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

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

Blood ◽

10.1182/blood.v126.23.4295.4295 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4295-4295 ◽

Cited By ~ 1

Author(s):

Aaron Foster ◽

Aruna Mahendravada ◽

Nicholas P Shinners ◽

Peter Chang ◽

An Lu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

T Cell Activation ◽

Cell Activation ◽

First Generation ◽

Car T Cell ◽

Car T ◽

Equity Ownership

Abstract Introduction: Adoptive transfer of T cells, genetically engineered to express chimeric antigen receptors (CARs) containing costimulatory domains, such as CD28 or 4-1BB, has yielded impressive clinical results in some blood cancers, but severe toxicities have been observed due to unchecked T cell activation. In contrast, CAR-T cells have demonstrated limited clinical efficacy, associated with poor engraftment, survival and proliferation of adoptively transferred cells when used to target a variety of solid tumors. Thus, technologies that can regulate T cell activation and proliferation in vivo should both mitigate toxicities and maximize anti-tumor efficacy, expanding their clinical utility to a wider range of indications. Here, we describe a novel T cell costimulation switch, inducible MyD88/CD40 (iMC), activated by a small molecule chemical inducer of dimerization, rimiducid, to enhance survival and drive T cell proliferation. Methods: T cells were activated with anti-CD3/28 and transduced with a retrovirus encoding tandem rimiducid-binding domains (FKBP12v36),cloned in-frame with MyD88 and CD40 signaling elements, and first generation CARs (CAR.ζ) targeting CD19 or PSCA (SFG-iMC-2A-CD19.ζ or SFG-iMC-2A-PSCA.ζ, respectively). iMC activation was measured by treating T cells with and without rimiducid and measuring cytokine production by ELISA and T cell activation markers by flow cytometry. Coactivation through iMC and CAR was tested in coculture assays with or without rimiducid using various tumor cells (CD19+, Raji and Daudi lymphoma; PSCA+, Capan-1 and HPAC pancreatic adenocarcinoma). Efficacy of iMC-modified CAR-T cells were assessed using an immune-deficient NSG mouse tumor model. For CD19-targeted CARs, 1x105 Raji tumor cells were injected i.v. followed on day 7 by a single i.v. injection at various doses of iMC-CD19.ζ-modified T cells. For PSCA-targeted CARs, 2x106 HPAC tumor cells were injected s.c. followed by iMC-PSCA.ζ-modified T cells on day 10. In both models, iMC was activated in vivo by weekly i.p. injections of rimiducid (5 mg/kg). In some experiments, iMC-CAR-modified T cells were engrafted into tumor-free mice. Tumor burden and CAR-T cell expansion in vivo was assessed using luciferase bioluminescent imaging and flow cytometry. Results: T cells transduced with either iMC-CD19.ζ or iMC-PSCA.ζ produce cytokines (e.g., IFN-γ and IL-6) in response to rimiducid; however, the key growth and survival cytokine, IL-2, was only produced when both iMC and CAR were activated simultaneously by rimiducid and tumor antigen, respectively. CD19+ Raji tumor-bearing mice treated with iMC-CD19.ζ-modified T cells with or without rimiducid administration increased survival compared to non-transduced T cells (p = 0.01). However, rimiducid treatment induced a 7.3-fold CAR-T cell expansion compared to mice infused with iMC-CD19.ζ, but untreated with dimer drug (p = 0.02). Additionally, treatment of NSG mice bearing large (>200 mm3) HPAC tumors with a single dose iMC-PSCA.ζ, resulted in complete elimination in 10/10 mice (100%) of tumors both with and without rimiducid treatment compared to mice receiving non-transduced T cells (p = 0.0003). Rimiducid administration again dramatically increased CAR-T cell levels, resulting in a 23-fold expansion of iMC-PSCA.ζ-modified T cells compared to mice not receiving rimiducid (p = 0.02), justifying ongoing experiments using larger tumors at baseline with fewer T cells. In addition, in tumor-free mice, rimiducid prolonged iMC-PSCA.ζ-modified T cell engraftment and survival for 28 days compared to those mice not treated with dimerizer (p = 0.03). Importantly, following rimiducid withdrawal, CAR-T cell numbers declined, consistent with the requirement of MC-mediated 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 (CD3ζ) domain from a potent, regulatable, signal 2 costimulation (iMC) in the novel platform, called "GoCAR-T", allows the expansion of T cells only in response to both rimiducid and tumor antigen, and their decrease in number by withdrawal of rimiducid-induced iMC costimulation. The "GoCAR-T" platform may allow the development of a new generation of more effective CAR-T cell therapies. Disclosures Foster: Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Morschl:Bellicum Pharmaceuticals: Employment. Shaw:Bellicum Pharmaceuticals: Employment. Saha:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: 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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Influence of DM-sensitivity on immunogenicity of MHC class II restricted antigens

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-002401 ◽

2021 ◽

Vol 9 (7) ◽

pp. e002401

Author(s):

Anna Luise Bernhardt ◽

Julia Zeun ◽

Miriam Marecek ◽

Hannah Reimann ◽

Sascha Kretschmann ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Lines ◽

T Cell Activation ◽

Allogeneic Stem Cell Transplantation ◽

Cell Activation ◽

T Cell Epitopes

BackgroundGraft-versus-host-disease (GvHD) is a major problem in allogeneic stem cell transplantation. We previously described two types of endogenous human leukocyte antigen (HLA)-II restricted antigens depending on their behavior towards HLA-DM. While DM-resistant antigens are presented in the presence of HLA-DM, DM-sensitive antigens rely on the expression of HLA-DO-the natural inhibitor of HLA-DM. Since expression of HLA-DO is not upregulated by inflammatory cytokines, DM-sensitive antigens cannot be presented on non-hematopoietic tissues even under inflammatory conditions. Therefore, usage of CD4+ T cells directed against DM-sensitive antigens might allow induction of graft-versus-leukemia effect without GvHD. As DM-sensitivity is likely linked to low affinity peptides, it remains elusive whether DM-sensitive antigens are inferior in their immunogenicity.MethodsWe created an in vivo system using a DM-sensitive and a DM-resistant variant of the same antigen. First, we generated murine cell lines overexpressing either H2-M or H2-O (murine HLA-DM and HLA-DO) to assign the two model antigens ovalbumin (OVA) and DBY to their category. Further, we introduced mutations within the two T-cell epitopes and tested the effect on DM-sensitivity or DM-resistance. Furthermore, we vaccinated C57BL/6 mice with either variant of the epitope and measured expansion and reactivity of OVA-specific and DBY-specific CD4+ T cells.ResultsBy testing T-cell recognition of OVA and DBY on a murine B-cell line overexpressing H2-M and H2-O, respectively, we showed that OVA leads to a stronger T-cell activation in the presence of H2-O demonstrating its DM-sensitivity. In contrast, the DBY epitope does not rely on H2-O for T-cell activation indicating DM-resistance. By introducing mutations within the T-cell epitopes we could generate one further DM-sensitive variant of OVA and two DM-resistant counterparts. Likewise, we designed DM-resistant and DM-sensitive variants of DBY. On vaccination of C57BL/6 mice with either epitope variant we measured comparable expansion and reactivity of OVA-specific and DBY-specific T-cells both in vivo and ex vivo. By generating T-cell lines and clones of healthy human donors we showed that DM-sensitive antigens are targeted by the natural T-cell repertoire.ConclusionWe successfully generated DM-sensitive and DM-resistant variants for two model antigens. Thereby, we demonstrated that DM-sensitive antigens are not inferior to their DM-resistant counterpart and are therefore interesting tools for immunotherapy after allogeneic stem cell transplantation.

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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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CTX-8573, an Innate-Cell Engager Targeting BCMA, is a Highly Potent Multispecific Antibody for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood-2019-128749 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3182-3182 ◽

Cited By ~ 2

Author(s):

Jennifer Watkins-Yoon ◽

Wilson Guzman ◽

Amanda Oliphant ◽

Sara Haserlat ◽

Alan Leung ◽

...

Keyword(s):

T Cells ◽

Cell Lines ◽

Cell Activation ◽

Γδ T Cells ◽

Therapeutic Window ◽

Target Cells ◽

Employment Equity ◽

Equity Ownership

Introduction: Current therapies for multiple myeloma (MM), such as immunomodulatory agents, proteasome inhibitors, stem-cell transplantation, and monoclonal antibodies against tumor-associated antigens have greatly improved patient survival. However, MM remains an incurable disease as most patients will eventually relapse. Recent advances in targeted T-cell therapies have shown promise in clinical trials but the adaptive immune system may be insufficient to eradicate all MM clones. In contrast, treatments harnessing the innate immune system have been relatively underdeveloped in MM despite evidence suggesting a role of innate immunity in the efficacy of existing therapies. Innate or innate-like cells, such as NK and γδ T cells, have the potential to display strong anti-tumor activity, and strategies aimed to improve or re-direct their cytotoxicity represent a new opportunity for cancer immunotherapies and a complementary approach to existing therapies. Here we describe the preclinical characterization of CTX-8573, a novel multispecific antibody that targets B-cell maturation antigen (BCMA) on tumor cells and promotes potent cytotoxicity by NK and γδ T cells through engagement of the activating receptors NKp30 and CD16a. Method: Bispecific constructs were generated by appending two common-light chain compatible anti-NKp30 Fab fragments to the C-terminus of an anti-BCMA IgG1 antibody containing an afucosylated Fc for enhanced CD16a engagement. To test the effects of targeting NKp30 alone, variants were expressed with an aglycosylated Fc to eliminate CD16a binding. In-vitro assays were performed with primary NK or γδ T cells to determine innate-cell activation, cytokine production, proliferation, and target-cell cytotoxicity against tumor cell lines with a range of BCMA expression levels. In-vivo efficacy studies were performed in multiple humanized mouse models and pharmacokinetics and safety were evaluated in Cynomolgus monkeys. Results: CTX-8573 is highly expressed in CHO cells with minimal aggregation and displays stability, solubility, and binding to BCMA and NKp30 equivalent to the parental monoclonal antibodies. By engaging NKp30 and CD16a, CTX-8573 promotes potent cytotoxicity of BCMA expressing target cells by NK and γδ T cells with >100 fold reduced EC50 compared to the corresponding BCMA monoclonal antibody control. CTX-8573 also demonstrates robust killing of low BCMA expressing cell lines including RPMI-8226 where monoclonal BCMA antibodies lack activity. An aglycosylated variant of CTX-8573 lacking CD16a binding maintains cell killing activity, demonstrating that engagement of NKp30 alone is sufficient to promote innate cell activation and cytotoxicity, although activity is enhanced by CD16A engagement. Furthermore, CTX-8573 maintains its cytotoxic activity in presence of soluble BCMA or BCMA ligands APRIL and BAFF. CTX-8573 does not induce innate cell activation, cytokine production, or killing in the absence of BCMA expressing target cells, supporting a wide therapeutic window. Additionally, unlike daratumumab, CTX-8573 does not induce NK-cell fratricide. In-vivo, CTX-8573 demonstrates anti-tumor efficacy in multiple humanized mouse models including killing of low BCMA expressing cell lines. In Cynomolgus monkeys, CTX-8573 displays standard biphasic pharmacokinetics with a 16 day β-phase half-life and has no evidence of systemic immune activation as measured by C-reactive protein levels. Lastly, NKp30 expression is maintained on bone marrow NK cells from MM patients including the presence of a significant NKp30+CD16a- subpopulation. Conclusion: CTX-8573 represents a novel class of bispecific antibodies that promote potent tumor cell killing by NK and γδ T-cells through engagement of the activating receptors NKp30 and CD16a. CTX-8573 demonstrates strong anti-tumor efficacy in vitro and in vivo, a wide therapeutic window with no evidence of systemic toxicity, and monoclonal-like pharmacokinetics and manufacturability. Together, these data highlight the potential of CTX-8573 as a novel treatment for MM either alone or as a complement to existing therapies. Disclosures Watkins-Yoon: Compass therapeutics LLC: Employment, Equity Ownership. Guzman:Compass therapeutics LLC: Employment, Equity Ownership. Oliphant:Compass therapeutics LLC: Employment, Equity Ownership. Haserlat:Compass therapeutics LLC: Employment, Equity Ownership. Leung:Compass therapeutics LLC: Employment, Equity Ownership. Chottin:University of Louisiana at Lafayette: Employment. Ophir:Compass therapeutics LLC: Employment, Equity Ownership. Vekeria:Compass therapeutics LLC: Employment, Equity Ownership. Nanjappa:Compass therapeutics LLC: Employment, Equity Ownership. Markrush:Compass therapeutics LLC: Employment, Equity Ownership. McConaughy:Compass therapeutics LLC: Employment, Equity Ownership. Wang:Compass therapeutics LLC: Employment, Equity Ownership. Schilling:Compass therapeutics LLC: Employment, Equity Ownership. Kim:Compass therapeutics LLC: Employment, Equity Ownership. Wu:Compass Therapeutics LLC: Employment, Equity Ownership. Liu:Compass therapeutics LLC: Employment, Equity Ownership. Rogers:University of Louisiana at Lafayette: Employment. Villinger:University of Louisiana at Lafayette: Employment. Gong:Compass therapeutics LLC: Employment, Equity Ownership. Hamilton:Compass therapeutics LLC: Employment, Equity Ownership. Bobrowicz:Compass therapeutics LLC: Employment, Equity Ownership. Schuetz:Compass therapeutics LLC: Employment, Equity Ownership. Schmidt:Compass therapeutics LLC: Employment, Equity Ownership. Draghi:Compass therapeutics LLC: Employment, Equity Ownership.

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

Blood ◽

10.1182/blood-2018-99-114469 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4133-4133

Author(s):

Yumi Nakayama ◽

Matthew E. Stokes ◽

Michelle Waldman ◽

Patrick R. Hagner ◽

Anita K. Gandhi

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Synergistic Effects ◽

Single Agent ◽

Differentially Expressed ◽

Cytotoxic T Cell ◽

Employment Equity ◽

Equity Ownership

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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Epstein Barr Virus (EBV)-Specific Cytotoxic T Lymphocytes (CTL) Expressing an Anti-CD30 Chimeric T Cell Receptor (CTCR) for the Treatment of Hodgkin’s Disease (HD).

Blood ◽

10.1182/blood.v104.11.745.745 ◽

2004 ◽

Vol 104 (11) ◽

pp. 745-745

Author(s):

B. Savoldo ◽

C. M. Rooney ◽

H. E. Heslop ◽

H. Abken ◽

A. Hombach ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Tumor Cells ◽

Cell Lines ◽

T Cell Activation ◽

Cell Activation ◽

Cell Receptor ◽

Barr Virus

Abstract HD may be a suitable target for immunotherapy, and in patients with EBV-associated HD, adoptive transfer of EBV-CTL has produced disease responses. An alternative target is the CD30 molecule, which is present on the malignant cells of almost all patients with HD. CD30 is a member of the TNF superfamily and monoclonal antibodies directed to this antigen are currently under investigation in patients with relapsed HD. An alternative way to target CD30 is by the construction of T cells expressing cTcR specific for the antigen. T lymphocytes engineered to express this cTcR can specifically kill CD30+ HD cell lines {Cancer Res,1998;58:1116}. However, these chimeric molecules connect the antigen-recognition properties of CD30 antibodies with the endodomain of CD3ζ, which is insufficient to fully activate resting T cells to proliferate and release cytokines. As a consequence chimeric T cells that express these endodomains divide infrequently, lose activity and have performed poorly in-vivo. Full T cell activation requires receptor engagement to be accompanied by a sequence of co-stimulatory stimuli. We have shown that EBV-CTL can fulfill this need, since the co-stimulatory signals delivered by EBV-infected B cells after native receptor engagement ensure full functionality when the CTL subsequently bind to tumor cells through their cTcR. We first evaluated whether EBV-CTL can be redirected to kill CD30+ HD cell lines and whether they retain their specificity and antigen repertoire. EBV-CTLs were prepared from 8 EBV+ healthy donors using weekly stimulation with irradiated autologous EBV-transformed lymphoblastoid cell lines (LCL) in the presence of IL-2 (50U/mL). CTL were transduced after the 3rd stimulation and further expanded with 3–4 weekly LCL/IL-2 stimulations. The expansion rate of the transduced CTL was similar to that of control EBV-CTL. Transduced CTL retained killing of their autologous LCL targets through their native receptor (64.4±16% at 20:1 E:T ratio), and became able to lyse CD30+ malignant lymphoma targets through their cTcR (e.g. HDLM-2=45.4±16% and Karpas-299=42.5±17%). Killing of CD30+ tumor cells was significantly inhibited by preincubation with an anti-CD30 blocking antibody (16.5±12%). Of potential concern, however, is that CD30 is expressed by activated normal T lymphocytes: expression was undetectable on resting T cells, but increased to 3–32% on day 4–7 after stimulation with LCL. Fortunately, expression dwindles to 3–6% by two weeks as an EBV-specific line emerges, suggesting that CD30 is expressed only in the early phases of T cell activation. As anticipated from these data, therefore, expression of a CD30 cTcR did not impair the antigenic repertoire of the EBV-CTL, which retained the same pattern of immunodominant MHC class I epitopes (detected by tetramer) as control cells. We also performed co-culture experiments to evaluate whether infusion of CTL-CD30 cTcR could cross-compromise the primary reactivation of other virus-specific CTL. Autologous EBV-CTLs engineered to express the CD30-cTcR were added to cultures of PBMC stimulated to reactivate cytomegalovirus- or adenovirus-specific CTL. In 4/4 donors, the percentage of CMV pp65+ T cells did not change, while generation of adenovirus-specific T cells (Hexon-tetramer+) was significantly reduced in only 1/3 donor. These data support the feasibility of using EBV-CTL bearing a cTcR for CD30 to treat both EBV+ and EBV− HD.

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Redirecting T-Cells Against AML in a Multidimensional Targeting Space Using T-Cell Engaging Antibody Circuits (TEAC)

Blood ◽

10.1182/blood-2019-127634 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2653-2653 ◽

Cited By ~ 1

Author(s):

Eleanor Minogue ◽

David Millar ◽

Yan Chuan ◽

Songfa Zhang ◽

Korneel Grauwet ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

T Cell Activation ◽

Cleavage Site ◽

Cell Activation ◽

Surface Antigens ◽

Research Support ◽

Car T ◽

Equity Ownership

Background T cell redirection strategies, such as CAR-T and bispecific antibodies (bsAb), are rapidly changing the way in which we approach and treat cancer. While CAR-T and bsAb have shown impressive clinical efficacy in a limited number of cancers, both strategies are ultimately limited by on-target toxicity that currently restricts application to B-cell lineage tumors as the number of genuinely tumor-specific surface antigens is extremely limited. BsAb also suffer from off-target toxicity relating their ability to directly active T-cells severely restricting the therapeutic window. We sought to solve these inherent problems with the current generation bsAb by re-designing the molecule to alter the mechanism of T-cell activation. By splitting the T-cell engaging VHVL antibody paratope between two separate molecules we created two molecules that formed an active T-cell engaging unit through protein domain complementation following proteolytic activation. Each antibody could target independent surface antigens vastly increasing targeting permutations. Thus, these two antibodies functioned as an "antibody circuit" permitting Boolean type logic to precisely control T-cell activation in multi-dimensional targeting space. We selected AML as model cancer to develop T-cell Engaging Antibody Circuits (TEACs) due to the highly characterized surface antigen landscape and the clear challenges and limitations of single-antigen targeting approaches. Results We first screened 10 AML cell lines for candidate surface antigens based upon prior studies of surface antigen display (Perna F et al, 2016) and identified CD33, CD123, CD49d, CD70, CD71, CD38, CLEC12A, Flt3, CD24, CD244, TIM3 and CCR1 as promising targets. We developed a secondary TEACs screening assay where the two TEAC molecules contained either a FITC or biotin binding domain and paired these to commercial FITC or biotin conjugated antibodies targeting the antigens above. We screened 72 TEAC pairs against the 10 cell lines which identified optimal antigen target combinations which included CD33xCD123, CD33xCLEC12A, CD33xCD49d and CD33xCD24. Using a FRET-based fluorescent peptide assay to identify peptide linkers susceptible to proteases we found MMP2 to be highly expressed in AML samples and thus designed all our TEACs with this cleavage site. We next generated IgG4 format TEACs targeting CD33, CD123, CLEC12A and CD24 that included the MMP2 cleavage activation site and tested these as TEAC pairs in vitro. This screen identified the CD123xCD33 as the most active TEAC pair which was active in 9/10 cells lines. To assess potential safety concerns, we tested TEACs and CD123 and CD33 BiTEs individually and as pairs on PBMCs and on plate-immobilized molecules. These data demonstrated that BiTEs were extremely active against healthy monocytes and also activate T-cells non-specifically once plate-immobilized. In contrast CD123xCD33 IgG TEACs pairs did not activate T-cells when plate-immobilized and did not target healthy monocytes.Finally, we examined the activity of both CD33 BiTEs and CD123xCD33 TEACs on primary patient AML samples. We conducted FRET based assays which confirmed high activity of MMP2 cleavage site on all primary AML samples. When we examined T-cell activation, CD123xCD33 TEACs were active in all CD123+ CD33+ AML samples evaluated with an EC50 of 30ug/ml. Conclusion These data suggest T-cell engaging antibody circuits is a new approach that could be safely applied toward AML. TEAC agents do not directly activate T-cells and CD123xCD33 TEAC pairs do not activate PBMC or monocytes. However, CD123xCD33 TEACs show strong activity against AML cell lines and primary CD123+CD33+ AML cells. Disclosures Millar: Revitope Oncology: Equity Ownership. Minshull:Atum Biotechnology: Employment, Equity Ownership. Narayan:Takeda: Other: Employment (spouse); Genentech: Other: Equity ownership (spouse); Merck: Other: Equity ownership (spouse). Graubert:Biogen: Other: Spouse Employee; Calico Life Sciences: Other: Research Support; Janssen Pharmaceuticals: Other: Research Support. Cobbold:Gritstone Oncology: Equity Ownership; Revitope Oncology: Consultancy; Revitope Oncology: Equity Ownership.

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