scholarly journals Multi-Modal Targeting of FLT3 with Chimeric Antigen Receptor T Cell Immunotherapy and Tyrosine Kinase Inhibition in High-Risk Pediatric Leukemias

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 404-404
Author(s):  
Lisa M Niswander ◽  
Zachary Graff ◽  
Asen Bagashev ◽  
Lillie Leach ◽  
Terry J. Fry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T ◽  
T Cell Immunotherapy

Abstract Background: Clinical outcomes for children with FLT3-mutant AML and infants with KMT2A-rearranged (KMT2A-R) B-ALL remain dismal. These leukemias share a common feature of aberrant activation of FLT3 kinase signaling, which occurs by activating FLT3 mutations in AML and by overexpression of wild-type FLT3 in KMT2A-R ALL. Several FLT3 tyrosine kinase inhibitors (FLT3i) are approved for adults with FLT3-mutant AML, but potential efficacy against KMT2A-R ALL remains incompletely characterized and may differ from responses in AML. We previously developed and preclinically validated chimeric antigen receptor (CAR) T cells directed against FLT3 (FLT3CART), which importantly showed potent anti-leukemia activity in preclinical models of both childhood FLT3-mutant AML and infant KMT2A-R ALL (Chien CD et al. ASH 2016). In the current studies, we hypothesized that combinatorial targeting of these two high-risk leukemia subtypes with FLT3CART and the selective next-generation FLT3i gilteritinib would have superior activity and potentially mitigate therapeutic resistance now known to occur with kinase inhibitors or CAR T cell immunotherapy. Methods and Results: We first assessed in vitro sensitivity of human FLT3-mutant AML and KMT2A-R ALL cell lines to gilteritinib, a second-generation selective FLT3i with established clinical activity in FLT3-mutant AML and unknown activity in KMT2A-R ALL. As detrimental effects of kinase inhibitors (e.g., dasatinib, ruxolitinib) upon CAR T cells have been reported, we evaluated for similar effects with gilteritinib co-incubated in vitro with CD3/CD28-bead activated healthy human donor T cells. However, we observed minimal deleterious effects of gilteritinib on normal T cell viability, immunophenotype, and IL-2 and interferon-gamma (IFNg) production. We validated combinatorial effects of gilteritinib and FLT3CART-induced cytotoxicity against FLT3-mutant AML and KMT2A-R ALL cell lines in vitro without impairment of IL-2/IFNg production. We then assessed this dual therapy approach in luciferase+ FLT3-mutant AML (MOLM14) and KMT2A-R ALL (SEM) cell line murine xenograft models. As predicted, both FLT3CART and gilteritinib monotherapies transiently inhibited in vivo leukemia proliferation, although leukemia progression eventually occurred. Conversely, FLT3CART and gilteritinib combination therapy strikingly induced enhanced and sustained leukemia clearance in all assessed AML and ALL cell line xenograft models (Figure 1). Confirmatory studies in our established childhood FLT3-mutant AML and KMT2A-R ALL patient-derived xenograft (PDX) models have also demonstrated potent anti-leukemia efficacy of combined FLT3CART and gilteritinib therapy. Earlier-generation FLT3i have been reported to increase cell surface FLT3 expression on FLT3-mutant AML cells. Given the known importance of target antigen site density for CAR T cell efficacy, we reasoned that a sequential approach to dual therapy with FLT3i 'priming' followed by FLT3CART may be superior to a simultaneous treatment strategy. In vitro studies with leukemia cell lines and in vivo studies with PDX models indeed confirmed gilteritinib-induced increases in FLT3 surface antigen density in FLT3-mutant AML cells. Intriguingly, we observed contrasting effects in KMT2A-R ALL cell lines and PDX with decreased surface FLT3 expression upon gilteritinib exposure. Ongoing studies are currently validating gilteritinib priming for FLT3CART given these initial data suggesting potentially divergent sequencing approaches in FLT3-mutant AML versus KMT2A-R ALL. Conclusions: Taken together, our preclinical studies demonstrate that dual targeting with FLT3CART immunotherapy and gilteritinib is a promising therapeutic strategy in FLT3-mutant AML and, importantly, also in KMT2A-R ALL. Notably, we also report minimal negative effects of gilteritinib on FLT3CART, suggesting that FLT3i may be used to enhance CAR T cell immunotherapy without inhibiting T cell function. Phase 1 clinical trials of FLT3CART will open soon for adults and children with FLT3-mutant AML and/or KMT2A-R ALL. Figure 1 Figure 1. Disclosures Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company; ElevateBio: Research Funding. Tasian: Kura Oncology: Consultancy; Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

scholarly journals Preclinical Development of FLT3-Redirected Chimeric Antigen Receptor T Cell Immunotherapy for Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1072-1072 ◽  
Author(s):  
Christopher Daniel Chien ◽  
Christopher Tor Sauter ◽  
Kazusa Ishii ◽  
Sang Minh Nguyen ◽  
Feng Shen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Hematologic Toxicity ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T ◽  
T Cell Immunotherapy

Abstract Background: Outcomes for adults and children with acute myeloid leukemia (AML) are dismal with 20-40% and 60% 5-year event-free survival, respectively. Alternative therapeutic strategies for AML are thus needed to improve outcomes. Chimeric antigen receptor (CAR) T cell immunotherapy has induced remarkable clinical responses in multiple phase 1 clinical trials for patients with relapsed or chemorefractory B cell leukemias, encouraging great interest in developing similar approaches for AML. Prior studies have demonstrated efficacy of CD33 or CD123-redirected CAR T cells in AML models, although the genetic heterogeneity of AML will likely require identification of additional therapeutic targets. In the current studies, we report preliminary in vitro and in vivo efficacy of new CAR T cells targeting the FMS-like tyrosine kinase 3 (FLT3) in human AML. FLT3 mutations via internal tandem duplication or kinase domain point mutations occur in approximately 25% of AML and result in FLT3 surface protein overexpression, suggesting potential efficacy of FLT3-targeting therapies. Both types of FLT3 alterations induce ligand-independent activation of FLT3 signaling, further demonstrating a critical role of FLT3 in AML pathogenesis. Hypothesis: FLT3 is a promising target for CAR T cell immunotherapy based treatment of AML. Results: Quantitative flow cytometric analysis of human AML cell lines demonstrated FLT3 surface expression ranging from 1338 (MOLM-13), 2594 (MOLM-14), and 2710 (MV4;11) receptors/cell versus 623 receptors/cell on negative control U937 cells. We first generated FLT3-redirected CAR construct consisting of a single chain variable fragment (scFv) derived from a well-characterized anti-human FLT3 antibody coupled to T cell 4-1BB (CD137) costimulatory and CD3-zeta activation domains. CD33 CAR T cells based on Gemtuzumab created by identical methodologies were also used as AML CAR T cell controls. In vitro studies verified that human T cells transduced with the FLT3 CAR construct induced interferon-gamma and interleukin-2 production after co-culture with AML cell lines MOLM-13, MOLM-14, and MV4;11. One dose of FLT3 CAR T cells inhibited leukemia proliferation in vivo in NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with FLT3-mutant MOLM-13 or MOLM14 cell lines. These first data demonstrate potent preclinical activity of FLT3 CAR T cells and warrant further study in additional AML models. However, on target/off tumor toxicities can occur with AML antigen-targeted immunotherapies, as previously reported in studies of CD33 and CD123 CAR T cells. Normal expression of FLT3 has been mainly described on CD34+ hematopoietic progenitor stem cell populations, and FLT3-targeted therapies have potential to induce aplastic anemia. To address this question of hematologic toxicity of FLT3 CAR T cells, we created normal human hematopoiesis xenograft models in NOD scid gamma Il3-GM-SF (NSGS) mice engrafted with CD34+ cord blood cells for treatment with anti-AML CAR T cells. No difference in human granulocyte numbers was observed in marrows of engrafted mice treated with FLT3 CAR T cells, CD33 CAR T cells, or non-transduced T cells. A significant reduction in monocytes was observed in FLT3 CAR T cell-treated animals, however (p<0.05 by t test). To determine potential for increased hematologic toxicity in the presence of greater target antigen levels, we injected MOLM-14 into CD34+ cell-engrafted mice, then treated animals with control or anti-AML CAR T cells. We surprisingly found no decrement in defined hematopoietic stem cell (HSC) or granulocyte macrophage progenitor (GMP) populations, but did observe increased multipotent and common myeloid progenitor (MPP, CMP) cell numbers and an increase in total human cell engraftment 5 days after FLT3 CAR treatment in comparison to non-transduced T cell-treated animals. Relative to CD33 CAR T cells, FLT3 CAR T cells induced less toxicity to HSCs and MPPs and equivalent toxicity to CMPs and GMPs, indicating lower hematologic toxicity with FLT3 targeting. Conclusions: Taken together, these initial data demonstrate potent in vitro and in vivo anti-AML activity with limited hematopoietic toxicity of FLT3 CAR T cell immunotherapy. Future studies are focused on testing the effectiveness on other AML cell lines with varying expression of FLT3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparison of Efficacy and Toxicity of CD19-Specific Chimeric Antigen Receptor T-Cells Alone or in Combination with Ibrutinib for Relapsed and/or Refractory CLL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 299-299 ◽  
Author(s):  
Jordan Gauthier ◽  
Alexandre V. Hirayama ◽  
Kevin A. Hay ◽  
Daniel Li ◽  
James Lymp ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T ◽  
Equity Ownership ◽  
T Cell Immunotherapy

Abstract Background We reported durable responses to CD19-specific chimeric antigen receptor-modified T-cell therapy (JCAR014) in relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) patients (pts) after prior failure of ibrutinib (Turtle, JCO 2017; NCT01865617). In those pts, ibrutinib was not administered during CAR-T cell immunotherapy. Continuation of ibrutinib through leukapheresis, lymphodepletion and CAR-T cell therapy may prevent tumor progression after ibrutinib withdrawal, mobilize tumor into the blood, improve CAR-T cell function, and decrease cytokine release syndrome (CRS). Methods We conducted a phase 1/2 study of CD19 CAR-T cell immunotherapy in R/R CLL pts and established a regimen of cyclophosphamide and fludarabine (Cy/Flu) lymphodepletion followed by JCAR014 at 2 x 106 CAR-T cells/kg (Turtle, JCO 2017). We then compared outcomes of these pts (No-ibr cohort) with a subsequent cohort that received Cy/Flu with 2 x 106/kg JCAR014 CAR-T cells with concurrent ibrutinib (420 mg/d) from at least 2 weeks prior to leukapheresis until at least 3 months after JCAR014 infusion (Ibr cohort). Dose reduction was permitted for toxicity. CRS was graded by consensus criteria (Lee, Blood 2014) and neurotoxicity and other adverse events were graded by CTCAE v4.03. Response was evaluated according to 2008 IWCLL criteria. Results Seventeen and 19 pts were treated in the Ibr and No-ibr cohorts, respectively. Pt characteristics were comparable (Table 1). Progression on ibrutinib was noted in 16 (94%) and 18 pts (95%) in the Ibr and No-ibr cohorts, respectively, and prior ibrutinib intolerance was reported in 1 pt in each cohort. The time to intolerance or failure of ibrutinib prior to treatment with JCAR014 was longer, and the pre-leukapheresis LDH was lower in the Ibr compared to the No-ibr cohort. The median follow-up in responders was 98 and 764 days in the Ibr and No-ibr cohorts, respectively. Administration of ibrutinib with Cy/Flu and JCAR014 was well tolerated in most pts; ibrutinib was reduced or discontinued in 6 pts (35%) at a median of 21 days after JCAR014 infusion. In the Ibr cohort, 1 pt with grade 2 CRS developed fatal presumed cardiac arrhythmia and 1 pt developed a subdural hematoma in the setting of trauma and thrombocytopenia. No differences in the incidences of grade ≥3 cytopenias were observed. Concurrent ibrutinib administration did not appear to affect the frequency or severity of neurotoxicity. Although the proportions of pts with grade ≥1 CRS were similar between cohorts (76% vs 89%, P = 0.39), the severity of CRS (grade ≥3 CRS: Ibr, 0%; No-Ibr, 26%; P = 0.05) and serum peak IL-8 (P = 0.04), IL-15 (P = 0.003) and MCP-1 (P = 0.004) concentrations were lower in the Ibr cohort. However, we found comparable CD8+ (P = 0.29) and higher CD4+ (P = 0.06) CAR-T cell counts in blood in the Ibr cohort. Sixteen pts (94%) and 18 pts (95%) in the Ibr and No-ibr cohorts, respectively, have completed response assessment. We observed a higher proportion of responders (complete and partial remission) by IWCLL criteria in the Ibr compared to the No-ibr cohort (88% vs 56%, respectively, P = 0.06). Ten of 12 pts (83%) with lymph node disease before treatment with Cy/Flu and JCAR014 in the Ibr cohort achieved CR or PR by IWCLL imaging criteria, compared to 10/17 pts (59%) in the No-ibr cohort (P = 0.23). The proportion of pts with pretreatment bone marrow (BM) disease who had no disease by flow cytometry after CAR-T cell immunotherapy was similar in the Ibr compared to the No-ibr cohort (75% vs 65%, P = 0.71). However, among pts with no disease by BM flow cytometry after CAR-T cell immunotherapy, a higher proportion of pts in the Ibr cohort had no malignant IGH sequences at 4 weeks (83% vs 60%, respectively, P = 0.35). We performed univariate logistic regression analysis for response by IWCLL criteria and variables with P < 0.10 were considered for stepwise multivariable analysis (Table 2). In the multivariable analysis, the Ibr cohort and a lower pre-treatment SUVmax on PET imaging were each associated with a higher probability of response by IWCLL criteria (Ibr cohort, OR = 14.02, 95%CI [0.52-379.61], P = 0.05; SUVmax, OR = 1.31 per SUV unit decrease, 95%CI [1.05-1.67], P < 0.001). Conclusion Administration of ibrutinib from 2 weeks before leukapheresis until 3 months after JCAR014 was well tolerated in most pts. This approach might decrease the incidence of severe CRS and improve responses in pts with R/R CLL. Disclosures Hirayama: DAVA Oncology: Honoraria. Hay:DAVA Oncology: Honoraria. Li:Juno Therapeutics: Employment, Equity Ownership. Lymp:Juno Therapeutics: Employment, Equity Ownership. Till:Mustang Bio: Patents & Royalties, Research Funding. Kiem:Magenta: Consultancy; Homology Medicine: Consultancy; Rocket Pharmaceuticals: Consultancy. Shadman:TG Therapeutics: Research Funding; Celgene: Research Funding; Gilead: Research Funding; Qilu Puget Sound Biotherapeutics: Consultancy; AstraZeneca: Consultancy; Verastem: Consultancy; Beigene: Research Funding; Mustang: Research Funding; Genentech: Consultancy, Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; Abbvie: Consultancy. Cassaday:Merck: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Seattle Genetics: Other: Spouse Employment, Research Funding; Incyte: Research Funding; Kite Pharma: Research Funding; Adaptive Biotechnologies: Consultancy; Jazz Pharmaceuticals: Consultancy. Acharya:Juno Therapeutics: Research Funding; Teva: Honoraria. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; NOHLA: Consultancy; Cell Medica: Membership on an entity's Board of Directors or advisory committees. Maloney:GlaxoSmithKline: Research Funding; Juno Therapeutics: Research Funding; Seattle Genetics: Honoraria; Roche/Genentech: Honoraria; Janssen Scientific Affairs: Honoraria. Turtle:Nektar Therapeutics: Consultancy, Research Funding; Juno Therapeutics / Celgene: Consultancy, Patents & Royalties, Research Funding; Aptevo: Consultancy; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Consultancy; Adaptive Biotechnologies: Consultancy; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Consultancy; Gilead: Consultancy.

scholarly journals CARTmath—A Mathematical Model of CAR-T Immunotherapy in Preclinical Studies of Hematological Cancers

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2941
Author(s):  
Luciana R. C. Barros ◽  
Emanuelle A. Paixão ◽  
Andrea M. P. Valli ◽  
Gustavo T. Naozuka ◽  
Artur C. Fassoni ◽  
...  
Keyword(s):  
Mathematical Model ◽  
T Cells ◽  
T Cell ◽  
Mouse Models ◽  
In Silico ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T ◽  
T Cell Immunotherapy

Immunotherapy has gained great momentum with chimeric antigen receptor T cell (CAR-T) therapy, in which patient’s T lymphocytes are genetically manipulated to recognize tumor-specific antigens, increasing tumor elimination efficiency. In recent years, CAR-T cell immunotherapy for hematological malignancies achieved a great response rate in patients and is a very promising therapy for several other malignancies. Each new CAR design requires a preclinical proof-of-concept experiment using immunodeficient mouse models. The absence of a functional immune system in these mice makes them simple and suitable for use as mathematical models. In this work, we develop a three-population mathematical model to describe tumor response to CAR-T cell immunotherapy in immunodeficient mouse models, encompassing interactions between a non-solid tumor and CAR-T cells (effector and long-term memory). We account for several phenomena, such as tumor-induced immunosuppression, memory pool formation, and conversion of memory into effector CAR-T cells in the presence of new tumor cells. Individual donor and tumor specificities are considered uncertainties in the model parameters. Our model is able to reproduce several CAR-T cell immunotherapy scenarios, with different CAR receptors and tumor targets reported in the literature. We found that therapy effectiveness mostly depends on specific parameters such as the differentiation of effector to memory CAR-T cells, CAR-T cytotoxic capacity, tumor growth rate, and tumor-induced immunosuppression. In summary, our model can contribute to reducing and optimizing the number of in vivo experiments with in silico tests to select specific scenarios that could be tested in experimental research. Such an in silico laboratory is an easy-to-run open-source simulator, built on a Shiny R-based platform called CARTmath. It contains the results of this manuscript as examples and documentation. The developed model together with the CARTmath platform have potential use in assessing different CAR-T cell immunotherapy protocols and its associated efficacy, becoming an accessory for in silico trials.

scholarly journals Innovative CAR-T Cell Therapy for Solid Tumor; Current Duel between CAR-T Spear and Tumor Shield

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2087
Author(s):  
Yuna Jo ◽  
Laraib Amir Ali ◽  
Ju A Shim ◽  
Byung Ha Lee ◽  
Changwan Hong
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T Cell Therapy ◽  
Car T ◽  
T Cell Immunotherapy

Novel engineered T cells containing chimeric antigen receptors (CAR-T cells) that combine the benefits of antigen recognition and T cell response have been developed, and their effect in the anti-tumor immunotherapy of patients with relapsed/refractory leukemia has been dramatic. Thus, CAR-T cell immunotherapy is rapidly emerging as a new therapy. However, it has limitations that prevent consistency in therapeutic effects in solid tumors, which accounts for over 90% of all cancer patients. Here, we review the literature regarding various obstacles to CAR-T cell immunotherapy for solid tumors, including those that cause CAR-T cell dysfunction in the immunosuppressive tumor microenvironment, such as reactive oxygen species, pH, O2, immunosuppressive cells, cytokines, and metabolites, as well as those that impair cell trafficking into the tumor microenvironment. Next-generation CAR-T cell therapy is currently undergoing clinical trials to overcome these challenges. Therefore, novel approaches to address the challenges faced by CAR-T cell immunotherapy in solid tumors are also discussed here.

scholarly journals CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Lena Andersch ◽  
Josefine Radke ◽  
Anika Klaus ◽  
Silke Schwiebert ◽  
Annika Winkler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Lines ◽  
T Cell Therapy ◽  
Retinoblastoma Cell ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. Methods CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. Results All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. Conclusion Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

Chimeric Antigen Receptor T Cell Immunotherapy for Tumor: A Review of Patent Literatures

2019 ◽  
Vol 14 (1) ◽  
pp. 60-69
Author(s):  
Manxue Fu ◽  
Liling Tang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Limiting Factors ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T ◽  
T Cell Immunotherapy

Background: Chimeric Antigen Receptor (CAR) T cell immunotherapy, as an innovative method for tumor immunotherapy, acquires unprecedented clinical outcomes. Genetic modification not only provides T cells with the antigen-binding function but also endows T cells with better immunological functions both in solid and hematological cancer. However, the CAR T cell therapy is not perfect because of several reasons, such as tumor immune microenvironment, and autologous limiting factors of CAR T cells. Moreover, the safety of CAR T cells should be improved.Objective:Recently many patents and publications have reported the importance of CAR T cell immunotherapy. Based on the patents about CAR T cell immunotherapy, we conclude some methods for designing the CAR which can provide information to readers.Methods:In this review, we collect recent patents and publications, summarize some specific antigens for oncotherapy from patents and enumerate some approaches to conquering immunosuppression and reinforcing the immune response of CAR T cells. We also sum up some strategies for improving the safety of CAR T cell immunotherapy.Results:CAR T cell immunotherapy as a neotype cellular immunotherapy has been proved effective in oncotherapy and authorized by FDA. Improvements in CAR designing enhance functions of CAR T cells.Conclusion:This review, summarizing antigens and approaches to overcome defects of CAR T cell immunotherapy from patents and publications, might contribute to a broad readership.

scholarly journals Targeting the Membrane-Proximal C2-Set Domain of CD33 for Improved CD33-Directed CAR T Cell Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2776-2776
Author(s):  
Salvatore Fiorenza ◽  
George S. Laszlo ◽  
Tinh-Doan Phi ◽  
Margaret C. Lunn ◽  
Delaney R. Kirchmeier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Set Domain ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Privately Held

Abstract Background: There is increasing interest in targeting CD33 in malignant and non-malignant disorders, but available drugs are ineffective in many patients. As one limitation, therapeutic CD33 antibodies typically recognize the membrane-distal V-set domain. Likewise, currently tested CD33-directed chimeric antigen receptor (CAR) T cells likewise target the V-set domain and have thus far shown limited clinical activity. We have recently demonstrated that binding closer to the cell membrane enhances the effector functions of CD33 antibodies. We therefore raised antibodies against the membrane-proximal C2-set domain of CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain ("CD33 PAN antibodies"). Here, we tested their properties as targeting moiety in CD33 PAN CAR T cell constructs, using a clinically validated lentiviral backbone. Methods: To generate CAR T cells, negatively selected CD8 + T cells were transduced with an epHIV7 lentivirus encoding the scFv from a CD33 PAN antibody (clone 1H7 or 9G2) linked to either a short (IgG 4 hinge only), intermediate (hinge plus IgG 4 CH3 domain), or long (hinge plus IgG 4 CH3 domain plus IgG 4 CH2 domain) spacer, the CD28-transmembrane domain, CD3zeta and 4-1BB intracellular signaling domains, and non-functional truncated CD19 (tCD19) as transduction marker. Similar constructs using scFvs from 2 different V-set domain-targeting CD33 antibodies, including hP67.6 (My96; used in gemtuzumab ozogamicin), were generated for comparison. CAR-T cells were sorted, expanded in IL-7 and IL-15, and used in vitro or in vivo against human AML cell lines endogenously expressing CD33 and cell lines engineered to lack CD33 (via CRISPR/Cas9) with/or without forced expression of different CD33 variants. Results: CD33 V-set-directed CAR T cells exerted significantly more cytolytic activity against AML cells expressing an artificial CD33 variant lacking the C2-set domain (CD33 ΔE3-4) than cells expressing full-length CD33 at similar or higher levels, consistent with the notion that CD33 CAR T cell efficacy is enhanced when targeting an epitope that is located closer to the cell membrane. CD33 PAN CAR T cells were highly potent against human AML cells in a strictly CD33-dependent fashion, with constructs containing the short and intermediate-length spacer demonstrating robust cytokine secretion, cell proliferation, and in vitro cytolytic activity, as determined by 51Cr release cytotoxicity assays. When compared to optimized CD33 V-set CAR T cells, optimized CD33 PAN CAR T cells were significantly more potent in cytotoxicity, proliferation, and cytokine production without appreciably increased acquisition of exhaustion markers. In vivo, CD33 PAN CAR T cells extended survival in immunodeficient NOD.SCID. IL2rg -/- (NSG) mice bearing significant leukemic burdens from various cell line-derived xenografts (HL-60, KG1α and MOLM14) with efficient tumor clearance demonstrated in a dose-dependent fashion. Conclusion: Targeting the membrane proximal domain of CD33 enhances the anti-leukemic potency of CAR T cells. Our data provide the rationale for the further development of CD33 PAN CAR T cells toward clinical testing. Disclosures Fiorenza: Link Immunotherapeutics: Consultancy; Bristol Myers Squibb: Research Funding. Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Turtle: Allogene: Consultancy; Amgen: Consultancy; Arsenal Bio: Consultancy; Asher bio: Consultancy; Astrazeneca: Consultancy, Research Funding; Caribou Biosciences: Consultancy, Current holder of individual stocks in a privately-held company; Century Therapeutics: Consultancy, Other; Eureka therapeutics: Current holder of individual stocks in a privately-held company, Other; Juno therapeutics/BMS: Patents & Royalties, Research Funding; Myeloid Therapeutics: Current holder of individual stocks in a privately-held company, Other; Nektar therapeutics: Consultancy, Research Funding; PACT Pharma: Consultancy; Precision Biosciences: Current holder of individual stocks in a privately-held company, Other; T-CURX: Other; TCR2 Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

scholarly journals Clinical Development of a Non-Gene-Edited Allogeneic Bcma-Targeting CAR T-Cell Product in Relapsed or Refractory Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-28
Author(s):  
A. Samer Al-Homsi ◽  
Sebastien Anguille ◽  
Jason Brayer ◽  
Dries Deeren ◽  
Nathalie Meuleman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Gene Editing ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Background Autologous CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) has shown impressive objective response rates in patients with advanced multiple myeloma (MM). Clinical grade manufacturing of autologous CAR T-cells has limitations including vein-to-vein delivery time delay and potentially sub-optimal immunological capability of T-cells isolated from patients with advanced disease. Allogeneic CAR T-cell products, whereby cells from healthy third-party donors are used to generate an "off-the-shelf" CAR T-cell product, have the potential to overcome some of these issues. To circumvent the primary potential risk of graft-versus-host disease (GvHD) associated with the use of allogeneic T-cells, abrogation of the T-cell receptor (TCR) expression in the CAR T-cells, via gene editing, is being actively pursued. To avoid the potential safety risks and manufacturing challenges associated with gene editing, the allogeneic CYAD-211 CAR T-cell product exploits short hairpin RNA (shRNA) interference technology to down-regulate TCR expression thus avoiding the risk of life-threatening GvHD. Aim The aim is to generate a BCMA-specific allogeneic CAR T-cell product using a non-gene editing approach and study its activity both in vitro and in vivo. CYAD-211 combines a BCMA-specific CAR with a single optimized shRNA targeting the TCR CD3ζ subunit. Downregulation of CD3ζ impairs the TCR expression on the surface of the donor T-cells, preventing their reactivity with the normal host tissue cells and potential GvHD induction. Maintaining all the elements required for the therapy within a single vector (all-in-one vector) provides some significant manufacturing advantages, as a solitary selection step will isolate cells expressing all the desired traits. Results CYAD-211 cells produce high amounts of interferon-gamma (IFN-γ) during in vitro co-cultures with various BCMA-expressing MM cell lines (i.e., RPMI-8226, OPM-2, U266, and KMS-11). Cytotoxicity experiments confirmed that CYAD-211 efficiently kills MM cell lines in a BCMA-specific manner. The anti-tumor efficacy of CYAD-211 was further confirmed in vivo, in xenograft MM models using the RPMI-8226 and KMS-11 cell lines. Preclinical data also showed no demonstrable evidence of GvHD when CYAD-211 was infused in NSG mice confirming efficient inhibition of TCR-induced activation. Following FDA acceptance of the IND application, IMMUNICY-1, a first-in-human, open-label dose-escalation phase I clinical study evaluating the safety and clinical activity of CYAD-211 for the treatment of relapsed or refractory MM patients to at least two prior MM treatment regimens, is scheduled to begin recruitment. IMMUNICY-1 will evaluate three dose-levels of CYAD-211 (3x107, 1x108 and 3x108 cells/infusion) administered as a single infusion after a non-myeloablative conditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classical Fibonacci 3+3 design. Description of the study design and preliminary safety and clinical data from the first cohort will be presented at ASH 2020. Conclusion CYAD-211 is the first generation of non-gene edited allogeneic CAR T-cell product based on shRNA technology. The IMMUNICY-1 clinical study seeks to provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without GvHD-inducing potential. We anticipate that subsequent generations of this technology will incorporate multiple shRNA hairpins within a single vector system. This will enable the production of allogeneic CAR T-cells in which multiple genes of interest are modulated simultaneously thereby providing a platform approach that can underpin the future of this therapeutic modality. Figure 1 Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Brayer:Janssen: Consultancy; Bristol-Myers Squibb, WindMIL Therapeutics: Research Funding; Bristol-Myers Squibb, Janssen, Amgen: Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution. Sotiropoulou:Celyad Oncology: Current Employment. Twyffels:Celyad Oncology: Current Employment. Bolsee:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

scholarly journals Clinical Predictors of T Cell Fitness for CAR T Cell Manufacturing and Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1886-1886 ◽  
Author(s):  
Ehren Dancy ◽  
Alfred L. Garfall ◽  
Adam D. Cohen ◽  
Joseph A Fraietta ◽  
Megan Davis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Expansion ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T Cell ◽  
Car T ◽  
T Cell Expansion

Abstract Introduction: The optimal clinical setting and cell product characteristics for chimeric antigen receptor (CAR) T cell therapy in multiple myeloma (MM) are uncertain. In CLL patients treated with anti-CD19 CAR T cells (CART19), prevalence of an early memory (early-mem) T cell phenotype (CD27+ CD45RO- CD8+) at time of leukapheresis was predictive of clinical response independently of other patient- or disease-specific factors and was associated with enhanced capacity for in vitro T cell expansion and CD19-responsive activation (Fraietta et al. Nat Med 2018). T cell fitness is therefore a major determinant of response to CAR T cell therapy. In an accompanying abstract (Cohen et al.), we report that higher percentage of early-mem T cells and CD4/CD8 ratio within the leukapheresis product are associated with favorable clinical response to anti-BCMA CAR T cells (CART-BCMA) in relapsed/refractory MM. Here, we compare leukapheresis samples from MM patients obtained at completion of induction therapy (post-ind) with those obtained in relapsed/refractory (rel/ref) patients for frequency of early-mem T cells, CD4/CD8 ratio, and in vitro T cell expansion. Methods: Cryopreserved leukapheresis samples were analyzed for the percentage of early-mem T cells and CD4/CD8 ratio by flow cytometry and in vitro expansion kinetics during anti-CD3/anti-CD28 bead stimulation. Post-ind samples were obtained between 2007 and 2014 from previously reported MM trials in which ex-vivo-expanded autologous T cells were infused post-ASCT to facilitate immune reconstitution (NCT01245673, NCT01426828, NCT00046852); rel/ref samples were from MM patients treated in a phase-one study of CART-BCMA (NCT02546167). Results: The post-ind cohort includes 38 patients with median age 55y (range 41-68) and prior exposure to lenalidomide (22), bortezomib (21), dexamethasone (38), cyclophosphamide (8), vincristine (2), thalidomide (8), and doxorubicin (4); median time from first systemic therapy to leukapheresis was 152 days (range 53-1886) with a median of 1 prior line of therapy (range 1-4). The rel/ref cohort included 25 patients with median age 58y (range 44-75), median 7 prior lines of therapy (range 3-13), and previously exposed to lenalidomide (25), bortezomib (25), pomalidomide (23), carfilzomib/oprozomib (24), daratumumab (19), cyclophosphamide (25), autologous SCT (23), allogeneic SCT (1), and anti-PD1 (7). Median marrow plasma cell content at leukapheresis was lower in the post-ind cohort (12.5%, range 0-80, n=37) compared to the rel/ref cohort (65%, range 0-95%). Percentage of early-mem T cells was higher in the post-ind vs rel/ref cohort (median 43.9% vs 29.0%, p=0.001, left figure). Likewise, CD4/CD8 ratio was higher in the post-ind vs rel/ref cohort (median 2.6 vs 0.87, p<0.0001, mid figure). Magnitude of in vitro T cell expansion during manufacturing (measured as population doublings by day 9, or PDL9), which correlated with response to CART19 in CLL, was higher in post-ind vs rel/ref cohort (median PDL9 5.3 vs 4.5, p=0.0008, right figure). Pooling data from both cohorts, PDL9 correlated with both early-mem T cell percentage (Spearman's rho 0.38, multiplicity adjusted p=0.01) and CD4/CD8 ratio (Spearman's rho 0.42, multiplicity adjusted p=0.005). Within the post-ind cohort, there was no significant association between early-mem T cell percentage and time since MM diagnosis, duration of therapy, exposure to specific therapies (including cyclophosphamide, bortezomib, or lenalidomide), or bone marrow plasma cell content at time of apheresis. However, in the post-ind cohort, there was a trend of toward lower percentage early-mem phenotype (29% vs 49%, p=0.07) and lower CD4/CD8 ratio (median 1.4 vs 2.7, p=0.04) among patients who required >2 lines of therapy prior to apheresis (n=3) compared to the rest of the cohort (n=35). Conclusion: In MM patients, frequency of the early-mem T cell phenotype, a functionally validated biomarker of fitness for CAR T cell manufacturing, was significantly higher in leukapheresis products obtained after induction therapy compared to the relapsed/refractory setting, as was CD4/CD8 ratio and magnitude of in vitro T cell expansion. This result suggests that CAR T cells for MM would yield better clinical responses at early points in the disease course, at periods of relatively low disease burden and before exposure to multiple lines of therapy. Figure. Figure. Disclosures Garfall: Novartis: Research Funding; Kite Pharma: Consultancy; Amgen: Research Funding; Bioinvent: Research Funding. Cohen:GlaxoSmithKline: Consultancy, Research Funding; Kite Pharma: Consultancy; Oncopeptides: Consultancy; Celgene: Consultancy; Novartis: Research Funding; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Janssen: Consultancy; Seattle Genetics: Consultancy. Fraietta:Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Levine:CRC Oncology: Consultancy; Brammer Bio: Consultancy; Cure Genetics: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Siegel:Novartis: Research Funding. Stadtmauer:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding. Vogl:Karyopharm Therapeutics: Consultancy. Milone:Novartis: Patents & Royalties. June:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding; Incyte: Research Funding; Tmunity: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy; CASI Pharmaceuticals: Consultancy.

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