Durable Remissions in Children with Relapsed/Refractory ALL Treated with T Cells Engineered with a CD19-Targeted Chimeric Antigen Receptor (CTL019)

BACKGROUND CARs combine a targeting antibody (scFv) domain with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR, which have resulted in up to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 53 pts with relapsed/refractory (r/r) ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with r/r CD19+ ALL. 48/53 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 3.6-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, and not their allo donors. RESULTS We treated 53 children and young adults with CD19+ ALL, median age 11y, (4-24y). To assess disease burden after lymphodepleting chemotherapy, pts had BM aspirations performed 1D prior to 1st CTL019 infusion: 41/53 pts had detectable ALL while 12 were MRD(-). A median of 4.3x106 CTL019 cells/kg (1-17.4x106/kg) were infused over 1-2D (1 pt got cells over 3D). There were no infusional toxicities >gr2, although pts who developed fevers within 24h of infusion did not receive a planned 2nd infusion of CTL019 cells. 50 pts (94%) achieved a CR, including a patient with CD19+ T ALL, 3 did not respond. MRD measured by clinical flow cytometry was <0.01% at D28 in 45 responding pts and positive at 0.024%-1.1% in 5 pts, with 2 patients becoming negative by 3 mo with no further therapy. With median follow up 10.6 mo (1-39 mo), 29 pts have ongoing CR, with only 6 receiving subsequent treatment such as donor lymphocyte infusion or SCT, EFS is 70% at 6 mo (95% CI, 58-85%) and 45% at 12 mo (95% CI, 31-66%), RFS is 72% at 6 mo (95% CI, 59-87%) and 44% at 12 mo (95% CI, 30-65%), and OS is 78% at 12 mo (95% CI, 67-91%). CTL019 was detected by qPCR in the CSF of 46/47 pts and 4 pts with CNS2a ALL experienced a CR in CSF. 20 pts with a CR at 1 mo have subsequently relapsed, with 3 relapses occurring after subsequent therapy (i.e. SCT) and 13 with CD19(-) blasts. 4/5 pts previously refractory to CD19-directed blinatumomab went into CR with CTL019, 3 subsequently relapsed with CD19(-) disease. All but 5 (90%) of pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 28% of patients and was reversed in all cases with the IL6-receptor antagonist tocilizumab, together with short courses of corticosteroids in 9 pts. Although T cells collected from the 35 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells can be detected by flow cytometry and/or QPCR, and results in the pharmacodynamic marker of CTL019 function, B cell aplasia, which continued for 3-39 months after infusion in pts with ongoing responses. B cell aplasia has been treated with IVIg without significant infectious complications. CONCLUSIONS: CTL019 cells can undergo robust in vivo expansion and can persist for 3 years or longer in children and young adults with r/r ALL, allowing for the possibility of long-term disease control without subsequent therapy such as SCT. This approach also has promise as salvage therapy for patients who relapse after allo SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with r/r ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CTL019 resistance. Rapid loss of CTL019 cells (prior to 3 months) is associated with a high risk of CD19+ relapse. CTL019 therapy has received Breakthrough Therapy designation from the FDA in pediatric and adult ALL, and phase 2 multicenter registration trials are well underway. Disclosures Grupp: Novartis: Consultancy, Research Funding. Maude:Novartis: Consultancy, Research Funding. Shaw:Novartis: Research Funding. Aplenc:Sigma Tau: Consultancy. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy; Endo: Other: Husband's employer, has equity interest. Teachey:Novartis: Research Funding. Wood:Novartis Pharmaceuticals Corporation: Employment. Porter:Novartis: Other: IP interest, Research Funding; Genentech: Other: Spouse employment. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding.

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T Cells Engineered with a Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Have Long Term Persistence and Induce Durable Remissions in Children with Relapsed, Refractory ALL

Blood ◽

10.1182/blood.v124.21.380.380 ◽

2014 ◽

Vol 124 (21) ◽

pp. 380-380 ◽

Cited By ~ 9

Author(s):

Stephan A. Grupp ◽

Shannon L Maude ◽

Pamela Shaw ◽

Richard Aplenc ◽

David M. Barrett ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cell ◽

Research Funding ◽

Dose Range ◽

Single Chain

Abstract BACKGROUND CARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 30 pts with relapsed, refractory ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with relapsed or refractory CD19+ ALL. 26/30 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 11-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, not allo donors. RESULTS 30 children median age 10y (5-22y) with CD19+ ALL were treated. 25/30 pts had detectable disease on the day before CTL019 cell infusion, while 5 were MRD(-). A median of 3.6x106 CTL019 cells/kg (1.1-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 9 pts developed fevers within 24 hrs of infusion and did not receive a planned 2nd infusion of CTL019 cells. 27 pts (90%) achieved a CR, including a patient with T cell ALL aberrantly expressing CD19+. 3 did not respond. MRD measured by clinical flow cytometry was negative in 23 responding pts and positive at 0.1% (negative at 3 mo), 0.09%, 0.22%, and 1.1% in 4 pts. With median follow up 8 mo (1-26 mo), 16 pts have ongoing CR, with only 3 patients in the cohort receiving subsequent treatment such as donor lymphocyte infusion or SCT, 6-month EFS measured from infusion is 63% (95% CI, 47-84%), and OS is 78% (95% CI, 63-95%). CTL019 cells were detected in the CSF of 17/19 pts and 2 pts with CNS2a disease experienced a CR in CSF. 10 pts with a CR at 1 mo have subsequently relapsed, half with CD19(-) blasts. 2/5 pts who relapsed with CD19(-) disease had previously been refractory to CD19-directed blinatumomab and subsequently went into CR with CTL019. Figure 1 Figure 1. All responding pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 37% of patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab, together with corticosteroids in 5 pts. Although T cells collected from the 21 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was strongly associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells detected by flow cytometry and/or QPCR, and accompanied by B cell aplasia, continued for 1-26 months after infusion in pts with ongoing responses. QPCR showed very high levels of CTL019 proliferation, with all patients achieving peak levels >5000 copies/ug gDNA and 26 patients with peak levels >15,000 copies/ug gDNA. B cell aplasia has been treated with IVIg without significant infectious complications. Probability of 6-mo CTL019 persistence by flow was68% (95% CI, 50-92%) andrelapse-free B cell aplasia was 73% (95% CI, 57-94%). CONCLUSIONS: CTL019 cells can undergo robust in-vivo expansion and can persist for 2 years or longer in pts with relapsed ALL, allowing for the possibility of long-term disease response without subsequent therapy such as SCT. This approach also has promise as a salvage therapy for patients who relapse after allo-SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CLT019 resistance. CTL019 therapy has received Breakthrough Therapy designation from the FDA in both pediatric and adult ALL, and phase II multicenter trials have been initiated. Disclosures Grupp: Novartis: Consultancy, Research Funding. Barrett:Novartis: Research Funding. Chew:Novartis: Research Funding. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy. Shen:Novartis: Employment. Wood:Novartis Pharma: Employment. Porter:Novartis: managed according to U Penn Policy Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties.

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Efficacy of Humanized CD19-Targeted Chimeric Antigen Receptor (CAR)-Modified T Cells in Children and Young Adults with Relapsed/Refractory Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v128.22.217.217 ◽

2016 ◽

Vol 128 (22) ◽

pp. 217-217 ◽

Cited By ~ 28

Author(s):

Shannon L Maude ◽

David M. Barrett ◽

Susan R. Rheingold ◽

Richard Aplenc ◽

David T Teachey ◽

...

Keyword(s):

T Cells ◽

Young Adults ◽

T Cell ◽

B Cell ◽

Research Funding ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Previously Treated ◽

Children And Young Adults

Abstract Background Targeted immunotherapy with CTL019, CD19-specific chimeric antigen receptor (CAR)-modified T cells, can produce potent and sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (ALL). However, a subset of patients has limited persistence, which can increase the risk of relapse. Most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin; therefore, anti-mouse reactivity is one potential cause of immune-mediated rejection that may be overcome by fully human or humanized CAR designs. We developed a humanized anti-CD19 scFv domain and now report on treatment with humanized CD19-directed CAR T cells (CTL119). Design A pilot/phase 1 study of CAR-modified T cells containing a humanized anti-CD19 scFv domain (CTL119) enrolled children and young adults with relapsed/refractory B-ALL with or without prior exposure to a CAR T cell product. Patients previously treated with CD19-specific CAR-modified T cells were eligible if they met 1 of 3 criteria: 1) CD19+ relapse 2) no response to prior CAR T cell therapy or 3) early B cell recovery indicating poor persistence of CAR T cells. Patient-derived T cells were transduced ex vivo with a lentiviral vector encoding a CAR composed of CD3z, 4-1BB, and humanized anti-CD19 scFv domains and activated/expanded with anti-CD3/CD28 beads. The humanized scFv domain was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Patients received lymphodepletion with cyclophosphamide and fludarabine 1 week prior to infusion with CTL119. Results Thirty children and young adults aged 29 mo-24 yr were infused with CTL119. Eighteen patients had received prior allogeneic stem cell transplant (SCT). Eleven patients who previously received murine-derived CD19-specific CAR-modified T cells (CTL019, n=7; other, n=4) were retreated for B cell recovery (n=5), CD19+ relapse (n=5), or no response to prior CAR T cells (n=1). CNS disease or other extramedullary disease was the indication for enrollment in 6 and 3 patients, respectively. At assessment 1 month after infusion, 26/30 patients (87%) achieved a complete response (CR), defined as morphologic remission with B cell aplasia. Of 11 patients previously treated with murine CD19-specific CAR-modified T cells, 7 (64%) achieved a CR at 1 month, 4 demonstrated no response. Multiparameter flow cytometry for minimal residual disease (MRD) was negative at a detection level of 0.01% in 5/7 responding patients. Two responding patients with positive MRD progressed to CD19+ relapse at 1.6 and 3 mo. In patients with no prior exposure to a CD19 CAR T cell product, MRD-negative CR was achieved in 19/19 patients (100%). One patient relapsed with CD19+ extramedullary disease at 2.8 mo. With a median follow-up of 4.2 mo (range, 1.0-14.1 mo) for all responding patients in both cohorts, 23/26 remain in remission with 1 proceeding to SCT in remission. B cell aplasia, a functional marker of CD19-targeted CAR T cell persistence, continued for 3 months or more in 11/18 patients with adequate follow-up: 1/6 retreatment, 10/12 CAR-naïve. Cytokine release syndrome (CRS) was observed in 28/30 patients and mild in most patients (grade 1, n=6; grade 2, n=18). Three patients experienced grade 3 CRS requiring supplemental oxygen or low-dose vasopressor support and 1 experienced grade 4 CRS requiring high-dose vasopressor and ventilatory support. Severe CRS was successfully managed with the IL6R antagonist tocilizumab in 3 patients. Neurologic toxicity included encephalopathy (n=5) and seizure (n=4) and was fully reversible. Conclusion In the first study of humanized anti-CD19 CAR T cells, CTL119 induced remissions in children and young adults with relapsed/refractory B-ALL, including 64% of patients previously treated with murine CD19-directed CAR T cells and 100% of CAR-naïve patients. Further investigation into CAR T cell persistence and anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. Disclosures Maude: Novartis: Consultancy. Barrett:Novartis: Research Funding. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding; Vitality Institute: Research Funding. Brogdon:Novartis: Employment. Scholler:Novartis: Patents & Royalties: Royalties, Research Funding. Marcucci:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Frey:Amgen: Consultancy; Novartis: Research Funding. Porter:Novartis: Patents & Royalties, Research Funding; Genentech: Employment. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Celldex: Consultancy, Equity Ownership; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Johnson & Johnson: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership. Grupp:Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Research Funding.

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Superior Immunogenicity of Idiotype Fab Fragments As Compared to Entire Immunoglobulin for Active Lymphoma Immunotherapy

Blood ◽

10.1182/blood.v118.21.1633.1633 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1633-1633

Author(s):

Marcelo A. Navarrete ◽

Benjamin Kisser ◽

Hendrik J. Veelken

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cell ◽

B Cell Lymphomas ◽

Cd8 Cells ◽

Fab Fragments ◽

The Individual

Abstract Abstract 1633 Introduction: The individual collection of epitopes within the variable regions of the unique immunoglobulin expressed by every mature B-cell lymphoma (idiotype, or Id) represents a tumor-specific antigen and lends itself as a target for therapeutic vaccination strategies. Immunization with tumor Id has the capacity to elicit polyclonal antibody responses as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II HLA molecules, respectively. Due to a perceived low immunogenicity of lymphoma-derived Id, most Id vaccines tested in clinical trials so far have been formulated as conjugates with the strongly immunogenic carrier keyhole limpet hemocyanin (KLH). In contrast, we have consistently observed high rates of humoral and cellular anti-Id immune responses in consecutive trials of active immunization with unconjugated recombinant Fab fragments of Id in indolent B-cell lymphomas (Bertinetti et al., Cancer Res. 2006; Navarrete et al., BLOOD 2011). We therefore hypothesized that Id Fab fragment might be intrinsically more immunogenic than entire Id Ig and tested this hypothesis by comparative in vitro experiments. Methods: Monocyte-derived dendritic cells (DC) where loaded with human monoclonal IgG, papain-digested Fab fragments, Fc fragments, or recombinant lymphoma-derived Fab fragments. Functional DC phenotypes were assessed by flow cytometry of crucial maturation and activation markers. IL-10 and IL-12 was measured in DC culture supernatants by ELISA. Antigen-loaded DC where subsequently used for priming of CFSE-labeled autologous peripheral blood mononuclear cells. Stimulated T cell populations were analyzed by multicolor flow cytometry. Results: Loading of DC with Fab, Fc, IgG, or mixtures of Fab and Fc fragments did not alter surface expression of CD11c, CD80, CD83, CD86, HLA-DR, PDL-1 and PDL-2 on DC. Likewise, the various antigens did not influence the cytokine release by DC during the loading or maturation process. DC loaded with isolated Fab fragments induced significantly higher proliferation of both CD4+ and CD8+ T cells than undigested IgG. The mean proliferation rate of CD4+ cells stimulated with Fab fragments was 18.5% versus 5.6% for undigested IgG stimulation (p=0.021); proliferation rates of CD8+ cells were 14.2% versus 6.2% (p=0.034). These results were reproduced for 4 different monoclonal IgGs tested on 4 different donors. The addition of Fc fragments to Fab reduced the proliferation rates of CD4+ and CD8+ cells to 10.2% and 8.6% respectively. In addition, DC loaded with undigested IgG induced a relative increase in the number of CD25high/FoxP3+ regulatory T cells compared with Fab stimulation (8.2% versus 1.4%; p<0.01). Conclusions: Isolated Fab fragments, i.e. the Id portions that contain the individual candidate antigenic epitopes of B-cell lymphomas, prime autologous T cells in vitro more efficiently than entire IgG. This finding is consistent with the high immune response rate against recombinant unconjugated Fab fragments observed in vivo in our clinical vaccination trials. Peptide sequences shared between Ig molecules that are predominantly located in the IgG Fc fragment appear to exert an inhibitory effect on T-cell priming. In accordance with our recent in vivo data in a syngeneic mouse model of Id vaccination (Warncke et al., Cancer Immunol. Immunother. 2011), this effect may be mediated by effective activation of Treg. Fab fragments therefore appear to be the more immunogenic and therefore preferable Ig antigenic format for active anti-Id immunotherapy. Furthermore, the inhibitory effects of IgG Fc offers a potential explanation for the recently reported lack of efficacy of Id vaccination in IgG-expressing follicular lymphomas in a randomized phase III trial, in which patients with IgM-expressing lymphomas, in contrast, had a significant benefit from Id vaccination in intention-to-treat analyses (Schuster et al., JCO 2011). Disclosures: No relevant conflicts of interest to declare.

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Phase 1 Study of CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR) Therapy in Children and Young Adults with B Cell Acute Lymphoblastic Leukemia (ALL)

Blood ◽

10.1182/blood-2018-99-117445 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 898-898 ◽

Cited By ~ 26

Author(s):

Liora M Schultz ◽

Kara L. Davis ◽

Christina Baggott ◽

Christie Chaudry ◽

Anne Cunniffe Marcy ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Research Funding ◽

Disease Burden ◽

Pediatric Patients ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Peak Car

Abstract Chimeric Antigen Receptor (CAR) therapy targeting CD19 achieves complete remission (CR) rates of 70%-90% in relapsed/refractory B-ALL. Relapse due to loss of the CD19 targeted epitope presents a therapeutic challenge as evidenced by the largest global pediatric CD19-CAR experience which showed 15 of 16 relapses to be explained by CD19 downregulation (Maude et al, NEJM 2018). Alternatively targeting CD22 using CD22-CAR therapy has demonstrated a CR rate of approximately 70% in both CD19+ and CD19- B-ALL, however relapse due to CD22 downregulation limits the curative potential of singularly-targeting CD22 (Fry et al, Nat Med. 2018). We hypothesized that simultaneous targeting of CD19 and CD22 via a bispecific CAR-T cell would be a safe and tolerable treatment strategy in relapsed/refractory B-cell ALL and address immune evasion. Here, we report the first clinical experience in pediatric patients using bispecific CD19-CD22 CAR T cells. We describe a single institution phase I dose escalation study in pediatric patients with relapsed or refractory B cell ALL. We utilized lentiviral transduction of a bivalent CAR construct incorporating the fmc63 CD19 and m971 CD22 single chain variable fragments (scFvs) used in clinically tested CAR constructs and a 41BB costimulatory endodomain (Fry et al, Nat Med. 2018). Our primary objectives are feasibility of production of this bivalent CAR and safety at 3 dose escalation levels (1x106, 3x106 and 1x107 CAR T cells/kg). Clinical response assessment is evaluated as a secondary aim. All patients described received lymphodepletion with fludarabine (25mg/m2 x 3 days) and cyclophosphamide (900mg/m2 x 1) followed by fresh or cryopreserved CAR T cell infusion after a 7-9 day production time. Patients were prospectively monitored at predefined intervals for disease response and correlative assessments. Four pediatric patients with precursor-B ALL, age 2-17, have been enrolled and treated with CD19/CD22 bispecific CAR T cells at dose level 1 (1x106) [Table 1]. Three patients entered CAR therapy with low disease burden detected by minimal residual disease (MRD) alone and 1 patient initiated therapy with 12% bone marrow blasts. All patients were CNS1 at time of treatment. The toxicity profile mirrored that of the singular CD19 and CD22 CAR experience with 3 patients experiencing reversible CRS (2 Grade I, 1 Grade II), onset day 3-8, and 2 patients experiencing grade I neurotoxicity, onset day 3-9. In our cohort, we experienced lower grade toxicities than previously reported, likely due to a mean lower disease burden. Only 1 patient with CRS met criteria for tocilizumab and this patient was the singular study patient treated with higher burden disease. Neurotoxicity was managed with supportive care and fully reversible. Peripheral blood flow cytometry analysis detects circulating CAR by day 6 in all patients and demonstrates peak CAR expansion between day 6-10. Peak CAR T expansion reached levels of 10-25% of total T cells with inter-patient variability in CD4 and CD8 predominance, favoring CD8 expansion in 3 of 4 patients. Clinical symptoms and inflammatory markers expectedly correlate with peak CAR expansion. Four of 4 patients achieved complete remission (CR) at day 28 post-CD19/CD22 bispecific CAR therapy. Three of 4 patients demonstrated MRD- remissions by flow cytometry and of these, next generation sequencing (NGS) was negative where available (N=2). Multi-parametric CyTOF studies permitting CAR T cell phenotyping in conjunction with single cell TCR tracking, proteomics, epigenomics and cytokine profiling are ongoing and will be used to further characterize persisting CAR T cells and define inter-product and inter-patient variability. In this phase I study, we demonstrate safety and tolerability of this bispecific CD19/CD22 CAR at a dose of 1x106 CAR T cells/kg in pediatric patients with relapsed/refractory B cell ALL. The CD19/22-bispecific CAR mediated antileukemic activity in 100% of patients studied thus far. Long-term follow up and further accrual will be required to inform the effect of bispecific CAR targeting on surface antigen remodeling. Disclosures Muffly: Adaptive Biotechnologies: Research Funding; Shire Pharmaceuticals: Research Funding. Miklos:Genentech: Research Funding; Kite - Gilead: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

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TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Blood ◽

10.1182/blood-2019-123226 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4070-4070

Author(s):

Harbani Malik ◽

Ben Buelow ◽

Udaya Rangaswamy ◽

Aarti Balasubramani ◽

Andrew Boudreau ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cell ◽

Tumor Cells ◽

Cytokine Secretion ◽

Employment Equity ◽

Equity Ownership

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

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Activation of T and NK Cells Following Lenalidomide Therapy in Patients with Follicular Lymphoma.

Blood ◽

10.1182/blood.v120.21.2766.2766 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2766-2766

Author(s):

Seema Rawal ◽

Nathan Fowler ◽

Min Zhang ◽

Zhiqiang Wang ◽

Tariq Muzzafar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Nk Cells ◽

Research Funding ◽

Memory T Cells ◽

T Cell Subsets ◽

Central Memory T Cells

Abstract Abstract 2766 Background: Lenalidomide plus rituximab therapy is a highly effective and well-tolerated therapy in patients (pts) with follicular lymphoma (FL). In a Phase II trial, this combination induced a complete remission rate of 87% in pts with advanced stage untreated FL (Fowler et al, Ann Oncol, 2011; 22; suppl 4:137). A randomized Phase III trial was recently initiated to compare this combination with current standard of care therapies in pts with FL. Although lenalidomide is known to be an immunomodulatory drug with effects on a variety of immune cells in vitro, its effects have not been well studied in vivo in humans. Understanding the in vivo effects of lenalidomide could lead to novel combination strategies to enhance the efficacy and improve clinical outcome in FL and other malignancies. Methods: Pts received lenalidomide 20 mg/day on days 1–21 of each 28-day cycle and rituximab was given at 375 mg/m2on day 1 of each cycle. Peripheral blood mononuclear cells (PBMC) were phenotyped by multiparametric flow cytometry at baseline, on cycle 2 day 15 (C2D15), and at the end of cycle 6. In addition, peripheral blood (PB) samples were collected in PAXgene Blood RNA tubes at baseline and on C2D15 for whole genome gene expression profiling (GEP). Results: Immunophenotyping of baseline and end of cycle 6 PBMC (n=17) showed that the percentages and absolute numbers of CD3+, CD4+, CD8+, TCRgd, and Foxp3+ regulatory T cells; and NK, NKT, and myeloid dendritic cells were not significantly different between the two time points. However, a significant increase in CD4+CD45RO+ (p<0.01) and CD8+CD45RO+ (p=0.04) memory T cells was observed post-therapy. Further characterization of CD4+ T cells showed a significant increase in central memory T cells (p<0.001) and a decrease in naïve (p<0.01) and terminally differentiated (p<0.01) T cells, but no change in effector memory T cells. The increase in CD8+ central memory T cells was marginally significant (p=0.06). Plasmacytoid dendritic cells (PDC) were also significantly increased (p=0.02). In contrast, no such changes in T cell subsets or PDC were observed in FL pts (n=9) treated with 6 cycles of R-CHOP chemotherapy that received equal number of rituximab doses and analyzed at similar time points (baseline and end of cycle 6). To understand lenalidomide-induced changes on a molecular level, we compared GEP data at C2D15 vs. baseline for 7 pairs of PB samples. The paired significance analysis of microarrays method, based on Student's t test, identified 1,748 differentially expressed genes (DEG; 713 up, 1035 down), without a fold-change threshold, in C2D15 samples vs. baseline. Results were influenced by rituximab-induced depletion of B cells in C2D15 samples, but there were many changes that suggested altered PBMC physiology. Noteworthy up-regulated genes (>1.5 fold) included genes associated with T and NK cell activation including BATF, CCR2, CD1B, CD2, CD160, CTLA4, CXCR3, ICOS, and LAG3; and CD163 and CD209, phagocytic receptors expressed on monocytes/macrophages. Down-regulated genes (>1.5 fold) included CXCR5, which mediates B cell migration into follicles; and IL1B and TNFSF13B (BAFF), which are produced by activated macrophages and induce B cell proliferation. Gene set enrichment analysis of all GEP results, and Ingenuity Pathway Analysis of DEGs, indicated up regulation of multiple pathways and processes including ribosomal and mitochondrial components involved in translation and oxidative phosphorylation, CTLA4 signaling in cytotoxic T cells, and differentiation and signaling by ICOS and CD28 in T helper cells. We confirmed up regulation of CTLA4, ICOS, and LAG3 at the protein level in C2D15 PBMC by flow cytometry. Furthermore, treatment of PBMC derived from untreated FL pts with lenalidomide in vitro resulted in up regulation of these molecules in T and/or NK cells consistent with our in vivo results. Conclusions: In FL pts, lenalidomide induced multiple changes in the immune system including increases in PDC and memory T cell subsets, activation of T and NK cells, and down-regulation of certain genes mediating B cell migration and proliferation. These results provide insights into the mechanism of action of lenalidomide and suggest that it can be combined with other immunostimulatory agents such as therapeutic vaccines, adoptive T cell therapy strategies, and immune checkpoint inhibitors to further enhance its efficacy in FL and other malignancies. Disclosures: Fowler: Celgene: Research Funding. Heise:Celgene Corporation: Employment, Equity Ownership. Lacerte:Celgene: Honoraria. Samaniego:Celgene: Research Funding. Neelapu:Celgene Corporation: Research Funding.

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T Cells Engineered With a Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Produce Significant In Vivo Proliferation, Complete Responses and Long-Term Persistence Without Gvhd In Children and Adults With Relapsed, Refractory ALL

Blood ◽

10.1182/blood.v122.21.67.67 ◽

2013 ◽

Vol 122 (21) ◽

pp. 67-67 ◽

Cited By ~ 15

Author(s):

Stephan A Grupp ◽

Noelle V. Frey ◽

Richard Aplenc ◽

David M Barrett ◽

Anne Chew ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Employment Equity ◽

Equity Ownership ◽

Cell And Gene Therapy ◽

Active Disease

Abstract Background CARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains into a single chimeric protein. We previously reported on CTL019 cells expressing a CAR with intracellular activation plus costimulatory domains. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including 1 sustained CR in a patient with ALL (Grupp, et al. NEJM 2013). We now report on outcomes and longer follow up from our pilot studies treating 20 pts (16 children and 4 adults) with relapsed, refractory ALL. Methods T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into pts with relapsed or refractory CD19+ ALL. 17/20 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency (TE) of 11-45%. On the adult protocol, the target dose was 5 x 109 total cells split over 3 days with a TE of 6-31%. 11 pts had relapsed ALL after a prior allogeneic SCT. T cells were collected from the pt, regardless of prior SCT status, and not from allo donors. All pts s/p allo SCT had to be 6 mos s/p SCT with no GVHD or GVHD treatment. Results 16 children median age 9.5 y (5-22y) and 4 adults median age 50y (26-60y) with CD19+ ALL were treated. One child had T cell ALL aberrantly expressing CD19. 14/16 pediatric pts had active disease or +MRD after chemotherapy on the day prior to CTL019 cell infusion, while 2 were MRD(-). 3 of 4 adults had active disease prior to lymphodepleting chemotherapy, while 1 was in morphologic CR. Lymphodepleting chemotherapy varied with most receiving a Cytoxan-containing regimen the week prior to CTL019. A median of 3.7x106 CTL019 cells/kg (0.7-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 5 pts developed fevers within 24 hrs of infusion and did not receive planned subsequent infusions of CTL019 cells. 14 patients (82%) achieved a CR, including the patient with CD19+ T ALL, 3 did not respond, and 3 are pending evaluation. 11/17 evaluable pts have ongoing BM CR with median follow up 2.6 mo (1.2-15 mo). Three patients with a CR at 1 month have subsequently relapsed, 1 with CD19(-) disease. Median follow-up as of August 1, 2013 was 2.6 mo (1-15 mo) for all pts. All responding pts developed some degree of delayed cytokine release syndrome (CRS), concurrent with peak T cell expansion, manifested by fever, with variable degrees of myalgias, nausea, anorexia. Some experienced transient hypotension and hypoxia. Detailed cytokine analysis showed marked increases from baseline values of IL6 and IFNγ (both up to 1000x), and IL2R, with mild or no significant elevation in systemic levels of TNFα or IL2. Treatment for CRS was required for hemodynamic or respiratory instability in 7/20 patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab (7 pts), together with corticosteroids in 4 pts. Although T cells collected from the 11 pts who had relapsed after allo SCT were generally 100% of donor origin, no GVHD has been seen. Persistence of CTL019 cells detected by flow cytometry and/or QPCR in pts with ongoing responses continued for 1-15 months after infusion, resulting in complete B cell aplasia during the period of CTL019 persistence. Pts have been treated with IVIg without any unusual infectious complications. One child who entered a CR subsequently developed MDS with a new trisomy 8 in ALL remission and has gone to SCT, and 1 child developed a single leukemia cutis lesion at 6 mo, still BM MRD(-). Conclusions CTL019 cells are T cells genetically engineered to express an anti-CD19 scFv coupled to CD3ζ signaling and 4-1BB costimulatory domains. These cells can undergo robust in-vivo expansion and can persist for 15 mo or longer in pts with relapsed ALL. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. This approach has promise as a salvage therapy for patients who relapse after allo-SCT, and collection of tolerized cells from the recipient appears to have a low risk of GVHD. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL. Multicenter trials are being developed to test this therapy for ALL in the phase 2 setting. Disclosures: Grupp: Novartis: Research Funding. Chew:Novartis: Patents & Royalties. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Litchman:Novartis Phamaceuticals: Employment, Equity Ownership. Rheingold:Novartis: Research Funding. Shen:Novartis Pharmaceuticals: Employment, Equity Ownership. Wood:Novartis Pharmaceuticals: Employment, Equity Ownership. June:Novartis: Patents & Royalties, Research Funding.

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BRAFV600E Accelerates Disease Progression and Increases Immune Suppression in a Mouse Model of B-Cell Leukemia

Blood ◽

10.1182/blood.v128.22.1206.1206 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1206-1206 ◽

Cited By ~ 1

Author(s):

Yo-Ting Tsai ◽

Aparna Lakshmanan ◽

Amy M. Lehman ◽

Ellen J. Sass ◽

Minh Tran ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

T Cells ◽

B Cell ◽

Immune Suppression ◽

Research Funding ◽

Cell Leukemia ◽

Wild Type ◽

B Cell Leukemia

Abstract Background: Mitogen-activated protein kinase (MAPK) pathway components are frequently mutated in cancer, and abnormal MAPK signaling can drive malignancy by promoting tumor survival and proliferation. Furthermore, work in solid tumors shows that the presence of BRAFV600E further enhances tumor-induced immune suppression, but if and how this occurs in a disseminated disease such as leukemia is unknown. MAPK mutations occur in nearly 100% of classic hairy cell leukemia (HCL) and approximately 10% of chronic lymphocytic leukemia (CLL). Furthermore, up to 30% of B cell lymphomas exhibit activated MAPK signaling via expression of a BRAF pseudogene. However, the pathomechanisms of these abnormalities and the clinical utility of MAPK inhibitors in hematologic malignancies are unclear, and few models are available to evaluate this. To study these aspects, we developed in vitro and in vivo models of BRAF-mutated B cell malignancy. Methods: To mechanistically interrogate the role of BRAFV600E in malignant B cells, we transfected the CLL cell line OSUCLL with doxycycline-inducible constructs containing normal BRAF or BRAFV600E and incubated the cells -/+ doxycycline. These cells were also co-cultured with healthy donor T cells pre-incubated with anti-CD3/anti-CD28, and T cell proliferation was measured by flow cytometry. Cytokines and surface proteins were assessed by flow cytometry. To mimic human HCL, we developed a transgenic mouse model of BRAFV600E B cell leukemia. Mice with Cre-activatable BRAFV600E expression were crossed with mice carrying Cre under the control of the CD19 promoter. CD19-Cre x BRAFV600E or CD19-Cre only mice were then crossed with the well-characterized Eµ-TCL1 model of CLL to generate mice with spontaneous B cell leukemia expressing either wild-type BRAF (CD19-Cre x TCL1) or BRAFV600E (BRAFVE x CD19-Cre x TCL1) under the native BRAF promoter. B cell restricted expression of BRAFV600E was confirmed by immunoblots of purified B and T cells using a BRAFV600E specific antibody. Blood and spleen cells were examined by flow cytometry. For adoptive transfer experiments, leukemia cells (2x10^7) from transgenic mice were introduced intravenously into syngeneic healthy adult animals. Results: OSUCLL cells expressing BRAFV600E showed no significant changes in growth vs. wild-type BRAF expressing cells, but more strongly inhibited anti-CD3/CD28-induced proliferation of normal donor T cells. Transwell assays showed this effect was due both to soluble and contact-dependent factors. Expression of PD-L1 was not different between cells expressing normal and mutated BRAF, indicating this common checkpoint molecule is not the reason for greater immune suppression in this context. TNF levels were higher in BRAFV600E expressing cells and reduced by vemurafenib, but a TNF neutralizing antibody did not alter the inhibitory effect of BRAFV600E expressing cells on T cell proliferation. BRAFV600E x CD19-Cre x TCL1 mice developed B cell leukemia significantly earlier (median 4.9 vs. 8.1 months; P<0.001) and had significantly shorter lifespan (median 7.3 vs. 12.1 months; P<0.001) than their wild-type BRAF counterparts. In contrast to effects of BRAFV600E described in some solid tumors, BRAFV600E expression in B cells had no impact on the rate of B cell proliferation in vivo and only modestly reduced spontaneous apoptosis. To study the effects of this activating mutation on tumor-mediated immune suppression in vivo, leukemia cells from BRAFV600E x CD19-Cre x TCL1 or CD19-Cre x TCL1 mice were adoptively transferred into syngeneic mice. In 3 separate studies, tumor cells from BRAFV600E mice produced leukemia (>10% CD5+/CD19+ cells in blood) sooner than CD19-Cre x TCL1 tumor cells. In mice matched for disease load, BRAFV600E B cells produced a greater negative impact on T cells as evidenced by lower overall percentage of T cells, increased expression of T cell exhaustion markers PD-1, CD244, and CD160, and higher percentage of CD44+ memory T cells. Current studies are investigating the mechanism of these effects in vivo as well as the potential for pharmacologic reversal. Conclusions: Together, these results demonstrate the immune-suppressive impact of BRAFV600E in B-cell leukemias and introduce a novel mouse model to develop rational combination strategies to both directly target the tumor cell and overcome tumor-mediated immune evasion. Disclosures Lozanski: Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding; Boehringer Ingelheim: Research Funding.

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Diffuse Large B Cell Pdtx in Humanized Mice Are Valuable Models to Study Host-Lymphoma Interactions and Immune-Modulating Agents

Blood ◽

10.1182/blood-2021-151598 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2406-2406

Author(s):

Giorgia Zanetti ◽

Giuseppina Astone ◽

Luca Cappelli ◽

William Chiu ◽

Maria Teresa Cacciapuoti ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

B Cell ◽

Research Funding ◽

Ex Vivo ◽

Human Immune System ◽

Human T Cells ◽

Human Immune

Abstract Introduction: Immunotherapy is a promising therapeutic intervention for cancer treatment. Activation of the immune system via checkpoint blockade has been shown to produce antitumor responses in patients with both solid and hematological tumors. However, many patients do not respond to checkpoint inhibitors, and additional therapies are needed to treat these patients. Testing immunotherapies requires a functional human immune system; thus, it is difficult to evaluate their effectiveness using conventional experimental models. For this reason, establishing in vivo models that closely reproduce not only human tumors, but also their interactions with the human immune system, has become mandatory. Methods: We developed a humanized mouse model and combined it with a patient-derived tumor xenograft (PDTX). Humanized mice (HuMice) were generated by transplantation of cord blood or mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells into preconditioned immunodeficient mice. We compared human engraftment in 3 different mouse strains: NSG (NOD.Cg-Prkdc scidIl2rg tm1Wjl/SzJ), NSGS (NOD.Cg-Prkdc scidIl2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) and NBSGW (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl/ThomJ). Immune cell profiling and distribution was performed using flow cytometry and immunohistochemistry. The B cell receptor (BCR) repertoire was evaluated using an RNA-based NGS assay. To evaluate the maturation and functionality of T cells developing in HuMice we performed proliferation, degranulation and intracellular cytokine staining. Results: Two months after CD34+ cell transplantation, we observed high levels of human hematopoietic chimerism in all the 3 strains. NSGS mice supported high-level chimerism as early as 1 month after transplantation, with more than 25% of human CD45+ cells in the blood. In all mice the majority of human circulating leukocytes were CD19+ B cells. An early appearance of CD3+ human T cells was detected in NSGS mice as compared to the other strains. Notably, the T cell expansion correlated with a decrease in relative B cell abundance while the myeloid cell contribution to the graft remained steady. We documented the differentiation of CD4+ and CD8+ human T cells at a 2:1 ratio. The characterization of the T cell subsets revealed that the majority was represented by CD45RA-CCR7- effector memory cells in both the spleen and the blood of HuMice. Nevertheless, recipient mice did not exhibit overt signs of graft-versus-host disease. We also evaluated the cytotoxic potential of T cells isolated from the spleen of HuMice: ex vivo peptide antigen (i.e. EBV) presentation let to generation of effective and specific cytotoxic T-cells. After assessing a functional human immune system reconstitution in HuMice, we challenged them in vivo with low-passage tumor fragments from a diffuse large B cell lymphoma (DLBCL) PDTX. All tumor implants were successfully engrafted in both HuMice and non-humanized controls. Remarkably, all the 3 HuMice strains showed a significant reduction in the tumor volume and/or eradication compared to matched non-humanized controls. Flow cytometry analysis of the peripheral blood of humanized PDTX revealed that the tumor engraftment elicited a significant expansion of CD3+ T cells and cytotoxic CD8+ lymphocytes. Moreover, tumors developing in HuMice exhibited intermediate to high levels of tumor infiltrating T lymphocytes commingling with the neoplastic B cells, as determined by immunohistochemistry. Large areas of necrosis were often observed in PDTX of HuMice. Infiltrating CD3+ cells were TIGIT, PD-1 and Lag-3 positive, and did not efficiently proliferate ex vivo: all features consistent with an exhaustion phenotype. PDTX of HuMice often displayed larger areas of necrosis. Conclusions: Collectively, our data demonstrate that a robust reconstitution can be achieved in different strains of immunocompromised mice and that HuMice elicit effective anti-lymphoma responses. PDTX HuMice represent a powerful platform to study host-tumor interactions, and to test novel immune-based strategies (CAR-T, bifunctional Abs) and new pharmacological approaches to counteract T-cell exhaustion. Figure 1 Figure 1. Disclosures Scandura: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Constellation: Research Funding; MPN-RF (Foundation): Research Funding; CR&T (Foudation): Research Funding; European Leukemia net: Honoraria, Other: travel fees . Roth: Janssen: Consultancy; Merck: Consultancy.

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Venetoclax Enhances Anti-Leukemia Activity of CD123-Specific BiTE-Secreting T-Cells in AML

Blood ◽

10.1182/blood-2020-139625 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-13

Author(s):

Hong Mu-Mosley ◽

Lauren B Ostermann ◽

Ran Zhao ◽

Challice L. Bonifant ◽

Stephen Gottschalk ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Vehicle Control ◽

Advisory Committees ◽

Cellular Therapies

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p<0.001). In the Molm13 luciferase xenograft model, NSGS mice were randomized into 5 groups after AML engraftment was confirmed: 1) vehicle control, 2) Venetoclax (Ven) only, 3) CD123-ENG T-cells only, 4) Ven+CD123-ENG T-cells, 5) Ven+CD123-ENG T-cells/2-day-off Ven post T-cell infusion (Ven[2-day-off]+CD123-ENG). Venetoclax treatment (100 µg/kg daily via oral gavage) was started on day 4 post Molm13 injection, and on day 7, mice received one i.v. dose of CD123-ENG T-cells (5x106 cells/mouse). Venetoclax or CD123-ENG T-cell monotherapy reduced leukemia burden compared to the control group, and combinational treatments further inhibited leukemia progression as judged by BLI and circulating AML cells (%GFP+mCD45-/total live cells) by flow cytometry on day 15 post MOLM-13 injection: vehicle control: 19.6%; Ven+: 3.4%; CD123-ENG T-cells:1.2 %; Ven+CD123-ENG T-cells: 0.3%; Ven[2-day-off]+CD123-ENG T-cells (p<0.01 Ven+ or CD123-ENG T-cells versus control; p<0.001 Ven+CD123-ENG or Ven[2-day-off]+CD123-ENG T cells versus CD123-ENG T cells, n=5). The enhanced anti-AML activity of combining venetoclax and CD123-ENG T-cells translated into a significant survival benefit in comparison to single treatment alone (Fig. 2). However, while Ven+CD123-ENG and Ven[2-day-off]+CD123-ENG T-cell treated mice had a survival advantage, they had reduced circulating numbers of human CD3+ T cells on day 8 post T-cells infusion compared to mice that received CD123-ENG T-cells, indicative of potential adverse effect of venetoclax on T-cell survival in vivo. Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

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