Target Expression, Preclinical Activity and Mechanism of Action of EM801: A Novel First-in-Class Bcma T-Cell Bispecific Antibody for the Treatment of Multiple Myeloma

Abstract Background. T-cell bispecific antibodies (TCBs) simultaneously binding CD3 on T-cells and individual tumor antigens, activate T-cells and destroy tumor antigen carrying cells. B-cell maturation antigen (BCMA), a surface antigen reported to be expressed on normal and malignant plasma cells (PCs), could represent a potentially promising target for TCBs in multiple myeloma (MM). The Aim of our study was to: i) assess expression of BCMA in normal and malignant PCs as well as cells of the bone marrow (BM) microenvironment by gene expression profiling and flow cytometry to validate it as potential clinical target for TCBs; ii) to evaluate activity of EM801 as member of a novel class of BCMA-TCBs in vitro on primary myeloma cells and in vivo in the H929-xenograft reconstituted NOG mouse model; and iii) to delineate its mechanism of action. Results. Expression. We investigated the expression of BCMA in CD138-purified PCs from BM aspirates obtained from 726 patients including MGUS (n=62), asymptomatic (n=59) and symptomatic MM (605), as well as different BM cellular subsets from healthy donors (n=10 PCs; plasmablasts, memory B-cells, T-cells, CD34+, CD14+, CD15+, n=5 each; n=8 mesenchymal stromal cells) using Affymetrix DNA-microarrays. BCMA expression was observed in malignant PC from 723/726 (99.5%) MGUS and MM patients, 10/10 normal PCs and 5/5 plasmablasts; gene expression of BCMA was undetectable in all other normal BM subsets. Using multiparameter flow cytometry, BCMA surface expression on malignant PCs was confirmed in 40/40 patients while being absent on normal BM cells. BCMA is thus a potential target in virtually all myeloma patients. Activity. In vitro, EM801 induced concentration dependent significant cell death in malignant plasma cells in BM-samples of 21/28 (75%) previously untreated and 8/10 (80%) relapsed/refractory MM patients in concentrations ranging from 10pM to 30nM. No or only minor unspecific toxicity on cells of the BM microenvironment was observed. In vivo efficacy of EM801 was studied in a subcutaneous H929 myeloma cell line xenograft model in NOG (NOD/Shi-scid/IL-2Rγnull) mice reconstituted with human PBMCs. Three doses of EM801, i.e. 0.026, 0.26 and 2.6 nM/kg, the same doses of a BCMAxCD3-(scFv)2 and two control groups were investigated (n=9 mice/group). Three weekly intravenous doses were given, starting on day 19 after tumor cell injection when tumor volumes were 293±135 mm3. On day 47, all mice from control groups had their tumors grown beyond 2000 mm3 and were euthanized for ethical reasons. In contrast, at 2.6 nM/kg (0.5 mg/kg) EM801 tumor regression was already observed after the second i.v. injection in 6/9 animals and the tumor regressed to 16±3 mm3 on day 47. BCMAxCD3-(scFv)2 bispecific antibody without Fc did not show any efficacy at all doses studied. Regarding the mechanism of action, we first demonstrated that EM801 effectively binds myeloma cells and T-cells with a strength of 1622±410 pN (5-10 fold of control) as measured by atomic force microscopy. Secondly, increasing concentrations (0.03-30nM) of EM801 led to progressive T-cell activation in primary BM samples, with significantly increased levels of CD69 (P<0.001), CD25 (P<0.001) and HLADR (P=0.001) expression in both CD4 and CD8 T-cells as compared to an unspecific TCB. Thirdly, EM801 induced significant secretion of interferon-γ (19-3000 pg/ml), granzyme B (68-2986 pg/ml), and perforin (145-3712 pg/ml) as measured by ELISA, together explaining the strong in vitro and in vivo activity of EM801. Conclusions. BCMA is selectively expressed at the RNA (723/726) and protein (40/40) levels on malignant PCs from virtually all MM patients, and thus represents a promising TCB-target. The novel BCMA-TCB EM801 was effective in vitro in 29/38 (76%) primary MM patients' BM samples at picomolar to low nanomolar concentrations, easily achievable in vivo in patients, as well as in the H929-xenograft reconstituted NOG mouse model at 0.5 mg/kg once a week. Neither in vitro (the BM microenvironment) nor in vivo the compound shows significant toxicity or side effects. EM801 confers cytotoxicity by effectively coupling T-cells with malignant PCs, inducing T-cell activation, secretion of interferon-γ, granzyme B and perforin, and thereby effectively killing malignant PCs. EM801 is thus a promising new compound for the treatment of multiple myeloma to be investigated in clinical phase I/II trials. Disclosures Seckinger: EngMab AG: Research Funding; Takeda: Other: Travel grant. Neuber:EngMab AG: Research Funding. Vu:EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Strein:BB Biotech AG: Membership on an entity's Board of Directors or advisory committees; Novimmune SA: Membership on an entity's Board of Directors or advisory committees; EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hundemer:EngMab AG: Research Funding. San Miguel:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Janssen-Cilag: Honoraria; Millennium: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria; Onyx: Honoraria. Hose:Takeda: Other: Travel grant; EngMab AG: Research Funding. Paiva:Celgene: Consultancy; Janssen: Consultancy; Binding Site: Consultancy; BD Bioscience: Consultancy; EngMab AG: Research Funding; Onyx: Consultancy; Millenium: Consultancy; Sanofi: Consultancy.

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Clinical Efficacy of Anti-CD22 Chimeric Antigen Receptor T Cells for B-Cell Acute Lymphoblastic Leukemia Is Correlated with the Length of the Scfv Linker and Can be Predicted Using Xenograft Models

Blood ◽

10.1182/blood.v130.suppl_1.807.807 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 807-807

Author(s):

Marco Ruella ◽

Shannon L Maude ◽

Boris Engels ◽

David M. Barrett ◽

Noelle Frey ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

Lymphoblastic Leukemia ◽

Advisory Committees ◽

Xenograft Models

Abstract Introduction. Anti-CD19 chimeric antigen receptor T cells (CART19 or CTL019) have shown impressive clinical activity in B-cell acute lymphoblastic leukemia (B-ALL) and are poised to receive FDA approval. However, some patients relapse after losing CD19 expression. Since CD22 remains highly expressed in relapsed/refractory (r/r) B-ALL even in these patients, anti-CD22 CART (CART22) have been developed. The National Cancer Institute (NCI) reported 4/9 complete remission (CR) in patients receiving CART22, with 100% CR at the highest T cell dose (NCT02315612)(S hah NN, ASH 2016 #650). Patients and Methods. We generated a second-generation CAR22 differing from that used by the NCI only by the use of a longer linker [4x(GGGGS); LL vs. 1x(GGGGS); SL] between the light and heavy chains of the scFv (Fig. 1 A). This construct was tested in two pilot clinical trials in adults (NCT02588456)and children with r/r-ALL (NCT02650414). CART22 cells were generated using lentiviral transduction as in our previous studies. The protocol-specified CART22 dose was 2x106-1x107 cells/kg for pediatric patients <50kg and 1-5x108 for pediatric patients ≥50kg and adult patients,. infused after lymphodepleting chemotherapy. Patient characteristics are described in Table 1. For the adult trial, 5 patients were screened, 4 enrolled (1 patient withdrew consent) and 3 infused (1 manufacturing failure). For the pediatric trial, 9 patients were screened, 8 enrolled (1 screen failure) and 6 infused (two patients were not infused for disease progression). For the preclinical studies, we generated CART22LL and CART22SL and tested them in vivo using xenograft models. NOD-SCID gamma chain deficient (NSG) mice were engrafted with either a luciferase+ standard B-ALL cell line (NALM6) or primary B-ALL cells obtained from a patient relapsing after CART19 (CHP110R). We also used 2-photon imaging to study the in vivo behavior and immune synapse formation and flow cytometry to asses T cell activation. Results. CART22 cells were successfully manufactured for 10/12 patients. In the adult cohort 3/3 patients developed CRS (gr.1-3) and no neurotoxicity was observed; in the pediatric cohort out of 5 evaluable patients (1 discontinued for lineage switch to AML on pre-infusion marrow), 3/5 developed cytokine-release syndrome (CRS) (all grade 2) and 1 patient had encephalopathy (gr.1). CART22 cells expanded in the PB with median peak of 1977 (18-40314) copies/ug DNA at day 11-18. Interestingly, in an adult patient who had previously received CART19 a second CART19 re-expansion was observed following CART22 expansion (Fig 1 B). At day 28, in the adult cohort the patient who was infused in morphologic CR remained in CR, while the other 2 had no response (NR); in the pediatric cohort 2/5 patients were in CR, 1 in partial remission (PR) that then converted to CR with incomplete recovery at 2 months, and 2 NR. No CD22-negative leukemia progression was observed. Since our results with a long linker appeared inferior compared to the previously reported CART22 trial (short linker), we performed a direct comparison of the 2 different CAR22 constructs. In xenograft models, CART22SL significantly outperformed CART22LL (Fi 1 C) with improved overall survival. Moreover, CART22SL showed higher in vivo proliferation at day 17 (Fig 1 D). Mechanistically, intravital 2-photon imaging showed that CART22SL established more protracted T cell:leukemia interactions than did CART22LL, suggesting the establishment of productive synapses (Fig 1 E). Moreover, in vivo at 24 hrs higher T cell activation (CD69, PD-1) was observed in CART22SL from the BM of NALM-6-bearing mice. Conclusions. Here we report the results of two pilot clinical trials evaluating the safety and feasibility of CART22 therapy for r/r B-ALL. Although feasible and with manageable toxicity CART22LL led to modest clinical responses. Preclinical evaluation allowed us to conclude that shortening the linker by 15 amino acids significantly increases the anti-leukemia activity of CART22, possibly by leading to more effective interactions between T cells and their targets. Finally, with the caveats of cross-trial comparison, our data suggest that xenograft models can predict the clinical efficacy of CART products and validate the use of in vivo models for lead candidate selection Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Maude: Novartis Pharmaceuticals: Consultancy, Other: Medical Advisory Boards. Engels: Novartis: Employment. Frey: Novartis: Research Funding. Lacey: Novartis: Research Funding; Genentech: Honoraria. Melenhorst: Novartis: Research Funding. Brogdon: Novartis: Employment. Young: Novartis: Research Funding. Porter: Incyte: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Immunovative Therapies: Other: Member DSMB; Genentech/Roche: Employment, Other: Family member employment, stock ownship - family member; Servier: Honoraria, Other: Travel reimbursement. June: WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Grupp: Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; University of Pennsylvania: Patents & Royalties; Adaptimmune: Consultancy. Gill: Novartis: Patents & Royalties, Research Funding.

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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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CLL B Cells Develop Resistance to Ibrutinib By Reinvigorating the IL-4R - IL-4 Axis Blocked By Bruton's Tyrosine Kinase Inhibitors Including Acalabrutinib and Zanubrutinib

Blood ◽

10.1182/blood-2019-127255 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 477-477

Author(s):

Shih-Shih Chen ◽

Constantine S. Tam ◽

Alan G. Ramsay ◽

Priyadarshini Ravichandran ◽

Natalia C. Couto-Francisco ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Subset ◽

Advisory Committees

Bruton's tyrosine kinases inhibitors (BTKis) represent major advances in CLL therapy. However resistance to this form of therapy is emerging, and such patients often progress more rapidly. Hence there is an important need for therapies that address resistance. Microenvironmental input like IL-4 is critical for CLL disease progression. Compared with normal B cells, CLL cells exhibit significantly higher levels of surface membrane (sm) IL-4 receptor (IL4-R) and contain increased amounts of pSTAT6, a downstream mediator of IL-4R signaling. IL-4 stimulation of CLL B cells suppresses smCXCR4 and increases smIgM, thus promotes CLL cell retention and expansion. In this study, we aimed to examine if smIL-4R expression, IL4R signaling, and IL-4-producing cells are altered in patients sensitive or resistant to BTKis. To do so, T and B cell subset changes were studied overtime in 12 acalabrutinib-treated CLL patients, 6 zanubrutinib-treated CLL patients, 30 ibrutinib-sensitive and 5 ibrutinib-resistant CLL patients, 4 of which exhibited BTK mutations. Consistent with only ibrutinib inhibiting T-cell kinase (ITK), T-cell subset analyses revealed no changes in Th1, Th2, Th17, Th9, and Th22 cells after zanubrutinib or acalabrutinib treatment. In contrast, a Th1-biased T-cell immunity was observed in patients responsive to ibrutinib. In patients progressing on ibrutinib, significantly reduced Th2 T cells were found during the resistant as well as sensitive periods. In an in vitro T-cell function assay using T cells collected before and after the treatment with each BTKi, only ibrutinib treated patients exhibited a reduced ability of T cells to support CLL B cell survival. We next studied changes in CLL B cells, including numbers of IL-4, -10 and -13 producing B cells after BTKi treatment. IL-13 producing CLL B cells were not changed. IL-10 producing CLL B cells were reduced in both ibrutinib sensitive and resistant patients, but not in zanubrutinib or acalabrutinib treated patients. Importantly, IL-4 producing CLL B cells were significantly decreased in patients treated with all 3 BTKi. Significantly reduced smIL-4R levels, impaired IL-4R signaling, decreased smIgM and increased smCXCR4 were also seen in patients treated with each BTKi. To understand the mechanism responsible for inhibition of IL-4 production in CLL cells treated with BTKis, we stimulated CLL cells through IgM, Toll-like receptor and CD40L, finding that only anti-IgM stimulation significantly increased IL-4 production and p-STAT6 induction. We then explored the function of IL-4. IL-4 enhanced CLL B cell survival in vitro and this action was blocked by all 3 BTKis. Moreover, adhesion of CLL B cells to smIL-4R expressing stromal cells was decreased by IL-4 and IL-4R neutralizing antibodies, especially in M-CLL cases. In in vivo studies transferring autologous T cells and CLL PBMCs into alymphoid mice, we found less CLL B cells in mouse spleens post ibrutinib than zanubrutinib or acalabrutinib treatment. This might be due to the suppressed Th2 cells found only in ibrutinib, while IL-4 producing B cells were reduced in all 3 BTKi treated mice. These results support the idea that IL-4 promotes CLL B cell adhesion and growth in tissues. Finally, we investigated the IL-4/IL-4R axis in ibrutinib-resistant patients. Although IL-4 producing T cells remain reduced during the sensitive and resistant phases, CLL B cell production of IL-4 and expression of and signaling through smIL-4R returned when patients developed ibrutinib-resistance. When comparing paired ibrutinib-sensitive and -resistant CLL B cells collected from 3 patients in a xenograft model that requires T cell help, we found ibrutinib-resistant CLL B cells grew in vivo with only minimal (~15%) numbers of autologous T cells compared to B cells collected from ibrutinib-sensitive phase; this suggested a reduced requirement for T-cell help for growth of ibrutinib-resistant CLL cells. In summary, we found IL-4 is a key survival factor in the CLL microenvironment that also improves leukemia cell adhesion to stromal cells expressing smIL-4R. IL-4 production and signaling can be stimulated in CLL B cells through the B-cell receptor, and are consistently blocked by BTKis. Moreover, the recovered ability of ibrutinib-resistant CLL B cells to produce and respond to IL-4 leads to disease progression, suggesting blocking the IL-4/IL-4R axis is a potential treatment for ibrutinib-resistant CLL patients. Disclosures Chen: Pharmacyclics: Research Funding; Beigene: Research Funding; Verastem: Research Funding; ArgenX: Research Funding. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Kolitz:Boeringer-Ingelheim: Research Funding; Roche: Research Funding; Astellas: Research Funding. Zhou:BeiGene: Employment. Barrientos:Genentech: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Rai:Pharmacyctics: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees.

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Predictors of T Cell Expansion and Clinical Responses Following B-Cell Maturation Antigen-Specific Chimeric Antigen Receptor T Cell Therapy (CART-BCMA) for Relapsed/Refractory Multiple Myeloma (MM)

Blood ◽

10.1182/blood-2018-99-119665 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1974-1974 ◽

Cited By ~ 2

Author(s):

Adam D. Cohen ◽

J. Joseph Melenhorst ◽

Alfred L. Garfall ◽

Simon F Lacey ◽

Megan Davis ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Advisory Committees ◽

Car T ◽

Equity Ownership

Abstract Background: Relapsed/refractory (rel/ref) MM is associated with progressive immune dysfunction, including reversal of CD4:CD8 T cell ratio and acquisition of terminally-differentiated T cell phenotypes. BCMA-directed CAR T cells have promising activity in MM, but the factors that predict for robust in vivo expansion and responses are not known. In a phase 1 study of CART-BCMA (autologous T cells expressing a human BCMA-specific CAR with CD3ζ/4-1BB signaling domains) in refractory MM patients (median 7 priors, 96% high-risk cytogenetics), we observed partial response (PR) or better in 12/25 (47%) (Cohen et al, ASH 2017, #505). Recently, we demonstrated in CLL pts receiving CD19-directed CAR T cells that certain T cell phenotypes prior to generation of the CAR T product were associated with improved in vivo expansion and clinical outcomes (Fraietta et al, Nat Med 2018). We thus sought to identify pre-treatment clinical or immunological features associated with CART-BCMA expansion and/or response. Methods: Three cohorts were enrolled: 1) 1-5 x 108 CART cells alone; 2) cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART cells. Phenotypic analysis of peripheral blood (PB) and bone marrow (BM) mononuclear cells, frozen leukapheresis aliquots, and phenotype and in vitro kinetics of CART-BCMA growth during manufacturing were performed by flow cytometry. CART-BCMA in vivo expansion was assessed by flow cytometry and qPCR. Responses were assessed by IMWG criteria. Results: Responses (≥PR) were seen in 4/9 pts (44%, 1 sCR, 2 VPGR, 1 PR) in cohort 1; 1/5 (20%, 1 PR) in cohort 2; and 7/11 (64%, 1 CR, 3 VGPR, 3 PR) in cohort 3. As of 7/9/18, 3/25 (12%) remain progression-free at 11, 14, and 32 months post-infusions. As previously described, responses were associated with both peak in vivo CART-BCMA expansion (p=0.002) as well as expansion over first month post-infusion (AUC-28, p=0.002). No baseline clinical or MM-related characteristic was significantly associated with expansion or response, including age, isotype, time from diagnosis, # prior therapies, being quad- or penta-refractory, presence of del 17p or TP53 mutation, serum hemoglobin, BM MM cell percentage, MM cell BCMA intensity, or soluble BCMA concentration. Treatment regimen given before leukapheresis or CART-BCMA infusions also had no predictive value. We did find, however, that higher CD4:CD8 T cell ratios within the leukapheresis product were associated with greater in vivo CART-BCMA expansion (Spearman's r=0.56, p=0.005) and clinical response (PR or better; p=0.014, Mann-Whitney). In addition, and similar to our CLL data, we found that a higher frequency of CD8 T cells within the leukapheresis product with an "early-memory" phenotype of CD45RO-CD27+ was also associated with improved expansion (Spearman's r=0.48, p=0.018) and response (p=0.047); Analysis of manufacturing data confirmed that higher CD4:CD8 ratio at culture start was associated with greater expansion (r=0.41, p=0.044) and, to a lesser degree, responses (p=0.074), whereas absolute T cell numbers or CD4:CD8 ratio in final CART-BCMA product was not (p=NS). In vitro expansion during manufacturing did associate with in vivo expansion (r=0.48, p=0.017), but was not directly predictive of response. At the time of CART-BCMA infusion, the frequency of total T cells, CD8+ T cells, NK cells, B cells, and CD3+CD56+ cells within the PB or BM was not associated with subsequent CART-BCMA expansion or clinical response; higher PB and BM CD4:CD8 ratio pre-infusion correlated with expansion (r=0.58, p=0.004 and r=0.64, p=0.003, respectively), but not with response. Conclusions: In this study, we found that CART-BCMA expansion and responses in heavily-pretreated MM patients were not associated with tumor burden or other clinical characteristics, but did correlate with certain immunological features prior to T cell collection and manufacturing, namely preservation of normal CD4:CD8 ratio and increased frequency of CD8 T cells with a CD45RO-CD27+ phenotype. This suggests that patients with less dysregulated immune systems may generate more effective CAR T cell products in MM, and has implications for optimizing patient selection, timing of T cell collection, and manufacturing techniques to try to overcome these limitations in MM patients. Disclosures Cohen: Celgene: Consultancy; Novartis: Research Funding; Oncopeptides: Consultancy; Janssen: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Kite Pharma: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Seattle Genetics: Consultancy. Melenhorst:Parker Institute for Cancer Immunotherapy: Research Funding; novartis: Patents & Royalties, Research Funding; Casi Pharmaceuticals: Consultancy; Incyte: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Garfall:Amgen: Research Funding; Kite Pharma: Consultancy; Bioinvent: Research Funding; Novartis: Research Funding. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Vogl:Karyopharm Therapeutics: Consultancy. Pruteanu:Novartis: Employment. Plesa:Novartis: Research Funding. Young:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Consultancy, Patents & Royalties, Research Funding; CRC Oncology: Consultancy; Incysus: Consultancy; Tmunity Therapeutics: Equity Ownership, Research Funding; Brammer Bio: Consultancy; Cure Genetics: Consultancy. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AbbVie, Inc: Research Funding; Janssen: Consultancy. Milone:Novartis: Patents & Royalties.

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Pharmacologic Inhibition of SUMO-Activating Enzyme Potentiates Interferon Response and T Cell-Mediated Anti-Tumor Immunity in Chronic Lymphocytic Leukemia (CLL) and Lymphoma Models

Blood ◽

10.1182/blood-2021-146347 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3719-3719

Author(s):

Vi Lam ◽

Xiaoguang Wang ◽

Scott R Best ◽

Nur Bruss ◽

Tingting Liu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd4 T Cells ◽

Research Funding ◽

Foxp3 Expression ◽

Tcr Signaling ◽

Advisory Committees

Abstract Introduction: CLL is characterized by deficient immunity which clinically manifests as increased predisposition towards malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFκB signaling, IFN response and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP; Clariom S), RNA was harvested after 3 and 24 hours of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 µg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days prior to spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1x10 6 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement (αCD3/CD28). Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFκB and NFAT activation detected by immunoblotting, immunocytochemistry and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4 + T cells following 72 and 96 hours of TCR stimulation vs. control. Meanwhile, expression of CD25, HLA-DR and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced two-fold in TAK-981-treated cells after 24 hours and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 μM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL-2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNγ and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift towards Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4 + naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNγ and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in expression of FoxP3 and an increased production of IFNγ (Figure 1). In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNγ secretion in CD8+ TILs. Conclusion. Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies. Figure 1 Figure 1. Disclosures Siddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Danilov: SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Rigel Pharm: Honoraria; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding.

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Nedd8-Activating Enzyme Inhibition Enhances Anti-Tumor Immunity and PD1 Blockade in In Vivo lymphoma Models

Blood ◽

10.1182/blood-2021-146349 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2414-2414

Author(s):

Xiaoguang Wang ◽

Vi Lam ◽

Dan Vuong ◽

Tingting Liu ◽

Olga Danilova ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Tumor Immunity ◽

Research Funding ◽

Checkpoint Inhibitors ◽

Dependent Manner ◽

Advisory Committees

Abstract Introduction: Immune checkpoint inhibitors have limited single agent activity in B-cell non-Hodgkin lymphoma (NHL). Hence, it is important to develop strategies which will thwart immune evasion in this disease. Neddylation is a sequential enzyme-based process which ultimately regulates protein turnover. In the initial step, NAE activates NEDD8 in an ATP-dependent reaction in which a high-energy thioester bond is formed between NEDD8 and the catalytic cysteine of NAE. Active NEDD8 is then transferred to the NEDD8-specific E2 conjugating enzyme (UBE2M) and is conjugated to cullin proteins which are part of the Cullin-RING E3 ubiquitin ligases (CRL). Pevonedistat (pevo) forms a covalent adduct with NEDD8, thereby inhibiting NAE and thus reduces CRL activity and diminishes ubiquitination and proteasomal degradation of CRL substrates (IκB, HIF-1α, etc). We have recently reported that neddylation regulates T cell activation and polarization (Best et al, Leukemia 2021). Here we investigate how pharmacologic targeting of neddylation modulates anti-tumor immunity using NHL models. Methods: Peripheral blood mononuclear cells were isolated from patients with NHL and T cells were purified using Dynabeads. A20 cells were transplanted into flanks of syngeneic BALB/c mice. When tumors reached 100 mm 3, mice were randomized into groups and treated with pevo 60 mg/kg subcutaneously daily for 10 days or vehicle control. Once moribund, mice were sacrificed, tumors were processed into single-cell suspension and analyzed by flow cytometry. Pevo was provided by Takeda Development Center Americas Inc. (Cambridge, MA). Results: Primary patient-derived CD3/28-stimulated CD3 + T cells exhibited upregulation of TNFα and IFNγ in vitro in the presence of pevo. Concurrently, we observed increased expression of PD-1 and CTLA-4. Pre-treatment of T cells with pevo enhanced killing of NHL cell lines (JeKo-1, Mino, Maver-1 and VAL) in allogeneic cytotoxicity assays. Expectedly, treatment with pevo resulted in increased expression of HIF-1α in TCR-stimulated T cells. shRNA-mediated knockdown of HIF-1α abrogated the pevo effect, suggesting that NAE inhibition modulates T cell function in HIF-1α-dependent manner. While A20 cells showed resistance in vitro, treatment with pevo delayed lymphoma progression in A20 mice in vivo (Fig 1A). This was accompanied by an increase of tumor-infiltrating lymphocytes (TILs; Fig 1B). CD8 + TILs from pevo-treated mice exhibited activated phenotype as manifested by increased secretion of IFNγ (Fig 1C). Meanwhile, expression of the exhaustion molecules CTLA-4 and PD-1 by CD4/CD8 + TILs remained unchanged. To further investigate the role of T-cell immunity in this setting, we employed 1) CD8 depletion by pre-treatment with 12.5 mg/kg anti-CD8 antibody (IV); or 2) CRISPR/Cas9-mediated knockout of β2-microglobulin (MHC class I protein) in A20 cells. Either approach led to a partial decrease of pevo efficacy in vivo compared with respective controls. To exclude tumor-intrinsic effect of NAE inhibition, we knocked down UBE2M in A20 cells. Loss of UBE2M had no effect on growth of control tumors, or pevo therapeutic effect, implying that the anti-tumor efficacy of NAEi was T cell-mediated in this model. Since pevo modulates PD-1 on human T cells, we explored its effect on PD-L1 expression. Treatment with pevo upregulated PD-L1 expression in A20 cells in a MYC-dependent manner. Hence, we explored a combination of pevo and αPD-1 blockade in A20 model. Combination treatment significantly increased the CD4 + and CD8 + TILs. A decrease in tumor growth was significantly more pronounced than with either drug alone (Fig 1A). The combination benefit was fully reversed by loss of B2M, again highlighting the importance of immune mechanism . We observed expansion of IL-2, IL-4 and IL-17-secreting CD4+ TILs following the combined treatment, compared with either drug alone. In addition, CD4+ and CD8+ TILs derived from these mice secreted high levels of IFNγ (Fig. 1C). Conclusions: NAE inhibition enhanced T cell-mediated cytotoxicity in vitro. Treatment with pevo promoted activation of TILs and restricted tumor growth in an A20 mouse lymphoma model. Pevo-treated tumors were sensitized to αPD-1 . Thus, targeting NAE enhances anti-tumor immunity. Our data provide a strong rationale for future studies of pevo in combination with immune checkpoint inhibitors in lymphoma and other tumors. Figure 1 Figure 1. Disclosures Siddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding. Berger: Takeda Development Center Americas, Inc.: Current Employment. Danilov: Bayer Oncology: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; TG Therapeutics: Consultancy, Research Funding; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Takeda Oncology: Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria.

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Constitutive and Immunoproteasome Inhibitors Increase IκB and Decrease NF-κB Expression In Dendritic Cell

Blood ◽

10.1182/blood.v122.21.3493.3493 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3493-3493

Author(s):

Ahmad-Samer Samer Al-Homsi ◽

Zhongbin Lai ◽

Tara Sabrina Roy ◽

Niholas Kouttab

Keyword(s):

Multiple Myeloma ◽

T Cell ◽

Cell Lines ◽

Mechanism Of Action ◽

Peripheral Blood ◽

Research Funding ◽

Myeloma Cell ◽

Multiple Myeloma Cell

Abstract Introduction Constitutive and immunoproteasome inhibitors (C&IPI) were thought to suppress nuclear factor-κB (NF-κB) pathway by preventing IκB degradation, which prevents NF-κB translocation into the nucleus. This mechanism of action has since been questioned by a number of studies. First, bortezomib promoted constitutive NF-κB activity in endothelial cell carcinoma. Second, NF-κB constitutive activity was resistant to bortezomib in multiple myeloma cell lines. Third, bortezomib increased IκB mRNA but post-transcriptionally downregulated IκB in normal cells and in multiple myeloma cell lines resulting in induced canonical NF-κB activation. Lastly, bortezomib increased nuclear levels of IκB as opposed to lowering cytoplasmic levels in cutaneous T cell lymphoma cell line suggesting that nuclear translocation of IκB was possibly responsible for NF-κB inhibition. The inhibitory activity of C&IPI on dendritic cells (DC) is of interest in the prevention of graft versus host disease (GvHD). It has been shown that different C&IPI impede DC maturation and T cell priming both in vitro and in vivo. Herein we sought to understand the mechanism of action of proteasome and immunoproteasome inhibitors on DC and to test their effect on IκB and NF-IκB expression. Materials and Methods We first performed RT PCR on lysates of DC obtained from the peripheral blood of 7 patients who received post-transplant cyclophosphamide and bortezomib as prevention of GvHD on a phase I clinical trial. Patients received allogeneic transplantation from matched-related or unrelated donors. Patients received no other immunosuppressive therapy except for rabbit anti-thymocyte globulin for those receiving graft from unrelated donor. Steroids were not allowed on the study. Samples were obtained on days +1, +4, and +7. The results were analyzed in comparison to samples obtained on day 0 before stem cell infusion. We then performed the same experiment on lysates of DC obtained from the peripheral blood of healthy volunteer donors. DC were untreated or incubated with bortezomib (10 nM for 4 h), carfilzomib (30 nM for 1 h), oprozomib (100 nM and 300 nM for 4 h), ONX 0914 (200 nM for 1 h), PR-825 (125 nM for 1 h), or PR-924 (1000 nM for 1 h). The drug concentration and duration of exposure were chosen based on the IC50 on proteasome activity and to reproduce in vivo conditions. We also performed IκB western blot on DC isolated from peripheral blood of healthy volunteers, untreated or incubated with bortezomib (10 nM for 4 h) or oprozomib (300 nM for 4 h). Each experiment was performed at least in triplicate. Results We found that the combination of cyclophosphamide and bortezomib significantly and progressively increased IκB mRNA while decreasing NF-κB mRNA in DC studied ex vivo. We also found that all studied C&IPI increased IκB mRNA to a variable degree while only oprozomib (300 nM) decreased NF-κB mRNA in DC in vitro. Finally, both bortezomib and oprozomib increased IκB protein level in DC in vitro (figure). Conclusion Our data suggest that C&IPI increase IκB expression in DC. As opposed to the previously reported data in other cell types, the effect is not associated with post-transcriptional downregulation. Cyclophosphamide and bortezomib also decrease NF-κB expression in DC in vivo while only oprozomib had the same effect in vitro. The effect of C&IPI on IκB and NF-κB expression may represent a new mechanism of action and suggests their effect may be cell-type dependent. Disclosures: Al-Homsi: Millennium Pharmaceuticals: Research Funding. Off Label Use: The use of cyclophosphamide and bortezomib for GvHD prevention. Lai:Millennium Pharmaceuticals: Research Funding.

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Gabarap Loss Mediates Immune Escape in High Risk Multiple Myeloma

Blood ◽

10.1182/blood-2021-148621 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 891-891

Author(s):

Annamaria Gulla ◽

Eugenio Morelli ◽

Mehmet K. Samur ◽

Cirino Botta ◽

Megan Johnstone ◽

...

Keyword(s):

Immune Response ◽

T Cell ◽

Clinical Outcome ◽

Board Of Directors ◽

Research Funding ◽

Immune Escape ◽

Publicly Traded ◽

Advisory Committees

Abstract Immune therapies including CAR T cells and bispecific T cell engagers are demonstrating remarkable efficacy in relapsed refractory myeloma (MM). In this context, we have recently shown that proteasome inhibitor bortezomib (BTZ) results in immunogenic cell death (ICD) and in a viral mimicry state in MM cells, allowing for immune recognition of tumor cells. Induction of a robust anti-MM immune response after BTZ was confirmed both in vitro and in vivo: treatment of 5TGM1 MM cells with BTZ induced tumor regression associated with memory immune response, confirmed by ELISPOT of mouse splenocytes. We have confirmed the obligate role of calreticulin (CALR) exposure in phagocytosis and the ICD process, since BTZ-induced ICD is impaired in CALR KO MM cells both in vitro and in vivo. We further showed that the therapeutic efficacy of BTZ in patients was correlated with ICD induction: BTZ-induced ICD signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Together, these studies indicate that ICD is associated with long-term response after BTZ treatment. In this work, we reasoned that genomic or transcriptomic alterations associated with shorter survival of MM patients after BTZ treatment may impair activation of the ICD pathway. To this aim, we performed a transcriptomic analysis of purified CD138+ cells from 360 newly diagnosed, clinically-annotated MM patients enrolled in the IFM/DFCI 2009 study. By focusing on genes involved in the ICD process, we found that low levels of GABA Type A Receptor-Associated Protein (GABARAP) were associated with inferior clinical outcome (EFS, p=0.0055). GABARAP gene locus is located on chr17p13.1, a region deleted in high risk (HR) MM with unfavorable prognosis. Remarkably, we found that correlation of low GABARAP levels with shorter EFS was significant (p=0.018) even after excluding MM patients with del17p; and GABARAP is therefore an independent predictor of clinical outcome. GABARAP is a regulator of autophagy and vesicular trafficking, and a putative CALR binding partner. Interestingly, among a panel of MM cell lines (n=6), BTZ treatment failed to induce exposure of CALR and MM cell phagocytosis by DCs in KMS11 cells, which carry a monoallelic deletion of GABARAP. This effect was rescued by stable overexpression of GABARAP. Moreover, CRISPR/Cas9-mediated KO of GABARAP in 3 ICD-sensitive cell lines (AMO1, H929, 5TGM1) abrogated CALR exposure and ICD induction by BTZ. GABARAP add-back by stable overexpression in KO clones restored both CALR exposure and induction of ICD, confirming GABARAP on-target activity. Similarly, pre-treatment of GABARAP KO cells with recombinant CALR restored MM phagocytosis, further confirming that GABARAP impairs ICD via inhibition of CALR exposure. Based on these findings, we hypothesized that GABARAP loss may alter the ICD pathway via CALR trapping, resulting in the ICD resistant phenotype observed in GABARAP null and del17p cells. To this end, we explored the impact of GABARAP KO on the CALR protein interactome, in the presence or absence of BTZ. Importantly, GABARAP KO produced a significant increase of CALR binding to stanniocalcin 1 (STC1), a phagocytosis checkpoint that mediates the mitochondrial trapping of CALR, thereby minimizing its exposure upon ICD. Consistently, GABARAP KO also affected CALR interactome in BTZ-treated cells, which was significantly enriched in mitochondrial proteins. Importantly, co-IP experiments confirmed GABARAP interaction with STC1. These data indicate a molecular scenario whereby GABARAP interacts with STC1 to avoid STC1-mediated trapping of CALR, allowing for the induction of ICD after treatment with ICD inducers; on the other hand, this mechanism is compromised in GABARAP null or del17p cells, and the STC1-CALR complex remains trapped in the mitochondria, resulting in ICD resistance. To functionally validate our findings in the context of the immune microenvironment, we performed mass Cytometry after T cell co-culture with DCs primed by both WT and GABARAP KO AMO1 clones. And we confirmed that treatment of GABARAP KO clones with BTZ failed to activate an efficient T cell response. In conclusion, our work identifies a unique mechanism of immune escape which may contribute to the poor clinical outcome observed in del17p HR MM patients. It further suggests that novel therapies to restore GABARAP may allow for the induction of ICD and improved patient outcome in MM. Disclosures Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Richardson: AstraZeneca: Consultancy; Regeneron: Consultancy; Protocol Intelligence: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; Karyopharm: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Munshi: Legend: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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

Blood ◽

10.1182/blood-2018-99-115319 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1886-1886 ◽

Cited By ~ 5

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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The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-130197 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3202-3202

Author(s):

Cameron S. Bader ◽

Henry Barreras ◽

Casey O. Lightbourn ◽

Sabrina N. Copsel ◽

Dietlinde Wolf ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Activation ◽

Cd8 T Cell ◽

Advisory Committees ◽

Hematopoietic Stem

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells, leading to damage of particular host tissues characteristic of GVHD. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to pro-inflammatory cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) aHSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of MUD C3H.SW bone marrow (BM) + T cells into irradiated B6 wildtype (WT) or STING-/- recipients. Colon tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs WT. MUD STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 wks post-HSCT vs WT. Double chimerism studies showed that the absence of STING in non-hematopoietic cells was responsible for GVHD amelioration. Conversely, a single dose of the highly specific STING agonist DMXAA given in vivo increased IFNβ, TNFα and IL-6 mRNA expression in WT, but not STING-/-, colon tissue 48 hrs after transplant and increased GVHD scores and lethality post-HSCT. Post-transplant cytoxan treatment abolished the ability of DMXAA to augment GVHD, supporting the notion that STING signaling increases donor T cell activation during aHSCT. To evaluate the potential impact of STING in the clinical setting, we transplanted C3H.SW BM + T cells into mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) and found that these mice also exhibited diminished GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD aHSCT contrasts to reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) aHSCT (Fischer J, et al, Sci. Transl. Med. 2017). Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we hypothesized that STING's effect on the predominant T cell subset in each model may explain these seemingly paradoxical results in STING-/- vs WT recipients. Therefore, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING-/- mice and found that - in contrast to MMUD recipients of combined CD4+ and CD8+ T cells - STING-/- recipients developed lower GVHD clinical scores, reduced skin pathology and had lower frequencies of activated T cells 8 wks post-HSCT vs WT, supporting a role for STING in the promotion of CD8+ T cell-mediated GVHD. Next, we investigated if recipient APCs played a role in STING's enhancement of CD8+ T cell-mediatedGVHD. We found that STING-/- mice had greater frequencies and numbers of recipient splenic CD11b+CD11c+ APCs 1 day after MMUD B6 into BALB/c aHSCT (Fig. A). BALB/c-STING-/- APCs also expressed reduced MHC class I protein levels (Fig. B). Moreover, STING-/- recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C). These data support the hypothesis that STING contributes to early activation of donor CD8+ T cells and elimination of recipient APCs. Next, to identify if the loss of host MHC II+ APCs affected subsequent donor CD4+ T cell activation, B6-Nur77GFP transgenic donor T cells were used to explicitly monitor T cell receptor signaling. Consistent with increased numbers of host MHC II+ APCs in the spleens of STING-/- recipients 1 day post-aHSCT, we found greater frequencies and numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ in STING-/- spleens 6 days after transplant (Fig. D). In summary, our studies demonstrate that STING plays an important role in regulating aHSCT and provide one potential mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Overall, our studies suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Blazar: BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding. Levy:Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

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