Phase 1/2 Study of Nexi-002 Autologous Multi-Antigen-Specific CD8+ T Cells for the Treatment of Relapsed or Refractory Multiple Myeloma

Abstract The NEXI-002 study is a prospective, multicenter, open-label phase 1/2 trial designed to characterize the safety, immunologic, and preliminary anti-myeloma activity of the NEXI-002 antigen specific CD8+ T cell product. Multiple myeloma (MM) is an incurable malignancy that occurs predominantly in older patients and is characterized by the growth of malignant plasma cells in the bone marrow. Despite substantial advances in therapy, virtually all patients relapse after treatment, emphasizing the unmet medical need for additional effective treatments. The NEXI-002 product is an autologous non-genetically engineered therapy of CD8+ T cells that recognize HLA 02.01-restricted peptides from the WT1, CD138, CS1, and NY-ESO-1 antigens. This T-cell product includes key memory phenotypes such as stem-like memory, central memory, and effector memory cells. Eligible patients have relapsed or refractory multiple myeloma (RRMM) who have received at least three prior lines of treatment that included at least an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 agent. Three patients were enrolled into the Safety Evaluation phase and received a single infusion of 80 million (M) to 100M cells of NEXI-002 product. In this phase of the study the primary endpoint is safety and secondary endpoints include expansion, persistence, and trafficking of the NEXI-002 cells. Bridging anti-MM treatment was permitted during the manufacture of the cellular product with a wash-out period of at least 14 days prior to lymphodepletion (LD) chemotherapy (intravenous fludarabine 30 mg/m 2 and cyclophosphamide 300 mg/m 2), which was administered on Days -5, -4, and -3 prior to the infusion of the NEXI-002 product up to 72 hours later (Day1). Treatment-related adverse events, including infusion reactions, events that prolong hospitalization post infusion, CRS, and neurotoxicity (ICANS) have not developed in these patients who received the NEXI-002 product. Lymphocyte recovery to baseline levels occurred within a few days after the infusion of the NEXI-002 product, demonstrating robust CD4 and CD8 T cell reconstitution following LD chemotherapy. NEXI-002 antigen specific T cells were detected in peripheral blood (PB) by multimer staining and proliferated over time and trafficked to the bone marrow (BM). The phenotype composition of detectable antigen specific T cells at both sites maintained that of the infused product. These NEXI-002 T cells persisted in PB and BM during follow-up. T-cell receptor (TCR) sequencing assays revealed T cell clones in the NEXI-002 product that were not detected in PB of patients tested at baseline. These clones subsequently expanded and persisted over time in the PB and BM. In conclusion, these results show that infusion of the NEXI-002 product is safe, well tolerated, and capable of generating a cell-mediated immune response that may lead to clinical activity. RNA Seq transcriptional profiling of the CD8+ T cells is planned. Additional patients have recently received NEXI-002 infusions and the trial will continue to be expanded to gain additional safety, immunologic, and clinical activity experience. Disclosures Knight: Neximmune, Inc: Current Employment. Varela: Nexlmmune: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite: Speakers Bureau. Edavana: Neximmune, Inc: Current Employment. Lu: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Suarez: Neximmune, Inc: Current Employment. Oelke: Neximmune, Inc: Current Employment. Bednarik: Neximmune, Inc: Current Employment.

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Phase 1/2 Study of Nexi-001 Donor-Derived Multi-Antigen Specific CD8+ T Cells for the Treatment of Relapsed Acute Myeloid Leukemia (AML) after Allogeneic Hematopoietic Transplantation

Blood ◽

10.1182/blood-2021-152419 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4819-4819

Author(s):

Monzr M. Al Malki ◽

Sumithira Vasu ◽

Dipenkumar Modi ◽

Miguel-Angel Perales ◽

Lucy Y Ghoda ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Phase 1 ◽

Clinical Activity ◽

Advisory Committees ◽

Over Time

Abstract Patients who relapse after allogeneic HCT have a poor prognosis and few effective treatment options. Responses to salvage therapy with donor lymphocyte infusions (DLI) are driven by a graft versus leukemia (GvL) effect. However, relapses and moderate to severe graft versus host disease (GVHD) are common. Therapies that increase the GvL effect without inducing GVHD are needed. The NEXI-001 study is a prospective, multicenter, open-label phase 1/2 trial designed to characterize the safety, immunogenic, and antitumor activity of the NEXI-001 antigen specific T-cell product. This product is a donor-derived non-genetically engineered therapy that consists of populations of CD8+ T cells that recognize HLA 02.01-restricted peptides from the WT1, PRAME, and Cyclin A1 antigens. These T cells consist of populations with key memory phenotypes, including stem-like memory, central memory, and effector memory cells, with a low proportion (<5%) of potentially allogeneic-reactive T-naïve cells. Patients enrolled into the first cohort of the dose escalation phase received a single infusion of 50 million (M) to 100M cells of the NEXI-001 product. Bridging anti-AML treatment was permitted during the manufacture of the cellular product with a wash-out period of at least 14 days prior to lymphodepletion (LD) chemotherapy (intravenous fludarabine 30 mg/m 2 and cyclophosphamide 300 mg/m 2) that was administered on Days -5, -4, and -3 prior to the infusion of the NEXI-001 product up to 72 hours later (Day1). Lymphocyte recovery to baseline levels occurred as early as three days after the NEXI-001 product infusion with robust CD4 and CD8 T cell reconstitution after LD chemotherapy. NEXI-001 antigen specific T cells were detectable in peripheral blood (PB) by multimer staining and were found to proliferate over time and to traffic to bone marrow. The phenotype composition of detectable antigen specific T cells at both sites was that of the infused product. T-cell receptor (TCR) sequencing assays revealed T cell clones in the NEXI-001 product that were not detected in PB of patients tested at baseline. These unique clones subsequently expanded in PB and bone marrow (BM) and persisted over time. Neutrophil recovery, decreased transfusion burden of platelets and red blood cells, and increased donor chimerism were observed. Decreases in myeloblasts and reduction in the size of an extramedullary myeloid sarcoma were suggestive of clinical activity. One patient, a 23-year- old with MRD+ disease at baseline, received two doses of 200M NEXI-001 cells separated by approximately 2 months. Following the first infusion, antigen specific CD8+ T cells increased gradually in PB to 9% of the total CD3+ T cell population just prior to the second infusion and were found to have trafficked to bone marrow. By Day 2 following the second infusion, which was not preceded by LD chemotherapy, the antigen specific CD8+ T cells again increased to 9% of the total CD3+ T cell population in PB and remained at ≥5% until the end of study visit a month later. The absolute lymphocyte count increased by 50% highlighting continued expansion of the NEXI-001 T cells. These cells also maintained significant Tscm populations. Treatment related adverse events, including infusion reactions, GVHD, CRS, and neurotoxicity (ICANS), have not developed in these patients who have received 50M to 200M T cells of the NEXI-001 product either as single or repeat infusions. In conclusion, these results show that infusion of the NEXI-001 product is safe and capable of generating a cell-mediated immune response with early signs of clinical activity. A second infusion is associated with increasing the level of antigen specific CD8+ T cells and their persistence in PB and BM. TCR sequencing and RNA Seq transcriptional profiling of the CD8+ T cells are planned, and these data will be available for presentation during the ASH conference. At least two cycles of 200M NEXI-001 cells weekly x 3 weeks of a 4-week cycle is planned for the next dose-escalation cohort. Early data suggest that the NEXI-001 product has the potential to enhance a GvL effect with minimal GVHD-associated toxicities. Disclosures Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Neximmune: Consultancy; Hansa Biopharma: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy. Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: travel support; Kiadis, Inc.: Research Funding; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Modi: MorphoSys: Membership on an entity's Board of Directors or advisory committees; Seagen: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding. Perales: Sellas Life Sciences: Honoraria; Novartis: Honoraria, Other; Omeros: Honoraria; Merck: Honoraria; Takeda: Honoraria; Karyopharm: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; MorphoSys: Honoraria; Kite/Gilead: Honoraria, Other; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Medigene: Honoraria; NexImmune: Honoraria; Cidara: Honoraria; Nektar Therapeutics: Honoraria, Other; Servier: Honoraria; Miltenyi Biotec: Honoraria, Other. Edavana: Neximmune, Inc: Current Employment. Lu: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Suarez: Neximmune, Inc: Current Employment. Oelke: Neximmune, Inc: Current Employment. Bednarik: Neximmune, Inc: Current Employment. Knight: Neximmune, Inc: Current Employment. Varela: Kite: Speakers Bureau; Nexlmmune: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Spontaneous CD4 and CD8 Memory T Cell Responses Against MUC1 and Carcinoembryonic Antigen in Bone Marrow of Multiple Myeloma Patients.

Blood ◽

10.1182/blood.v110.11.3533.3533 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3533-3533

Author(s):

Mathias Witzens-Harig ◽

Dirk Hose ◽

Michael Hundemer ◽

Simone Juenger ◽

Anthony D. Ho ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Carcinoembryonic Antigen ◽

Cd8 T Cells ◽

T Cell Responses ◽

Cd8 T Cell ◽

Cd4 T Cell ◽

Cell Responses

Abstract Introduction: The bone marrow (BM) is a site of induction of tumour antigen specific T cell responses in many malignancies. We have demonstrated in the BM of myeloma patients high frequencies of spontaneously generated CD8 memory T cells with specificity for the myeloma-associated antigen MUC1, which were not detectable in the peripheral blood (PB). Besides MUC1, carcinoembryonic antigen was recently identified as a tumour-associated antigen in a patient with multiple myeloma. Up to now, spontaneous CD4 T cell responses against myeloma-associated antigens have not been reported. We undertook this study to evaluate to what extent spontaneous CD4 T cell responses against myeloma antigens occur during myeloma progression and if MUC1 or carcinoembryonic antigen represent immunogenic targets of spontaneous CD4 and CD8 T cell responses. Methods: Altogether, 78 patients with multiple myeloma were included into the study. Presence of functionally competent antigen specific T cells was evaluated by ex vivo short term (40 h) IFN-γ Elispot analyses. CD4 T cell responses against MUC1 were assessed by stimulation of purified CD4 T cell fractions with antigen pulsed, autologous dendritic cells (DCs) pulsed with two synthetic 100 meric polypeptides (pp1-100ss and (137–157)5 tr) that can be processed and presented via multiple HLA-II alleles. CD4- or CD8 T cell reactivity against carcinoembryonic antigen was assessed on purified CD4- and CD8 T cell fractions by pulsing DCs with highly purified CEA derived from culture supernatants of an epithelial carcinoma cell line. CD8 responses against MUC1 were analyzed by stimulation of HLA-A2+ patients derived purified T cells with DCs loaded with HLA-A2 restricted MUC1-derived nonameric peptide LLLLTVLTV. As negative control antigen for MUC1 polypeptides and CEA human IgG was used for pulsing DCs at identical concentrations while HLA-A2-restricted peptide SLYNTVATL derived from HIV was used as control antigen for LLLLTVLTV. Test antigen specific reactivity was defined by significantly increased numbers of IFN-γ spots in triplicate test wells compared to control wells (p<0.05, students T test). Results: 8 out of 19 tested patients (42%) contained MUC1 specific CD8 T cells in their bone marrow, while MUC1 specific CD4 T cells were detected in the BM of 30% of the cases (3/10). Interestingly, in peripheral blood (PB) CD8 reactivity against MUC1 was detectable in only 1 out of 10 patients while CD4 reactivity in PB was not detectable at all (0/10). CEA was specifically recognized by BM CD8 T cells from 5 out of 30 patients (17%) and by BM CD4 T cells from 5 out of 18 patients (28%). CEA was not recognized by CD4 and CD8 T cells in the PB of the same patients (0/13). Conclusion: Spontaneous T helper responses against tumour-associated antigens occur in the BM at similar levels as antigen specific CD8 T cells responses while they are virtually undetectable in the PB. Compared to CEA, MUC1 induces CD8 T cell responses in a much higher proportion of myeloma patients. Nevertheless, our data suggest that CEA may trigger spontaneous T cell responses against multiple myeloma in a considerable number of patients. Thus, systematic functional analyses of this potential tumour antigen in multiple myeloma appears to be justified.

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Increased Population Of Myeloid-Derived Suppressor Cells In Patients With Myelodysplastic Syndromes Overexpress ARG1 and Mediate CD8+ T Cell Inhibition

Blood ◽

10.1182/blood.v122.21.5212.5212 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5212-5212 ◽

Cited By ~ 1

Author(s):

Zonghong Shao ◽

Huijuan Jiang ◽

Rong Fu

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Myelodysplastic Syndromes ◽

Suppressor Cells ◽

Cd8 T Cell ◽

Myeloid Derived Suppressor Cells ◽

Normal Controls ◽

Apoptosis Ratio

Abstract Objective To investigate the proportion and activation of myeloid- derived suppressor cells (MDSC) in bone marrow from patients with myelodysplastic syndromes (MDS). Methods The proportion of MDSC (Lin-HLA-DR-CD33+) in bone marrow of 30 MDS patients and 19 normal controls were measured by flow cytometry assay(FCM). MDSC and CD8+ T cell were isolated from bone marrow of 14 MDS patients and 14 normal controls among them by FCM and microbeads. The expressions of arginase 1(ARG1) and inducible nitric oxide synthase (iNOS) were analyzed by qPCR and western bolting. Co-cultures with CD8+ T cell were proved the MDSC-mediated inhibition of CD8+ T cell. Results MDS patient’s median MDSC were 7.29% which was higher than that of controls (2.91%). The expression of ARG1 and iNOS mRNA in MDSC of high-risk MDS patients was higher than that of low-risk MDS patients. But the protein of ARG1 was overexpressed rather than that of iNOS. After co-cultured, the apoptosis ratio of CD8+ T cells of MDS((64.17±4.86) %) was increased compared to pure CD8+ T cells ( (54.58±9.95)%). Further more, the production of IFN-γsecreted by CD8+ T cells co-cultured with MDSC ((551.94±47.39) pg/ml)was lower than that of pure CD8+ T cells ((586.04±46.65) pg/ml) There was no significant difference in level of TNF-βbetween co-cultured with MDSC and pure CD8+ cells. Conclusion The proportion of MDSC in bone marrow was increased significantly in MDS. MDSC overexpressed ARG1 in patients with MDS and correlated to the malignant degree of this disease. Further more, MDSC can increased the apoptosis ratio of CD8+ T cell, and inhibited the secretion of IFN-γ. These findings suggested MDSC mediated the response of immunosuppression in MDS. Disclosures: No relevant conflicts of interest to declare.

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Dominant Expression of PVRIG and TIGIT Inhibitory Pathways in Bone Marrow of Multiple Myeloma Patients

Blood ◽

10.1182/blood-2021-148052 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4717-4717

Author(s):

Masha Frenkel ◽

Zoya Alteber ◽

Ning Xu ◽

Mingjie Li ◽

Haiming Chen ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Plasma Cells ◽

Myeloma Cell ◽

Inhibitory Receptor ◽

Preclinical Models ◽

Cell Numbers

Abstract Introduction Blocking inhibitory immune checkpoints holds promise to treat multiple myeloma (MM) patients. However, currently available checkpoint inhibitors have not shown significant clinical benefits for MM patients, warranting the need for alternative checkpoint blockers. The immune checkpoint TIGIT was recently shown to be the most upregulated immune inhibitory receptor on CD8+ T cells in MM patients' bone marrow (BM), compared to other checkpoints (Guillerey C., Blood. 2018). Preclinical models demonstrated the dominant effects of TIGIT blockade or depletion, by significantly improving mice survival, reducing myeloma cell numbers and exhausted T cell hallmarks (Minnie S., Blood. 2018). As a result, several clinical trials using anti-TIGIT monoclonal antibodies have been recently initiated in MM patients. The DNAM-1 family, in addition to TIGIT, also includes the inhibitory receptor PVRIG, that competes with the co-activating receptor DNAM-1 for the binding to the shared ligand PVRL2, similarly to the TIGIT/PVR/DNAM-1 interaction. Accordingly, TIGIT and PVRIG co-blockade were shown to synergize in enhancing T cell activity and anti-tumor activity in preclinical models (Whelan S., Cancer Immunol. Res. 2019). PVRL2 together with PVR (ligand of TIGIT) were shown to be highly expressed on plasma cells and on CD14+ cells in BM of MM patients (Lozano E., Clin. Cancer Res. 2020). This study aimed at evaluating DNAM-1 axis receptors expression in MM patients' BM. Methods Fresh BM aspirates were collected from 21 MM patients with progressive disease (PD) or in complete response (CR) after obtaining IRB approval. BM mononuclear cells were isolated and single cell suspensions were obtained followed by staining with anti-human antibodies to evaluate DNAM-1 axis members and PD-1 expression. BM biopsies from 6 MM patients (each patient had 4 core on the Tissue Micro-Array T291 USBiomax) were stained for PVRL2 expression by immuno-histochemistry (IHC). Results Flow cytometry analysis of PD-1 and DNAM-1 axis receptors revealed a significant lower fraction of PD1+ cells among cell populations examined compared with other receptors. TIGIT expression was the highest on NK, CD8+ and NKT cells compared to CD4+ T cells, which is in line with previous published data (Lozano E. Clin. Cancer Res. 2020). In contrast, DNAM-1 was expressed on CD8+ T, NK and NKT cells with prominent high expression on CD4+ T cells (Fig 1A). The highest expression among the receptors was of PVRIG on all lymphoid populations, except CD4+ where DNAM-1 was more highly expressed. Importantly, 50% of CD8+ T cells co-expressed TIGIT and PVRIG, supporting a combinatorial therapeutic approach (Fig. 1B). Additionally, the expression of the PVRL2 ligand on MM plasma and endothelial cells was demonstrated by IHC. FACS analysis further supported PVRL2 expression on plasma cells in MM BM (Fig 2). A higher expression of PVRIG, TIGIT and PD-1 was present on DNAM-1 negative CD8+ T cells (Fig 3A, B), suggesting accumulation of exhausted cells in MM tumor microenvironment (TME) as previously described (Minnie S., Blood. 2018). PVRIG had significantly higher expression on DNAM+ cells, compared to PD-1 and TIGIT (Fig 3C), suggesting the potential of its blockade to enhance DNAM-1 activation and subsequent proliferation of earlier differentiated memory cells in MM TME. Finally, CR patients had a trend for higher DNAM-1 expression on CD8+ T cells compared to those with PD (Fig 3D). This is consistent with other reports in mice showing that the expression of DNAM-1 negatively correlates with BM myeloma cell numbers (Minnie S., Blood. 2018). Conclusions DNAM-1 axis receptors are dominantly expressed on lymphocytes in BM of MM patients, with PVRIG exhibiting the most prominent expression. The reduced expression of DNAM-1 in PD patients' TME, compared to CR patients, suggests a link between DNAM-1 axis and clinical outcomes. Recent data suggest TIGIT is an attractive target for blockade in MM. Our new findings highlight for the first time the dominant expression of PVRIG, as well as TIGIT, and suggest that combined blockade of TIGIT with PVRIG may potentially benefit MM patients, placing the DNAM-1 axis as a dominant pathway in MM therapy. Figure 1 Figure 1. Disclosures Frenkel: Compugen Ltd.: Current Employment, Other: in the event of frontal participation, I will be reimbursed for my travel expenses by Compugen Ltd.. Alteber: Compugen Ltd.: Current Employment. Cojocaru: Compugen Ltd.: Current Employment. Ophir: Compugen Ltd.: Current Employment.

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Efficacy and Safety of CAR-T Therapy with Safety Switch Targeting Bcma for Patients with Relapsed/Refractory Multiple Myeloma in a Phase 1 Clinical Study

Blood ◽

10.1182/blood-2019-127608 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3154-3154 ◽

Cited By ~ 7

Author(s):

Weijun Fu ◽

Juan Du ◽

Hua Jiang ◽

Zhi CHENG ◽

Runhong Wei ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Conflicts Of Interest ◽

Phase 1 ◽

Cellular Immunotherapy ◽

Infusion Therapy ◽

Cell Product ◽

Car T Cell ◽

Car T

Background: Encouraging results are seen from several early phase clinical trials on the cellular immunotherapy based on chimeric antigen receptor (CAR)-engineered T (CAR-T) targeting B cell maturation antigen (BCMA) for the treatment of relapsed/refractory (RR) multiple myeloma (MM). We developed an anti-BCMA CAR-T cell product manufactured via gamma-retrovirus-mediated transduction of activated T cells to express a second-generation CAR with the 4-1BB costimulatory domain along with a truncated epidermal growth factor receptor (tEGFR) as a safety switch. The preclinical study confirmed its high reactivity against MM cells. Methods: A phase 1 clinical trial (NCT03093168) has been launched to evaluate the safety and feasibility of this BCMA CAR-T cell product for treating RRMM. The enrolled RRMM patients had received at least 2 prior treatment regimens, including a proteasome inhibitor and an immunomodulatory agent, or are double-refractory, and have over 5% BCMA expression on plasma cells (Nine patient with extramedullary plasmacytoma does not express BCMA). Patients were subjected to a lymphodepleting regimen with Cy (300 mg/m2, d-5 to d-3) and Flu daily for 3 days (25 mg/m2, d-5 to d-3) prior to the CAR-T infusion (d0) at a dose of 9×106CAR+ cells/kg. The efficacy was assessed by the International Uniform Response Criteria for Multiple Myeloma, and the toxicity is graded by CTCAE 4.03. Results: As of March 1th, 2019, 46 patients had been infused with this intended dose of the autologous BCMA CAR-T cells, and 44 patients had reached at least 1 month of follow-up. As of this data cut-off, the overall response rate (ORR) for the 44 evaluable patients was 79.6%, including 2sCRs, 16CRs, 8VGPRs and 8PRs, and 16 patients reached MRD-negative response. The CAR-T cell expansion and persistence were consistently observed throughout these patients. The medianPFS is 15mon, and the median OS result has not been reached (49.16% progression-free survival, and 53.95% overall survival at 24 months). Among the 44 infused patients, 22.7% had grade 1-2 Cytokine release syndrome (CRS ) and 6.8% (3 patients) had grade 3 CRS. No grade 4 CRS reactions developed and all toxicities were fully reversible. Conclusions: Our result demonstrates the high potential of this single CAR-T infusion therapy for RRMM, including 2sCRs, 16CRs and ongoing clinical responses for more than 26 months, with manageable CRS to date. These initial data provide strong evidence to support the further development of this anti-myeloma cellular immunotherapy. Disclosures No relevant conflicts of interest to declare.

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Tigit, CD226 and PD-L1/PD-1 Are Highly Expressed By Marrow-Infiltrating T Cells in Patients with Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.2102.2102 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2102-2102 ◽

Cited By ~ 5

Author(s):

Mahesh Yadav ◽

Cherie Green ◽

Connie Ma ◽

Alberto Robert ◽

Andrew Glibicky ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Plasma Cells ◽

Color Flow

Abstract Introduction:TIGIT (T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) is an inhibitory immunoreceptor expressed by T and natural killer (NK) cells that is an important regulator of anti-tumor and anti-viral immunity. TIGIT shares its high-affinity ligand PVR (CD155) with the activating receptor CD226 (DNAM-1). We have recently shown that TIGIT blockade, together with PD-L1/PD-1 blockade, provides robust efficacy in syngeneic tumor and chronic viral infection models. Importantly, CD226 blockade abrogates the benefit of TIGIT blockade, suggesting additional benefit of TIGIT blockade through elaboration of CD226-mediated anti-tumor immunity, analogous to CTLA-4/CD28 regulation of T-cell immunity. Whether TIGIT and CD226 are expressed in patients with multiple myeloma (MM) and how TIGIT expression relates to PD-L1/PD-1 expression is unknown. Here we evaluate expression of TIGIT, CD226, PD-1 and PD-L1 in patients with MM to inform novel immunotherapy combinations. Methods:We performed multi-color flow cytometry (n = 25 patients), and multiplex qRT-PCR (n = 7) on bone marrow specimens from patients with MM to assess expression of TIGIT, CD226, PD-1, and PD-L1 on tumor and immune cells. Cells were stained with fluorescently conjugated monoclonal antibodies to label T cells (CD3, CD4, CD8), NK cells (CD56, CD3), plasma cells (CD38, CD45, CD319, CD56), inhibitory/activating receptors (PD-1, TIGIT, PD-L1, CD226), and an amine-reactive viability dye (7-AAD). Stained and fixed cells were analyzed by flow cytometry using BD FACSCanto™ and BD LSRFortessa™. Results:TIGIT, CD226 and PD-L1/PD-1 were detectable by flow cytometry in all patients with MM who were tested, with some overlapping and distinct expression patterns. TIGIT was commonly expressed by marrow-infiltrating CD8+ T cells (median, 65% of cells), CD4+ T cells (median, 12%) and NK cells. In contrast, CD226 was more commonly expressed by marrow-infiltrating CD4+ T cells (median, 74%) compared with CD8+ T cells (median, 38%). PD-1 was expressed by marrow-infiltrating CD8+ T cells (median 38%) and CD4+ T cells (median, 16%). TIGIT was co-expressed with PD-1 on CD8+ T cells (67%-97% TIGIT+ among PD-1+), although many PD-1-negative CD8+ T cells also expressed TIGIT (39%-78% of PD-1-negative). PD-L1 was also expressed by CD8+ (median, 23%) and CD4+ (median, 8%) T cells in addition to MM plasma cells (median, 95%), albeit with significantly lower intensity on T cells compared with plasma cells. The expression of TIGIT and PD-L1 mRNA was highly correlated (R2 = 0.80). Analysis of PVR expression will also be presented. Conclusions: TIGIT, CD226, PD-1, and PD-L1 were commonly expressed in MM bone marrow, but with different patterns. Among CD8+ T cells, the frequency of TIGIT+ T cells was almost twice that of PD-1+ T cells, whereas the majority of CD4+ T cells expressed CD226. TIGIT blockade may complement anti-PD-L1/PD-1 immunotherapy by activating distinct T-cell/NK-cell subsets with synergistic clinical benefit. These results provide new insight into the immune microenvironment of MM and rationale for targeting both the PD-L1/PD-1 interaction and TIGIT in MM. Disclosures Yadav: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Glibicky:Makro Technologies Inc.: Employment; Genentech, Inc.: Consultancy. Nakamura:Genentech, Inc.: Employment. Sumiyoshi:Genentech, Inc.: Employment. Meng:Genentech, Inc.: Employment, Equity Ownership. Chu:Genentech Inc.: Employment. Wu:Genentech: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Adamkewicz:Genentech, Inc.: Employment. Grogan:Genentech, Inc.: Employment. Venstrom:Roche-Genentech: Employment.

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Identification and characterization of an immunodominant SARS-CoV-2-specific CD8 T cell response

10.21203/rs.3.rs-33197/v2 ◽

2020 ◽

Author(s):

Anastasia Gangaev ◽

Steven L. C. Ketelaars ◽

Olga I Isaeva ◽

Sanne Patiwael ◽

Anna Dopler ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Hla Class I ◽

Cd8 T Cell ◽

T Cell Response ◽

Clinical Activity ◽

Tcr Diversity ◽

And Migration

Abstract Global efforts are ongoing to develop vaccines against SARS-CoV-2 causing COVID-19. While there is accumulating information on antibody responses against SARS-CoV-2, less is known about CD8 T-cell recognized SARS-CoV-2 epitopes and the functional state of SARS- CoV-2-specific CD8 T cells. To address these issues, we analyzed samples from 18 COVID- 19 patients for CD8 T-cell recognition of 500 peptide HLA class I complexes, restricted by 10 common HLA alleles. Several epitopes derived from ORF1ab were identified, including an immunodominant epitope restricted by HLA-A*01:01. The immunodominance was further supported by high TCR diversity within the CD8 T cells specific for this epitope. Noteworthy, the ORF1ab is not included in the majority of vaccine candidates in development, which may influence their clinical activity. In-depth characterization of identified SARS-CoV-2-specific CD8 T cell responses revealed a lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining cell survival.

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PD-1 Blockade Reinvigorates Bone Marrow CD8+ T Cells from Patients with Multiple Myeloma in the Presence of TGF-β Inhibitors

Blood ◽

10.1182/blood-2019-128182 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3241-3241

Author(s):

Minsuk Kwon ◽

Eui-Cheol Shin ◽

Yoon Seok Choi

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Checkpoint ◽

Cytokine Production ◽

Plasma Cells ◽

Myeloma Cells ◽

Tgf Β1

Programmed cell death (PD)-1/PD-Ligand 1(PD-L1) blockade that reinvigorates exhausted T cells has been approved for the treatment of various solid tumors and hematological malignancies. However, in a clinical trial of multiple myeloma (MM) patients, anti-PD-1 monotherapy did not result in a clinical response. Furthermore, clinical trials of combining PD-1 blockade with immunomodulatory drugs or anti-CD38 monoclonal antibody failed to demonstrate clinical benefits in MM patients. To overcome the limitation of anti-PD-1 therapy in MM, the phenotype and differentiation of CD8+ T cells need to be characterized in the bone marrow (BM) of MM patients, particularly by analyzing myeloma antigen-specific CD8+ T cells. In addition, the role of immunosuppressive factors abundant in the MM microenvironment should be considered, including TGF-β. First, we confirmed the upregulation of PD-1 and PD-L1 expression in CD8+ T cells and myeloma cells, respectively, from the BM of MM patients. PD-1-expressing CD8+ T cells from the BM of MM patients co-expressed other checkpoint inhibitory receptors including Tim-3, LAG-3, and TIGIT. We also investigated the expression of T-cell transcription factors, such as T-bet, and EOMES, which are related to T-cell differentiation. In BM from MM patients, PD-1+CD8+ T cells had a higher percentage of EomeshiT-betlo cells than PD-1-CD8+ T cells. These data demonstrate that PD-1-expressing CD8+ T cells from the BM of MM patients exhibit a terminally differentiated phenotype with co-expression of multiple immune checkpoint inhibitory receptors. These results were also observed in BM CD8+ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. Next, we investigated proliferation and cytokine production of BM CD8+ T cells from MM patients. BM CD8+ T cells from MM patients exhibited reduced proliferation and cytokine production upon T cell receptor (TCR) stimulation, compared to BM CD8+ T cells from other control group such as of undetermined significance. However, both anti-PD-1 alone and combined blockade of PD-1 with other immune checkpoint receptors, such as Tim-3, Lag-3, or TIGIT, did not increase the proliferation of BM CD8+ T cells from MM patients. Likewise, anti-PD-1 treatment failed to induce reinvigoration of BM CD8+ T cells stimulated with HLA-A*0201-restricted myeloma antigen peptides, including NY-ESO-1157-165 and HM1.2422-30 peptides. These data demonstrate that blocking PD-1 is not sufficient to restore the function of BM CD8+ T cells from MM patients. It has been known that TGF-β, which is actively secreted by malignant plasma cells and BM stromal cells, can inhibit T-cell responses. We confirmed that the major source of TGF- β1 is plasma cells including myeloma cells among BMMCs from MM patients, and the number of TGF- β1-producing plasma cells, including myeloma cells, is increased in the BM of MM patients. We investigated whether blocking TGF-β signaling enhances reinvigoration of BM CD8+ T cells from MM patients. The combined blockade of PD-1 and TGF- β significantly increased the proliferation of BM CD8+ T cells from MM patients in the presence of TCR stimulation. The production of IFN-γ and TNF by BM CD8+ T cells was also rescued by combined blockade of PD-1 and TGF-β. Moreover, combination of anti-PD-1 antibody and TGF-β inhibitors increased proliferative responses of BM CD8+ T cells from HLA-A2+ MM patients stimulated with a mixture of HLA-A*0201-restricted myeloma antigen peptides (NY-ESO-1157-165 and HM1.2422-30 peptides). Thus, PD-1 blockade reinvigorates BM CD8+ T cells from MM patients in the presence of TGF-β inhibitors. Taken together, BM CD8+ T cells and myeloma antigen-specific CD8+ T cells express increased levels of PD-1 and have a terminally exhausted phenotype in MM patients. Under TGF-β inhibition, anti-PD-1 reinvigorates BM CD8+ T cells from MM patients, but PD-1 blockade alone does not restore the function of BM CD8+ T cells. Blocking both TGF-β and PD-1 can be a promising therapeutic strategy for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

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Superior Efficacy of CAR-T Cells Using Marrow-Infiltrating Lymphocytes (MILsTM) As Compared to Peripheral Blood Lymphocytes (PBLs)

Blood ◽

10.1182/blood-2019-130629 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4437-4437 ◽

Cited By ~ 2

Author(s):

Eric R. Lutz ◽

Srikanta Jana ◽

Lakshmi Rudraraju ◽

Elizabeth DeOliveira ◽

Jing Zhou ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Employment Equity ◽

Car T Cells ◽

Car T ◽

Equity Ownership

Background The type of T cell used in generating chimeric antigen receptor (CAR) T cells is an important choice. Evidence suggests that T cells that are early in the effector/memory differentiation pathway with more stemness and greater potential to persist are better than more differentiated T cells with less stemness that are more readily exhausted and have less potential to persist. Marrow-infiltrating Lymphocytes (MILsTM) is a novel form of adoptive T cell therapy composed of patient-autologous, polyclonal CD4 and CD8 T cells that are activated and expanded from the bone marrow. Genetically unmodified MILsTM have demonstrated antitumor activity in patients with multiple myeloma and are being developed for several other tumor types, including non-small cell lung cancer and other solid tumors. Distinguishing features of bone marrow T cells used to produce MILsTM include their memory phenotype, inherent tumor antigen-specificity, higher CD8:CD4 ratio and ability to persist long-term when compared to peripheral blood lymphocytes (PBLs) which is the T cell source used to produce currently approved CAR-T therapies. Based on these differences, we hypothesize that MILsTM provide a more robust and better fit platform for CAR-T therapy compared to PBLs. Using a CD38-specific, 4-1BB/CD3z-signaling CAR as an initial model, we have demonstrated the feasibility of producing CAR-modified MILsTM (CAR-MILsTM) and showed that CAR-MILsTM demonstrate superior killing in vitro compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs). Herein, we build on our previous data and add a second BCMA-specific CAR model. We use the two multiple myeloma model systems to compare cytolytic potential, functionality, and expression of phenotypic markers of memory, stemness and exhaustion between patient-matched CAR-MILsTM and CAR-PBLs. Methods Matched pairs of CAR-MILsTM and CAR-PBLs were produced from the bone marrow and blood of multiple myeloma patients. Two different in vitro cytotoxicity assays, the RTCA xCelligence real-time impedance and FACS assays, were used to evaluate antigen-specific killing of target tumor cells. Functionality of CD4 and CD8 CAR-T cells, at the single-cell level, was evaluated by measuring the secretion of 32 cytokines and chemokines following in vitro antigen-specific stimulation using IsoPlexis IsoCode chips and analyzed using IsoPeak. Expression of markers of T cell memory (CD45RO & CCR7/CD62L), stemness (CD27) and exhaustion (PD1 & TIM3) on CAR-MILsTM and CAR-PBLs prior to and following antigen-specific stimulation was evaluated by flow-cytometry (FACS). Results CAR-MILsTM demonstrated superior killing of tumor target cells in vitro, regardless of the antigen specificity of the CAR, when compared to matched CAR-PBLs and this superiority persisted even upon repeated antigen encounter - a factor that may be critical in guaranteeing better anti-tumor efficacy and persistence. CAR-MILsTM demonstrated increased polyfunctionality (secretion of 2+ cytokines per cell) and an increased polyfunctional strength index (PSI) following antigen-stimulation compared to CAR-PBL in both CD4 and CD8 T cells. The enhanced PSI in CAR-MILsTM was predominately mediated by effector, stimulatory and chemoattractive proteins associated with antitumor activity including Granzyme B, IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and enhanced secretion of these same proteins was reported to be associated with improved clinical responses in patients with Non-Hodgkin lymphoma treated with CD19-specific CAR-T therapy. Expression of memory markers on CD4 and CD8 T cells were similar in CAR-MILsTM and CAR-PBLs both prior to and following antigen-stimulation. Although expression of CD27, PD1 and TIM3 were similar at baseline, CAR-MILs maintained higher levels of CD27 and lower levels of PD1 and TIM3 compared to CAR-PBLs following antigen-stimulation in both CD4 and CD8 T cells. Conclusions Collectively, our data suggest that CAR-MILsTM have several advantages over CAR-PBLs, including increased cytolytic potential, enhanced polyfunctionality, increased stemness and less exhaustion. Based on these differences and the inherent antitumor properties of MILsTM, we speculate that CAR-MILsTM would be more potent and effective than currently approved CAR-T products derived from PBLs. Disclosures Lutz: WindMIL Therapeutics: Employment, Equity Ownership. Jana:WindMIL Therapeutics: Employment, Equity Ownership. Rudraraju:WindMIL Therapeutics: Employment, Equity Ownership. DeOliveira:WindMIL Therapeutics: Employment, Equity Ownership. Zhou:Isoplexis: Employment, Equity Ownership. Mackay:Isoplexis: Employment, Equity Ownership. Borrello:Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX; BMS: Consultancy; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau. Noonan:WindMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties; Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX.

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CD8+ T Cells Specific for Cancer-Testis Antigens Are Found in Many Patients with Multiple Myeloma and Correlate with Disease Burden.

Blood ◽

10.1182/blood.v104.11.2460.2460 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2460-2460

Author(s):

Oliver C. Goodyear ◽

Karen Piper ◽

Julie Arrazi ◽

Naeem Khan ◽

Premini Mahendra ◽

...

Keyword(s):

Multiple Myeloma ◽

Immune Response ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Specific Immune Response ◽

Functional Responses ◽

Cancer Testis Antigens ◽

Cancer Testis

Abstract Proteins from the family known as ‘cancer-testis antigens’ (CTAg) are expressed in some cases of multiple myeloma and subsets of acute myeloid leukaemia. CTAg can stimulate CD8+ T cell responses in patients with melanoma but there are no reports of CTAg-specific immune response in patients with haematological malignancy. Such information is critical to assess whether or not these antigens act as targets for tumour-specific immunity or if they could be used as targets for immunotherapy. We have used twelve peptide epitopes from a range of cancer-testis antigens which have been previously defined as epitopes for CD8+ T cells. These were used to screen for tumour-specific T-cells in blood of patients with multiple myeloma at various stages of their disease. The IFNγ cytokine secretion assay was used to detect functional responses and magnetic selection was employed to increase the sensitivity of detection. FACS analysis was used to quantitate the frequency of responding cells. 37 patients were screened with an age range of between 45 and 88 years. Blood samples were taken at monthly intervals and the percentage of CD8+ T cells responding to each peptide was calculated. 13 patients responded to 1 or more of the peptides with a range between 0.01% and 0.7% of the total CD8+ T cell pool. The frequency of the tumour-specific response fluctuated during treatment in individual patients. Analysis of the CTAg-specific immune response in relation to disease course revealed that the immune response was generally correlated with tumour burden as revealed by the paraprotein level. CTAg HLA-peptide tetramers incorporating peptides from LAGE-1 and MAGE-2 were able to directly visualize CTAg-reactive T cells in PBMC. CTAg-specific CD8+ T cells may have been primed and expanded by expression of CTAg on tumour cells or following ‘cross presentation’ through dendritic cells. In conclusion, T cells specific for cancer-testis antigens are present in the blood of a subset of patients with multiple myeloma. The clinical significance of this observation is currently being addressed.

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