CD19+CD22+ rrB-ALL, with the primary objectives to determine the feasibility of manufacturing products with dual specificity, to assess the safety of the cryopreserved product infusion, and to describe the full toxicity profile.Methods:Subjects undergo apheresis, after which the CD4 and CD8 T cell subsets are immunomagnetically selected and seeded at a prescribed ratio for co-culture in a closed-system G-Rex bioreactor. Following anti-CD3xCD28 bead stimulation, T cells are transduced with two separate SIN lentiviral vectors that direct the expression of a CD19-specific FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ CAR with an Her2tG tag and expression of a CD22-specific m971scFv:IgG4hinge:CH2(L235D)-CH3:CD28tm:4-1BB:ζ CAR with an EGFRt tag, creating three distinct populations of CAR T cells (anti-CD19, anti-CD22, and anti-CD19x 22). Transduced cells are expanded in serum free media formulation with IL-7, IL-15, and IL-21. Following lymphodepleting chemotherapy, cryopreserved products are thawed and infused at the protocol-prescribed dose level. Cytokine release syndrome (CRS) is graded according to Lee et al. (Blood 2014) and is treated according to our early intervention strategy of tocilizumab and dexamethasone for persistent, mild CRS.Results:Seven subjects (ages 1-26 yr) with rrB-ALL have been enrolled with 4 treated at dose level 1 (1 x 106 CAR T cells/kg) and 3 treated at dose level 2 (3 x 106 CAR T cells/kg). The mean culture time was 7.9 days (range 7-11) and subjects received infusions with a mean CD8:CD4 ratio of 1.7 (range 0.2 - 3.1). CD8 CAR composition, on average, consisted of 21.6 % CD19 CAR, 37.8 % CD22 CAR, and 40.6 % CD22xCD19 CAR T cells. CD4 CAR composition, on average, consisted of 25.8 % CD19 CAR, 30.6 % CD22 CAR, and 43.6 % CD22xCD19 CAR T cells (Figure). Peak engraftment occurred between days 7 and 14 for all patients and was predominantly composed of the CD19 CAR population with median peak values for CD19 CAR, CD22 CAR, and CD19xCD22 CAR T cell populations of 9.1%, 1.2%, and 2.4%, respectively. A CR was achieved in 5/7 (71%) subjects by day 21, 4 of which were minimal residual disease negative. The two subjects without a CR did not exhibit evidence of CAR T cell engraftment; one had previously received CD19 CAR T cells, and the other had progressive disease and pursued alternative therapy at day 10. Therapy was well tolerated with no dose limiting toxicities. CRS occurred in 5 subjects (Grade 1) with 2 of these subjects experiencing mild neurotoxicity (Grade 1). Four subjects received tocilizumab +/- dexamethasone, and two of these received multiple doses of dexamethasone.Conclusions:Preclinical testing showed superior efficacy against both CD19 and CD22 when using two separate CARs and dual transduction, compared to a single bi-specific CAR. Preliminary analysis of PLAT-05 supports feasibility of product manufacturing, and toxicity and response rates that are consistent with the reported CD19 CAR T cell experience. While the infused SCRI-CAR19x22v1 products consist of a near-uniform distribution of the 3 distinct populations, we observed selective in vivo expansion of the CD19 CAR T cell population. Further investigation is required to understand the mechanism of CD19 CAR dominance in vivo. Continued accrual of subjects is ongoing to further assess the impact of dual antigen targeting on the prevention of antigen escape and the potential to provide a more durable remission. [Display omitted] DisclosuresPark:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector.

B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) Tcell therapy. In CAR Tcell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR Tcell expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR Tcell therapy with properties, including rapid invivo expansion (>10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR Tcell vectors stemming from this work are under clinical investigation.

Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia

BackgroundCD19-specific chimeric antigen receptor (CAR) T cells induce high rates of initial response among patients with relapsed B-cell acute lymphoblastic leukemia (ALL) and long-term remissions in a subgroup of patients.MethodsWe conducted a phase 1 trial involving adults with relapsed B-cell ALL who received an infusion of autologous T cells expressing the 19-28z CAR at the Memorial Sloan Kettering Cancer Center (MSKCC). Safety and long-term outcomes were assessed, as were their associations with demographic, clinical, and disease characteristics.ResultsA total of 53 adults received 19-28z CAR T cells that were manufactured at MSKCC. After infusion, severe cytokine release syndrome occurred in 14 of 53 patients (26%; 95% confidence interval [CI], 15 to 40); 1 patient died. Complete remission was observed in 83% of the patients. At a median follow-up of 29 months (range, 1 to 65), the median event-free survival was 6.1 months (95% CI, 5.0 to 11.5), and the median overall survival was 12.9 months (95% CI, 8.7 to 23.4). Patients with a low disease burden (<5% bone marrow blasts) before treatment had markedly enhanced remission duration and survival, with a median event-free survival of 10.6 months (95% CI, 5.9 to not reached) and a median overall survival of 20.1 months (95% CI, 8.7 to not reached). Patients with a higher burden of disease (≥5% bone marrow blasts or extramedullary disease) had a greater incidence of the cytokine release syndrome and neurotoxic events and shorter long-term survival than did patients with a low disease burden.ConclusionsIn the entire cohort, the median overall survival was 12.9 months. Among patients with a low disease burden, the median overall survival was 20.1 months and was accompanied by a markedly lower incidence of the cytokine release syndrome and neurotoxic events after 19-28z CAR T-cell infusion than was observed among patients with a higher disease burden. (Funded by the Commonwealth Foundation for Cancer Research and others; ClinicalTrials.gov number, NCT01044069.)

Long-Term Outcomes Following CD19 CAR T Cell Therapy for B-ALL Are Superior in Patients Receiving a Fludarabine/Cyclophosphamide Preparative Regimen and Post-CAR Hematopoietic Stem Cell Transplantation

▪Relapsed pre-B acute lymphoblastic leukemia (ALL) portends a poor prognosis even with hematopoietic stem cell transplantation (HSCT). CD19 chimeric antigen receptor (CAR) T cells have shown promise in early studies although morbidity related tohigh gradecytokine release syndrome (CRS) and/or neurotoxicity could limit its wide applicability in patients with high disease burden. The lympho depleting chemotherapy regimen may affect both toxicity and response and has not been well studied. Relapse rates among complete responders to CD19 CAR therapy occur in nearly half of patients in the first year. We report outcomes from our completed clinical trial of 53 children and young adults with relapsed/refractory ALL (n=51) or lymphoma (n=2) with a median follow up (mF/U) of 18.7 months.The first 21 patients received a low dose fludarabine (25 mg/m2/day Days -4 to -2) and cyclophosphamide (900 mg/m2 Day -2) preparative regimen (LDflu/cy) and results are reported in Lancet 385:517-28. The regimen for the subsequent 32 patients, who all received 1x106 CAR+ T cells/kg, was stratified based on disease burden. Subjects with low burden ALL (lowALL; <25% marrow blasts) received LDflu/cy while those with high burden disease (highALL; >25% marrow blasts or lymphomatous disease) received an alternative regimen [FLAG (n=6), ifosfamide/etoposide per AALL0031 (n=2) or fludarabine (30mg/m2/day Days -6 to -3) and cyclophosphamide (1200 mg/m2/day Days -4 and -3) (HDflu/cy; n=8)] in an attempt to mitigate severe CRS risk and improve response. Four highALL subjects received LDflu/cy due to comorbidities including Trisomy 21. CRS was graded and anti-cytokine therapy was instituted as per Blood 124:188-95. Date for data cutoff was July 31, 2016.Of the 53 subjects 11 had primary refractory ALL, 5Ph+, 3 with Trisomy 21, 4 with CNS2 and 2 with CNS3 ALL including one with extensive leptomeningeal and parenchymal involvement. Cells were manufactured in 7-11 days and none underwent a test expansion. One patient was not infused due to rapidly progressive fungal pneumonia but was accounted for in all analyses.Of 51 ALL patients, 31 (60.8%) achieved a complete response (CR) with 28/31 (90%) of responders negative for minimal residual disease (MRD-). All 6 subjects with CNS ALL were rendered into CNS1 status with resolution of leptomeningeal enhancement, where appropriate, and CAR cells in CSF. The median leukemia free survival (mLFS) of MRD- CR responders is 18 months with a 49.5% probability of LFS beginning at 18 months (mF/U 22.6 months). Grade 3 (n=5) and 4 (n=2) CRS combined for a severe CRS incidence of 13.5%. Three grade 3 neurotoxicities(1 each: dysphasia, delirium, headache) and 2 seizures (one grade 1, one grade 2) occurred. There were no grade 4 neurotoxicities, even in the subject with extensive CNS disease.Subjects with low ALL had a significantly higher CR rate (18/21; 85.7%) than those with high ALL (13/32; 40.6%) (p=0.0011) and use of a flu/cy regimen correlated with higher response (29/44; 65.9% vs 2/8; 25%; p=0.0301). Overall survival in all subjects receiving a flu/cy regimen was 13.3 months with a 34.7% probability of survival beginning at 38 months (mF/U 18.7 months), which is significantly longer than those who did not receive a flu/cy regimen (5.5 months, no survivors beyond 11 months). The hazard ratio (HR) of not receiving a flu/cy regimen was 6.35 (1.906-21.14; p=0.0026). mLFS of subjects with MRD- CR who received a flu/cy regimen was not reached with a 53.3% probability of LFS beginning at 18 months (mF/U 22.6 months).Of the 28 subjects achieving MRD- CR, 21 had a subsequent HSCT with a median time to HSCT of 54 days from CAR infusion. 8/28 (28.6%) relapsed with CD19+ (n=2), CD19-/dim (n=5), CD19 unknown (n=1) blasts. Relapse was significantly more common in subjects who did not have a HSCT after CAR therapy (6/7; 85.7%) compared to those who did (2/21; 9.5%) (p=0.0001). Even accounting for transplant related mortality, them LFS in the HSCT group was not reached with a 62% probability of LFS beginning at 18 months. This is significantly longer than them LFS of 4.9 months in MRD- CR subjects who did not proceed to HSCT (p=0.0006) with a HR of 16.9 (3.37-85.1) of not having a subsequent HSCT.In all, CD19 CAR T cell therapy was effective and safe with a low incidence of severe CRS and neurotoxicity. In this nonrandomized series, the rate of durable remission was higher when a flu/cy preparative regimen was used and consolidation HSCT was employed. [Display omitted] [Display omitted] DisclosuresLee:Juno: Honoraria. Kochenderfer:bluebird bio: Patents & Royalties, Research Funding; Kite Pharma: Patents & Royalties, Research Funding. Rosenberg:Kite pharma: Research Funding. Mackall:NCI: Patents & Royalties: B7H3 CAR.

CD19 CAR immune pressure induces B-precursor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity.

Adoptive immunotherapy using chimeric antigen receptor (CAR) expressing T cells targeting the CD19 B lineage receptor has demonstrated marked success in relapsed pre-B-cell acute lymphoblastic leukaemia (ALL). Persisting CAR-T cells generate sustained pressure against CD19 that may drive unique mechanisms of resistance. Pre-B ALL originates from a committed pre-B cell or an earlier progenitor, with potential to reprogram into other hematopoietic lineages. Here we report changes in lineage markers including myeloid conversion in patients following CD19 CAR therapy. Using murine ALL models we study the long-term effects of CD19 CAR-T cells and demonstrate partial or complete lineage switch as a consistent mechanism of CAR resistance depending on the underlying genetic oncogenic driver. Deletion of Pax5 or Ebf1 recapitulates lineage reprogramming occurring during CD19 CAR pressure. Our findings establish lineage switch as a mechanism of CAR resistance exposing inherent plasticity in genetic subtypes of pre-B-cell ALL.

From T-cell Engineering to CAR therapy: Progress and Prospects

The genetic engineering of T cells provides a means to rapidly generate anti-tumor T cells for any cancer patient. This approach is predicated on gene transfer technology that enables the expression of receptors for antigen and other gene products in primary T cells. Tumor targeting may be achieved through the transfer of a physiological receptor for antigen, which is known as the T cell receptor (TCR), or synthetic fusion receptors, which we grouped under the general term of chimeric antigen receptor (CAR). CARs are recombinant receptors for antigen, which, in a single molecule, redirect T cell specificity and eventually enhance anti-tumor potency. Functional augmentation is achieved through the design of second generation CARs, which not only redirect cytotoxicity, but also reprogram T cell function and longevity through their costimulatory properties. The combined activating and costimulatory domains incorporated in second-generation CARs critically determine the function, differentiation, metabolism and persistence of engineered T cells. CD19 CARs that incorporate CD28 or 4-1BB signalling domains are the best known to date. Two decades ago, we selected CD19 as the prime target for developing our CAR technology and provided the first proof-of-principle that CD19-targeted human peripheral blood T cells could eradicate a broad range of B cell malignancies in immunodeficient mice (Brentjens RJ, Riviere I, et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. 2003;9(3):279-86). CD19 has since become the poster child for CAR therapies. Complete remissions have been reported from several centers in patients with non-Hodgkin lymphoma, chronic lymphocytic leukemia and, most dramatically, acute lymphoblastic leukemia. Two types of second generation CARs, utilizing either CD28 or 4-1BB as their costimulatory signaling components, have been used in ALL patients. Both have yielded dramatic outcomes, in adults as well as in children. Our data indicate that CD28-based CARs direct a brisk proliferative response and boost effector functions, while 4-1BB-based CARs direct a gradual T cell accumulation that may eventually overcome lesser functional potency. These distinct kinetic features can be exploited to further develop CAR T cell therapies for a variety of cancers. We have now modeled CD19 CAR therapy for ALL in a "stress test", wherein we purposefully lower the infused T cell doses to challenge the CAR therapy. We have compared novel CAR designs intended to recruit both CD28 and 4-1BB signaling. These quantitative analyses reveal striking disparities that hinge on subtle variations in the structural design of CARs and co-expressed costimulatory molecules. Remarkably, we find that some of the most effective engineering strategies activate and sustain the recruitment of the IFNβ pathway through the induction of IRF7, while lowering the induction of exhaustion markers relative to second generation CARs activating either CD28 or 4-1BB alone. The field is thus poised to move beyond the CD28 vs 4-1BB debate, which will be rendered obsolete by the emergence of superior CAR designs that coopt the use of costimulatory ligands, cytokines and/or checkpoint blockade inhibitors. A new field of immunopharmacology is emerging. Disclosures Sadelain: Juno Therapeutics: Consultancy, Equity Ownership, Other: Co-Founder, stockholder, Patents &amp; Royalties: Licensed patents on CARs.

Feasibility and safety of cCD20 RNA CAR-bearing T cell therapy for the treatment of canine B cell malignancies

CD19 chimeric antigen receptor (CAR) T cell therapy has shown great promise in treating human hematological malignancies. However, the full potential of CAR technology has not been reached in solid malignancies and limitations exist in utilizing genetically identical murine models to interrogate the safety and efficacy of anti-tumor immune therapies. Canines are an outbred population that naturally develop spontaneous disease. Here we have established a treatment model for CAR T cell therapy in outbred dogs with spontaneous B cell lymphoma. We developed a protocol to isolate, activate, and express CAR in canine T cells. We characterized a methodology utilizing anti-cCD3 antibody with a K562 antigen-presenting cell (APC) line that expresses human CD32 and canine CD86 to activate canine lymphocytes. This method yields robust (50-120x) expansion of cCD4+ or cCD8+ T cells from normal or lymphoma-diseased dogs. Canine T cells were then engineered utilizing mRNA electroporation to express a CAR targeting the B cell antigen CD20. An anti-canine CD20 (cCD20) CAR was selected for proof of principle studies to establish feasibility of efficient CAR expression and redirected effector function in canine T cells from a healthy and lymphoma-diseased dogs. The cCD20-Z CAR construct was efficiently, but transiently, expressed in canine T cells after mRNA electroporation. cCD20 CAR T cells exhibited specific lysis and IFN-γ secretion in response to cCD20+ target cells. These data indicate successful establishment of a method to isolate, activate, and express a fully functional CAR in canine T cells for use in adoptive immunotherapy. These results rationalized testing of this CAR approach in canines with advanced CD20+ malignancies. In an IACUC approved protocol, we administered three doses of autologous cCD20-Z RNA CAR+ T cells to a companion canine with advanced B cell lymphoma. Treatment was well-tolerated and no evidence of serious adverse events was observed. The anti-tumor effect, however, was transient, suggesting a need for product optimization. Future study will establish a lenti- or retroviral-based method to modify canine T cells to promote T cell persistence and durable anti-tumor effects in vivo. In conclusion, our study establishes the methodologies and feasibility of anti-cCD20 CAR therapy in canines with spontaneous B cell lymphoma that may represent a future clinical option for such companion pets. This work also lays the foundation for study of a large, outbred animal model that better resembles spontaneous, human malignancies to allow for improved assessment for potential toxicity risks prior to translation into human studies.

Abstract 4705: Late ALL relapse following CD19 CAR immune-pressure demonstrates reversible pan-antigen loss

Abstract Despite remarkable remission rates in relapsed and refractory acute lymphoblastic leukemia (ALL) following CD19 chimeric antigen receptor (CAR)-T cells or CD3-CD19 bispecific constructs, relapse of CD19 negative leukemic blasts occurs in approximately 10-20% of cases. The mechanism of antigen loss under CD19-targeted immune pressure on pre-B ALL has yet to be elucidated. Although xenograft models have been excellent tools for pre-clinical studies, the development of xenogeneic GVHD has precluded the ability to study late leukemic relapse under CD19-targeted immunotherapeutic pressure. Thus, we performed such studies in a murine transplantable pre-B ALL (CD19+ B220+ CD22+ sIgM- CD127+ CD43+) in immunocompetent mice that induces lethality of recipients within 15-25 days without therapy. Treating ALL bearing mice with a murine CD19 CAR T cells (CART), resulted in prolonged remissions followed by CART dose-dependent relapses varying between 50 days to &amp;gt;200 days post treatment. All relapses were negative for CD19 expression with earlier relapses maintaining similar pre-B cell surface marker phenotype of the parent ALL. However, relapses longer than 100 days consistently showed a dramatic alteration in phenotype, with loss of CD22, B220 and CD127, stability of CD45 and CD43, and partial gain of cKIT, resembling a more immature B cell or common lymphoid progenitor. While early relapses had normal mRNA levels of CD19 despite loss of surface antigen, late antigen loss correlated with low CD19 mRNA, along with other alterations in pre-B cell signature on microarray. This late antigen negative leukemia took longer time to engraft in secondary recipients, compared to the parent cell line. However, serial transplantation without immune-pressure resulted in regain of CD19 and CD22 antigens with tertiary recipients demonstrating leukemia phenotype and time to engraftment similar to the parent cell line. Interestingly, in-vivo reacquisition of CD22 always preceded regain of CD19. To further study loss of B cell antigen, we co-cultured CAR T cells with both human and murine leukemia. Using CD19 CART, we observed rapid CART dose-dependent loss of surface CD19 followed by partial loss of surface CD22, but not other leukemia markers, in both systems. Interestingly, antigen loss was not seen when co-culturing leukemia with anti-CD22 CART. Suboptimal dose of CD22 CART in an NSG mouse model of human leukemia did generate a CD22-CD19- late relapse. We conclude that loss of CD19 may be an early isolated phenomenon, while prolonged immune-pressure drives an mRNA-regulated loss of CD19 accompanied by fundamental alterations in leukemia antigens. We also demonstrated that CD22 loss requires previous loss of CD19. Altogether, as antigen-directed immunotherapies become more common in the clinic, these findings have implications for the optimization of CAR therapy, particularly if antigen loss can be regulated therapeutically. Citation Format: Elad Jacoby, Yinmeng Yang, Chris D. Chien, Waleed Haso, Haiying Qin, Terry J. Fry. Late ALL relapse following CD19 CAR immune-pressure demonstrates reversible pan-antigen loss. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4705. doi:10.1158/1538-7445.AM2015-4705

CD19 CAR Therapy for Acute Lymphoblastic Leukemia.

Chimeric antigen receptor (CAR) therapy is an emerging immunotherapy that shows great promise for cancer, in particular acute lymphoblastic leukemia (ALL). CARs are recombinant receptors for antigen, which, in a single molecule, redirect the specificity and function of T lymphocytes. Following their genetic transfer to patient T cells, the latter acquire the ability to recognize leukemia cells and destroy them. Several years ago, we identified CD19 as an attractive target for CAR therapy for most B cell malignancies, including ALL. We and others have reported remarkable clinical outcomes in adults and children with ALL, achieving a high complete remission rate irrespective of age, prior treatments, or other prognostic markers. Severe cytokine release may develop in patients with high tumor burdens. Several interventions are available to curb the cytokine release syndrome when it occurs. Based on the impressive results obtained with CD19 CAR therapy for ALL, it is realistic to expect that CD19 CARs will become part of the armamentarium for B cell-ALL and other B cell malignancies.

Adoptive Therapy With Chimeric Antigen Receptor–Modified T Cells of Defined Subset Composition

The ability to engineer T cells to recognize tumor cells through genetic modification with a synthetic chimeric antigen receptor has ushered in a new era in cancer immunotherapy. The most advanced clinical applications are in targeting CD19 on B-cell malignancies. The clinical trials of CD19 chimeric antigen receptor therapy have thus far not attempted to select defined subsets before transduction or imposed uniformity of the CD4 and CD8 cell composition of the cell products. This review will discuss the rationale for and challenges to using adoptive therapy with genetically modified T cells of defined subset and phenotypic composition.