Mechanism of Synergistic Effects on T Cell Activation By Avadomide (CC-122) and Nivolumab

Abstract Background: Avadomide (CC-122) is a cereblon modulator that promotes ubiquitination and degradation of the hematopoietic transcription factors Ikaros and Aiolos, leading to immunomodulation, such as T cell activation and increased interleukin-2 (IL-2) production in primary peripheral blood mononuclear cells (PBMCs). The immune checkpoint inhibitor nivolumab (nivo), an anti-PD-1 antibody, induces immune activation and can enhance immune response against various solid tumors. Previously, we have shown that the combination of avadomide and nivo synergistically enhance IL-2 production, T cell proliferation, and immune-mediated cytotoxicity, relative to single agent activity. To understand molecular mechanisms underlying these synergistic effects, we compared the effects of avadomide, nivo, or the combination on gene expression in primary human T cells using whole transcriptome RNA sequencing and differential pathway analysis. Methods: PBMCs were isolated from healthy donors (N=6), treated with DMSO/IgG, avadomide 50 nM, nivo 10 µg/mL, or avadomide and nivo for 1 hour, then stimulated with 0.5 ng/mL staphylococcus enterotoxin B for 48 hours. Culture supernatants were collected for cytokine analysis; T cells were isolated by magnetic cell separation for RNA extraction. RNA was sequenced by Illumina HiSeq v4; data was filtered to transcripts ≥10 counts across all samples and processed by DESeq2. Significantly differentially expressed genes (FDR-adj. P values <0.05) were computed for each treatment group relative to DMSO/IgG controls. Pathway analysis was performed with the GSEA Molecular Signatures Database (MSigDB) using the Hallmark and C2 curated gene sets, which comprise a diverse set of biological pathways, including KEGG and Reactome, to provide unbiased enrichment analysis. T cell-related pathways from the MSigDB C5 (Gene Ontology) collection were used to investigate specific effects on immune function. Synergy was defined by the fractional product method. Results: Avadomide, nivo, and the combination enhanced IL-2 production in SEB-stimulated PBMCs by 282%, 47%, and 586% respectively, compared with DMSO/IgG controls, confirming the synergistic effects on cytokine production. The top pathways upregulated in each treatment group included the following T cell related pathways: for avadomide - T cell receptor (TCR) signaling, JAK/STAT, cytokine/receptor interaction; for nivo - CD8 TCR pathway and HIF1A targets; and for the combination - TCR signaling, JAK/STAT, and HDAC3 targets which are required for T cell maturation and cytokine production. Combination treatment uniquely upregulated pathways including the Biocarta cytotoxic T cell pathway and calcium signaling in CD4+ T cells. Among the 7732 genes modified by any treatment, 1949 were uniquely differentially expressed by the combination. A targeted enrichment analysis using only T cell-related pathways from the C5 collection revealed that these uniquely differentially expressed genes represent processes such as T cell differentiation, proliferation, and activation. Conclusions: At the gene expression level, many T cell-related pathways were upregulated by one or more single agent and/or the combination. However, the presence of uniquely differentially regulated genes suggests that combination treatment induced broader effects in pathways involved in T cell differentiation, proliferation, and activation than either avadomide or nivo alone. Interestingly, the cytotoxic T cell pathway was uniquely upregulated by the combination, consistent with our previous finding of a significant increase in cytotoxicity with avadomide/nivo in combination. Calcium signaling in CD4+ T cell genes were also uniquely upregulated, suggesting that avadomide/nivo combination effects may involve nuclear factor of activated T cells (NFAT)-dependent T cell immune regulation. These data provide molecular support for the in vitro phenotypic activity of avadomide and checkpoint blockade on enhancing T cell activity. Avadomide is now under investigation in combination with checkpoint blockade in solid tumors (NCT02859324) and with CAR T therapy in lymphoma (NCT03310619). Disclosures Nakayama: Celgene Corporation: Employment, Equity Ownership. Stokes:Celgene Corporation: Employment, Equity Ownership. Waldman:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership.

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Clinical Dosing Regimen of Selinexor Maintains Normal Immune Homeostasis and T Cell Effector Function in Mice: Implications for Combination with Immunotherapy

Blood ◽

10.1182/blood.v128.22.2525.2525 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2525-2525

Author(s):

Paul M Tyler ◽

Mariah M Servos ◽

Boris Klebanov ◽

Trinayan Kashyap ◽

Sharon Shacham ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Cell Activation ◽

T Cell Development ◽

Immune Homeostasis ◽

Dosing Regimen ◽

Employment Equity ◽

Equity Ownership

Abstract Selinexor (KPT-330) is a first in class nuclear transport inhibitor of exportin-1(XPO1) currently in advanced clinical trials to treat patients with solid and hematological malignancies. To determine how selinexor might impact anti-tumor immunity, we analyzed immune homeostasis in mice treated with high selinexor doses (15 mg/kg, three times a week: M, W, F) and found disruptions in T cell development, a progressive loss of CD8 T cells and increases in inflammatory monocytes. Antibody production in response to immunization was mostly normal. Precursor populations in bone marrow and thymus were unaffected by high doses of selinexor, suggesting that normal immune homeostasis could recover. We found that high dose of selinexor given once per week preserved nearly normal immune functioning, whereas a lower dose given 3 times per week (7.5 mg/kg, M, W, F) was not able to restore immune homeostasis. Both naïve and effector CD8 T cells cultured in vitro showed impaired activation in the presence of selinexor. These experiments suggest that XPO1 function is required for T cell development and function. We then determined the minimum concentration of selinexor required to block T cell activation, and showed that T cell inhibitory effects of selinexor occur at levels above 100nM, corresponding to the first 24 hours post-oral dosing of 10 mg/kg. In a model of implantable melanoma, we used selinexor treatment at the clinically relevant dosing regimen of 10 mg/kg with a 5-day drug holiday (M, W selinexor treatment). After two weeks of treatment, tumors were harvested and tumor infiltrating leukocyte (TIL) populations were analyzed. This treatment led to intratumoral IFNg+, granzyme B+ cytotoxic CD8 T cells that were comparable to vehicle treated mice. Overall, selinexor treatment leads to transient inhibition of T cell activation but the clinically relevant once and twice weekly dosing schedules that incorporate sufficient drug holidays allow for normal CD8 T cell functioning and development of anti-tumor immunity. These results provide additional support to the recommended selinexor phase 2 dosing regimen, as was determined recently (Razak et al. 2016). Disclosures Klebanov: Karyopharm Therapeutics: Employment, Equity Ownership. Kashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics: Employment, Equity Ownership. Dougan:Karyopharm Therapeutics: Consultancy. Dougan:Karyopharm Therapeutics: Consultancy.

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

Blood ◽

10.1182/blood-2019-123979 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5319-5319 ◽

Cited By ~ 2

Author(s):

Ann-Marie E Bröske ◽

Ian James ◽

Anton Belousov ◽

Enrique Gomez ◽

Marta Canamero ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Board Of Directors ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

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

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Therapeutic Candidate Alpn-101, a Dual ICOS/CD28 Antagonist, Potently Suppresses Human/NSG Mouse Xenograft Graft Vs. Host Disease (GvHD) in a Dose Ranging Study and Reduces Disease Activity in a Mouse Model of Hemophagocytic Lymphohistiocytosis (HLH)

Blood ◽

10.1182/blood-2018-99-110001 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2037-2037

Author(s):

Stacey R. Dillon ◽

Katherine E. Lewis ◽

Katherine Verbist ◽

Paige Tedrick ◽

Sabrin Albeituni ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

Inflammatory Diseases ◽

Employment Equity ◽

Fc Fusion Protein ◽

Dose Ranging ◽

Equity Ownership

Abstract Background/Purpose: ALPN-101 is a potent dual inhibitor of the ICOS and CD28 T cell costimulatory pathways designed for therapeutic application in inflammatory diseases. CD28 and ICOS bind CD80/CD86 and ICOS ligand (ICOSL), respectively, and play critical roles in T cell activation and adaptive immunity. ALPN-101 has previously been demonstrated to have potent efficacy - superior to wild type ICOSL-Fc - in models of graft versus host disease (GvHD), a disease reflecting immune-mediated attack of recipient tissue by donor T cells. Here, we examined the efficacy of a single dose of ALPN-101 or repeat dosing with different dose levels in GvHD. We also explored the potential therapeutic benefit of ALPN-101 in another T cell-driven inflammatory disease, hemophagocytic lymphohistiocytosis (HLH), a spectrum of disorders of the immune system characterized by the excessive production of cytokines by activated T cells and macrophages accumulating in organs such as the liver, spleen, bone marrow, and brain, which mediate significant tissue damage. Methods: ALPN-101 was generated using our proprietary variant Ig domain (vIgD™) platform and is an effector-function negative Fc-fusion protein with an engineered variant Ig ICOSL domain capable of binding both ICOS and CD28 with high affinity. ALPN-101 blocks the interaction of these T cell costimulatory molecules with their respective receptors, downregulating T cell activation. The dose ranging GvHD study was executed with ALPN-101 (3x weekly/4 weeks, 20 ug - 500 ug) treatment of NSGTM mice engrafted with human peripheral blood mononuclear cells (PBMC) in comparison to belatacept, a CTLA-4-Fc fusion protein CD28 pathway inhibitor. Mice were monitored daily for clinical signs of GvHD. In a model of primary (inherited) HLH in which perforin-deficient (Prf1(-∕-)) mice are infected with lymphocytic choriomeningitis virus (LCMV), we evaluated both prophylactic (days 0, 3, and 6 post LCMV infection) and delayed (days 3, 5, and 7) treatment with ALPN-101 (400ug/dose). Results: ALPN-101 significantly attenuated T cell activation in the human PBMC-NSG GvHD model at a single 100ug dose and at all multiple doses tested, protecting mice from the effects of xenogeneic T cell activation in vivo. Treated animals exhibited enhanced survival and reduced disease scores compared to control mice treated with saline or belatacept. Flow cytometric analyses of blood collected at 1-2 weeks post cell transfer demonstrated ALPN-101 reduced both the number and activation state of the transferred human CD4+ and CD8+ T cells. In the HLH model, ALPN-101 lessened several of the clinical and laboratory manifestations of HLH, including organomegaly, anemia, CD8+ T cell expansion, and liver inflammation. Conclusion: ALPN-101 is a potent T cell inhibitor capable, even with a single dose, of preventing T cell activation, such as that observed in the huPBMC-NSGTM GvHD and the LCMV-induced HLH models, and thus is a promising novel therapeutic candidate for GvHD and other inflammatory diseases. Preclinical development is underway to support clinical studies of this potentially first-in-class dual ICOS and CD28 inhibitor. Disclosures Dillon: Alpine Immune Sciences: Employment, Equity Ownership. Lewis:Alpine Immune Sciences: Employment, Equity Ownership. Swanson:Alpine Immune Sciences: Employment, Equity Ownership. Evans:Alpine Immune Sciences: Employment, Equity Ownership. Levin:Alpine Immune Sciences: Employment, Equity Ownership. Rixon:Alpine Immune Sciences: Employment, Equity Ownership. Peng:Alpine Immune Sciences: Employment, Equity Ownership. Nichols:Incyte: Research Funding; Alpine Immune Sciences: Research Funding. Swiderek:Alpine Immune Sciences: Employment, Equity Ownership.

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Avadomide (CC-122) Alters T Cell Repertoire and Enhances Infiltration of Lymphocytes into Tumor Microenvironment in DLBCL Patients

Blood ◽

10.1182/blood-2018-99-114481 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4211-4211

Author(s):

Patrick R. Hagner ◽

Fadi Towfic ◽

Frank Schmitz ◽

Xuehai Wang ◽

Andrew P. Weng ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Tumor Microenvironment ◽

T Cell Activation ◽

Cell Activation ◽

Cell Populations ◽

Employment Equity ◽

Equity Ownership

Abstract Background : Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, constituting 30-40% of all new cases. Avadomide, a small molecule cereblon modulator currently being developed in DLBCL, binds to cereblon in the CRL4CRBN E3 ligase, leading to ubiquitination and subsequent proteasomal degradation of transcription factors Aiolos and Ikaros. This results in decreased proliferation and increased apoptosis of DLBCL cells, independent of cell-of-origin, and immunostimulatory effects in T and NK cells, as measured by increased cytokine production, cell surface activation markers, and enhanced antibody-dependent cellular cytotoxicity. A novel gene expression-based classifier, which detects DLBCL patients with T cell and macrophage infiltration within the tumor microenvironment, has been shown to enrich for responders to avadomide. Avadomide, as a single agent and in combination with rituximab, is currently being investigated in relapsed/refractory DLBCL (NCT01421524 and NCT02031419). Methods : Eighty-one DLBCL patients were enrolled in the expansion phase of the CC-122-ST-001 study (NCT01421524). Peripheral blood T cell subsets were enumerated at screening (baseline), cycle 1 day 15 (C1D15) and cycle 2 day 15 (C2D15) by flow cytometric immunophenotyping. Ex vivo production of IL-2 and IFNγ, as a measure of T cell activation, was determined using the α-CD3 TruCulture Assay. Changes from baseline were evaluated using the t-test with P<0.05 considered significant. T cell receptor (TCR) repertoire analysis through TCRB CDR3 region sequencing was done to derive metrics of population diversity and composition. RNAseq was performed on screening and on-treatment (C1D10/15) biopsies; gene expression deconvolution analyses were used to identify immune cell populations within the tumor microenvironment. Results : Avadomide treatment results in decreased peripheral CD4+ and CD8+ naïve (CD45RA+/CD45RO-) T cells and increased memory (CD45RA-/CD45RO+) and activated (HLA-DR+) T cells, without significantly affecting the absolute numbers of total CD3+, CD4+ or CD8+ populations (Table). High-dimensional single-cell mass cytometry of longitudinally collected peripheral blood samples confirmed the significant increase in CD8+ memory T cells and identified an increase in Treg populations and decreases in CD16+ monocytes and dendritic cells (adj. P<0.02). A single dose of avadomide on C1D1 significantly activated T cells, as indicated by a 300% increase in IL-2 (P=0.018) and 185% increase in IFNγ (P=0.003) secretion. Assessment of TCR B clonotypes revealed that avadomide increases the TCRB repertoire breadth, while reducing its clonality. To understand the influence of avadomide treatment on the tumor microenvironment, we performed RNA sequencing on tumor biopsies collected at screening and two weeks after initiating avadomide treatment (n=18 patients). Deconvolution analyses identified an increase in the expression of genes indicative of various T cell populations, dendritic cells and macrophages, while B cell associated gene expression decreased in on-treatment biopsies compared to screening biopsies. Gene set enrichment analysis (GSEA) revealed significantly increased expression of genes associated with "HALLMARK Interferon Alpha Response" (adj. P=0.04), indicative of an increase in Type I/II interferon production by cells such as T and NK cells. Buttressing the in vitro observations of avadomide-mediated inhibition of DLBCL cell proliferation, GSEA identified a decrease in "E2F targets" (adj. P=0.007) consistent with decreased proliferation of malignant B cells. Conclusion : Avadomide is a potent immunomodulating agent with multiple immune activating properties, including positive effects on T cell activation, as well as a broad expansion of T cell populations as defined by an increase in the richness of the T cell repertoire in blood. In addition, our data demonstrate decreased proliferation of malignant B cells in the tumor, with concomitant increased trafficking of immune cells, such as dendritic cells and macrophages, to the tumor microenvironment. These data further delineate the immune enhancing activity of avadomide in DLBCL patients beyond T-cell activation and provide rational combination strategies. Table. Table. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership.

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Inducible MyD88/CD40 Allows AP1903-Dependent Costimulation to Control Proliferation and Survival of Chimeric Antigen Receptor-Modified T Cells

Blood ◽

10.1182/blood.v124.21.1121.1121 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1121-1121 ◽

Cited By ~ 1

Author(s):

Aaron Foster ◽

Aruna Mahendravada ◽

Peter Chang ◽

Nicholas Shinners ◽

Kevin Slawin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

T Cell Activation ◽

Cell Activation ◽

First Generation ◽

Employment Equity ◽

Co Activation ◽

Equity Ownership

Abstract Introduction: Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) has begun to show impressive clinical results. The efficacy of T cell therapy is dependent not only on tumor recognition, but also on the survival and expansion of T cells following infusion. T cells modified with CAR constructs encoding costimulatory domains such as CD28 or 4-1BB have the capacity to rapidly proliferate in vivo, but severe toxicities have been observed due to unchecked T cell activation. Thus, strategies to regulate T cell activation in vivowould allow physicians to prevent toxicities and maximize anti-tumor efficacy. Here, we describe a novel T cell costimulation switch, inducible MyD88/CD40 (iMC), that can be activated by a small molecule chemical inducer of dimerization, AP1903, to enhance survival and drive T cell proliferation. Methods: T cells were activated with anti-CD3/28 antibodies and subsequently transduced with a biscistronic retrovirus encoding myristolated tandem AP1903 binding domains (FKBPv36), cloned in-frame with MyD88 and CD40 cytoplasmic signaling molecules, and truncated CD19 to identify transduced T cells (SFG-iMC.2A.ΔCD19). Control vectors without signaling elements, or with only MyD88 or cytoplasmic CD40 were also used to generate gene-modified T cell lines. iMC activation was measured by treating T cells with and without AP1903 and measuring cytokine production by ELISA, and assessing cell surface activation markers by flow cytometry. Co-activation of T cells through CD3ζ in combination with iMC was accomplished using anti-CD3 antibodies, or by co-transducing T cells with first generation CAR constructs recognizing prostate stem cell antigen or CD19 (PSCA.ζ or CD19.ζ, respectively), and coculturing T cells with PSCA+ (Capan-1) or CD19+ tumor cell lines (Raji, Daudi and Nalm-1) with and without AP1903. Efficacy of iMC-modified CAR T cells were assessed using NOD scid gamma (NSG) immune deficient mice engrafted with tumor cell lines followed by intravenous injection of T cells. The iMC costimulatory molecule was subsequently activated in vivo by intraperitoneal injection of AP1903 (5 mg/kg). Tumor burden was assessed and T cell expansion in vivowas measured by bioluminescent imaging using an IVIS instrument. Results: T cells transduced with iMC produce cytokines (e.g. IFN-γ, TNF-α, IL-6) in response to AP1903. iMC activation permits T cell survival in the absence of growth cytokines, such as IL-2, but by itself is not sufficient to induce IL-2 production or autonomous growth. Interestingly, AP1903-induction of MyD88 or cytoplasmic CD40 alone showed minimal T cell activation, suggesting potential synergy of the two signaling molecules. However, co-activation of the T cell receptor (TCR) with soluble anti-CD3 and iMC with AP1903 upregulated CD25 expression, induced IL-2 production and promoted T cell expansion. Importantly, endogenous TCR signaling could be substituted by a PSCA-specific CAR linked to the CD3 ζ endodomain (PSCA.ζ CAR), where co-activation of iMC by AP1903, and CAR by tumor cells expressing PSCA (Capan-1) induced high levels of IL-2 secretion, CD25 upregulation and rapid T cell proliferation. Similar results were achieved using T cells transduced with iMC-enabled CD19 CAR (SFG-iMC.2A.CD19.ζ) when cocultured with CD19+lymphoma cell lines. Treatment of tumor bearing immunodeficient mice with T cells modified with iMC and PSCA.ζ CAR showed enhanced antitumor efficacy when mice were administered with AP1903 dimerizer. Bioluminescence imaging also demonstrated marked proliferation and persistence of iMC-transduced T cells in response to AP1903 administration. Following AP1903 withdrawal, T cell levels declined, consistent with the requirement for costimulation in combination with CAR activation. Summary: Inducible MyD88/CD40 represents a novel activation switch that can be used to provide a controllable costimulatory signal to T cells transduced with a first generation CAR. The separation of the cytolytic signal 1 (CD3 ζ) domain from signal 2 costimulation (iMC) provides a unique mechanism by which T cells can be expanded only in response to both AP1903 and tumor antigen, or reduced in number by withdrawal of AP1903-induced iMC costimulation. Disclosures Foster: Bellicum Pharmaceuticals: Employment, Patents & Royalties. Mahendravada:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

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Tuning T Cell Affinity Improves Efficacy and Safety of Anti-CD38 × Anti-CD3 Bispecific Antibodies in Monkeys - a Potential Therapy for Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.1798.1798 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1798-1798 ◽

Cited By ~ 14

Author(s):

Gregory L. Moore ◽

Sung-Hyung Lee ◽

Suzanne Schubbert ◽

Yvonne Miranda ◽

Rumana Rashid ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Bispecific Antibodies ◽

Employment Equity ◽

High Affinity ◽

Cell Immunotherapy ◽

Equity Ownership

Abstract CD38 is highly expressed on plasma cells and is an attractive target for multiple myeloma (MM) therapies. Several anti-CD38 antibodies including daratumumab and SAR650984 show promising results in clinical development, though such antibodies are not able to stimulate T cell-mediated killing of myeloma cells. To exploit a T cell immunotherapy mechanism while retaining the favorable drug properties of therapeutic antibodies, we designed bispecific antibodies that recruit T cells to CD38+ MM cells. Such bispecifics act via redirected T cell-cytotoxicity (RTCC) to stimulate T cell-mediated target cell killing regardless of T cell receptor antigen specificity. These anti-CD38 × anti-CD3 antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain lacks binding to Fcγ receptors to minimize nonselective T cell activation, yet retains binding to FcRn to maintain long serum half-life. We have previously reported that XmAb13551, a humanized and affinity-optimized anti-CD38 × anti-CD3 antibody, stimulates killing of the CD38+ MM cell line RPMI8226 by human T cells and suppresses human Ig levels in SCID mice engrafted with human PBMCs, showing much greater efficacy than daratumumab in these models (Blood 2014 124:4727). We also investigated efficacy of XmAb13551 in monkeys given a single dose of 2, 5, and 20 μg/kg. Within 1 hr after dosing, CD25 and CD69 activation markers were upregulated on T cells and within 8 hr, circulating CD38+ cells were depleted by > 95% at the 20 μg/kg dose. However, depletion of peripheral CD38+ cells was not sustained, suggesting that a large antigen sink was limiting drug exposure. Although higher dosing might overcome an antigen sink, higher doses of XmAb13551 (0.2 mg/kg or higher) resulted in a T cell-mediated cytokine release syndrome (CRS) in monkeys. We reasoned that an anti-CD38 × anti-CD3 antibody with reduced CD3 affinity would stimulate sufficient RTCC to deplete MM cells, yet would attenuate the acute T cell activation (and associated CRS) induced by high-affinity coengagement of T cells with CD38+ target cells. Using XmAb13551 as a starting point, we engineered a series of bispecifics retaining the same high-affinity (0.2 nM) binding to CD38, but with reduced affinity to CD3. We selected two antibodies - XmAb15426 and XmAb14702 - that have significantly reduced CD3 affinity. As expected, these molecules showed reduced potency in RTCC assays using T cells to kill RPMI8226 cells, with potency correlating with CD3 affinity (XmAb13551 > XmAb15426 >> XmAb14702). We next tested XmAb15426 and XmAb14702 at single doses of 0.5 mg/kg and 3 mg/kg, respectively, in cynomolgus monkeys. Both antibodies were well-tolerated at these higher doses, consistent with the moderate levels of IL6 observed in serum from the treated monkeys. Moreover, XmAb15426, with intermediate CD3 affinity, more effectively depletes CD38+ cells at 0.5 mg/kg compared to the original high-affinity XmAb13551 dosed at 2, 5 or 20 µg/kg. Depletion by XmAb15426 was more sustained compared to the highest dose of XmAb13551 in the previous study (7 vs. 2 days, respectively). Notably, although target cell depletion was greater for XmAb15426, T cell activation (CD69, CD25 and PD1 induction) was much lower in monkeys treated with XmAb15426 even dosed 25-fold higher than the 20 µg/kg XmAb13551 group. XmAb14702, with very low CD3 affinity, had little effect on CD38+ cells and T cell activation. Our results demonstrate that modulating T cell activation by attenuating CD3 affinity is a promising method to improve the therapeutic window of T cell-engaging bispecific antibodies. This strategy has potential to expand the set of antigens amenable to targeted T cell immunotherapy by improving tolerability and enabling higher dosing to overcome antigen sink clearance with targets such as CD38. We have shown that by reducing affinity for CD3, XmAb15426 effectively depletes CD38+ cells while minimizing the CRS effects seen with comparable doses of its high-affinity counterpart XmAb13551. Our preclinical data for XmAb15426 provide a rationale for clinical testing of this bispecific antibody in patients with multiple myeloma and other CD38+ malignancies. Disclosures Moore: Xencor, Inc.: Employment, Equity Ownership. Lee:Xencor, Inc.: Employment, Equity Ownership. Schubbert:Xencor, Inc.: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Ardila:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Chu:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

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EP.WE.805From bench to bedside; A rationale for Immunotherapy in the adjuvant setting for Oesophageal cancer

British Journal of Surgery ◽

10.1093/bjs/znab308.094 ◽

2021 ◽

Vol 108 (Supplement_7) ◽

Author(s):

Noel Donlon ◽

Maria Davern ◽

Andrew Sheppard ◽

John Reynolds ◽

Joanne Lysaght

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Immune Checkpoint ◽

Cell Activation ◽

Single Agent ◽

T Cell Subsets ◽

Effector Memory ◽

Post Surgery ◽

Post Operative Period

Abstract Background Immunotherapy is being intensively investigated for its utilisation in the curative setting as a single agent and in the multimodal setting, however, the most appropriate time to incorporate ICIs remains unknown. Our study profiles systemic anti-tumour immunity perioperatively to provide a rationale for adjuvant immunotherapy. Methods Systemic immunity was immunophenotyped pre and post-oesophagectomy on days 0, 1, 3, 7 and week 6 by flow cytometry (n = 14). The frequency of circulating lymphocytes, T cells, cytotoxic and helper T lymphocytes was profiled longitudinally including the proportion of T cell subsets in circulation. This study also profiled immune checkpoint expression on circulating T cells including: PD-1, CTLA-4, TIGIT, TIM-3, LAG-3, PD-L1 and PD-L2. Markers of immunogenicity (calreticulin, HMGB1 and MIC-A/B) were also assessed. Results The frequency of circulating CD27 + T cells increases sequentially in the immediate post-operative period peaking on day 7 in OAC patients. (p < 0.01) There is a sequential decrease in the percentage of effector memory and central memory T cells in circulation and an increase in the percentage of naïve T cells in peripheral circulation of OAC patients in the immediate post-operative period. The expression of CTLA-4 on the surface of circulating CD4 + T cells decreases 6 weeks post-operatively in OAC patients. Conclusions We observed increased T cell activation and immune checkpoints immediately post-surgery with returns to baseline by week 6. These results suggest that immune checkpoint inhibitors such as anti-PD-1 may be beneficial immediately post-surgery to maintain T cell activation and prevent exhaustion of this increased population of activated T cells observed immediately post-surgery.

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Diacylglycerol kinase ζ limits IL-2-dependent control of PD-1 expression in tumor-infiltrating T lymphocytes

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001521 ◽

2020 ◽

Vol 8 (2) ◽

pp. e001521

Author(s):

Javier Arranz-Nicolás ◽

Miguel Martin-Salgado ◽

Cristina Rodríguez-Rodríguez ◽

Rosa Liébana ◽

Maria C Moreno-Ortiz ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Therapeutic Potential ◽

Interleukin 2 ◽

Tumor Rejection ◽

Cytotoxic T Cell ◽

Adenocarcinoma Cells ◽

The Impact

BackgroundThe inhibitory functions triggered by the programmed cell death-1 (PD-1) receptor following binding to its ligand (PD-L1) protect healthy organs from cytotoxic T cells, and neutralize antitumor T cell attack. Antibody-based therapies to block PD-1/PD-L1 interaction have yielded notable results, but most patients eventually develop resistance. This failure is attributed to CD8+ T cells achieving hyporesponsive states from which recovery is hardly feasible. Dysfunctional T cell phenotypes are favored by a sustained imbalance in the diacylglycerol (DAG)- and Ca2+-regulated transcriptional programs. In mice, DAG kinase ζ (DGKζ) facilitates DAG consumption, limiting T cell activation and cytotoxic T cell responses. DGKζ deficiency facilitates tumor rejection in mice without apparent adverse autoimmune effects. Despite its therapeutic potential, little is known about DGKζ function in human T cells, and no known inhibitors target this isoform.MethodsWe used a human triple parameter reporter cell line to examine the consequences of DGKζ depletion on the transcriptional restriction imposed by PD-1 ligation. We studied the effect of DGKζ deficiency on PD-1 expression dynamics, as well as the impact of DGKζ absence on the in vivo growth of MC38 adenocarcinoma cells.ResultsWe demonstrate that DGKζ depletion enhances DAG-regulated transcriptional programs, promoting interleukin-2 production and partially counteracting PD-1 inhibitory functions. DGKζ loss results in limited PD-1 expression and enhanced expansion of cytotoxic CD8+ T cell populations. This is observed even in immunosuppressive milieus, and correlates with the reduced ability of MC38 adenocarcinoma cells to form tumors in DGKζ-deficient mice.ConclusionsOur results, which define a role for DGKζ in the control of PD-1 expression, confirm DGKζ potential as a therapeutic target as well as a biomarker of CD8+ T cell dysfunctional states.

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Molecular transfer of CD40 and OX40 ligands to leukemic human B cells induces expansion of autologous tumor–reactive cytotoxic T lymphocytes

Blood ◽

10.1182/blood-2004-07-2556 ◽

2005 ◽

Vol 105 (6) ◽

pp. 2436-2442 ◽

Cited By ~ 42

Author(s):

Ettore Biagi ◽

Gianpietro Dotti ◽

Eric Yvon ◽

Edward Lee ◽

Martin Pule ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Antibody Therapy ◽

Interferon Γ ◽

Lymphocytic Leukemia ◽

Cytotoxic T Cell ◽

Autologous Tumor ◽

Molecular Transfer

AbstractClinical benefits from monoclonal antibody therapy for B-chronic lymphocytic leukemia (B-CLL) have increased interest in developing additional immunotherapies for the disease. CD40 ligand is an accessory signal for T-cell activation and can overcome T-cell anergy. The OX40-OX40 ligand pathway is involved in the subsequent expansion of memory antigen-specific T cells. We expressed both CD40L and OX40L on B-CLL cells by exploiting the phenomenon of molecular transfer from fibroblasts overexpressing these ligands. We analyzed the effects of the modified B-CLL cells on the number, phenotype, and cytotoxic function of autologous T cells in 7 B-CLL patients. Transfer of CD40L and OX40L was observed in all and was followed by the up-regulation of B7-1 and B7-2. The culture of CD40L/OX40L-expressing B-CLL cells with autologous T cells generated CD4+/CD8+ cytotoxic T-cell lines, which secreted interferon-γ (IFN-γ) and granzyme-B/perforin in response to autologous, but not to allogeneic, B-CLL cells or to autologous T-cell blasts. CD40L or OX40L alone was insufficient to expand tumor-reactive T cells. The combination of CD40L and OX40L on B-CLL cells may allow the generation of therapeutic immune responses to B-CLL, either by active immunization with modified tumor cells or by adoptive immunotherapy with tumor-reactive autologous T cells.

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