Targeting PRAME with TCR-Mimic CAR T Cells in AML

Abstract Chimeric antigen receptor (CAR) Ts have been effective in pre-B ALL, but their efficacy in AML has yet to be established. A significant barrier to effective CAR T therapy for AML is the substantial overlap of cell surface antigens expressed on AML and normal hematopoietic cells. To overcome this barrier, we profiled the transcriptome of over 3000 AML cases in children and young adults and contrasted this to normal hematopoietic tissues in search for AML-restricted targets (high expression in AML, silence in normal hematopoiesis). This led to the discovery of over 200 AML-restricted genes. Of these, Preferentially Expressed Antigen in Melanoma (PRAME) is among one of the highest expressing AML-restricted genes (Figure 1A) and, given its previous track record as a target for a variety of cancers, we selected this target for further assessment and therapeutic development in AML. However, PRAME is intracellular and therefore is inaccessible for targeting with conventional CAR T. Recently, a novel approach to target intracellular antigens was developed using TCR mimic (mTCR) antibodies, which recognize peptide/human leukocyte antigen (HLA) complexes on the tumor cell surface in a similar mode of recognition as authentic T Cell Receptors (TCRs). The Pr20 antibody was developed to recognize the PRAME ALY peptide in the context of HLA-A*02. Utilizing this Pr20 antibody, we developed a mTCR CAR T targeting PRAME and evaluated its preclinical efficacy in AML. The VL and VH sequences from Pr20 were used to construct the single-chain fragment variable domain of the 41-BB/CD3ζ CAR vector. We evaluated PRAME mTCR CAR T cells against OCI-AML-2 and THP-1 AML cell lines (PRAME +/HLA-A*02 +), K562 CML cell line (PRAME +/HLA-A*02 -) and HEK293T (293T) (PRAME -/HLA-A*02 +). Using a PE-conjugated Pr20 antibody, we confirmed that OCI-AML2 and THP-1 express PRAME ALY: HLA-A*02 but not K562 and 293T by flow cytometry (Figure 1B). As further confirmation, AML blasts in primary patient samples also stained with the Pr20 antibody (Figure 1C). For in-vivo studies, leukemia-bearing mice were treated with unmodified T or PRAME mTCR CAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse 1 week following leukemia injection. Leukemia burden was measured weekly by bioluminescence IVIS imaging. Cells were treated with 10ng/mL of IFN-γ prior to co-incubation with T cells for 16 hours. PRAME mTCR CAR T cells demonstrated potent cytolytic activity against OCI-AML2 and THP1 but not against K562 or 293T cells, following co-incubation with target cells for 24 hours (Figure 1D). Consistent with potent, target-specific reactivity against PRAME ALY: HLA-A*02 positive cells, increased levels of IFN-γ, IL-2 and TNF-α were detected in cocultures of CAR T cells with OCI-AML2 and THP1 but not with K562 and 293T cells (Figure 1D). The cytolytic activity of PRAME mTCR CAR T cells extended to primary AML specimens expressing the PRAME ALY: HLA-A*02 antigen (data not shown). In-vivo efficacy of PRAME mTCR CAR T was demonstrated in OCI-AML2 and THP-1 CDX models (Figure 1E). Treatment with CAR T cells induced leukemia clearance and significantly reduced leukemia burden in OCI-AML2 and THP-1 xenograft mice, respectively, while treatment with unmodified T cells exhibited leukemia progression (Figure 1E). The anti-leukemia activity of CAR T cells resulted in enhanced survival in OCI-AML2 (p=0.0035) and THP-1 (p=0.0047) xenografts (Figure 1F). The in-vivo activity of PRAME mTCR CAR T cells was target specific, as treatment with CAR T cells did not affect leukemia burden and survival in K562 xenograft mice (Figure 1F). Given that IFN-γ promotes PRAME presentation, we investigated whether treatment of IFN-γ would enhance cytolytic activity of PRAME mTCR CAR T cells. OCI-AML2 and THP-1 cells pretreated with IFN-γ were more sensitive to cytolysis compared to untreated controls (Figure 1G). In this study, we demonstrate the therapeutic potential of targeting PRAME with mTCR CAR T cells in AML. We show potent, target-specific reactivity of PRAME mTCR CAR T cells against PRAME ALY: HLA-A*02 positive AML cells, both in-vitro and in-vivo. We further demonstrate that the activity of PRAME mTCR CAR T cells can be enhanced with IFN-γ treatment, providing a useful strategy to increase efficacy. Thus, the results presented provide a novel approach to target PRAME with CAR T cells and compelling data to evaluate PRAME mTCR CAR T cells in AML clinical trials. Figure 1 Figure 1. Disclosures Pardo: Hematologics, Inc.: Current Employment. Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Scheinberg: Eureka Therapeutics: Current equity holder in publicly-traded company.

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Tri-Specific CD19xCD20xCD22 VHH CAR-T Cells (LCAR-AIO) Eradicate Antigen-Heterogeneous B Cell Tumors, Enhance Expansion, and Prolong Persistence in Preclinical In Vivo Models

Blood ◽

10.1182/blood-2021-150650 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1700-1700

Author(s):

Zhe (joy) Zhou ◽

Yue Han ◽

Hong-Bo Pan ◽

Cai-Jun Sang ◽

Dong-Lin Shi ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Treatment Efficacy ◽

Cytolytic Activity ◽

Publicly Traded ◽

Car T Cells ◽

Car T ◽

Ifn Γ

Abstract Introduction: Anti-CD19 CAR-T therapy has achieved remarkable treatment efficacy in B cell lymphoma. However, targeting CD19 antigen alone can only benefit about half of patients with B cell malignancies. The FDA-approved CD19 CAR-T therapies all use same binder, which is murine FMC63 scFv targeting CD19 and up to 39%-88% of patients have relapsed. Possible mechanisms of relapse include mutations or downregulation of the targeted antigen, CD19, however, the targetable expression of CD20 and CD22 is preserved. In addition, immunogenicity against murine FMC63 scFv could have a negative impact on possible re-dosing regimen. To overcome these limitations, we designed and developed a novel tri-specific VHH CAR-T, targeting three antigens that include CD19, CD20 and CD22, for treating patients who relapsed from prior CAR-T therapies. Methods: We engineered mono-, bi-, or tri-specific VHH CAR constructs targeting CD19, CD20 and/or CD22 respectively in a lentiviral vector. The mono-, bi- or tri-specific CAR-T cells were tested against tumor lines expressing single, dual or triple antigens in an in vitro cytotoxicity assay. In addition, we evaluated the contribution of different CAR backbones, and possible combinations of scFv, VH or VHH to CAR design. We hypothesized that our lead tri-specific VHH CAR-T, LCAR-AIO, would potently inhibit tumors with heterogeneous Ag expression and prevent Ag escape. To validate this, we compared in vitro cytolytic activity and cytokine production of LCAR-AIO CAR-T to anti-CD19 FMC63 CAR-T against CD19 +CD20 +CD22 + Raji.Luc and CD19KOCD20 +CD22 +Raji.Luc cells . In vivo treatment efficacy and CAR-T persistence were also investigated in NCG murine model xenografted with Raji tumor line. 0.3x10 6 CAR +T cells or dose-matched untransduced T cells were given to NCG mice four days post i.v. implantation of Raji.Luc tumor cells. Tumor growth was monitored weekly by bioluminescence imaging until achieved endpoint (55 days), and CAR-T persistence was determined using genomic DNA level. Results: Tri-specific VHH CAR-T cells can mediate dose-dependent cytotoxicity against Raji tumor lines. Compared to mono- or bi-specific VHH or scFv CAR-T, tri-specific VHH CAR-T demonstrated equal or better cytolytic activity. Our lead tri-VHH CAR-T, LCAR-AIO, was able to specifically lyse K652 over-expressing single target such as CD19, CD20 or CD22, at the similar level to mono-specific CD19, CD20 or CD22 VHH CAR-T. Since no blocking effect of recognition against these three antigens was observed, our result suggested that all three VHHs in LCAR-AIO are functional. In comparison to anti-CD19 FMC63 scFv CAR-T, LCAR-AIO exhibited higher lytic activity and IFN-γ production against Raji.Luc tumor lines in vitro. In addition, LCAR-AIO retained its robust lytic activity and IFN-γ production when co-cultured with CD19KO-Raji.Luc cells while anti-CD19 FMC63 scFv CAR-T could not, suggesting LCAR-AIO may prevent tumor escape due to loss of CD19. Furthermore, comparison of LCAR-AIO to mono-scFv CAR-T (anti-CD19 FMC63-BBz, anti-CD20 Leu16-BBz or anti-CD22 m971-BBz) was performed in NCG mice xenografted with Raji cell line, LCAR-AIO exhibited better T cell expansion, longer persistence, and superior efficacy in eliminating tumors. Conclusions: Based on in vitro and in vivo preclinical data, tri-specific CD19xCD20xCD22 VHH CAR-T can be effective targeting tumors lack of CD19 expression, therefore, it has the potential of treating relapsed patients with prior CD19 CAR-T therapy. The feasibility of making tri-specific CAR-T would help to extend this technology to solid cancers where heterogeneity poses a major challenge at current stage. Figure 1 Figure 1. Disclosures Zhou: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Han: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Pan: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Sang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Shi: Legend Biotech: Current Employment. Feng: Legend Biotech: Current Employment. Xiao: Legend Biotech: Current Employment. Zhuang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Wang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Fan: Legend Biotech: Current Employment, Current equity holder in publicly-traded company.

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Project Stella: Development and Preclinical Assessment of FOLR1-Directed Chimeric Antigen Receptor T Cells in CBF2AT3-GLIS2/RAM AML

Blood ◽

10.1182/blood-2021-151836 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2788-2788

Author(s):

Quy Le ◽

Sommer Castro ◽

Thao T. Tang ◽

Cynthia Nourigat-Mckay ◽

LaKeisha Perkins ◽

...

Keyword(s):

T Cells ◽

High Risk ◽

Cell Line ◽

Chimeric Antigen Receptor ◽

Cytolytic Activity ◽

Target Specificity ◽

Publicly Traded ◽

Car T Cells ◽

Car T

Abstract Background: A rare but highly aggressive type of AML that is only seen in infants with a unique immunophenotype (RAM phenotype) is caused by cryptic CBFA2T3-GLIS2 (CBF/GLIS) fusion. This infant AML is highly refractory to conventional chemotherapy with near uniform fatality despite highly intensive and myeloablative therapy (PMID 23153540). Transcriptome profiling of CBF/GLIS AML has revealed new insights into the pathogenesis of the fusion and uncovered fusion-specific molecular biomarkers that could be used for risk stratification and to inform treatment (PMID 30592296). Studying the largest cohort of these high-risk infants, we demonstrated several alterations in gene expression and transcriptional networks in these CBF/GLIS-positive patient samples that have potential for therapeutic targeting (PMID 31719049). FOLR1, which encodes for folate receptor alpha, was highly and uniquely expressed in CBF/GLIS AML but was entirely absent in AML with other cytogenetics abnormalities and in normal hematopoietic cells. Furthermore, we recently demonstrated that forced expression of CBF/GLIS enhances the proliferation and alters differentiation in cord blood (CB) CD34+ early precursors towards megakaryocytic lineage that recapitulates acute megakaryocytic leukemia seen in infants (PMID 31719049). Of significance, we showed that FOLR1 surface expression is causally linked to CBF/GLIS-induced malignant transformation, thus making it an attractive antigen for targeted therapies against CBF/GLIS AML cells. Given that chimeric antigen receptor (CAR) T cells are extremely effective at eradicating relapsed/refractory B-ALL malignancies, we developed FOLR1-directed CAR T cells for pre-clinical evaluation in CBF/GLIS AML. Methods: We generated a FOLR1-directed CAR using anti-FOLR1 binder (Farletuzumab), IgG4 intermediate spacer and 41-BB/CD3zeta signaling domains. The pre-clinical efficacy of FOLR1 CAR T cells was evaluated against CBF/GLIS AML cell lines in vitro and in vivo. CBF/GLIS AML models include CB CD34+ cells transduced with CBF/GLIS expression construct (CBF/GLIS-CB) and WSU-AML cell line. We also engineered Kasumi-1 cell line to express FOLR1 (Kasumi-1 FOLR1+) to evaluate target specificity (Figure 1A). Results: We tested the target specificity of FOLR1-directed CAR T cells against FOLR1-positive (CBF/GLIS-CB, WSU-AML, Kasumi-1 FOLR1+) and FOLR1-negative (Kasumi-1) cells. CD8 FOLR1 CAR T cells demonstrated cytolytic activity against FOLR1 positive but not FOLR1 negative cells (Figure 1B). Furthermore, both CD8 and CD4 FOLR1 CAR T cells produced higher levels of IL-2, IFN-γ, and TNF-α and proliferated more robustly than did unmodified T cells when co-incubated with FOLR1 positive but not FOLR1 negative cells (Figure 1C). These results indicate highly specific reactivity of FOLR1 CAR T cells against AML cells expressing FOLR1. We next investigated the in vivo efficacy of FOLR1-directed CAR T cells. In CBF/GLIS-CB, WSU-AML, and Kasumi-1 FOLR1+ xenograft models, treatment with FOLR1 CAR T cells induced leukemia clearance, while disease progression occurred in all mice that received unmodified T cells (Figure 1D). Activity of FOLR1 CAR T cells in vivo was target specific, as they did not limit the leukemia progression nor extend the survival of Kasumi-1 xenografts (Figure 1D). To determine whether FOLR1 is expressed on normal HSPCs, we characterized FOLR1 expression in normal CB CD34+ samples. FOLR1 expression was entirely silent in HSPC subsets (Figure 1E). Consistent with lack of expression, no cytolytic activity was detected against HPSCs Moreover, FOLR1 CAR T cells did not affect the self-renewal and multilineage differentiation capacity of normal HSPCs as compared to unmodified control T cells (Figure 1F), whereas significant eradication of colonies were detected in the CBF/GLIS-CB cells (Figure 1G). Conclusion: In this study, we demonstrate that FOLR1 CAR T effectively eradicates CBF/GLIS AML cells without compromising normal HSPCs, providing a promising approach for the treatment of high-risk CBF/GLIS AML. Transition of this CAR T to clinical development for infant AML is underway. Figure 1 Figure 1. Disclosures Hylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

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Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-141540 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Pinar Ataca Atilla ◽

Mary K McKenna ◽

Norihiro Watanabe ◽

Maksim Mamonkin ◽

Malcolm K. Brenner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Tumor Control ◽

Publicly Traded ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T

Introduction: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia associated antigen with chimeric antigen receptor T (CAR T) cells have had limited success. We determined whether combinatorial expression of chimeric antigen receptors directed to two different AML associated antigens would augment tumor eradication and prevent relapse in targets with heterogeneous expression of myeloid antigens. Methods: We generated CD123 and CD33 targeting CARs; each containing a 4-1BBz or CD28z endodomain. We analyzed the anti-tumor activity of T cells expressing each CAR alone or in co-transduction with a CLL-1 CAR with CD28z endodomain and CD8 hinge previously optimized for use in our open CAR-T cell trial for AML (NCT04219163). We analyzed CAR-T cell phenotype, expansion and transduction efficacy by flow cytometry and assessed function by in vitro and in vivo activity against AML cell lines expressing high, intermediate or low levels of the target antigens (Molm 13= CD123 high, CD33 high, CLL-1 intermediate, KG1a= CD123 low, CD33 low, CLL-1 low and HL60= CD123 low, CD33 intermediate, CLL-1 intermediate/high) For in vivo studies we used NOD.SCID IL-2Rg-/-3/GM/SF (NSGS) mice with established leukemia, determining antitumor activity by bioluminescence imaging. Results: We obtained high levels of gene transfer and expression with both single (CD33.4-1BBʓ, CD123.4-1BBʓ, CD33.CD28ʓ, CD123.CD28ʓ, CLL-1 CAR) and double transduction CD33/CD123.4-1BBʓ or CD33/CD123.CD28ʓ) although single-transductants had marginally higher total CAR expression of 70%-80% versus 60-70% after co-transduction. Constructs containing CD28 co-stimulatory domain exhibited rapid expansion with elevated peak levels compared to 41BB co-stim domain irrespective of the CAR specificity. (p<0.001) (Fig 1a). In 72h co-culture assays, we found consistently improved anti-tumor activity by CAR Ts expressing CLL-1 in combination either with CD33 or with CD123 compared to T cells expressing CLL-1 CAR alone. The benefit of dual expression was most evident when the target cell line expressed low levels of one or both target antigens (e.g. KG1a) (Fig 1b) (P<0.001). No antigen escape was detected in residual tumor. Mechanistically, dual expression was associated with higher pCD3ʓ levels compared to single CAR T cells on exposure to any given tumor (Fig 1c). Increased pCD3ʓ levels were in turn associated with augmented CAR-T degranulation (assessed by CD107a expression) in both CD4 and CD8 T cell populations and with increased TNFα and IFNɣ production (p<0.001 Fig 1d). In vivo, combinatorial targeting with CD123/CD33.CD28ʓ and CLL-1 CAR T cells improved tumor control and animal survival in lines (KG1a, MOLM13 and HL60) expressing diverse levels of the target antigens (Fig 2). Conclusion: Combinatorial targeting of T cells with CD33 or CD123.CD28z CARs and CLL-1-CAR improves CAR T cell activation associated with superior recruitment/phosphorylation of CD3ʓ, producing enhanced effector function and tumor control. The events that lead to increased pCD3ʓ after antigen engagement in the dual transduced cells may in part be due to an overall increase in CAR expression but may also reflect superior CAR recruitment after antigen engagement. We are now comparing the formation, structure, and stability of immune synapses in single and dual targeting CARs for AML. Disclosures Brenner: Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Atilla:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: founder; Marker Therapeuticsa: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen: Membership on an entity's Board of Directors or advisory committees; KUUR: Membership on an entity's Board of Directors or advisory committees.

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Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9

Nature Communications ◽

10.1038/s41467-020-19672-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Lintao Liu ◽

Enguang Bi ◽

Xingzhe Ma ◽

Wei Xiong ◽

Jianfei Qian ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Effector Memory ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Ifn Γ

AbstractCAR-T cell therapy is effective for hematologic malignancies. However, considerable numbers of patients relapse after the treatment, partially due to poor expansion and limited persistence of CAR-T cells in vivo. Here, we demonstrate that human CAR-T cells polarized and expanded under a Th9-culture condition (T9 CAR-T) have an enhanced antitumor activity against established tumors. Compared to IL2-polarized (T1) cells, T9 CAR-T cells secrete IL9 but little IFN-γ, express central memory phenotype and lower levels of exhaustion markers, and display robust proliferative capacity. Consequently, T9 CAR-T cells mediate a greater antitumor activity than T1 CAR-T cells against established hematologic and solid tumors in vivo. After transfer, T9 CAR-T cells migrate effectively to tumors, differentiate to IFN-γ and granzyme-B secreting effector memory T cells but remain as long-lived and hyperproliferative T cells. Our findings are important for the improvement of CAR-T cell-based immunotherapy for human cancers.

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Opposing Effects of PD-1/PD-L1/L2 Engagement and IFN-γ/TNF-α in the Treatment of AML w/ Anti-CD33 Chimeric Antigen Receptor-Modified T Cells

Blood ◽

10.1182/blood.v128.22.5891.5891 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5891-5891

Author(s):

Jacob Halum Basham ◽

Terrence L. Geiger

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Chimeric Antigen Receptor ◽

Short Term ◽

Car T Cells ◽

Tnf Α ◽

Car T ◽

Ifn Γ

Abstract Chimeric antigen receptor-modified T lymphocytes (CART cells) have shown benefit as an adjuvant immunotherapy in the treatment of B cell malignancies. This success of re-targeted T cells has not been extended to other hematologic malignancies. We have developed an immunotherapeutic approach to treat acute myeloid leukemia (AML) using CAR T cells re-directed against the myeloid-specific antigen CD33 (CART-33). CART-33 cells are potent and specific in eliminating AML cells in vitro and in vivo. Despite this, CART-33 cells have shown poor in vivo expansion and persistence in NOD-SCID IL2rγ (-/-) (NSG) AML xenograft models. To address the reason for this, we assessed the impact of AML-expressed programmed death ligands 1 & 2 (PD-L1/2) on CART-33 cell activity. PD-L1 inhibits T cell functions upon binding PD-1, which is upregulated with T cell activation. Less is known about PD-L2's effect. Interferon-gamma (IFN-γ), a primary effector cytokine secreted by CD4+ and CD8+ effector T cells, is a known potent inducer of PD-L1 on AML blasts. Using AML cell lines U937, Oci-AML3, CMK, and MV4-11 we show that IFN-γ, TNF-α, and activated CART-33 supernatant can induce up-regulation of PD-L1 and PD-L2 on AML. IFN-γ and TNF-α synergize strongly in up-regulating PD-1 ligands on AML. The kinetics and induction of PD-L2 are distinct from that of PD-L1. Although PD-L1 is well documented to suppress T cell function via ligation of T cell expressed PD-1, induction of PD-L1/L2 had no effect on the cytolytic activity of CART-33 cells against AML in short term (<48 h) cultures. Paradoxically, 24 hr pre-treatment of AML with either IFN-γ or CART-33 supernatant increased AML susceptibility to killing by CART-33 cells despite elevated expression of PD-L1/L2 by AML. Our results highlight the regulatory complexity of AML cytolysis by re-targeted T lymphocytes, and argue that tumor-expressed PD-L1 and PD-L2 impacts the sustainability, but not short-term killing activity, of adoptively transferred CAR T cells in the treatment of AML. Disclosures No relevant conflicts of interest to declare.

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IL-18 Secreting CAR T Cells Enhance Cell Persistence, Induce Prolonged B Cell Aplasia and Eradicate CD19+ Tumor Cells without Need for Prior Conditioning

Blood ◽

10.1182/blood.v128.22.816.816 ◽

2016 ◽

Vol 128 (22) ◽

pp. 816-816 ◽

Cited By ~ 10

Author(s):

Mauro P. Avanzi ◽

Dayenne G. van Leeuwen ◽

Xinghuo Li ◽

Kenneth Cheung ◽

Hyebin Park ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Tumor Cells ◽

Car T Cells ◽

Car T Cell ◽

Cytokine Analysis ◽

Car T ◽

Ifn Γ

Abstract Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed. The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5. We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system. To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively) Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006). Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p<0.0001). 19m28mz-P2A-mIL18 CAR T cells were detected in peripheral blood for up to 30 days after injection, whereas the 19m28mz CAR T cells were not detectable at any time point. In addition, 19m28mz-P2A-mIL18 CAR T cells were capable of inducing B cell aplasia for greater than 70 days, whereas 19m28mz treatment was not capable of inducing B cell aplasia. In vivo serum cytokine analysis demonstrated that 19m28mz-P2A-mIL18 CAR T cells, as compared to 19m28mz, significantly increased the levels of IFN-γ and TNF-α in the peripheral blood for up to 14 days after injection (p<0.0001 and 0.01, respectively). Despite the increase in IFN-γ and TNF-α cytokines, there was no increase in IL-6 levels. Our findings demonstrate that anti-CD19 CAR T cells that constitutively secrete IL-18 significantly increase serum cytokine secretion, enhance CAR T cell persistence, induce long-term B cell aplasia and improve mouse survival, even without any prior preconditioning. To our knowledge, this is the first description of an anti-CD19 CAR T cell that constitutively secretes IL-18 and that induces such high levels of T cell proliferation, persistence and anti-tumor cytotoxicity. We are currently investigating other mechanisms by which this novel CAR T cell functions, its interactions with the endogenous immune system, as well as testing its applicability in other tumor types. We anticipate that the advances presented by this new technology will expand the applicability of CAR T cells to a wider array of malignancies. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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CD4-Targeted Fusosomes Are Capable of Transducing Resting T Helper Cells to Generate Highly Potent CAR-T Cells

Blood ◽

10.1182/blood-2021-148469 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2942-2942

Author(s):

Christie Ciarlo ◽

Zach Frye ◽

Andre DeGroot ◽

Walter Flores ◽

Kutlu Elpek ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cd4 T Cells ◽

Transduction Efficiency ◽

Publicly Traded ◽

Car T Cells ◽

Car T ◽

Integrating Vector

Abstract Introduction: Chimeric antigen receptor T cell therapy (CAR T) is a successful treatment for B cell malignancies; however, the time, complexity and cost of manufacturing autologous CAR T products limits the availability of these therapies to patients. Furthermore, ex vivo manipulation of T cells is likely to have a negative impact on quality. In vivo gene delivery of CAR T transgenes by systemic infusion of standard lentiviral vectors may increase therapeutic accessibility but is limited by off-target transduction and the requirement for T cell activation. Here, we demonstrate that a paramyxovirus-based integrating vector (fusosome) engineered with a CD4 re-targeted envelop (CD4 fusogen) can efficiently and specifically transduce resting and activated CD4+ T cells to generate functional CD4+ CD19-specific CAR T cells capable of eliminating CD19+ lymphoma cells. Methods: Anti-CD4 single chain variable fragments () and single variable domain (VHHs) were screened for CD4 binding, specificity, and NHP cross-reactivity and inserted into receptor binding paramyxovirus fusogen. CD4-targeted fusosomes expressing GFP were screened for high on-target titer against the CD4+ SupT1 cell line and low off-target transduction on non-CD4 expressing cells. Subsequently, a CD19-specific CAR encoding 4-1BB and the CD3z endo-domains (CD19 CAR) was generated to examine CD4+ CAR T transduction efficiency and functionality. PBMCs were thawed and activated with anti-CD3/anti-CD28 beads and exposed to GFP, CD4-targeted fusosomes and specificity of targeting CD4+ T cells was measured by flow cytometry. Subsequently, CD19 CAR fusosomes targeting CD4 were used to test transduction efficiency against activated (CD3/CD28 or IL-7 treated) or resting T cells, and to measure T cell function against CD19+ and CD19 knockout (CRISPR/Cas9-edited) Nalm-6 lymphoma cells (e.g., tumor co-culture and rechallenge assays and cytokine production) in vitro. Vector copy number (VCN) was determined by a multiplex ddPCR assay and reported as copies per diploid genome (c/dg). Results: To target CD4+ T cells, we generated fusogens encoding scFvs and VHHs specific to the CD4 T cell co-receptor (n = 399). Using fusosomes carrying the GFP transgene, NHP cross-reactive CD4-targeted fusogens that efficiently transduced CD4+ SupT1 cells were selected (n = 12 with crude SupT1 titers >1E6). Activated PBMCs transduced with a CD4-targeted fusosomes exhibited specific CD4 T cell transduction whereas VSV-G pseudotyped vectors showed broad transduction including CD4+ and CD8+ T cells. CD4-targeted CD19 CAR fusosomes could efficiently transduce both activated (34% ± 1.5% CD4+CAR+; 0.54 ± 0.18 c/dg) and resting T cells, albeit at a lower expression and integration rate (20% ± 0.5% CD4+CAR+; 0.28 ± 0.14 c/dg). Resting CD4-transduced CAR T cells demonstrated specific cytotoxicity and cytokine production (GM-CSF, IFN-g, TNF-a, IL-2, IL-6, and IL-10) against CD19+ Nalm-6 but did not recognize CD19 knockout tumor cells. In long-term co-culture assays with repetitive stimulation with fresh tumor cells, resting CD4+ CD19 CAR T cells continued to show potent tumor cell killing. Future experiments will evaluate the efficacy of CD4 fusosomes against CD19+ tumors in vivo. Summary: CD4-specific fusosomes can efficiently deliver an integrating CAR payload to resting and activated CD4+ T cells. Modified CD4+ CAR T cells demonstrate potent anti-tumor activity against CD19+ tumor cells. These data suggest that targeting the CD4 co-receptor through in vivo delivery using a novel pseudotyped integrating vector can produce functional CAR T cells to target cancer. Disclosures Ciarlo: Sana Biotechnology: Current Employment. Frye: Sana Biotechnology: Current Employment. DeGroot: Sana Biotechnology: Current Employment. Flores: Sana Biotechnology: Current Employment. Elpek: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Johnson: Sana Biotechnology: Current Employment. Shah: Sana Biotechnology: Current Employment. Foster: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

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Preservation of T-Cell Stemness with a Novel Expansionless CAR-T Manufacturing Process, Which Reduces Manufacturing Time to Less Than Two Days, Drives Enhanced CAR-T Cell Efficacy

Blood ◽

10.1182/blood-2021-146246 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2848-2848

Author(s):

Boris Engels ◽

Xu Zhu ◽

Jennifer Yang ◽

Andrew Price ◽

Akash Sohoni ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

Antitumor Efficacy ◽

Publicly Traded ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract Background: Extended T-cell culture periods in vitro deplete the CAR-T final product of naive and stem cell memory T-cell (T scm) subpopulations that are associated with improved antitumor efficacy. YTB323 is an autologous CD19-directed CAR-T cell therapy with dramatically simplified manufacturing, which eliminates complexities such as long culture periods. This improved T-Charge™ process preserves T-cell stemness, an important characteristic closely tied to therapeutic potential, which leads to enhanced expansion ability and greater antitumor activity of CAR-T cells. Methods: The new T-Charge TM manufacturing platform, which reduces ex vivo culture time to about 24 hours and takes <2 days to manufacture the final product, was evaluated in a preclinical setting. T cells were enriched from healthy donor leukapheresis, followed by activation and transduction with a lentiviral vector encoding for the same CAR used for tisagenlecleucel. After ≈24 hours of culture, cells were harvested, washed, and formulated (YTB323). In parallel, CAR-T cells (CTL*019) were generated using a traditional ex vivo expansion CAR-T manufacturing protocol (TM process) from the same healthy donor T cells and identical lentiviral vector. Post manufacturing, CAR-T products were assessed in T-cell functional assays in vitro and in vivo, in immunodeficient NSG mice (NOD-scid IL2Rg-null) inoculated with a pre-B-ALL cell line (NALM6) or a DLBCL cell line (TMD-8) to evaluate antitumor activity and CAR-T expansion. Initial data from the dose escalation portion of the Phase 1 study will be reported separately. Results: YTB323 CAR-T products, generated via this novel expansionless manufacturing process, retained the immunophenotype of the input leukapheresis; specifically, naive/T scm cells (CD45RO -/CCR7 +) were retained as shown by flow cytometry. In contrast, the TM process with ex vivo expansion generated a final product consisting mainly of central memory T cells (T cm) (CD45RO +/CCR7 +) (Fig A). Further evidence to support the preservation of the initial phenotype is illustrated by bulk and single-cell RNA sequencing experiments, comparing leukapheresis and final products from CAR-Ts generated using the T-Charge™ and TM protocols. YTB323 CAR-T cell potency was assessed in vitro using a cytokine secretion assay and a tumor repeat stimulation assay, designed to test the persistence and exhaustion of the cell product. YTB323 T cells exhibited 10- to 17-fold higher levels of IL-2 and IFN-γ secretion upon CD19-specific activation compared with CTL*019. Moreover, YTB323 cells were able to control the tumor at a 30-fold lower Effector:Tumor cell ratio and for a minimum of 7 more stimulations in the repeat stimulation assay. Both assays clearly demonstrated enhanced potency of the YTB323 CAR-T cells in vitro. The ultimate preclinical assessment of the YTB323 cell potency was through comparison with CTL*019 regarding in vivo expansion and antitumor efficacy against B-cell tumors in immunodeficient NSG mouse models at multiple doses. Expansion of CD3+/CAR+ T-cells in blood was analyzed weekly by flow cytometry for up to 4 weeks postinfusion. Dose-dependent expansion (C max and AUC 0-21d) was observed for both YTB323 and CTL*019. C max was ≈40-times higher and AUC 0-21d was ≈33-times higher for YTB323 compared with CTL*019 across multiple doses. Delayed peak expansion (T max) of YTB323 by at least 1 week compared with CTL*019 was observed, supporting that increased expansion was driven by the less differentiated T-cell phenotype of YTB323. YTB323 controlled NALM6 B-ALL tumor growth at a lower dose of 0.1×10 6 CAR+ cells compared to 0.5×10 6 CAR+ cells required for CTL*019 (Fig B). In the DLBCL model TMD-8, only YTB323 was able to control the tumors while CTL*019 led to tumor progression at the respective dose groups. This ability of YTB323 cells to control the tumor at lower doses confirms their robustness and potency. Conclusions: The novel manufacturing platform T-Charge™ used for YTB323 is simplified, shortened, and expansionless. It thereby preserves T-cell stemness, associated with improved in vivo CAR-T expansion and antitumor efficacy. Compared to approved CAR-T therapies, YTB323 has the potential to achieve higher clinical efficacy at its respective lower doses. T-Charge™ is aiming to substantially revolutionize CAR-T manufacturing, with concomitant higher likelihood of long-term deep responses. Figure 1 Figure 1. Disclosures Engels: Novartis: Current Employment, Current equity holder in publicly-traded company. Zhu: Novartis: Current Employment, Current equity holder in publicly-traded company. Yang: Novartis: Current Employment, Patents & Royalties. Price: Novartis: Current Employment. Sohoni: Novartis: Current Employment. Stein: Novartis: Current Employment. Parent: Novartis: Ended employment in the past 24 months; iVexSol, Inc: Current Employment. Greene: iVexSol, Inc: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Niederst: Novartis: Current Employment, Current equity holder in publicly-traded company. Whalen: Novartis: Current Employment. Orlando: Novartis: Current Employment. Treanor: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties: no royalties as company-held patents. Brogdon: Novartis Institutes for Biomedical Research: Current Employment.

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Novel CAR T Modules (Tmod) to Target HLA-Class I Loss in Lymphoma

Blood ◽

10.1182/blood-2020-134041 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 23-24

Author(s):

Agnes E. Hamburger ◽

Breanna DiAndreth ◽

Mark E. Daris ◽

Melanie L. Munguia ◽

Kiran Deshmukh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Publicly Traded ◽

Private Company ◽

Car T Cells ◽

The Past ◽

Car T Cell ◽

Car T

Background: Chimeric Antigen Receptor (CAR) T-cell therapy is a proven, powerful clinical modality. However, it is still limited by the fundamental obstacle of cancer therapy: discriminating cancer from normal cells. Current FDA-approved CAR T-cell therapies eliminate normal B cells, leaving patients with B cell aplasia, hypogammaglobulinemia, and susceptible to infection. HLA-Class I loss of heterozygosity (LOH) occurs at an average frequency of ~13% among cancers and specifically ~13% in DLBCL (Broad Institute TCGA database). These losses are irreversible and distinguish the cancer from normal cells. To exploit LOH at the HLA locus, we target the remaining allelic product in tumors with LOH. We evaluated a novel AND NOT Boolean logic gate CAR T module (Tmod) T-cell system to target HLA-A*02 (A2) LOH in lymphoma using both in vitro and in vivo models. Methods: To model tumor cells that have lost A2 via LOH, we used CD19+ Raji lymphoma tumor cells. To model the corresponding "normal" cells, we established CD19+ Raji cells stably expressing A2 (CD19+/A2+ Raji). We then engineered human primary T cells to express a modular signal-integration circuit designed to be activated only by CD19+ lymphoma that do not express A2 (CD19+/A2- Raji). Each primary Tmod CAR T cell expresses both a CD19 activator (A) module using a CD19-targeting 3rd generation CAR, and a separate A2-targeting blocker (B) module using a novel A2-targeting inhibitory receptor. Human primary Tmod CAR T cells were engineered to co-express the A/B modules. First, T cells were stimulated via CD3/CD28 activation, followed by A/B module lentivirus transduction, and enriched for the B module. In vitro Tmod CAR T cells were evaluated for selective killing of CD19+/A2- Raji compared with CD19+/A2+ Raji. For in vivo proof of concept, both CD19+/A2- Raji and CD19+/A2+ Raji cell lines were injected and established into flanks of immunocompromised NGS mice and challenged with adoptive transfer of engineered human primary Tmod CAR T cells. Results: Engineered primary Tmod CAR T cells selectively killed CD19+/A2- Raji and spared CD19+/A2+ Raji (Figure 1). Tmod CAR T cells reversibly cycled from a state of non-killing, "block", to cytotoxicity and back, depending on the CD19+/A2- Raji vs. CD19+/A2+ Raji cells to which they were exposed. Importantly, primary Tmod CAR T cells selectively eliminated only the CD19+/A2- Raji cells in mixed cultures. In vivo, Tmod CAR T cells selectively eradicated CD19+/A2- Raji. More importantly, Tmod CAR T cells did not eradicate CD19+/A2+ Raji in vivo. Conclusions: CD19-targeting Tmod CAR T cells demonstrated robust and selective killing, distinguishing Raji lymphoma lines, one with A2 (CD19+/A2+) and one without (CD19+/A2-), both in vitro and in vivo. A critical requirement for Tmod CAR T-cell therapy is to determine reversibility and lack of anergy in the kill-"block"-kill and "block"-kill-"block" scenarios. This result demonstrates that Tmod CAR T cells do not terminally differentiate into one state (blockade or activation), but rather can switch back and forth as they integrate signals from "normal" and tumor cells. Furthermore, because Tmod CAR T cells can selectively target malignant B cells, it may increase the clinical therapeutic window for CAR T. Tmod CAR T cells may provide a powerful system to address hematologic malignancies and solid tumors with HLA-Class I LOH. Disclosures Hamburger: A2 Biotherapeutics: Current Employment, Current equity holder in private company. DiAndreth:A2 Biotherapeutics: Current Employment. Daris:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Munguia:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Deshmukh:A2 Biotherapeutics: Current Employment. Mock:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Asuelime:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Lim:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Kreke:A2 Biotherapeutics: Current Employment, Current equity holder in private company; Gilead: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Tokatlian:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Maloney:A2 Biotherapeutics: Consultancy, Current equity holder in publicly-traded company, Honoraria; Bioline Rx: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria; Gilead Science: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Patents & Royalties, Research Funding. Go:A2 Biotherapeutics: Current Employment, Current equity holder in private company; Amgen: Current equity holder in publicly-traded company; Allogene: Divested equity in a private or publicly-traded company in the past 24 months; Gilead: Current equity holder in publicly-traded company; Illumina: Divested equity in a private or publicly-traded company in the past 24 months. Kamb:A2 Biotherapeutics: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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A Small Number of HER2 Redirected CAR T Cells Significantly Improves Immune Response of Adoptively Transferred Mouse Lymphocytes against Human Breast Cancer Xenografts

International Journal of Molecular Sciences ◽

10.3390/ijms21031039 ◽

2020 ◽

Vol 21 (3) ◽

pp. 1039 ◽

Cited By ~ 1

Author(s):

Gábor Tóth ◽

János Szöllősi ◽

Hinrich Abken ◽

György Vereb ◽

Árpád Szöőr

Keyword(s):

T Cells ◽

Complete Remission ◽

Cytolytic Activity ◽

Scid Mice ◽

Target Cells ◽

Her2 Positive ◽

Car T Cells ◽

Car T

HER2 positive JIMT-1 breast tumors are resistant to trastuzumab treatment in vitro and develop resistance to trastuzumab in vivo in SCID mice. We explored whether these resistant tumors could still be eliminated by T cells redirected by a second-generation chimeric antigen receptor (CAR) containing a CD28 costimulatory domain and targeting HER2 with a trastuzumab-derived scFv. In vitro, T cells engineered with this HER2 specific CAR recognized HER2 positive target cells as judged by cytokine production and cytolytic activity. In vivo, the administration of trastuzumab twice weekly had no effect on the growth of JIMT-1 xenografts in SCID mice. At the same time, a single dose of 2.5 million T cells from congenic mice exhibited a moderate xenoimmune response and even stable disease in some cases. In contrast, when the same dose contained 7% (175,000) CAR T cells, complete remission was achieved in 57 days. Even a reduced dose of 250,000 T cells, including only 17,500 CAR T cells, yielded complete remission, although it needed nearly twice the time. We conclude that even a small number of CAR T lymphocytes can evoke a robust anti-tumor response against an antibody resistant xenograft by focusing the activity of xenogenic T cells. This observation may have significance for optimizing the dose of CAR T cells in the therapy of solid tumors.

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