scholarly journals 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 ◽  
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.

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

Blood ◽  
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.

scholarly journals 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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 596-596
Author(s):  
Benjamin Watkins ◽  
Yvonne Suessmuth ◽  
Kayla Betz ◽  
Alison Yu ◽  
Brandi Bratrude ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Costimulation Blockade ◽  
Research Support ◽  
Advisory Committees ◽  
Highly Correlated

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.

scholarly journals Development of Novel Second Generation DC/Tumor Fusion Vaccine in Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 392-392 ◽  
Author(s):  
Shira Orr ◽  
Marzia Capelletti ◽  
Haider Ghiasuddin ◽  
Dina Stroopinsky ◽  
Jessica Liegel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Research Funding ◽  
Second Generation ◽  
Advisory Board ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Cells Cultured

Introduction: We have pioneered a personalized cancer vaccine in which patient derived tumor cells are fused with autologous dendritic cells (DCs) such that a broad array of shared and neo-tumor antigens is presented in the context of DC mediated co-stimulation, limiting the risk of antigen escape. In clinical trials of patients with hematologic malignancies, vaccination with DC/tumor fusions induced an expansion of tumor-specific T cells, and resulted in prolonged remissions in a subset of patients. In the current study, we have developed a novel second generation vaccine, whereby a DC/lymphoma fusion vaccine is presented in the context of a unique biomatrix that expresses high levels of the 41BB costimulatory molecule, to further accentuate T cell activation and prevent the establishment of tumor tolerance. In this study, we demonstrate efficacy of DC/lymphoma fusion cell vaccination in a preclinical lymphoma model, and show enhanced potency of the second-generation vaccine. Methods/Results: We first demonstrated the potency of the DC/tumor fusion vaccine in generating anti-tumor immunity in the A20 lymphoma model. Murine DC/A20 fusions were generated from bone marrow derived mononuclear cells cultured with GM-CSF and IL-4 then fused to syngeneic A20 lymphoma cells. DC/A20 fusion cells effectively induced tumor specific immunity as manifested by potent lysis of A20 T cells in vitro as compared to unstimulated T cells in a standard CTL assay. Consistent with this observation, vaccination with DC/A20 fusions effectively induced lymphoma specific immunity in an immunocompetent murine model. Balb/C mice (30 animals) underwent IV inoculation with 750,000 syngeneic, luciferase and mCherry transduced, A20 cells. 24 hours after tumor cells challenge, 15 mice were treated subcutaneously with 105 DC/A20 fusions. Tumor burden was detected using BLI imaging. 10 days post inoculation, within the untreated cohort all 15/15 mice had detectable tumor whereas within the treated group, 5 mice did not demonstrate any evidence of disease and 5 mice demonstrated minimal disease. We subsequently demonstrated that patient derived autologous DC/lymphoma fusions stimulated T cell mediated lysis of primary lymphoma cells. DC were generated from patient derived peripheral blood mononuclear cells cultured with GM-CSF and IL-4 and matured with TNFa. Primary lymphoma cells were isolated from resected tumor and fused with DC at a ratio of 10:1. Fusion stimulated T cells potently lysed autologous tumor cells as compared to unstimulated T cells (25.7% as compared to 12.66%) in a standard CTL assay. To further enhance vaccine potency, we developed a biomatrix substrate expressing the costimulatory molecule 41BB. Using carbodiimide chemistry we covalently bonded RGD peptide and 41BBL protein to an alginate (Alg)-based scaffold. The Alg/RGD/41BBL scaffold can serve as a supporting microenvironment for the co-culture of T cells and fusion vaccine. We cultured syngeneic T cells with DC/A20 fusion vaccine within a scaffold with or without bound 41BBL and examined the T cells cytotoxicity by a CTL assay as described above. Vaccine mediated stimulation of T cells in the context of the Alg/RGD/41BBL scaffold demonstrated higher levels of tumor lysis as compared to the percent T cells cultured within an Alg/RGD scaffold (22.95% and 13.95% respectively). Conclusion: In the current study we assessed the efficacy of the DC/Lymphoma fusion vaccine to elicit a tumor specific immune response. We succeeded in demonstrating the capacity of DC/Lymphoma fusion vaccine to generate tumor specific T cell cytotoxicity in vitro as well as in vivo in an immunocompetent murine model. Accordingly, we presented patient derived primary tumor results supporting the applicable nature of the DC/Lymphoma vaccine in lymphoma patients. In addition, we developed a second-generation fusion vaccine comprised of the original DC/Tumor vaccine presented to the T cells in an Alg/RGD/41BBL scaffold acting as a nurturing microenvironment for T cell immune specific response against the tumor cells. Our initial results exhibit promising potential and an in vivo experiment with the second-generation fusion vaccine is ongoing. Disclosures Arnason: Celgene/Juno: Consultancy; Regeneron Pharmaceuticals, Inc.: Consultancy. Kufe:Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Hillstream BioPharma: Equity Ownership; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Canbas: Consultancy, Honoraria. Rosenblatt:Dava Oncology: Other: Education; BMS: Research Funding; Partner Tx: Other: Advisory Board; Merck: Other: Advisory Board; Parexel: Consultancy; Imaging Endpoint: Consultancy; Celgene: Research Funding; BMS: Other: Advisory Board ; Amgen: Other: Advisory Board. Avigan:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: 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; Janssen: Consultancy; Parexel: Consultancy; Takeda: Consultancy.

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 ◽  
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.

scholarly journals 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 ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5319-5319 ◽  
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.

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

Blood ◽  
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.

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 ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 117-117 ◽  
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.

scholarly journals Changes in Tumor Immune Micro-Environment in Diffuse Large B-Cell Lymphoma (DLBCL): A Comparative Study of Relapsed Versus Diagnostic DLBCL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3968-3968
Author(s):  
Raoul Santiago ◽  
Nathalie Johnson ◽  
Svetlana Dmitrienko ◽  
Andreas Papadakis ◽  
Naciba Benlimame ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Board Of Directors ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Research Funding ◽  
Immune Escape ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
Pre Treatment

Introduction The tumor immune microenvironment (TiME) of DLBCL at diagnosis bears many biomarkers known to predict sensitivity to checkpoint blockade therapy (CBT). Approximately 25% of DLBCL, harboring PDL1 gene alteration, exhibit an immune inflamed phenotype. However, CBT studies in relapsed DLBCL show very low response rates. The immune landscape of DLBCL in the setting of relapse has not yet been characterized. We hypothesized that the TiME can be influenced by the therapy, explaining the CBT lack of efficacy. We explored the TiME evolution by comparing the tumor infiltrating lymphocytes (TIL) and the immune evasion signature by protein and gene expression (GE) in paired biopsies of DLBCL at diagnosis and at relapse. Method This study included patients with DLBCL treated with first-line R-CHOP-like regimen, in which we had access to nodal biopsies obtained pre-treatment and at relapse. Assessment of TIL and PDL1+ cell content was carried out by CD3, CD4, CD8 immunostaining and with PDL1 (Ventana, SP263) and PAX5 double staining. These were performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections cut at 4µm. The proportion of each cell-type was expressed in relation to total nucleated cells. PDL1+ cell proportion was expressed for both total cells and tumor cells. The paired non-parametric Wilcoxon test, double-sided with p=0.05, was used for comparison. GE using a panel of 760 cancer immune genes was performed with the digital hybridization NanoString platform on paired FFPE specimens. NSolver software was used for normalization on housekeeping genes, differential expression (DE) and cell-type profiling. The correlation between IHC and GE variation were computed using the Spearman test. Analysis of enriched pathways on combined patient data were performed with GSEA software, using two gene sets for T-cell activation and immune evasion validated in B-cell lymphoma, and Ingenuity Pathway Analysis (IPA) for the 339 highest changed genes. The pathway enrichment analysis was reported for false discovery rate (FDR) <0.05. Results Twenty DLBCL patients with paired diagnostic and relapse biopsies were studied. IHC was performed on 19 patients (necrotic tissue for one patient) and GE by NanoString on 6 patients. The median number of treatments received prior to obtaining the relapsed biopsy was 3 (1-6). Three patients had prior exposure to CBT, none from the patients in whom GE was performed. Our population was composed of 2/3 germinal center B-cell (GCB) DLBCL. Six patients had transformed DLBCL. At relapse, CD4 and CD8 cells density was significantly reduced compared to diagnosis. The median percentages of CD3, CD4 and CD8 were 15%, 7.5% and 5% at diagnosis and 7.5%, 2.5% and 2.5% at relapse, respectively (Figure 1A). The median proportion of PDL1+ cells (n=15) did not differ at relapse compared to pre-treatment (5.25% and 5%, respectively) (Figure 1B). At diagnosis, 87% of biopsies were found to be positive for PDL1 (≥1% of positive cells) and 73% at relapse. The PDL1 positive cells were mostly immune cells; only 3 samples had tumor cells expressing PDL1 at pre-treatment and 4 at relapse with <1% of positive tumor cells in all cases. The GE profile detected 63 genes that were significantly different in relapse vs pre-treatment samples (p<0.005), including 8 up-regulated and 55 down-regulated. The correlation for T-cell abundance variation between IHC and GE was high (r=0.94, p=0.017). For the subset of 6 patients in whom GE was performed, the T-cells were not significantly reduced as measured by both IHC and GE. However, the GSEA for immune escape genes and T-cell activation signatures were both down-regulated (FDR=0.033 and 0.010, respectively) (Figure 2). IPA predicted decreased activation of leukocytes (z-score=-6.317) and Th2 activation pathway (z-score=-3.182). Conclusion At relapse, a proportion of DLBCL have a significant reduction in T cell infiltration. As well, they acquire a non-T cell-inflamed TiME. The T-cell inhibition seems to be independent of local T-cell immunoregulatory mechanisms as the immune escape signature is also reduced. This non immunogenic, "cold" condition could explain the lack of efficacy of CBT in the relapsed setting. Prior exposure to steroid containing therapy might be a trigger for intra-tumor T-cell desertion and downregulation. This observation must be confirmed on a larger population. Further analyses will be presented. Disclosures Johnson: BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Seattle Genetics: Honoraria; Merck: Consultancy, Honoraria; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Assouline:Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria.

scholarly journals Evaluation of T Cell and Monocyte Immune Subsets in the Stem Cell Product of Patients Developing Engraftment Syndrome Following Autologous Stem Cell Transplantation for Multiple Myeloma and Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4604-4604
Author(s):  
Dante B. Descalzi-Montoya ◽  
Zheng Yang ◽  
Kira Goldgirsh ◽  
Rena Feinman ◽  
David S Siegel ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Board Of Directors ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Activation ◽  
Advisory Committees ◽  
Classical Monocytes

Abstract BACKGROUND: High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is now standard of care for newly diagnosed patients with multiple myeloma (MM) and is used for some forms of non-Hodgkin lymphoma, providing improved outcomes. ASCT has been associated with a high incidence of engraftment syndrome (ES), which clinically presents as skin rashes, diarrhea, non-infectious fevers, and capillary leak syndrome in the peri-engraftment period. ES can be a severe and potentially lethal complication in patients who do not respond to corticosteroid therapy. RATIONALE: Prior to stem cell collection, MM and lymphoma patients are typically exposed to chemotherapy and immunomodulatory agents in order to reduce the cancer cell burden and other drugs to mobilize stem cells. It has been hypothesized that these agents may be involved with ES development in that they can disrupt the balance of immune regulatory and effector cell subsets. OBJECTIVE: To analyze T cell activation/memory and Treg cell compartments as well as classical, intermediate, and non-classical monocytes in the autologous apheresis product of MM and lymphoma patients undergoing ASCT. STUDY DESIGN: Samples were collected under IRB approval from 28 patients undergoing ASCT for the treatment of MM (n=21) or lymphoma (n=7) at the time of peripheral blood stem cell collection (the apheresis product); Peripheral blood mononuclear cells (PBMC) were isolated by sucrose gradient centrifugation and frozen until needed for phenotyping by multi-parametric flow cytometry. MM patients were conditioned for ASCT with melphalan alone (n=14) or in combination with bortezomib (n=7); lymphoma patients received conditioning with BEAM (carmustine, etoposide, cytarabine, and melphalan) chemotherapy. RESULTS: Of the 28 patients undergoing ASCT, 10 (35.7%) developed ES within the first four weeks post-transplantation. We analyzed by flow cytometric phenotyping the following immune cell subsets: overall CD4/CD8 T cells, CD4/CD8 naïve and memory cells, HLA-DR expression on memory T Cells, memory CD4 Tregs, as well as overall classical, intermediate, and non-classical monocytes. The absolute numbers (PBMC conc. (cell/ml of blood) x (% of cells in PBMC/100) and percentage population (# of events in population gate/# of total PBMC events x 100) data were transformed with log and square root functions, respectively. All data were tested for normality with a Shapiro-Wilks online test and p-values were obtained by performing an unpaired Student T-test. From our analysis of the apheresis product, the main cell compartments that were significantly increased in the ES+ group were the % CD8+ T cells [5.41 mean ± 0.51 s.e. vs. 3.34 ± 0.36 (ES-), p=0.003] and naïve CD8+ T cells [3.76 ± 0.56 vs. 1.61 ± 0.18 (ES-), p<0.001; In addition, although the memory CD8 T cell subset was not significantly increased in the apheresis product, the % of those memory CD8 T cells expressing HLA-DR significantly increased [2.0 ± 0.22 (ES+) vs. 1.2 ± 0.15 (ES-); p<0.01], suggesting an increase in CD8 T cell activation in the ES+ group. No major differences were observed in the CD4 memory Treg compartment or CD25 expression on CD4 T cells. In the monocyte compartment, the main subset that was significantly increased was the non-classical CD16+CD14low cells in the ES+ group [0.40 ± 0.08 vs. 0.25 ± 0.03 (ES-), p=0.02]. In addition, the % of CD25+ and CD163+ non-classical and intermediate monocytes were favorably increased in the ES+ group (non-classical monocytes, CD25+ [0.14 ± 0.03 vs. 0.07 ± 0.02 (ES-), p=0.03] and CD163+ [0.33 ± 0.06 vs. 0.18 ± 0.02 (ES-), p=0.03]; and for intermediate monocytes, CD25+ [0.44 ± 0.1 vs. 0.22 ± 0.04 (ES-), p=0.02] and CD163+ (p=0.02). No major differences were observed between ES+ and ES- groups for CD64 and PDL-1 expression. CONCLUSIONS: The development of ES correlated with the observation of a significant increase of naïve and activated CD8 T cells in the autologous apheresis product. In contrast, no significant differences were found between the ES+ and ES- groups in the CD4 T cell or memory Treg subsets, suggesting that they do not contribute to the etiology of the syndrome. Moreover, an increased presence of non-classical monocytes with higher expression of both CD163 and CD25 was found, suggesting increased potential for both M2 and M1 activity. Further investigation is needed to determine the implications of these findings for the development of ES. Disclosures Siegel: Merck: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Novartis: Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Biran:BMS: Research Funding; Merck: Research Funding; Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau. Feldman:Johnson and Johnson: Speakers Bureau; KITE: Speakers Bureau; Seattle Genetics: Research Funding, Speakers Bureau; Pharmacyclics: Speakers Bureau; Janssen: Speakers Bureau; Portola: Research Funding; Celgene: Speakers Bureau. Skarbnik:Gilead Sciences: Honoraria, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

scholarly journals Leukemic Cell Expressed CTLA-4 Suppresses T Cells Via Down-Modulation of CD80 By Trans-Endocytosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3221-3221 ◽  
Author(s):  
Priscilla Do ◽  
Kyle A. Beckwith ◽  
Larry Beaver ◽  
Brittany G. Griffin ◽  
Xiaokui Mo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cell Surface ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Research Funding ◽  
Leukemic Cell ◽  
Advisory Committees

Abstract The function of CTLA-4 on non-T cells is largely ignored and currently ill defined despite rapidly growing interest in targeting this immune checkpoint protein in several cancers. While anti-CTLA-4 therapy is proposed to work through inhibition of the immunosuppressive effect of CTLA-4 on T cells, multiple examples of non-T cell expressed CTLA-4 have been reported. These cells include tumor cells of hematological and non-hematological origin and normal B cells. In this study, we have defined a novel immune suppressive role for non-T cell, tumor expressed CTLA-4 in Chronic Lymphocytic Leukemia (CLL). We have detected by microarray that CTLA-4 is in the top 5 most differentially expressed genes between pooled samples of healthy donor normal B cells (N=6) and pooled CLL leukemic B cells (N=5). Upregulation of CTLA-4 by CLL B cells compared to normal B cells was validated by RT-qPCR and flow cytometry. CTLA-4 was predominantly intracellular (42/46 CTLA-4+) and not on the cell surface (2/48 CTLA-4+) in primary CLL samples. B cell activating factors (CD40L, PMA/Ionomycin, LPS, IL4, LPS+IL4, CD40L+IL4, CpG, and anti-IgM) could not induce surface expression of CTLA-4; however, co-culture with anti-CD3/anti-CD28 or ConA activated T cells (autologous or allogeneic) resulted in detectable CTLA-4 on the cell surface of leukemic B cells. This induction did not occur with resting T cells. This finding suggests a role for CTLA-4+ tumor cells in sites of T cell activation, such as the lymph node, a site of leukemic cell proliferation in CLL. To mechanistically study leukemic B cell expressed CTLA-4, we generated CLL-derived Mec1 and OSU-CLL that inducibly express CTLA-4 upon doxycycline (dox) treatment. Mec1 and OSU-CLL cells highly express the ligands for CTLA-4, CD80 and CD86. Dox-induction of CTLA-4 resulted in decreased expression of Mec1 and OSU-CLL expressed CD80, a critical T cell co-stimulatory protein (N=3). Blockade of CTLA-4 using the anti-CTLA-4 therapeutic antibody, Ipilimumab, could restore CD80 on Mec1 and OSU-CLL cells (N=3). Because T cell-expressed CTLA-4 has been previously shown by others to down-modulate CD80 via trans-endocytosis, we co-cultured CTLA-4+ Mec1 and CTLA-4+ primary CLL cells with stably transfected CD80-GFP or CD86-GFP Hek293 cell lines to assess uptake of CD80/CD86 into CTLA-4 expressing tumor cells as the mechanism of CD80 down-modulation. Transfer of CD80-GFP and CD86-GFP was detected by flow cytometry in primary CLL cells and the Mec1 cell line, consistent with the ability of T cell expressed CTLA-4 to trans-endocytose CD80 and CD86. Furthermore, uptake of CD80-GFP or CD86-GFP by primary tumor cells was CTLA-4 dependent, demonstrated by inhibition of GFP uptake in the presence of Ipilimumab. Following determination of decreased CD80, we found that co-culture of primary T cells with Mec1 CTLA-4+ cells resulted in decreased IL2 production measured by Cytokine Bead Array. The loss of IL2 signified decreased co-stimulation as a result of tumor expressed CTLA-4. Studies are ongoing regarding dependence on CD80 or CD86. A minor subset of T cells, Tregs, are known to exert profound immunosuppressive effects through their expression of CTLA-4. Due to our results, tumor expressed CTLA-4 has an overlapping function with Treg CTLA-4, and it is imperative that we define the immunosuppressive effects as, in patients, the leukemic cells may comprise a much larger proportion of white blood cells than T cells. Efforts are now underway to address the effect of tumor expressed CTLA-4 in suppressing anti-tumor immunity in vivo utilizing a novel mouse model. Suppression of T cells by tumor expressed CTLA-4 is a novel finding that is broadly applicable to fields within and outside of cancer research as the pathway and mechanism described here are potentially applicable to CTLA-4 in diverse disease contexts and to the general biology of CTLA-4. [Funding: This work was supported by P01 CA95426. PD received the Pelotonia Graduate Fellowship. Any opinions, findings, and conclusions expressed in this material are those of the author(s) and do not necessarily reflect those of the Pelotonia Fellowship Program] Disclosures Jones: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Sign in / Sign up

Export Citation Format

Share Document