scholarly journals 795 APVO603: a dual 4-1BB and OX40 bispecific approach utilizing ADAPTIRTM technology designed to deliver a conditional T cell/NK response against solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A830-A830
Author(s):  
Michelle Nelson ◽  
Ashly Lucas ◽  
Rebecca Gottschalk ◽  
Catherine McMahan ◽  
Jane Gross ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Bispecific Antibody ◽  
Expression Profiles ◽  
T Cell Subsets ◽  
Dependent Manner ◽  
Dose Dependent

BackgroundAPVO603 is a dual targeting bispecific antibody for 4-1BB (CD137) and OX40 (CD134), engineered with Aptevo's ADAPTIRTM technology. We have previously shown that the distinct characteristics of APVO603 may enable conditional agonism of 4-1BB and OX40 only when cross-linked through engagement of the other receptor via cis and/or trans cellular interactions. Thus, APVO603 is designed with the potential to overcome both the on-target toxicity and limited efficacy observed with 4-1BB and OX40 monoclonal antibody treatment in the clinic.MethodsGenevestigator Software was used to analyze curated transcriptomic data for the expression profiles of OX40 and 4-1BB across select human heme and solid cancer patient sample data sets, as well as, non diseased tissue. Primary inducible Treg (iTreg) cells were sub-optimally stimulated with an anti-CD3/CD28 antibody and cell proliferation was assessed using CFSE-labelled. Cytokines were measured using intracellular flow-based methods. For in vitro tumor lysis studies, activated T cells were co-cultured with Nuclight-labelled tumor cells expressing a tumor-associated antigen (TAA) and activated with TAA x CD3 bispecific protein. Live tumor cells were continually assessed using the Incucyte Live-Cell Analysis System and Cell-By-Cell Software Module.ResultsOX40 and 4-1BB displayed distinct tumor expression profiles, however, several tumor indications were identified with high co-expression and may aid in identifying indications for the clinical development of APVO603. In vitro, APVO603 favored activation of effector T cell subsets and had minimal impact in augmenting iTreg cells proliferation, cytokine production or expression of effector-related molecules, despite the fact that a portion of the iTreg cells expressed OX40 and 4-1BB. The mechanistic activity of APVO603 resulted in dose-dependent control of in vitro tumor growth when paired with a T-cell activating TAA x CD3 bispecific under standard conditions or those leading to T cell exhaustion. In preclinical assays using PBMCs sub-optimally stimulated with TAA x CD3, APVO603 enhanced TAA-expressing tumor cell lysis when compared to TAA x CD3 alone.ConclusionsAPVO603 is a dual-agonistic bispecific antibody that augments the effector function of activated CD4+ and CD8+ T cells and NK cells, but not iTreg cells, in a dose-dependent manner and reduces growth of tumors in vitro and in vivo. Further, mechanistic evaluation supports the ability of APVO603 to pair with T-cell modulating IO approaches to support a more fit T cell response and favorable TME. This preclinical data supports further development of APVO603, a promising immuno-oncology therapeutic with potential for benefit in hematologic and solid tumors.

scholarly journals 796 ALG.APV-527: a 5T4 tumor directed bispecific approach utilizing ADAPTIRTM technology designed for conditional 4-1BB T cell/NK agonism against solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A831-A831
Author(s):  
Michelle Nelson ◽  
Anette Sundstedt ◽  
Yago Pico de Coaña ◽  
Ashly Lucas ◽  
Anneli Nilsson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Immune Cell ◽  
Expression Profiles ◽  
Transcriptional Profiling ◽  
Sequencing Data ◽  
Multiple Tumor

Background4-1BB (CD137) is an activation-induced co-stimulatory receptor that regulates immune responses of activated CD8+ T cells and NK cells. Leveraging the therapeutic benefit of 1st generation 4-1BB monospecifics has been challenging due to dose limiting hepatotoxicity. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel 4-1BB x 5T4 bispecific antibody that stimulates 4-1BB function only when co-engaged with 5T4, a highly selective tumor-associated antigen. The combined preclinical dataset presented here provides an overview of the potential indication landscape, mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic.MethodsGenevestigator Software was used to analyze curated transcriptomic data from bulk tumor mRNA-sequencing data libraries and from single cell RNA-seq libraries for the expression profiles of CD8, 4-1BB and 5T4 across selected human solid tumor datasets. ADCC and ADCP reporter bioassays were utilized to assess Fc engagement by ALG.APV-527. For in vitro tumor lysis studies, human T cells were co-cultured with labelled tumor cells and sub-optimally activated with anti-CD3. Cytotoxicity of tumor cells were continually assessed using a Live-Cell Analysis System.ResultsDual expression of CD8 and 5T4 occurred in many tumor types and correlated well with indications that are pursued in the clinical development of ALG.APV-527. 4-1BB expression was observed in tumor-derived lymphoid subpopulations, especially in those with an exhausted phenotype. Since ALG.APV-527 is designed with a non-Fcγ receptor binding Fc, minimal ADCC & ADCP was induced in vitro. Additionally, ALG.APV-527 enhanced primary immune cell-mediated killing of 5T4-expressing tumor cells when compared to anti-CD3 alone, demonstrating the potential benefit of 4-1BB agonism for enhancing cytotoxic anti-tumor responses in the clinic.ConclusionsALG.APV-527 is designed to elicit safe and efficacious 4-1BB-mediated antitumor activity in a range of 5T4-expressing tumor indications. Transcriptional profiling of patient tumor samples demonstrates 4-1BB expression in multiple tumor-infiltrating lymphocyte subsets and identifies potential indications with 5T4 expression and CD8+ T cell infiltration. The unique design of the molecule minimizes systemic immune activation and hepatotoxicity, allowing for highly efficacious tumor-specific responses as demonstrated by potent activity in in vitro models. Based on these preclinical data, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of a variety of 5T4-expressing solid tumors and is progressing towards a phase I clinical trial in 2021.

scholarly journals Thiols decrease human interleukin (IL) 4 production and IL-4-induced immunoglobulin synthesis.

1995 ◽  
Vol 182 (6) ◽  
pp. 1785-1792 ◽  
Author(s):  
P Jeannin ◽  
Y Delneste ◽  
S Lecoanet-Henchoz ◽  
J F Gauchat ◽  
P Life ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Messenger Rna ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Dependent Manner ◽  
Dose Dependent

N-Acetyl-L-cysteine (NAC) is an antioxidant precursor of intracellular glutathione (GSH), usually given in human as a mucolytic agent. In vitro, NAC and GSH have been shown to act on T cells by increasing interleukin (IL) 2 production, synthesis and turnover of IL-2 receptors, proliferation, cytotoxic properties, and resistance to apoptosis. We report here that NAC and GSH decrease in a dose-dependent manner human IL-4 production by stimulated peripheral blood T cells and by T helper (Th) 0- and Th2-like T cell clones. This effect was associated with a decrease in IL-4 messenger RNA transcription. In contrast, NAC and GSH had no effect on interferon gamma and increased IL-2 production and T cell proliferation. A functional consequence was the capacity of NAC and GSH to selectively decrease in a dose-dependent manner IL-4-induced immunoglobulin (Ig) E and IgG4 production by human peripheral blood mononuclear cells. Interestingly, NAC and GSH also acted directly on purified tonsillar B cells by decreasing the mature epsilon messenger RNA, hence decreasing IgE production. In contrast, IgA and IgM production were not affected. At the same time, B cell proliferation was increased in a dose-dependent manner. Not all antioxidants tested but only SH-bearing molecules mimicked these properties. Finally, when given orally to mice, NAC decreased both IgE and IgG1 antibody responses to ovalbumin. These results demonstrate that NAC, GSH, and other thiols may control the production of both the Th2-derived cytokine IL-4 and IL-4-induced Ig in vitro and in vivo.

Development of human NKG2D-CD3ε chimeric antigen receptor (CAR) for T-cell-mediated cancer immunotherapy.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 150-150
Author(s):  
Sergei Kusmartsev ◽  
Johaness Vieweg ◽  
Victor Prima
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Adaptor Protein ◽  
T Cell Subsets ◽  
Human T Cells

150 Background: NKG2D is a lectin-like type 2 transmembrane receptor that expressed by natural killer cells and some T cell subsets. Stimulation of NKG2D receptor with specific agonistic ligands produces activating signals through signaling adaptor protein DAP10 leading to the enhanced cytokine production, proliferation, and cytotoxicity against tumor cells. There is strong evidence that NKG2D ligands are expressed in many human tumors, including melanoma, leukemia, myeloma, glioma, and carcinomas of the prostate, breast, lung, and colon. Recent studies also demonstrated that T cells bearing chimeric antigen receptor (CAR) NKG2D linked to CD3ζ (zeta) chain produce marked in vitro and in vivo anti-tumor effects. The aim of current study was to determine whether human T cells bearing chimeric antigen receptor (CAR) NKGD2 linked to CD3ε (epsilon) chain could be activated by the NKG2D-specific stimulation and able to kill human cancer cells. Given the important role of CD3ε in activation and survival of T cells, we hypothesized that NKG2D-CDε-bearing T cells could exert strong in vitro and in vivo anti-tumor effects. Methods: NKG2D CAR was produced by linking human NKG2D to DAP10 and the cytoplasmic portion of the CD3ε chain. Original full-length human cDNA clones were obtained from NIH Mammalian Gene Collection (MGC). Functional domain analysis and oligonucleotide design in the in-Fusion system of DNA cloning (Clontech) was used to generate the retroviral expression constructs. Results: Human PBMC-derived T cells were retrovirally transduced with newly generated NKG2D-CD3ε CAR DNA construct. These NKG2D CAR-expressing human T cells responded to NKG2D-specific activation by producing IFN-γ and exhibited significant cellular cytotoxicity against human tumor cells in vitro. In vivo studies demonstrated that NKG2D-CD3ε-bearing cells are capable of inhibiting growth of DU-145 human prostate cancer in the immunodeficient mice. Conclusions: Collectively, our data indicate the feasibility of developing chimeric antigen receptor NKG2D-CD3ε for T cells and suggest that adoptive transfer of T cells bearing NKG2D-CD3ε CAR could be potentially effective for immunotherapy of cancer patients.

Small Molecule Inhibitor of ITK Disrupts TCR Signaling and Demonstrates Anti-Tumor Efficacy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3932-3932
Author(s):  
Mary Faris ◽  
Uriel M Malyankar ◽  
Qingping Zeng ◽  
Gary A Flynn ◽  
Gerold Feuer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Production ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
T Cell Malignancies ◽  
Dose Dependent ◽  
Leukemias And Lymphomas

Abstract Abstract 3932 ITK (Interluekin-2 Inducible Tyrosine Kinase) is a member of the TEC family of intracellular protein tyrosine kinases. ITK is highly expressed in T cells and NK cells, with expression detected in mast cells. ITK plays a key role in several aspects of T cell biology, including T cell development, differentiation, migration, proliferation and activation. The function of ITK in immunity and allergy is well documented. T cells from ITK knock out mice show several developmental and functional defects, including defective signal transduction, altered CD4+ to CD8+ T cells ratios, reduced Th2 lineage differentiation, diminished IL4 and IL2 production and reduced T cell proliferation. Importantly ITK deficient mice fail to mount an immune response to infection and show reduced allergic asthma reactions. In contrast to its well described role in immune function, ITK's function in cancer biology is still emerging. Recent studies had reported enhanced ITK expression and activation of the ITK pathway in several types of leukemias and lymphomas. In addition, the dependence of T cell malignancies on an ITK-regulated pathway, namely the IL2/IL2R (CD25) pathway, has also been observed. Taken together, this information indicates that ITK is a therapeutic target, with applicability in leukemias and lymphomas. MannKind scientists have developed a series of selective small molecule ITK inhibitors, including the orally available tool compound described within, and evaluated their activity in enzyme, cell-based and in vivo studies. In cellular assays, the compounds showed significant inhibition of the T cell-receptor mediated activation of the ITK pathways and related downstream cytokine production. In addition to inhibiting the phosphorylation of ITK and its downstream mediator, PLCg, our tool compounds inhibited the production of IL2 and expression of CD25 in a dose dependent manner. Importantly, our compound regulated the in vitro growth of tumor T cells but not that of unrelated control cells. In vivo studies revealed that the tool compounds inhibited the growth and progression of patient derived ATL tumors in a xenograft pre-clinical model, and prolonged the survival of treated mice in a dose dependent manner, in addition to regulating cytokine production in vivo. In summary, our team has identified ITK selective compounds with demonstrated on-target and anti-tumor activity in vitro and preclinical T cell tumor models, and validated this pathway relative to T cell malignancies. This effort provides a platform for further compound optimization and evaluation for hematologic malignancies. Disclosures: Faris: MannKind Corp: Employment. Malyankar:MannKind Corp: Employment. Zeng:MannKind Corp: Employment. Kertesz:Mannkind Corporation: Employment, Equity Ownership. Vuga:MannKind Corp.: Employment. Rosario:MannKind Corp: Employment. Bot:MannKind Corp: Employment.

scholarly journals Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes

Blood ◽  
1995 ◽  
Vol 86 (11) ◽  
pp. 4199-4205 ◽  
Author(s):  
M Brunetti ◽  
N Martelli ◽  
A Colasante ◽  
M Piantelli ◽  
P Musiani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Interleukin 2 ◽  
Cell Number ◽  
T Cell Subsets ◽  
Dependent Manner ◽  
Cell Repertoire ◽  
Induced Apoptosis ◽  
And Function ◽  
Dose Dependent

Glucocorticoid (GC)-induced apoptosis is a well-recognized physiologic regulator of murine T-cell number and function. We have analyzed its mechanisms in human mature T cells, which have been thought to be insensitive until recently. Peripheral blood T cells showed sensitivity to GC-induced apoptosis soon after the proliferative response to a mitogenic stimulation, and were also sensitive to spontaneous (ie, growth factor deprivation-dependent) apoptosis. CD8+ T cells were more sensitive to both forms than CD4+ T cells. Acquisition of sensitivity to GC-induced apoptosis was not associated with any change in number or affinity of GC receptors. Both spontaneous and GC-induced apoptosis were increased by the macromolecular synthesis inhibitors, cycloheximide (CHX) and puromycin. A positive correlation between the degree of protein synthesis inhibition and the extent of apoptosis was observed. Interleukin-2 (IL-2) IL-4, and IL-10 protected (IL-2 > IL-10 > IL-4) T cells from both forms of apoptosis in a dose-dependent manner. Our data suggest that spontaneous and GC-induced apoptosis regulate the human mature T-cell repertoire by acting early after the immune response and differentially affecting T-cell subsets.

682 Antibody-mediated blockade of interleukin-10 receptor-alpha promotes the activation of immune cells from in vitro dissociated tumor samples

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A721-A721
Author(s):  
Alexey Berezhnoy ◽  
Kalpana Shah ◽  
Daorong Liu ◽  
Peter Lung ◽  
Vatana Long ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Expression Profiles ◽  
Interleukin 10 ◽  
Biologically Active ◽  
List Type ◽  
Receptor Alpha

BackgroundInterleukin-10 (IL-10) is a multifunctional cytokine that can mediate immune suppression or activation depending on the immunological context. Mouse studies have demonstrated that blockade of IL-10 enhances immune response against tumors and chronic viral infections;1 2 intriguingly, high concentrations of long-acting, pegylated IL-10 have also shown anti-tumor activity.3 Here we investigated IL-10 and IL-10 receptor-alpha (IL-10RA) expression profiles in normal and tumor tissues as well as the immunological effects of modulating the IL-10 pathway via antibody-mediated blockade of IL-10RA.MethodsIL-10 and IL-10RA mRNA are expressed by several tumors, including renal, lung, breast, and colon cancers. Fluorescent in-situ hybridization revealed that the majority of IL-10RA was expressed by CD3-negative tumor-infiltrating cells, localized in close proximity to T cells in the tumor microenvironment (TME). Immunohistochemistry studies confirmed expression of IL-10RA in the TME, while no expression was detected in healthy tissues. Furthermore, dissociated tumor cells produced biologically active levels of IL-10 in culture.ResultsMonoclonal antibodies (mAbs) against IL-10RA prevented IL-10 signaling and enhanced release of IL-12 by monocyte-derived dendritic cells activated with suboptimal LPS concentrations. The effect of IL-10RA blockade was greater than that observed with IL-10 neutralizing mAbs. In mixed lymphocyte reactions and superantigen-driven T-cell activation, IL-10RA blockade enhanced IL-2 secretion by T lymphocytes. Consistent with earlier observations in mouse models,4 the effect of IL-10RA blockade was nonredundant with blockade of the PD-1/PD-L1 axis, resulting in enhanced IL-2 and interferon-gamma secretion by T cells when both pathways were inhibited. Blockade of IL-10RA during CD3-redirected in vitro killing of tumor cells by PBMC induced IL-12 release as well as upregulation of CD86 and HLA-DR by CD3-negative cells. In in vitro dissociated tumor cells, IL-10RA blockade induced release of IL-2, interferon-gamma and other proinflammatory cytokines; additional PD-1/PD-L1 axis blockade further enhanced cytokine release.ConclusionsIn summary, antibody-mediated IL-10RA blockade can potentiate immune activation in the dissociated tumor cells and may be a valuable addition to cancer immunotherapies, including redirected T-cell killing and checkpoint blockade.ReferencesVicari A, et al. J. Exp. Med 2002;196:541–549.Ejrnaes M, et al. J Exp Med 2006;203:2461–72.Emmerich J, et al. Cancer Res 2012. 72:3570–3581.Brooks D, et al. Proc Natl Acad Sci U S A 2008;105:20428–33.

633 Dual-targeting of 4–1BB and OX40 with an ADAPTIR™ bispecific antibody enhances anti-tumor responses to solid tumor

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A669-A669
Author(s):  
Michelle Nelson ◽  
Robert Miller ◽  
Gabriele Blahnik-Fagan ◽  
Lauren Loh ◽  
Danielle Van Citters ◽  
...  
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Bispecific Antibody ◽  
Cytokine Production ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Binding Domains ◽  
Dose Dependent

Background4-1BB (CD137) and OX40 (CD134) are critical activation-induced co-stimulatory receptors that regulate immune responses of activated T and NK cells by enhancing proliferation, cytokine production, survival, and cytolytic activity. A superagonist 4-1BB antibody has shown clinical activity but severe toxicities. APVO603, is a 4-1BB x OX40 targeting bispecific antibody with conditional agonism, activating these receptors only when both are co-engaged. The Fc portion was mutated to eliminate FcγR-mediated interactions. Co-stimulation through 4-1BB and OX40 has the potential to amplify the cytotoxic function and the number of activated T and NK cells in multiple solid tumor indications.1–2Methods scFv binding domains to 4-1BB and OX40 were optimized to increase affinity, function and stability, and then incorporated into the ADAPTIR™ bispecific antibody platform to produce the APVO603 lead candidate. NF-κB/luciferase reporter cell lines expressing OX40 or 4-1BB were initially used to assess the agonistic function of APVO603’s binding domains. Primary PBMC were sub-optimally stimulated with an anti-CD3 antibody and T and NK cell proliferation was assessed using Cell TraceTM-labelled PBMC. Cytokine secretion was measured at 48 hrs using Luminex-based assays. For in vitro tumor lysis studies, PBMC were co-cultured with tumor cells expressing a tumor-associated antigen (TAA) and activated with TAA x CD3 bispecific protein. 7-AAD expression was assessed on tumor cells at 72 hrs. The in vivo therapeutic efficacy of APVO603 was evaluated using a murine MB49 bladder cancer model in human 4-1BB and OX40 double knock-in mice.ResultsAPVO603 stimulates 4-1BB and OX40 NF-κB/luciferase reporter activity in a dose-dependent manner, and is strictly dependent on engagement of the reciprocal receptor to elicit 4-1BB or OX40 activity. In primary PBMC assays, APVO603 induces synergistic proliferation of CD4+, CD8+ T and NK cells when compared to OX40 or 4-1BB monospecific molecules with a wt Fc, either individually or in combination. Additionally, APVO603 enhances proinflammatory cytokine production and granzyme B expression, and augments in vitro tumor cell lysis induced by a TAAx CD3 engager. In vivo, APVO603 reduces growth of established MB49 tumors in human 4-1BB and OX40 double knock-in mice.ConclusionsAPVO603 is a dual-agonistic bispecific antibody that augments the effector function of activated CD4+ and CD8+ T and NK cells in a dose-dependent manner, and reduces growth of established tumors in vivo. This preclinical data, demonstrates conditional dual stimulation of 4-1BB and OX40 and supports further development of APVO603, a promising immuno-oncology therapeutic with potential for benefit in solid tumors.Ethics ApprovalTreatment of study animals was in accordance with conditions specified in the Guide for the Care and Use of Laboratory Animals, and the study protocol (ACUP 20) was approved by the Institutional Animal Care and Use Committee (IACUC).ReferencesBandyopadhyay S, Long M, Qui H, Hagymasi A, Slaiby A, Mihalyo M, Aguila H, Mittler R, Vella A, Adler A. Self-antigen prevents CD8 T cell effector differentiation by CD134 and CD137 dual costimulation. J Immunol 2008;181(11):7728–37.Ryan J, Mittal P, Menoret A, Svedova J, Wasser J, Adler A, Vella A. A novel biologic platform elicits profound T cell costimuloaroty activity and antitumor immunity in mice. Cancer Immunol Immunother 2018;67(4):605–613.

scholarly journals Development of a CD123xCD3 Bispecific Antibody (JNJ-63709178) for the Treatment of Acute Myeloid Leukemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2824-2824 ◽  
Author(s):  
François Gaudet ◽  
Jennifer F Nemeth ◽  
Ronan McDaid ◽  
Yingzhe Li ◽  
Benjamin Harman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Stock Options ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
Tumor Regression

Abstract AML is a cancer of the myeloid lineage that is characterized by the accumulation of abnormal white blood cells in the bone marrow and blood. Existing therapies do not lead to cures, partially due to their inability to eliminate residual leukemic stem cells (LSCs) in the bone marrow. T-cell redirection has been shown to be an effective method of treatment for hematologic malignancies (eg, blinatumomab) and represents an attractive approach to treat AML. CD123 (α-chain of the interleukin-3 receptor) has been shown to be expressed on the surface of AML blasts and LSCs. To eradicate CD123+ cells, we developed a bispecific antibody (JNJ-63709178) using the Genmab DuoBody® technology that can bind both CD123 on tumor cells and CD3 on T cells. JNJ-63709178 is a humanized IgG4 bispecific antibody with silenced Fc function. This antibody is able to recruit T cells to CD123-expressing tumor cells and induce the killing of these tumor cells in vitro (MOLM-13, OCI-AML5 and KG-1; EC50 = 0.51-0.91 nM). In contrast, this antibody does not kill CD123- cell lines, demonstrating the specificity of cytotoxicity. Consistently, the degree of cell killing correlated with the level of T-cell activation (CD69 and CD25) and cytokine release (TGF-β and TNF-α). Control bispecific antibodies containing a null arm (viral epitope) paired with a CD123 arm (CD123xnull) or a CD3 arm (nullxCD3) did not induce cytotoxicity or T-cell activation in the assays tested. JNJ-63709178 had no effect on T-cell activation when incubated with T cells alone. In AML murine xenograft models, JNJ-63709178 was able to suppress tumor growth and induce tumor regression (MOLM-13 and KG-1, respectively) in the presence of human peripheral blood mononuclear cells (PBMCs) or T cells. Tumor regression correlated with the infiltration of T cells in the tumor and the expression of T-cell activation markers such as CD25, PD1 and TIM3. Furthermore, this antibody was able to induce the killing of primary CD123+ cancer cells from the blood of patients with AML without the need to supplement with fresh T cells (EC50 = 0.83 nM). These results indicate that JNJ-63709178 can potently and specifically kill CD123+ cancer cells in vitro, in vivo and ex vivo. Pharmacokinetic studies in cynomolgus monkeys support twice weekly dosing for human studies. JNJ-63709178 is currently being investigated in a Phase 1 clinical trial in relapsed and refractory AML (ClinicalTrials.gov ID: NCT02715011). Disclosures Gaudet: Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued. Nemeth:Janssen Pharmaceuticals R&D: Employment, Other: stock, Patents & Royalties: patent pending. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Harman:Janssen Pharmaceuticals R&D: Employment. Millar:Janssen Pharmaceuticals R&D: Employment, Other: stock options. Teplyakov:Janssen Pharmaceuticals R&D: Employment. Wheeler:Janssen Pharmaceuticals R&D: Employment. Luo:Janssen Pharmaceuticals R&D: Employment. Tam:Janssen Pharmaceuticals R&D: Employment, Other: stocks, Research Funding. Wu:Janssen Pharmaceuticals R&D: Employment. Chen:Janssen Pharmaceuticals R&D: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Chu:Janssen Pharmaceuticals R&D: Employment. Hughes:Janssen Pharmaceuticals R&D: Employment. Luistro:Janssen: Employment. Chin:Janssen: Employment. Babich:Janssen: Employment. Kalota:Janssen Pharmaceuticals R&D: Employment, Other: stock. Singh:Janssen Pharmaceuticals R&D: Employment, Other: stock options. Salvati:Janssen Pharmaceuticals R&D: Employment, Other: stock options, Patents & Royalties: patent. Elsayed:Janssen: Employment, Other: stock options. Attar:Janssen: Employment.

CAR-T cells to deliver engineered peptide antigens and reprogram antigen specific T cell responses against solid tumors.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 2530-2530
Author(s):  
Daniel Lee ◽  
Andy J Minn ◽  
Lexus R Johnson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
In Vivo Studies ◽  
Peptide Antigens ◽  
Car T Cells ◽  
Car T

2530 Background: Neoantigen depleted malignancies such as colorectal cancer demonstrate primary resistance to immune checkpoint blockade, and solid tumors in general have shown resistance to chimeric antigen receptor (CAR) T cell therapy. However, CAR-T cells have been shown to be capable of delivering various therapeutic molecules in a targeted fashion to the tumor microenvironment, in some cases through extracellular vesicles (EVs). In vivo studies have shown that the presentation of foreign viral peptides by solid tumors can reprogram bystander virus-specific cytotoxic T cells (CTLs) against tumor cells. In this study, we demonstrate that CAR-T cells can deliver engineered peptide antigens to solid tumors, leading to presentation on tumor cells and anti-tumor response. Methods: Second generation CAR-T cells (41BB endodomain) targeting human CD19 (19BBz) or human mesothelin (M5BBz) were generated via retroviral and lentiviral transduction respectively. CAR-T cells were engineered to co-express peptides such as SIINFEKL of ovalbumin and NLVPMVATV of CMV pp65 among others. Peptides were isolated from EVs via ultracentrifugation. For in vivo studies, C57BL/6 or NSG mice were injected on the flank with relevant tumors and treated with peptide-CAR-T cells. In vitro studies utilized flow cytometry and xCELLigence killing assays. Results: Murine 19BBz CAR-T cells expressing the SIINFEKL peptide of ovalbumin (ova-19BBz) were found to transfer SIINFEKL peptide to tumor cells via EVs in vitro and in vivo, leading to peptide presentation on MHC-I of tumor cells. This resulted in significantly delayed tumor growth in tumor bearing mice transfused with OT-I T cells to mimic an existing antigen specific T cell pool. We expanded on these findings by isolating EVs from human M5BBz CAR-T cells expressing CMV viral peptides. Peptide-CAR-T EVs were co-cultured with human ovarian cancer cells to assess presentation to Jurkat T cells. Finally, we utilized primary human T cells from CMV+ healthy donors to assess the clinical feasibility of our peptide delivery approach. Conclusions: CAR-T cells can be engineered to deliver peptides to tumor cells for presentation and subsequent targeting by antigen specific CTLs. This represents a novel strategy for the treatment of non-immunogenic tumors.

116 Multi-antigen targeting of heterogenous solid tumors using CAR T cells secreting bi-specific T-cell engagers

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A128-A128
Author(s):  
Martin Hosking ◽  
Bishwas Shrestha ◽  
Megan Boyett ◽  
Soheila Shirinbak ◽  
Angela Gentile ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Cytolytic Activity ◽  
Laboratory Animals ◽  
Car T Cells ◽  
Car T

BackgroundAlthough CAR T cells have been shown to be effective and potent in treating several hematologic malignancies, engineered T-cell therapies have had limited success in addressing solid tumors. Unlike liquid tumors where uniformly expressed antigens are accessible and can be effectively targeted, tumor access and antigen heterogeneity are a significant barrier to the successful development of CAR-T cells in solid tumors.MethodsHere we demonstrate that the combination of a bi-specific T-cell engager (BiTE) targeting EpCAM with a CAR T cell targeting HER2 enhances the in vitro and in vivo anti-tumor activity against heterogenous solid tumors.ResultsWe observed a dose-dependent enhancement of cytolytic activity when EpCAM-specific BiTEs were titrated alongside 4D5-based HER2-specific CAR T cells against HER2low tumors, enhancing maximal cytolysis by two-fold compared to CAR T cells alone (figure 1). Moreover, the escape of HER2low tumor cells in mixed heterogenous culture systems was circumvented by the combination of HER2-specific CAR T cells and EpCAM-specific BiTEs. The enhancement of efficacy was further demonstrated in an established HER2low MDA-MB-231 xenografts. HER2-specific CAR T cells were unable to contain Her2low tumors, whereas tumor growth was effectively controlled in mice receiving both EpCAM-specific BiTEs and HER2-specific CAR T cells.Abstract 116 Figure 1EpCAM specific BiTEs supplement CAR-T efficacy in vitro (A) HER2 and EpCAM expression of SKOV3, MDA-MB-231, and K562 tumor cells was assessed by flow cytometry. (B) HER2 specific CAR-T rapidly targeted and lysed HER2High SKOV3 tumor cells as measured via xCelligence RTCA assay. (C) SKOV3 were co-cultured with untransduced CD8+ T cells and the indicated concentrations of EpCAM BiTE and specific cytolysis was assessed. (D) MDA-MB-231 (HER2low) tumor cells were co-cultured with HER2 CAR-T ± EpCAM BiTE and specific cytolysis was determinedConclusionsCollectively, these data demonstrate that multi-antigen targeting mediated by BiTEs and CARs extends overall anti-tumor efficacy in preclinical models of heterogenous solid tumors. Fate Therapeutics is currently using its proprietary induced pluripotent stem cell (iPSC) product platform to generate iPSC-derived CAR T cells and iPSC-derived CAR NK cells that secrete BiTEs for the treatment of solid tumors.Ethics ApprovalThese studies were approved by Fate Therapeutics Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

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