CD73 Inhibition Overcomes Immunosuppression and Triggers Autologous T-Cell Mediated Multiple Myeloma Cell Lysis in the Bone Marrow Milieu

Abstract Introduction: Adenosine is an anti-inflammatory and immunosuppressive metabolite, that signals to diminish activation and proliferation of cytotoxic T-cells, impair activity of natural killer cells and CD4 + effector T-cells, and promote the expansion of immunosuppressive cell types. CD73, a cell surface ecto-5'-nucleotidase, is required to convert AMP to adenosine and is a major catalyst of adenosine generation in the tumor microenvironment. Overexpression of CD73 is observed in many tumors and correlates with unfavorable clinical outcome. Bone marrow (BM) aspirates from multiple myeloma (MM) patients have shown increased adenosine levels correspond with disease progression [Horenstein et al. Mol Med. 2016,22:694-704] In addition to the adenosine rich feature of MM, multiple cell types within the MM BM niche express the enzymes required for adenosine production from both NAD and ATP precursors, including CD38, CD203a, CD39 and CD73. Previously, we demonstrated that dysfunctional plasmacytoid dendritic cells (pDCs) predominantly found in the BM of MM patients contribute to MM cell growth, survival, and suppression of antitumor immunity [Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia 2015, 29:1441-1444]. We recently discovered that the interaction between pDCs and MM cells increased CD73 transcript and protein levels in both cell types, implicating a role for adenosine signaling via CD73 signaling axis in MM. Together, these MM disease features indicate that reducing the level of adenosine via inhibition of CD73 may represent a unique vulnerability and treatment strategy for MM. Methods: To understand the functional consequence of CD73 inhibition in MM, autologous ex vivo cell assays using freshly isolated BM aspirates from MM patients were used to detect changes in immune cell function and MM cell viability upon treatment with OP-5558, a potent and selective CD73 small molecule inhibitor which is an analog of the clinical candidate, ORIC-533. The majority of BM samples utilized were from patients with relapsed or refractory MM after at least three lines of therapy including immunomodulatory drugs, proteasome inhibitors, and anti-CD38 monoclonal antibodies, as well as a patient with relapsed MM post BCMA-CAR-T therapy. Results: In BM aspirates from MM patients with relapsed refractory MM, CD73 inhibition by OP-5558 triggered activation of MM pDCs, evidenced by increased expression of CD40/CD83/CD86 (1.2-1.5-fold, OP-5558-treated versus untreated; p < 0.05; n=3). This inhibition of CD73 reversed immunosuppression in MM BM. Specifically, CD73 inhibitor OP-5558 stimulated T-cell activation, associated with increased CD69 cell surface expression on CD3 + T-cells (CD69 MFI:20% increase, treated versus control; p = 0.0031; n = 3). Moreover, CD8 + T-cells from these co-cultures enhanced cytolytic activity against patient MM cells, significantly decreasing autologous MM cell viability (mean 42% decrease in viability; treated versus control; p=0.014; n=5). Of note, OP-5558 treatment did not directly affect viability of MM cells when treated in isolation, indicating that the observed decreased viability occurs via enhanced cytotoxic T-cell activity. Importantly, we show that OP-5558 triggered significant MM cell lysis even within autologous MM bone marrow mononuclear cell (BMNC) cultures, confirming that CD73 inhibition restores MM-specific cytolytic activity of autologous patient T-cells in the MM BM microenvironment. (mean 37% decrease in viability; treated versus control; p=0.009; n=3). Conclusions: This study therefore demonstrates that: 1. CD73-mediated adenosine activity suppresses the cytolytic activity of T-cells against tumor cells in the MM BM milieu; and 2. CD73 inhibition can overcome immune suppression and restore lysis of MM cells by autologous T-cells. A clinical trial of potent, selective, orally bioavailable CD73 inhibitor ORIC-533 will examine the utility of CD73 inhibition to improve outcome in patients with relapsed refractory MM. Disclosures Junttila: ORIC Pharmaceuticals: Current Employment. Sutimantanapi: ORIC Pharmaceuticals: Current Employment. Chen: ORIC Pharmaceuticals: Current Employment. Warne: ORIC Pharmaceuticals: Current Employment. Chang: ORIC Pharmaceuticals: Current Employment. Blank: ORIC Pharmaceuticals: Current Employment. Wu: ORIC Pharmaceuticals: Current Employment. Moore: ORIC Pharmaceuticals: Current Employment. Ndubaku: ORIC Pharmaceuticals: Current Employment. Zavorotinskaya: ORIC Pharmaceuticals: Current Employment. Nadeem: GSK: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Friedman: ORIC Pharmaceuticals: Current Employment. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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The Effects of BCMA Expression, Soluble BCMA, and Combination Therapeutics on the Anti-Tumor Activity of HPN217, a BCMA-Targeting Tri-Specific T Cell Engager Against Multiple Myeloma

Blood ◽

10.1182/blood-2021-151880 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1185-1185

Author(s):

Patrick P Ng ◽

Wade Aaron ◽

Evan Callihan ◽

Golzar Hemmati ◽

Che-Leung Law ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cell Surface ◽

Half Life ◽

Plasma Cells ◽

Target Cells ◽

Publicly Traded ◽

Anti Tumor Activity

Abstract Introduction B-cell maturation antigen (BCMA) is a cell surface receptor highly and selectively expressed on normal plasma cells and transformed plasma cells in multiple myeloma (MM) patients. Upon ligand binding, BCMA initiates signals that promote the survival of MM cells and the production of immunosuppressive factors. Therapeutics that target BCMA are being investigated in the clinic, with encouraging preliminary results. HPN217 is a Tri-specific T Cell-Activating Construct (TriTAC) specific to BCMA, to serum albumin for half-life extension, and to CD3ε for redirecting T cells against MM cells. It is currently being evaluated in a phase 1 /2 clinical trial for relapsed or refractory MM (NCT04184050). Herein, we describe translational studies to examine factors that may impact the therapeutic efficacy of HPN217, including the target BCMA, in membrane-bound or soluble form, and concomitant or combination therapeutics such as γ-secretase inhibitor (GSI) and dexamethasone. Results To evaluate the effects of HPN217 against primary MM cells, we used a patient-derived 3D-culture system (3DTEBM) designed to recapitulate the biology within the bone marrow microenvironment. 3DTEBM seeded with bone marrow accessory cells and autologous plasma recreate niches along an oxygen gradient that enable the survival and expansion of autologous MM cells without additional nutrient supplements. 3DTEBM's were established from 5 MM patients with varying ratios of autologous CD3+ T cells to MM cells (0.15-0.6). Although the functional competence of the T cells was unknown, HPN217 was able to mediate MM cell killing in 80% of the cultures with up to 71% of MM cells eliminated at a T cell/MM cell ratio of 0.45. The anti-tumor efficacy of HPN217 correlated strongly (R 2 = 0.99) with BCMA expression on the MM cells as measured by flow cytometry, suggesting the number of target receptors can be a limiting factor in efficacy. Consistent with this result, pre-incubation of target cells with 1 or 10 μg/mL anti-BCMA reduced the activity of HPN217 in T cell-dependent cellular cytotoxicity (TDCC) assays using healthy donor T cells and MM cell lines. Soluble BCMA (sBCMA) is produced when the extracellular domain of BCMA is cleaved by γ-secretase. It may act as a sink for HPN217. There was no correlation between the activity of HPN217 and the quantity of sBCMA in 3DTEBM. However, in TDCC assays, the addition of 6.25, 25 and 100 nM recombinant BCMA respectively led to 4-, 9- and 28-fold increases in the EC 50 of HPN217. Taken together, these data underscore the importance of preserving BCMA on MM cells and reducing sBCMA in circulation. Interestingly, treatment of MM cell line RPMI8226 with the GSI LY-3039478 for 24 hours increased the cell surface expression of BCMA by 3.6 folds. Using RPMI8226 as target cells in the 3DTEBM system, LY-3039478 increased the killing efficacy of HPN217-redirected primary T cells by 1.9 folds. Dexamethasone (Dex) is used with other therapeutics for treating MM. It is also commonly given to manage cytokine release syndrome (CRS) caused by T cell engagers. We conducted TDCC assays in the presence of 0.07-300 nM Dex to simulate plasma concentrations relevant to dose levels of Dex premedication for CRS. The highest Dex concentrations caused ≤3-fold increases in the EC 50 of HPN217. Considering this and the plasma half-life of i.v. injected Dex at <5 h, the suppressive effect of Dex on the anti-tumor activity of HPN217-redirected T cells may be limited. We then evaluated if MM.1S-Luc cell line xenografts in NCG mice would be a suitable model to extend the above in vitro findings to an in vivo setting. Lesions in the spine, skull and femur in NCG mice treated with vehicle could be detected by bioluminescent imaging. All mice succumbed to the disease within 40 days. By contrast, animals treated with HPN217 were protected in a dose-dependent manner. Mice that received the highest dose remained 100% disease-free at the end of the study (Figure 1). Conclusions We demonstrated HPN217 mediated BCMA-dependent primary MM cell killing by autologous T cells, and that the density of BCMA target on the surface of MM cells and sBCMA affected the efficacy of HPN217 in cultures. GSI, which increased the expression of BCMA on MM cells, enhanced the efficacy of HPN217. On the other hand, Dex had limited negative effect. HPN217 in combination with approved and experimental MM therapeutics is being evaluated in the 3DTEBM and MM.1S-Luc models. Figure 1 Figure 1. Disclosures Ng: Harpoon Therapeutics: Current Employment, Current equity holder in publicly-traded company. Aaron: Harpoon Therapeutics: Current Employment, Current equity holder in publicly-traded company. Callihan: Harpoon Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hemmati: Harpoon Therapeutics: Current Employment, Current equity holder in publicly-traded company. Law: Harpoon Therapeutics: Current Employment, Current equity holder in publicly-traded company. Azab: Cellatrix, LLC: Current Employment, Current holder of individual stocks in a privately-held company. Sun: Harpoon Therapeutics: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months.

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Clonally Expanded Bone Marrow T Cells Show Effector Differentiation and Rarely Recognize Disease-Associated Antigens in Multiple Myeloma

Blood ◽

10.1182/blood-2020-136991 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-7

Author(s):

Carlotta Welters ◽

Meng-Tung Hsu ◽

Christian Alexander Stein ◽

Livius Penter ◽

María Fernanda Lammoglia Cobo ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Cell Expansion ◽

Advisory Committees ◽

Myeloma Cells ◽

T Cell Expansion

Multiple myeloma is a malignancy of monoclonal plasma cells accumulating in the bone marrow. The critical influence of tumor-infiltrating T cells on disease control and therapeutic responses has been shown in a variety of malignancies, however, the role of multiple myeloma bone marrow-infiltrating T cells is incompletely understood. Although it has been shown that multiple myeloma neo-antigen-specific T cells can be expanded in vitro, little is known about functions and specificities of clonally expanded multiple myeloma-infiltrating bone marrow T cells. Here we asked at the single cell level whether clonally expanded T cells i) were detectable in multiple myeloma bone marrow and peripheral blood, ii) showed characteristic immune phenotypes, and iii) recognized antigens selectively presented on multiple myeloma cells. A total of 6,744 single bone marrow T cells from 13 treatment-naïve patients were index-sorted and sequenced using our methodologies for determination of paired T cell receptor (TCR) αβ sequences along with immune phenotype, transcription factor and cytokine expression. Clonal T cell expansion occurred predominantly within the CD8+ compartment. Phenotypes of clonally expanded T cells were distinctive of cytolytic effector differentiation and significantly different from non-expanded CD8+ T cells. Less than 25% of expanded CD8+ T cell clones expressed the immune checkpoint molecules programmed death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), or T cell immunoglobulin and mucin-domain containing-3 (TIM-3), while B and T lymphocyte attenuator (BTLA) was expressed on more than half of the expanded clones. Clonal T cell expansion did not correlate with neo-antigen load as determined by whole exome and RNA sequencing of purified multiple myeloma cells. Furthermore, peripheral blood TCRβ repertoire sequencing from five selected patients with substantial bone marrow T cell expansion identified 90% of expanded bone marrow T cell clones overlapping with peripheral blood. To determine whether clonally expanded bone marrow T cells recognized antigens selectively presented on multiple myeloma cells, 71 dominant TCRs from five selected patients with substantial clonal T cell expansion were re-expressed in 58α-β- T-hybridoma reporter T cells and co-incubated with CD38-enriched multiple myeloma cells from the same patients. Only one of these TCRs recognized antigens selectively presented on multiple myeloma cells and this TCR was not neo-antigen-specific. Hypothesizing that the target antigen was a non-mutated self-antigen, we could show that this TCR also recognized the plasma cell leukemia cell line U-266 in an HLA-A*02:01-restricted manner. In summary, clonally expanded T cells in multiple myeloma bone marrow of newly diagnosed patients show cytolytic effector differentiation. In the majority of patients, clonally expanded bone marrow T cells do not recognize antigens presented on multiple myeloma cells and are not neo-antigen-specific. Our findings are relevant for the design of future therapeutics and clinical trials. The identified TCR, which recognizes a multiple myeloma antigen shared with U-266 in an HLA-A*02:01-restricted manner, could be a promising candidate for T cell therapy. Disclosures Bullinger: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Abbvie: 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; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

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Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-141540 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Pinar Ataca Atilla ◽

Mary K McKenna ◽

Norihiro Watanabe ◽

Maksim Mamonkin ◽

Malcolm K. Brenner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Tumor Control ◽

Publicly Traded ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T

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

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Induction of T Cell Immunity Using a Multipeptide Cocktail Containing XBP1, CD138 and CS1 Peptides in Smoldering Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.5039.5039 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5039-5039

Author(s):

Jooeun Bae ◽

Rao H. Prabhala ◽

Weihua Song ◽

Yu-Tzu Tai ◽

Kenneth C. Anderson ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Board Of Directors ◽

Advisory Committees ◽

Smoldering Multiple Myeloma ◽

Human T Cell ◽

Cell Immunity ◽

Equity Ownership

Abstract Abstract 5039 Smoldering multiple myeloma (SMM) patients are at high risk for progression to active multiple myeloma (MM), making them candidates for novel immunotherapeutic strategies to prevent or delay disease progression. Among potential strategies, the ability to induce cytotoxic T lymphocytes (CTL) against multiple immunogenic epitopes provides a framework for overcoming major therapeutic challenges including heterogeneity of tumor associated antigen expression, frequent mutations of specific antigens, and variability of the human T-cell repertoire among individuals. In this study, we provide evidence that a cocktail of four immunogenic HLA-A2 specific peptides, heteroclitic XBP1 US184–192, heteroclitic XBP1 SP367–375, native CD138260–268 and native CS1239–247, induces specific CTL response in T cells from SMM patients. Following repeated rounds of multipeptide stimulation, we induced development of CD8+ CTL from SMM patients' T cells. The multipeptide specific-CTL demonstrated polyfunctional immune activities including high levels of IFN-g production, cell proliferation and cytotoxicity against MM cells in an HLA-A2 restricted manner. The multipeptide-specific CTL displayed increased memory (CD45RO+) and activated (CD69+) CD3+CD8+ T lymphocytes, suggesting that a multipeptide vaccine has the potential to induce durable memory by generating specific memory CTL with characteristics of effector T cells against MM cells. In addition, the multipeptide-specific CTL demonstrated peptide-specific responses to each of the relevant epitopes including heteroclitic XBP1 US184–192, heteroclitic XBP1 SP367–375, native CD138260–268 and native CS1239–247, but not against an irrelevant HLA-A2-specific MAGE-3271–279 peptide in various functional assays including antigen-triggered CD137 (4-1BB) expression, IFN-g production and CD107a up-regulation. Therefore, these results suggest the potential of inducing a broad spectrum of immune responses against selected XBP1 unspliced, XBP1 spliced, CD138 and CS1 target antigens in SMM using multipeptide vaccination. In conclusion, these studies provide the framework for clinical trials of vaccination in patients with SMM to delay or prevent progression to active MM. Disclosures: Bae: Oncopep Inc. : Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Anderson:Oncopep Inc. : Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Munshi:Oncopep Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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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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ADG152, a Novel CD20xCD3 T Cell Engager Prodrug with Enhanced Therapeutic Index, Demonstrates Strong Anti-Tumor Activity with Improved Safety

Blood ◽

10.1182/blood-2021-147573 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1204-1204

Author(s):

Bin Cai ◽

Aaron N Nguyen ◽

Songmao Zheng ◽

Jianfeng Shi ◽

Guizhong Liu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Tumor Cells ◽

Cytokine Release ◽

Publicly Traded ◽

Advisory Committees ◽

The Past

Abstract Recent clinical data illustrate the effectiveness of CD20xCD3 T cell engagers (TCEs) that redirect the patient's endogenous T cells to eliminate CD20-positive tumor cells. While several of these products have demonstrated promising clinical activities in B-cell malignancies, their potential therapeutic utility is limited by cytokine release syndrome (CRS), even after strategies such as step-up dosing are implemented. ADG152 is a novel CD20xCD3 TCE prodrug engineered using Adagene's SAFEbody technology to minimize or eliminate CRS and on-target/off-tumor toxicities. The anti-CD20 arm of ADG152 has been engineered for enhanced binding to CD20 compared to other clinical stage or approved antibodies, while its anti-CD3 arm has modulated affinity for CD3 and is also masked by a conditionally activable peptide. In normal tissues and in circulation, the masking moiety on the anti-CD3 arm can function to block the binding of ADG152 to T cells; however, in an activable condition such as the tumor microenvironment where protease activity has been reported to be elevated, the masked antibody can be activated, enabling the activated ADG152 to simultaneously engage T cells and neighboring CD20-expressing tumor cells. In vitro studies showed that ADG152 has enhanced binding to human B cells and CD20-positive Raji tumor cells compared with the benchmark CD20xCD3 TCE plamotamab. On the other hand, ADG152 has significantly reduced binding to the human CD3 δ/ε protein dimer and no binding to human CD3+, CD4+, and CD8+ T cells isolated from PBMCs of normal human donors. Consistent with these results, ADG152 shows significantly decreased ability (more than 100-fold) compared with the benchmark and the unmasked parental molecule to activate CD8+ T cells and to induce T cell-mediated killing in the presence of tumor cells in vitro. ADG152 demonstrated strong anti-tumor effects in vivo. In a human PBMC-engrafted Raji xenograft mouse tumor model, dosing with ADG152 resulted in almost complete tumor growth inhibition at 1.5 mg/kg. In exploratory toxicology studies in cynomolgus monkeys, ADG152 resulted in significantly less cytokine release in monkey blood compared with benchmark, giving ~100-fold safety margin for ADG152 for cytokine induction (Figure). In addition, ADG152 was as effective as the benchmark at inducing B cell depletion from peripheral blood of cynomolgus monkeys. In summary, the preclinical characterization of ADG152 demonstrates that our SAFEbody platform can be used to engineer safe and potent bispecific T cell engagers with increased therapeutic index by allowing for strong anti-tumor activities in mice at doses with minimal cytokine release in monkeys, thereby supporting its advancement to clinical development either as a single agent or in combination with other therapies for the treatment of CD20-expressing B cell malignancies. Figure 1 Figure 1. Disclosures Cai: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen: Sparcbio, LLC: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Zheng: Janssen Pharmaceuticals: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Shi: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Liu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Li: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Du: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Frankel: Cytovia Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company; Adagene Inc.: Consultancy, Current equity holder in publicly-traded company; Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; IMV: Consultancy; Precision Biosciences: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Immunai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Minerva Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Myeloid Therapeutics: Consultancy; RAPT Therapeutics: Consultancy; Syros: Consultancy. Luo: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Xu: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company.

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Baseline and on-Treatment Bone Marrow Microenvironments Predict Myeloma Patient Outcomes and Inform Potential Intervention Strategies

Blood ◽

10.1182/blood-2018-99-113169 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1882-1882 ◽

Cited By ~ 1

Author(s):

Samuel A Danziger ◽

Mark McConnell ◽

Jake Gockley ◽

Mary Young ◽

Adam Rosenthal ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Expression Profiles ◽

Cell Types ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Abstract Introduction The multiple myeloma (MM) tumor microenvironment (TME) strongly influences patient outcomes as evidenced by the success of immunomodulatory therapies. To develop precision immunotherapeutic approaches, it is essential to identify and enumerate TME cell types and understand their dynamics. Methods We estimated the population of immune and other non-tumor cell types during the course of MM treatment at a single institution using gene expression of paired CD138-selected bone marrow aspirates and whole bone marrow (WBM) core biopsies from 867 samples of 436 newly diagnosed MM patients collected at 5 time points: pre-treatment (N=354), post-induction (N=245), post-transplant (N=83), post-consolidation (N=51), and post-maintenance (N=134). Expression profiles from the aspirates were used to infer the transcriptome contribution of immune and stromal cells in the WBM array data. Unsupervised clustering of these non-tumor gene expression profiles across all time points was performed using the R package ConsensusClusterPlus with Bayesian Information Criterion (BIC) to select the number of clusters. Individual cell types in these TMEs were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results Our deconvolution approach accurately estimated percent tumor cells in the paired samples compared to estimates from microscopy and flow cytometry (PCC = 0.63, RMSE = 9.99%). TME clusters built on gene expression data from all 867 samples resulted in 5 unsupervised clusters covering 91% of samples. While the fraction of patients in each cluster changed during treatment, no new TME clusters emerged as treatment progressed. These clusters were associated with progression free survival (PFS) (p-Val = 0.020) and overall survival (OS) (p-Val = 0.067) when measured in pre-transplant samples. The most striking outcomes were represented by Cluster 5 (N = 106) characterized by a low innate to adaptive cell ratio and shortened patient survival (Figure 1, 2). This cluster had worse outcomes than others (estimated mean PFS = 58 months compared to 71+ months for other clusters, p-Val = 0.002; estimate mean OS = 105 months compared with 113+ months for other clusters, p-Val = 0.040). Compared to other immune clusters, the adaptive-skewed TME of Cluster 5 is characterized by low granulocyte populations and high antigen-presenting, CD8 T, and B cell populations. As might be expected, this cluster was also significantly enriched for ISS3 and GEP70 high risk patients, as well as Del1p, Del1q, t12;14, and t14:16. Importantly, this TME persisted even when the induction therapy significantly reduced the tumor load (Table 1). At post-induction, outcomes for the 69 / 245 patients in Cluster 5 remain significantly worse (estimate mean PFS = 56 months compared to 71+ months for other clusters, p-Val = 0.004; estimate mean OS = 100 months compared to 121+ months for other clusters, p-Val = 0.002). The analysis of on-treatment samples showed that the number of patients in Cluster 5 decreases from 30% before treatment to 12% after transplant, and of the 63 patients for whom we have both pre-treatment and post-transplant samples, 18/20 of the Cluster 5 patients moved into other immune clusters; 13 into Cluster 4. The non-5 clusters (with better PFS and OS overall) had higher amounts of granulocytes and lower amounts of CD8 T cells. Some clusters (1 and 4) had increased natural killer (NK) cells and decreased dendritic cells, while other clusters (2 and 3) had increased adipocytes and increases in M2 macrophages (Cluster 2) or NK cells (Cluster 3). Taken together, the gain of granulocytes and adipocytes was associated with improved outcome, while increases in the adaptive immune compartment was associated with poorer outcome. Conclusions We identified distinct clusters of patient TMEs from bulk transcriptome profiles by computationally estimating the CD138- fraction of TMEs. Our findings identified differential immune and stromal compositions in patient clusters with opposing clinical outcomes and tracked membership in those clusters during treatment. Adding this layer of TME to the analysis of myeloma patient baseline and on-treatment samples enables us to formulate biological hypotheses and may eventually guide therapeutic interventions to improve outcomes for patients. Disclosures Danziger: Celgene Corporation: Employment, Equity Ownership. McConnell:Celgene Corporation: Employment. Gockley:Celgene Corporation: Employment. Young:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Reiss:Celgene Corporation: Employment, Equity Ownership. Davies:MMRF: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; TRM Oncology: Honoraria; Abbvie: Consultancy; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria. Copeland:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Barlogie:Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend; Multiple Myeloma Research Foundation: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Millenium: Consultancy, Research Funding; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC. Trotter:Celgene Research SL (Spain), part of Celgene Corporation: Employment, Equity Ownership. Hershberg:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Dervan:Celgene Corporation: Employment, Equity Ownership. Ratushny:Celgene Corporation: Employment, Equity Ownership. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding.

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Phase 1 Study of CD19 Targeted 4-1BBL Costimulatory Agonist to Enhance T Cell (Glofitamab Combination) or NK Cell Effector Function (Obinutuzumab Combination) in Relapsed/Refractory B Cell Lymphoma

Blood ◽

10.1182/blood-2020-136571 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 16-17 ◽

Cited By ~ 1

Author(s):

Martin Hutchings ◽

Fritz C. Offner ◽

Francesc Bosch ◽

Giuseppe Gritti ◽

Carmelo Carlo-Stella ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Dose Escalation ◽

Research Funding ◽

Bispecific Antibody ◽

Nk Cell ◽

Publicly Traded ◽

Advisory Committees ◽

Anti Tumor Activity

Background: Up to 50% of patients suffering from Non-Hodgkin`s lymphoma (NHL) become refractory to or relapse after treatment (M. Crump, Blood 2017). With this, the lack of curative outcomes for patients with both indolent and aggressive NHL subtypes remains an unmet medical need. The CD20 CD3 T cell bispecific antibody glofitamab induces specific T-cell activation and has demonstrated significant single agent activity in r/r NHL patients (NP30179 study, M. Dickinson, EHA 2020, Abstract S241). RO7227166, a CD19 targeted 4-1BBL (CD137) costimulatory agonist has shown synergistic anti-tumor activity when combined with glofitamab in preclinical models (fig 1). RO7227166 is a bispecific antibody-like fusion protein composed of a split trimeric 4-1BB ligand, a tumor antigen-targeting moiety recognizing CD19, and a silent Fc part preventing Fc-mediated toxicity. 4-1BB is an inducible co-stimulatory molecule expressed by activated T-cells or NK cells. Through CD19-binding, the 4-1BB ligand moiety can deliver co-stimulatory signals to activated T- and NK-cell subsets in the tumor. The expected mode of action (MoA) for this molecule is to deliver a costimulatory signal 2 to enhance the effector function of tumor-infiltrating T cells or NK cells upon their activation (signal 1) by a T-cell bispecific antibody (e.g. glofitamab, RO7082859) or a tumor-targeted ADCC antibody (e.g. obinutuzumab). By delivering direct T-cell-target cell engagement followed by costimulatory activation the aim is to offer a highly active off-the-shelf immunotherapy combination. Methods: RO7227166 is being developed in combination with glofitamab and obinutuzumab in a phase I, open-label, dose-escalation study BP41072 (NCT04077723). The study is designed to evaluate the combination maximum tolerated dose (MTD), safety, tolerability, pharmacokinetic (PK), and/or pharmacodynamic (PD) profile of escalating doses of RO7227166, and to evaluate preliminary anti-tumor activity in participants with r/r NHL. The dose escalation stage is divided into Part I (combination with obinutuzumab) and Part II (combination with glofitamab) followed by an expansion stage (Part III). During Part I patients receive 1000mg obinutuzumab intravenously (IV) at a q3w schedule in combination with CD19 4-1BBL IV. During part II glofitamab is given in a q3w schedule with RO7227166 introduced at C2D8 and administered concomitantly from C3D1 onwards. A fixed dose of obinutuzumab (Gpt; pre-treatment) is administered seven days prior to the first administration of RO7227166 and seven days prior to the first administration of glofitamab (M. Bacac, Clin Cancer Res 2018; M. Dickinson, EHA 2020, Abstract S241). Patients will initially be recruited into part I of the study only using single-participant cohorts, where a rule-based dose-escalation is implemented, with dosing initiated at 5 μg (flat dose). As doses of RO7227166 increase, multiple participant cohorts will be recruited and dose-escalation will be guided by the mCRM-EWOC design for overdose control. Commencement of Part II including decision on the RO7227166 starting dose will be guided by safety and PK data from Part I. Patients with r/r NHL meeting standard organ function criteria and with adequate blood counts will be eligible. The maximum duration of the study for each participant will be up to 24 months in Part I (excluding survival follow-up) and up to 18 months in Part II and Part III. Tumor biopsies and peripheral blood biomarker analyses will be used to demonstrate MoA and proof of concept of an off the shelf flexible combination option providing signals 1 and 2. Disclosures Hutchings: Takeda: Honoraria; Takeda: Research Funding; Genmab: Honoraria; Roche: Honoraria; Genmab: Research Funding; Janssen: Research Funding; Novartis: Research Funding; Sankyo: Research Funding; Roche: Consultancy; Genmab: Consultancy; Takeda: Consultancy; Roche: Research Funding; Celgene: Research Funding; Daiichi: Research Funding; Sanofi: Research Funding. Bosch:Hoffmann-La Roche: Research Funding. Gritti:Italfarmaco: Consultancy; F. Hoffmann-La Roche Ltd: Honoraria; Jannsen: Other: Travel Support; Autolus: Consultancy; IQVIA: Consultancy; Kite: Consultancy; Takeda: Honoraria; Amgen: Honoraria. Carlo-Stella:Bristol-Myers Squibb, Merck Sharp & Dohme, Janssen Oncology, AstraZeneca: Honoraria; Servier, Novartis, Genenta Science srl, ADC Therapeutics, F. Hoffmann-La Roche, Karyopharm, Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics and Rhizen Pharmaceuticals: Research Funding; Boehringer Ingelheim and Sanofi: Consultancy. Townsend:Roche, Gilead: Consultancy, Honoraria. Morschhauser:Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Consultancy; Janssen: Honoraria; Epizyme: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy. Cartron:Celgene: Consultancy, Honoraria; F. Hoffmann-La Roche: Consultancy, Honoraria; Sanofi: Honoraria; Abbvie: Honoraria; Jansen: Honoraria; Gilead: Honoraria. Ghesquieres:CELGENE: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Roche: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Gilead: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Janssen: Honoraria. de Guibert:Gilead Sciences: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Herter:Roche Glycart AG: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Korfi:Roche Diagnostics GmbH: Consultancy. Craine:Roche: Current Employment. Mycroft:Roche: Current Employment. Whayman:Roche: Current Employment. Mueller:Roche: Current Employment. Dimier:Roche: Current Employment. Moore:Roche: Current Employment. Belli:Roche Pharma: Current Employment. Kornacker:Hoffmann-La Roche Ltd.: Current Employment, Current equity holder in publicly-traded company. Lechner:Roche Diagnostics GmbH: Current Employment, Current equity holder in publicly-traded company. Dickinson:Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck Sharp & Dohme: Consultancy; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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Characterization of T-Cell Exhaustion in Rapid Progressing Multiple Myeloma Using Cross Center Scrna-Seq Study

Blood ◽

10.1182/blood-2021-153863 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 401-401

Author(s):

William Pilcher ◽

Beena E Thomas ◽

Swati S Bhasin ◽

Reyka G Jayasinghe ◽

Adeeb H Rahman ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Surface Marker ◽

Cell Cluster ◽

Advisory Committees ◽

Surface Marker Expression

Abstract Introduction: Multiple myeloma (MM) is a complex hematological malignancy with the heterogenous immune bone marrow (BM) environment contributing to tumor growth, drug resistance, and immune escape. T-Cells play a critical role in the clearance of malignant plasma cells from the tumor environment. However, T-Cells in multiple myeloma demonstrate impaired cytotoxicity, proliferation, and cytokine production due to the activation of immune inhibitory receptors from ligands produced by the myeloma cells. In this study, we investigate the behavior of T-Cells in MM patients by using single-cell RNA-Seq (scRNA-Seq) to compare the transcriptomic profiles of BM T-Cells of patients with rapid progressing (FP; PFS < 18mo) and non-progressing (NP; PFS > 4yrs) disease. Methods: Newly diagnosed MM patients (n=18) from the Multiple Myeloma Research Foundation (MMRF) CoMMpass study (NCT01454297) were identified as either rapid progressors or non-progressors based on their progression free survival since diagnosis. To capture transcriptomic data, scRNA-Seq was performed on 48 aliquots of frozen CD138-negative BM cells at three medical centers/universities (Beth Israel Deaconess Medical Center, Boston, Washington University in St. Louis, and Mount Sinai School of Medicine, NYC). Samples were collected at diagnosis prior to treatment. Surface marker expression for 29 proteins was captured for at least one sample per patient using CITE-Seq. After integration and batch correction, clustering was performed to identify cells of T or NK lineage. Uniform Manifold Approximation and Projection (UMAP) and differential expression were used to identify T-Lymphoid subtypes, and differences in NP and FP samples. Results: In this study, single cell transcriptomic profiles were identified for ~102,207 cells from 48 samples of 18 MM patients. 40,328 T (CD3+) and NK (CD3-, NKG7+) cells were isolated, and subclustered for further analysis (Fig 1A). Using differentially expressed markers for each cluster, the T-Lymphoid subset was refined into seven subtypes, consisting of various CD4+ T-Cells, CD8+ T-Cells, and NK cells (Fig 1B). The CD8+ cells were divided into three distinct phenotypes, namely a GZMK-, GZMB- CD8+ T-Cell cluster, a GZMK+ CD8+ Exhausted T-Cell cluster enriched in TIGIT and multiple chemokines (CCL3, CCL4, XCL2), and a GZMB+ NkT cluster enriched in cytolytic markers (PRF1, GNLY, NKG7) (Fig 1C). Differential expression between NP and FP samples in this CD8+ subset showed enrichment of the NkT cytotoxic markers in NP samples, while FP samples were enriched in the CD8+ Exhausted chemokine markers (Fig 1D). Furthermore, the proportion of CD8+ Exhausted T-Cells was enriched in FP samples (p.val < 0.05) (Fig 1E). Exhaustion markers were measured through both RNA and surface marker levels. In RNA, TIGIT was uniquely associated with the FP-enriched CD8+ Exhausted T-Cell cluster, and CD160 was uniquely expressed in FP samples (Fig 1F). CITE-Seq surface marker expression confirms enrichment of both TIGIT and PD1 in the CD8+ Exhausted T-Cell cluster, and along with more exhaustion in FP samples (p.val < 0.01). Conclusion: In this study, we have identified significant differences in T-Cell activity in patients with non-progressing and rapid-progressing multiple myeloma. T-Cells in rapid progressing patients appear to be in a suppressed state, with low cytolytic activity and enriched exhaustion markers. This GZMK+ T-Cell population shows strong similarities with an aging-associated subtype of effector memory T-Cells found to be enriched in older populations (Mogilenko et al, Immunity 54, 2021). These findings will be further validated in an expanded study, consisting both of a larger number of samples, and multiple samples at different timepoints from the same patient. Figure 1 Figure 1. Disclosures Jayasinghe: MMRF: Consultancy; WUGEN: Consultancy. Vij: BMS: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; BMS: Honoraria; GSK: Honoraria; Oncopeptides: Honoraria; Karyopharm: Honoraria; CareDx: Honoraria; Legend: Honoraria; Biegene: Honoraria; Adaptive: Honoraria; Harpoon: Honoraria. Kumar: Carsgen: Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Bluebird Bio: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Oncopeptides: Consultancy; Antengene: Consultancy, Honoraria; Roche-Genentech: Consultancy, Research Funding; Merck: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Amgen: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: 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; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

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Rejuvenated BCMA-Specific CD8 + Cytotoxic T Lymphocytes Derived from Antigen-Specific Induced Pluripotent Stem Cells : Immunotherapeutic Application in Multiple Myeloma

Blood ◽

10.1182/blood-2021-153110 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 75-75

Author(s):

Jooeun Bae ◽

Shuichi Kitayama ◽

Laurence Daheron ◽

Zach Herbert ◽

Nikhil C. Munshi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

T Cell Differentiation ◽

Immune Checkpoints ◽

Publicly Traded ◽

Advisory Committees ◽

Induced Pluripotent

Abstract T cell regenerative medicine represents an emerging immunotherapeutic approach using antigen-specific Induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8 + cytotoxic T lymphocytes (CTL). Here we report on an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) against multiple myeloma (MM) via establishment of antigen-specific iPSC, followed by differentiation into highly functional BCMA-specific CD8 + CTL. The reprogrammed BCMA-specific iPSC displayed normal karyotypes and pluripotency potential as evidenced by expression of stem cell markers (SSEA-4, TRA1-60) and alkaline phosphatase, along with differentiation into three germ layers (Ectoderm, Mesoderm, Endoderm). During embryoid body formation, BCMA-specific iPSC further polarized into the mesoderm germ layer, evidenced by the activation of SNAI2, TBX3, PLVAP, HAND1 and CDX2 transcriptional regulators. Next, the BCMA-specific iPSC clones committed to CD8 + T cell differentiation were characterized by analyzing their hematopoietic progenitor cells (HPC; CD34 + CD43 +/CD14 - CD235a -) for specific transcriptional regulation. RNAseq analyses indicated a low variability and similar profiles of gene transcription within the iPSC clones committed to CD8 + CTL compared to increased transcriptional variability within iPSC clones committed to different cell types. The unique transcriptional profiles of the iPSC committed to CD8 + T cells included upregulation of transcriptional regulators controlling CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, and cytokine transporter/cytotoxic mediator development, as well as downregulation of regulators controlling B and T cell interactions, CD4 + Th cells, and inhibitory receptor development. Specifically, a major regulatory shift, indicated by upregulation of specific genes involved in immune function, was detected in HPC from the iPSC committed to CD8 + T cells. BCMA-specific T cells differentiated from the iPSC were characterized as displaying mature CTL phenotypes including high expression of CD3, CD8a, CD8b, TCRab, CD7 along with no CD4 expression (Fig. 1). In addition, the final BCMA iPSC-T cells were predominantly CD45RO + memory cells (central memory and effector memory cells) expressing high level of T cell activation (CD38, CD69) and costimulatory (CD28) molecules. Importantly, these BCMA iPSC-T cells lacked immune checkpoints (CTLA4, PD1, LAG3, Tim3) expression and regulatory T cells induction, distinct from other antigen-stimulated T cells. The rejuvenated BCMA iPSC-T cells demonstrated a high proliferative (1,000 folds increase) during the differentiation process as well as poly-functional anti-tumor activities and Th1 cytokine (IFN-g, IL-2, TNF-a) production triggered in response to MM patients' cells in HLA-A2-restricted manner (Fig. 2). Furthermore, the immune responses induced by these BCMA iPSC-T cells were specific to the parent heteroclitic BCMA 72-80 (YLMFLLRKI) peptide used to reprogram and establish the antigen-specific iPSC. Evaluation of 88 single cell Tetramer + CTL from the BCMA iPSC-T cells revealed a clonotype of unique T cell receptor (TCRa, TCRb) sequence. The BCMA-specific iPSC clones maintained their specific differentiation potential into the antigen-specific CD8 + memory T cells, following multiple subcloning in long-term cultures under feeder-free conditions or post-thaw after long-term (18 months) cryopreservation at -140 oC, which provides additional benefits to treat patients in a continuous manner. Taken together, rejuvenated CD8 + CTL differentiated from BCMA-specific iPSC were highly functional with significant (*p < 0.05) levels of anti-MM activities including proliferation, cytotoxic activity and Th-1 cytokine production. Therefore, the antigen-specific iPSC reprogramming and T cells rejuvenation process can provide an effective and long-term source of antigen-specific memory CTL lacking immune checkpoints and suppressors for clinical application in adoptive immunotherapy to improve patient outcome in MM. Figure 1 Figure 1. Disclosures Munshi: Amgen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Legend: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: 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; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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