Transcriptomic Analysis of CD4+ T Cell Dysfunction during Gvhd: Evidence for Profound Reprograming of T Cell Signaling during Acute Gvhd That Is Controlled during CD28:CD80/86 Costimulation Blockade with Abatacept

Although acute graft-versus-host-disease (AGVHD) is one of the major causes of non-relapse mortality after hematopoietic stem cell transplant (HCT), we are still unable to predict which patients will develop the most severe form of this disease, or which molecular pathways are dysregulated in the T cells that cause disease. Thus, understanding the molecular features of AGVHD is a critical unmet need. To address this, we have performed a companion mechanistic study as a part of our completed Phase 2 trial of abatacept, a CD28:CD80/86 costimulation blockade agent, for severe AGVHD prevention (Clinicaltrials.gov # NCT01743131, 'ABA2'). ABA2 has demonstrated significant improvement in AGVHD in patients prophylaxed with abatacept in addition to calcineurin inhibition (CNI) + Methotrexate (MTX) compared to controls receiving CNI/MTX alone. To begin to uncover mechanisms responsible for the control of AGVHD with abatacept, and given that CD4+ T cells have been consistently documented to be the main therapeutic target of this drug, we interrogated the transcriptome of CD4+ T cells reconstituting in patients prophylaxed with abatacept compared to CNI/MTX. To perform this analysis, we flow cytometrically sorted CD4+ T cells on Days 21-28 post-transplant from all patients on ABA2, as well as a cohort of 12 untransplanted healthy controls, and subsequently performed mRNA-sequencing on these cells. Weighted Gene Correlation Network Analysis (WGCNA) was performed on the top 6000 most variant transcripts from the resulting sequencing data. Hierarchical clustering of the WGCNA co-expression matrix enabled the identification of self-assembling modules (SAMs) that met a threshold of coexpression (Figure 1A). For the ABA2 dataset, we considered the following variables in the WGCNA model: patient cohort (7/8 patients, 8/8 patients, healthy controls), +/- prophylaxis with abatacept, CMV reactivation, EBV reactivation, Grade of GVHD (0-4), relapse, non-relapse mortality, and all-cause mortality. The WGCNA clustering analysis resulted in the identification of 4 discrete SAMs, which were highly correlated with clinical variable metamodules. This analysis revealed a strong positive correlation of a 476-gene SAM (the Turquoise module) in patients prophylaxed with CNI/MTX + placebo and anti-correlation of this module in patients prophylaxed with CNI/MTX + abatacept, as demonstrated in both the WGCNA heatmap and through Gene Set Enrichment Analysis (Figure 1 A-B). These opposing correlations suggested that interrogation of this module would reveal mechanistic correlates with standard prophylaxis that were decoupled by abatacept. Pathway analysis using the Reactome database (Figure 1C) revealed the turquoise SAM to be dominated by four types of pathways: (1) Those that define canonical cell-cycle pathways (2) Those involved in T cell metabolism (3) Those involved in apoptosis and (4) Those involved in T cell activation, consistent with upregulation of these transcripts in placebo versus abatacept patients. In addition to being highly correlated with patients receiving placebo, the expression of a subset of the transcripts in the Turquoise module were also directly correlated with the severity of AGVHD in these patients. Thus, linear regression analysis of the 476 transcripts in this module identified a subset of 93 genes for which transcript expression level was increased both in placebo compared to abatacept, and for which expression level also positively correlated with Grade of AGVHD. As with the Turquoise module as a whole, this subset of genes also formed a highly correlated network, linking transcripts involved in T cell proliferation, apoptosis, activation, metabolism as well as the T cell checkpoint (Figure 1D). This analysis represents the first comprehensive interrogation of the transcriptomic correlates of AGVHD. It identifies a novel set of transcripts which positively associate with the severity of AGVHD, and which costimulation blockade with abatacept down-regulates and de-couples from AGVHD severity. These results suggest a profound reprograming of T cell activation with abatacept that is correlated with the control of AGVHD. Disclosures Qayed: Bristol-Myers Squibb: Honoraria. Langston:Astellas Pharma: Other: Research Support; Incyte: Other: Research Support; Jazz Pharmaceuticals: Other: Research Support; Chimerix: Other: Research Support; Takeda: Other: Research Support; Kadmon Corporation: Other: Research Support; Novartis: Other: Research Support; Bristol Myers Squibb: Other: Research Support. Blazar: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; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: 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; Regeneron Pharmaceuticals: 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. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Kymab: Consultancy; Jazz: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding. OffLabel Disclosure: Abatacept: Approved for Rheumatoid Arthritis; used in this trial for prevention of GVHD.

Download Full-text

CD4+ T Cell Fate in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117674 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4494-4494

Author(s):

Rachel Elizabeth Cooke ◽

Jessica Chung ◽

Sarah Gabriel ◽

Hang Quach ◽

Simon J. Harrison ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Flow Cytometric Analysis ◽

Advisory Committees ◽

Mitochondrial Mass ◽

Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

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.

Download Full-text

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.

Download Full-text

CD20-TCB, a Novel T-Cell-Engaging Bispecific Antibody, Induces T-Cell-Mediated Killing in Relapsed or Refractory Non-Hodgkin Lymphoma: Biomarker Results From a Phase I Dose-Escalation Trial

Blood ◽

10.1182/blood-2019-123979 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5319-5319 ◽

Cited By ~ 2

Author(s):

Ann-Marie E Bröske ◽

Ian James ◽

Anton Belousov ◽

Enrique Gomez ◽

Marta Canamero ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Board Of Directors ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Introduction: CD20-TCB (RG6026) is a novel T-cell-engaging bispecific (TCB) antibody with a '2:1' molecular format that comprises two fragment antigen binding regions that bind CD20 (on the surface of B cells) and one that binds CD3 (on the surface of T cells). CD20-TCB offers the potential for increased tumor antigen avidity, rapid T-cell activation, and enhanced tumor cell killing versus other bispecific formats. The safety, tolerability, pharmacokinetics, biomarkers, and antitumor activity of CD20-TCB are currently being investigated in a multicenter Phase I dose-escalation trial (NP30179; NCT03075696). We recently presented preliminary clinical data demonstrating promising clinical activity in relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) patients with indolent or aggressive disease (Dickinson et al. ICML 2019). Here, we present preliminary blood and tissue biomarker analyses to explore modes of action, support optimal biological dose selection, and identify potential outcome predictors. Methods: For biomarker analyses, we performed immune profiling of peripheral blood by flow cytometry, analyzed plasma cytokine levels by ELISA, and characterized baseline and on-treatment tumor biopsies by immunohistochemistry/immunofluorescence assays and RNA sequencing. Biomarker data were obtained from 122 patients dosed with 0.005-25mg CD20-TCB. Results: CD20-TCB infusion led to a rapid and transient reduction in T cells in the peripheral circulation (T-cell margination) in all patients. T-cell margination reached nadir 6 hours after the first CD20-TCB infusion, and showed a strong association with CD20-TCB dose and receptor occupancy (RO%; as determined by Djebli et al. ASH 2019). Interestingly, rebound of T cells 160 hours after the first CD20-TCB infusion was associated with response to treatment. Responding patients showed long-term T-cell activation after the first infusion of CD20-TCB at doses from 0.6mg and above. T-cell activation was demonstrated by 2-4-fold elevation of T-cell activation markers such as Ki67, HLA-DR, PD-1, ICOS, OX40, and 4-1BB, which was sustained up to Cycle 5 (105 days). Analysis of paired pre- and on-treatment tumor biopsies (n=6) obtained before and 2-3 weeks after the first dose of CD20-TCB showed evidence of T-cell-mediated tumor cell killing. Analysis of archival and pre-treatment tumor biopsies (n=80) revealed that clinical responses were achieved irrespective of the amount of tumor T-cell infiltration at baseline. In contrast, preliminary baseline bulk tumor RNA sequencing data (n=46) showed upregulation of gene signatures associated with cell proliferation/Myc and T-cell subsets (effector vs exhausted-like) in non-responding patients. Conclusions: In this study, we demonstrated the mode of action of CD20-TCB, a novel bispecific antibody with promising clinical activity in R/R NHL. We also demonstrated that biomarker data on T-cell activation can support dose finding in conjunction with pharmacokinetics. Additional analysis is ongoing to evaluate response predictors and better characterize the population that will benefit most from T-cell mediated therapies. Disclosures Bröske: Roche: Employment, Equity Ownership. James:A4P Consulting Ltd: Consultancy. Belousov:Roche: Employment. Gomez:F. Hoffmann-La Roche Ltd: Employment. Canamero:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Ooi:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Grabole:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Wilson:F. Hoffmann-La Roche Ltd: Employment. Korfi:F. Hoffmann-La Roche Ltd: Consultancy. Kratochwil:F. Hoffmann-La Roche Ltd: Employment. Morcos:Roche: Employment, Equity Ownership. Ferlini:Roche: Employment, Equity Ownership. Thomas:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Dimier:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Moore:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Bacac:Roche: Employment, Equity Ownership, Patents & Royalties: Patents, including the one on CD20-TCB. Weisser:Pharma Research and Early Development Roche Innovation Center Munich: Employment, Equity Ownership, Patents & Royalties. Dickinson:Merck Sharpe and Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: CD20-TCB (also known as RG6026, RO7082859) is a full-length, fully humanized, immunoglobulin G1 (IgG1), T-cell-engaging bispecific antibody with two fragment antigen binding (Fab) regions that bind to CD20 (on the surface of B cells) and one that binds to CD3 (on the surface of T cells) (2:1 format). The 2:1 molecular format of CD20-TCB, which incorporates bivalent binding to CD20 on B cells and monovalent binding to CD3 on T cells, redirects endogenous non-specific T cells to engage and eliminate malignant B cells. CD20-TCB is an investigational agent.

Download Full-text

The Ratio of Costimulatory Vs Coinhibitory Molecules on AML Cells Determines the CD33-BiTE® Mediated T-Cell Response

Blood ◽

10.1182/blood-2018-99-116297 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1349-1349

Author(s):

Anetta Marcinek ◽

Bettina Brauchle ◽

Dragica Udiljak ◽

Roman Kischel ◽

Peter Kufer ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

T Cell Proliferation ◽

Target Cells ◽

Advisory Committees

Abstract Bispecific T-cell engagers (BiTE® antibody constructs) represent a novel immunotherapeutic strategy relying on the recruitment of T cells against tumor cells independent of TCR specificity. In Acute Myeloid Leukemia (AML), CD33 represents a suitable target antigen with high expression levels in >90 % of primary AML samples (Krupka et al, 2014). A CD33-BiTE® antibody construct (AMG 330) was developed mediating cytotoxicity against primary AML in vitro although to a variable degree (Krupka et al, 2016). Several parameters have been identified which modulate AMG 330-mediated cytotoxicity, including CD33 expression level as well as effector to target cell (E:T) ratio. However, the exact mechanism of T-cell activation through BiTE® antibody constructs is only partly understood. Physiological T-cell activation is based on engagement of the T-cell receptor complex together with costimulatory molecules whereas the absence of positive costimulation leads to T-cell anergy. In line with this concept, we hypothesized that BiTE®-mediated cytotoxicity requires positive costimulatory signals on the target cells for T-cell activation. We hypothesize that the ratio of costimulatory and coinhibitory molecules on AML cells determines the susceptibility to AMG 330-mediated cytotoxicity independent of target antigen expression level. A stable expression system was established utilizing murine Ba/F3 cells expressing human CD33 ± CD80 ± CD86 ± PD-L1. Co-cultures of Ba/F3 constructs and T cells were performed in presence of AMG 330 or a control BiTE® (cBiTE®) (5 ng/ml). For some experiments, T cells were separated into naive (CD45RA+/CCR7+) vs memory (CD45RADIM) cells using fluorescence-activated cell sorting. After 3 days, specific lysis was determined by flow cytometry and calculated as % specific lysis = 100 × (1 - live CD33+ cellsAMG 330 / live CD33+ cellscBiTE). T-cell proliferation was defined as number of CD2+ cells on day 3 compared to day 0. The expression pattern of CD33, CD80, CD86 and PD-L1 on primary AML cells was evaluated by specific fluorescence intensity (SFI) using multiparameter flow cytometry. A sample was considered positive at an SFI of > 1.5. Characterized primary AML patient samples were used in a long-term culture assay to determine the influence of the checkpoint molecule expression profile on AMG 330-mediated cytotoxicity. CD33 single positive Ba/F3 cells were not lysed upon the addition of AMG 330 and allogeneic T cells. Cytotoxicity could be restored by expression of CD80, CD86 and CD80+CD86 with following tendency: CD80+CD86 >> CD80 > CD86 (see table 1). There was a direct correlation of T-cell proliferation to AMG 330 mediated cytotoxicity. Memory T cells showed increased cytotoxicity compared to naive T cells against the different Ba/F3 cell lines. The influence of co-inhibition was investigated by additionally transducing PD-L1 into the different Ba/F3 cells. This led to a reduced AMG 330-mediated cytotoxicity in all PD-L1 expressing Ba/F3 cells (Table 1). This was accompanied by a reduction in T-cell proliferation. Looking at the expression profile of CD80 and CD86 in primary AML samples, we observed expression of CD80 in 7/123 and of CD86 in 188/226 of cases (respectively 5.7 % and 83.2 %). When comparing AMG 330-mediated cytotoxicity against primary AML cells for patient pairs with similar CD33 expression levels, a higher CD86/PD-L1 ratio led to an increased AMG 330-mediated cytotoxicity compared to patient samples with a lower CD86/PD-L1 ratio (exemplary data: SFI CD33+: 81.7; SFI-ratio CD86/PD-L1: 4; specific cytotoxicity: 64.2 % vs. SFI CD33+: 89.5; SFI-ratio CD86/PD-L1: 15.9; specific cytotoxicity: 96.4 %). In summary, this data supports the hypothesis that AMG 330-mediated cytotoxicity and T-cell proliferation are influenced by the ratio of costimulatory and coinhibitory molecules on AML cells. Our data supports the notion that the checkpoint profile on AML, rather than one molecule by itself, determines T-cell response to AMG 330. Prospective analyses in clinical trials are needed to validate the relevance of checkpoint molecules on target cells as a predictive biomarker for response. Disclosures Marcinek: AMGEN Research Munich: Research Funding. Brauchle:AMGEN Inc.: Research Funding. Kischel:AMGEN: Employment. Kufer:AMGEN Research Munich: Employment. Subklewe:Pfizer: Membership on an entity's Board of Directors or advisory committees; Roche AG: Research Funding; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text

Intra-Tumoral CD8+ T-Cells in Follicular Lymphoma Contain Large Clonal Expansions That Are Amenable to Dual-Checkpoint Blockade

Blood ◽

10.1182/blood-2019-130050 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2793-2793 ◽

Cited By ~ 1

Author(s):

Karthik Nath ◽

Soi C. Law ◽

Muhammed B. Sabdia ◽

Lilia Merida De Long ◽

Mohamed Shanavas ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd4 T Cells ◽

Research Funding ◽

Checkpoint Blockade ◽

T Cell Subsets ◽

Advisory Committees ◽

Cell Clones ◽

Tcr Repertoire

Introduction. Intra-tumoral T-cell infiltration is associated with R-CHOP responsiveness in aggressive B-cell lymphoma (Keane, Lancet Haem 2015). These patients also have a broad (i.e. diverse) intra-tumoral T-cell receptor (TCR) repertoire with a ~20% superior survival compared to those with a narrow (i.e. clonal) repertoire after R-CHOP therapy. Here, the major contributor to the TCR clonal expansion were CD8+ T cells (Keane, CCR 2017). Paradoxically, our recent results in Follicular Lymphoma (FL) (Tobin, JCO in press) found that clonal T-cell expansions were markedly enriched in those patients that experienced progression of disease within 24 months (POD24). Given that FL is a histological subtype associated with a tumor microenvironment distinct from DLBCL including numerous CD4+ T-follicular helper cells (TFH), we now expand upon these findings by comparing TCR repertoires across histological subtypes. We then established whether the TCR repertoire in FL is related to differential TCR clonal expansions between different T-cell subsets and immune checkpoints. Finally, the overlap between tissue and blood TCR repertoires was investigated. Methods. Firstly, unbiased, high-throughput TCRβ sequencing (ImmunoSEQ, Adaptive Biotechnologies) was compared in 164 FFPE tissues (12 healthy nodes, 40 FL, 88 DLBCL, and as a comparator tumor known to be sensitive to checkpoint blockade and to have a high neoantigen burden, 24 melanoma tissues). Next, to determine the contribution of individual T-cell subsets to overall clonality, a further 21 fresh de-aggregated/cryopreserved FL tumor samples were FACS sorted into four T-cell groupings: CD8+ cytotoxic T-lymphocytes (CTLs), CD4+ TFH, CD4+ regulatory T-cells (TREGs) and 'other' (non-TFH/TREG) CD4+ T-cells. Flow cytometry quantified the expression of the checkpoints LAG3, TIM3 and PD1. Then, 5 FL paired tissue/blood samples were tested for shared TCR clones. Results. FL exhibited strikingly reduced TCR repertoire clonality (higher diversity) compared to DLBCL, melanoma and healthy lymph nodes (Fig 1A). Analysis of de-aggregated sorted nodal T-cells revealed a more complex TCR repertoire. The outcome measure was median clonality index (CIx ranging from '0' or minimal, to '1' or maximal clonality). Large T-cell clones in FL (CIx=0.12) predominantly resided within the CTL subset (34% all T-cells). By contrast, there was marked T-cell diversity in TFH (CIx=0.04; 27% all T-cells), TREG (CIx=0.02; 7% all T-cells) and 'other' CD4+ T-cells (CIx=0.02; 32% all T-cells) (Fig 1B). The CTL population had a bimodal expression for PD1 (+51%/-49%), a marker in FL that has been shown to remain functionally active unless co-expressed with LAG3 and/or TIM3 (Yang, Oncotarget 2017). These dual-checkpoint expressing CTLs have reduced capacity to produce cytokines or lytic granules (i.e. they are 'exhausted'). Notably, 54% of the PD1+ CTLs co-expressed either LAG3 or TIM3. Put together, these results are consistent with expanded CTL clones that are frequently functionally exhausted. In contrast, TFH, TREG and 'other' CD4+ T-cells had a low expression of LAG3 and TIM3, although PD1 was frequently found (as expected, particularly in the TFH cells). Finally, in paired tissue/blood samples, there was weak overlap between the circulating and intra-tumoral TCR repertoire in CTLs and TFH T-cells. Conclusion. Although FL has a markedly less clonal TCR repertoire compared to DLBCL, melanoma and even healthy nodes, this result is misleading. Detailed analysis on sorted intra-tumoral T-cell subsets in FL revealed large clonal expansions in CTLs, with approximately half of these classified as functionally exhausted (dual-positive for PD1 and LAG3 and/or TIM3), a state potentially amenable to reversal by dual-checkpoint blockade. The explanation for TCR repertoire diversity lies in CD4+ T-cells (representing approximately two-thirds of T-cells, including the large TFH subset). T-cells in blood did not reflect FL tissue T-cell clones, further highlighting the need for sorted intra-tumoral nodal tissues to accurately assess TCR repertoires in FL. Further characterization of the neo-antigenic targets that CTL clones potentially recognize is required. These results have implications for therapeutic vaccine design and selective recruitment of patients for immune checkpoint blockade. Disclosures Keane: MSD: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Roche: Consultancy, Other: Travel Grant; BMS: Research Funding. Gandhi:Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding.

Download Full-text

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

Blood ◽

10.1182/blood.v126.23.117.117 ◽

2015 ◽

Vol 126 (23) ◽

pp. 117-117 ◽

Cited By ~ 8

Author(s):

Anja Seckinger ◽

Jose Antonio Delgado ◽

Laura Moreno ◽

Brigitte Neuber ◽

Anna Grab ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Mechanism Of Action ◽

Research Funding ◽

Advisory Committees

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.

Download Full-text

Galectin-3 Signaling in Donor T Cells Regulates Acute Graft Versus Host Disease (aGvHD) after Allogenic Transplantation

Blood ◽

10.1182/blood-2021-150777 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2765-2765

Author(s):

Hemn Mohammadpour ◽

Takemasa Tsuji ◽

Cameron R. MacDonald ◽

Joseph L. Sarow ◽

Jingxin Qiu ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd4 T Cells ◽

Research Funding ◽

T Cell Proliferation ◽

Advisory Committees ◽

Human T Cells ◽

Donor T Cells

Abstract Galectin-3 (Gal-3) is a unique member of the galectin family of lectins. Gal-3 possesses immune-regulatory functions depending on the immune cell and the immunologic situation. There are no studies that specifically delineate the role of Gal-3 in the setting of acute GvHD but mounting research suggests that dysregulation of pathways involving the galectin family may contribute to the pathogenesis of other immune disorders. Gal-3 is expressed by many types of immune cells, including T-cells. It suppresses signaling downstream of the TCR, decreases effector T-cell cytokine production, but increases the development and differentiation of memory T cells, myeloid cells, and macrophages. We investigated the mechanisms and downstream events of Gal-3 signaling in donor T cells after Allo-HCT, using Gal-3 knockout (Gal-3 -/-) mice. We further studied the effect of Gal-3 in controlling aGvHD incidence and severity while preserving the Graft-versus Leukemia (GvL) effect by overexpressing Gal-3 in human T cells. We utilized both a major MHC-mismatch (C57B/6 (H-2 b) into BALB/c (H-2 k) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2 b) into C3H/SW (H-2 b) model. Lethally irradiated recipient BALB/c and C3H/SW WT animals were injected with T cell depleted bone marrow alone (3 ×10 6) or with splenic T cells derived from allogeneic WT or Gal-3 -/- B6 donors (0.7 × 10 6 T cells in B6 → BALB/c and 1.5 × 10 6 in B6 → C3H/SW). We found that donor T cells express Gal-3 after Allo-HCT and that Gal-3 expression in WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, decreased clinical GvHD scores, and longer survival when compared to mice receiving Gal-3 -/- donor T cells (Figure 1A). We studied the mechanisms by which Gal-3 signaling controls the severity of aGvHD. Using flow cytometry analysis, we determined that Gal-3 plays a critical role in T cell proliferation and exhaustion. Gal-3 -/- T cells have a cytotoxic T phenotype with increased IFN-ℽ and GM-CSF production in T cells from the spleen and liver tissues on days 7 and 14 after Allo-HCT when compared to WT T cells (Figure 1B). There was a significant increase in T cell proliferation in Gal-3 -/- CD4 +T cells with a significantly higher level of IFN- ℽ mediated activation induced cell death (AICD) when compared to WT T cells. Gal-3 expression in T cells significantly increased the expression of exhaustion markers evidenced by a higher percentage of Slamf6 + Tim-3 + in WT T cells when compared to Gal-3 -/- T cells (Figure 1B). Gal-3 induced T cell exhaustion by through overactivation of NFAT signaling (data not shown). We sought to determine whether overexpression of Gal-3 in human T cells could control GvHD without affecting GVL. Gal-3 was overexpressed in human T cells using retrovirus containing Gal-3, vector alone and control T cells: Gal-3 T cells (T RV-Gal-3), GFP T cells (T RV-GFP) and control T cells were injected in irradiated NSG-HLA-A2 mice. All human cells expressed HLA-A2. Gal-3 overexpression in T cells effectively controlled the severity and mortality of GvHD after Allo-HCT in this humanized murine model of GvHD, evidenced by decreased body weight loss and decreased GvHD clinical scores in recipients transplanted with Gal-3 T cells when compared to control or GFP T cells (Figure 1C). Gal-3 overexpression did not impair the GvL effect when T cells cultured with Raji and THP-1 cell lines in vitro (data not shown). Gal-3 overexpression in T cells increased the frequencies of exhausted CD4 + T cells, and central memory CD4 + T cells while decreasing the percentage of effector CD4 T cell and INF-ℽ + CD4 + T cells. Clinical GI colon biopsies from patients undergoing allo-HCT were evaluated for Gal-3 expression in T cells using the multi-color Vectra 3 Automated Quantitative Pathology Imaging System. T cells in the colon biopsies expressed Gal-3. There was a significant correlation between Gal-3 MFI in CD4+ T cells, and GI histopathology score when analyzing Gal-3 intensity on Gal-3-expressing T cells. The Gal-3 MFI in CD4+ T cells was significantly lower in biopsies with higher colon GI histopathology scores (III-IV) compared to with lower colon GI histopathology scores I-II. In conclusion, these data reveal how Gal-3 can influence donor T cell proliferation and function in preclinical aGvHD models and point to the feasibility of manipulation of Gal-3 signaling to ameliorate aGvHD in the clinical setting. Figure 1 Figure 1. Disclosures Blazar: Rheos Medicines: Research Funding; Carisma Therapeutics, Inc: Research Funding; Equilibre Pharmaceuticals Corp: Research Funding; Tmunity Therapeutics: Other: Co-founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. McCarthy: Magenta Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Juno: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Duobody-CD3xCD20 Induces Potent Anti-Tumor Activity in Malignant Lymph Node B Cells from Patients with DLBCL, FL and MCL Ex Vivo, Irrespective of Prior Treatment with CD20 Monoclonal Antibodies

Blood ◽

10.1182/blood-2019-125765 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4066-4066

Author(s):

Hilma J Van Der Horst ◽

A. Vera de Jonge ◽

Ida H Hiemstra ◽

Anne T Gelderloos ◽

Daniella RAI Berry ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Tumor Cell ◽

Cytotoxic Activity ◽

Board Of Directors ◽

Tumor Cells ◽

T Cell Activation ◽

Research Funding ◽

Advisory Committees

DuoBody-CD3xCD20 (GEN3013) is a novel clinical-stage CD3 bispecific antibody (bsAb) targeting CD20-positive tumor cells. GEN3013 was previously shown to induce potent T cell-mediated cytotoxicity towards B cell Non-Hodgkin lymphoma (B-NHL) cell lines in vitro and in vivo. Here, we investigated the cytotoxic activity of GEN3013 in tumor cells obtained from lymph node (LN) biopsies of B-NHL patients, who were newly diagnosed (ND) or relapsed from/refractory to (RR) treatment regimens containing CD20 monoclonal antibodies. Moreover, we explored whether specific tumor microenvironment characteristics could be associated with sensitivity to GEN3013. To test the intrinsic susceptibility of B-NHL cells to GEN3013, independent of interpatient variation in tumor T cell frequency or activation status, single cell suspensions obtained from LN of B-NHL patients were incubated with GEN3013 in the presence of allogeneic PBMC from a single donor, at an effector to target (E:T) ratio 10:1. GEN3013 (30 ng/mL) induced median tumor cell lysis of 64% in Diffuse Large B Cell Lymphoma (DLBCL, n=14), 69% in Follicular Lymphoma (FL, n=14) and 84% in Mantle Cell Lymphoma (MCL, n=8) samples, with EC50 values ranging from 0.01-3.9 ng/ml. Importantly, cytotoxic activity of GEN3013 was comparable in ND (n=24) and RR (n=12) patients (Figure 1). In these assays considerable heterogeneity in T cell activation, as assessed by expression of CD25, CD69 and granzyme B release, was observed. Furthermore, high expression of T cell activation markers was not always associated with high levels of GEN3013 cytotoxic activity, suggesting tumor-intrinsic resistance mechanisms. In parallel, in all B-NHL samples GEN3013-mediated cytotoxicity was assessed without the addition of allogeneic PBMCs, thus purely relying on T cells present in the LN biopsy. In this setting, median tumor cell lysis was lower; 18% in DLBCL (range 0-46%), 17% in FL (range 0-46%) and 0% in MCL (range 0-11%), but strongly correlated with the number of T cells present in the single cell suspensions. Analysis of the tumor microenvironment by 7 color immunohistopathology of matched FFPE-embedded tumor biopsies (n=24), confirmed that the T cell frequency in the tumor biopsies was the major determinant of GEN3013 cytotoxic activity in DLBCL, FL and MCL. Moreover, experiments using (MACS) purified T cells from 4 DLBCL and 5 FL LN biopsies demonstrated that the intrinsic capacity of tumor LN T cells to induce GEN3013 mediated cytotoxicity was comparable to healthy donor T cells. Detailed tumor microenvironment analysis based on 7 color immunohistopathology staining, including relative frequency and spatial distribution of CD4 and CD8 T cells and macrophages, as well as the T cell activation status, in relation to sensitivity to GEN3013 mediated tumor cell lysis is ongoing and results will be presented. In conclusion, GEN3013 induced potent cytotoxicity in tumor cells of DLBCL, FL and MCL patients ex vivo, irrespective of prior treatment with CD20 monoclonal antibodies. Autologous T-cells at the tumor site were able to mediate GEN3013-induced cytotoxicity, and cytotoxic activity was enhanced in presence of PBMCs suggesting that optimal tumor cell kill by GEN3013 is dependent on T-cells in the tumor microenvironment. The cytotoxic capacity of B-NHL patient T cells within the tumor microenvironment was comparable to healthy donor peripheral blood T cells, emphasizing the therapeutic potential of CD3 bsAb in B-NHL. A First-in-Human trial to assess the safety and preliminary efficacy of GEN3013 in B-NHL patients is currently ongoing (NCT03625037). Figure 1 Cytotoxic activity induced by GEN3013 compared to CD3xcontrol bsAb (both 30ng/ml) towards tumor cells obtained from lymph node (LN) biopsies of newly diagnosed (ND) versus relapse or refractory (RR) DLBCL, FL and MCL patients. GEN3013 achieved comparable lysis in ND versus RR patients (Mann-Whitney U test; not significant). Error bars represent median ± interquartile range. Figure 1 Disclosures Van Der Horst: Genmab: Other: Financial Support. Hiemstra:Genmab: Employment, Equity Ownership, Other: Warrants. de Jong:Genmab: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Chamuleau:Genmab: Research Funding. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Breij:Genmab: Employment, Other: Warrants. Roemer:Genmab: Research Funding. Mutis:Celgene: Research Funding; Janssen Research and Development: Research Funding; Onkimmune: Research Funding; Genmab: Research Funding.

Download Full-text

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.

Download Full-text