16. Effective and Safe Gene-Modified T Cells Expressing High Affinity NYESO-1 Specific TCR Variants with Silencing Endogenous TCRs

BackgroundChimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity, including on-target off-tumor antigen binding, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CAR bearing a low and high affinity single-chain variable fragment (scFv,) binding to the same epitope and cross-reactive with murine GPC3. We characterized low and high affinity CAR-T cells immunophenotype and effector function in vitro, followed by in vivo efficacy and safety studies in hepatocellular carcinoma (HCC) xenograft models.ResultsCompared to the high-affinity construct, the low-affinity CAR maintained cytotoxic function but did not show in vivo toxicity. High-affinity CAR-induced toxicity was caused by on-target off-tumor binding, based on the evidence that high-affinity but not low-affinity CAR, were toxic in non-tumor bearing mice and accumulated in organs with low expression of GPC3. To add another layer of safety, we developed a mean to target and eliminate CAR-T cells using anti-TNFα antibody therapy post-CAR-T infusion. This antibody functioned by eliminating early antigen-activated CAR-T cells, but not all CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from anti-tumor efficacy.ConclusionsSelecting a domain with higher off-rate improved the quality of the CAR-T cells by maintaining cytotoxic function while reducing cytokine production and activation upon antigen engagement. By exploring additional traits of the CAR-T cells post-activation, we further identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that would eliminate early activated CAR-T following antigen engagement in vivo. By combining the reduced affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.Ethics ApprovalAll animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.

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721 AT1412, a patient-derived CD9 antibody in preclinical development promoting tumor immune infiltration and inducing tumor rejection

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0721 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A763-A763

Author(s):

Remko Schotte ◽

Julien Villaudy ◽

Martijn Kedde ◽

Wouter Pos ◽

Daniel Go ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

Tumor Cells ◽

Tumor Rejection ◽

Human Immune System ◽

Preclinical Development ◽

High Affinity ◽

Human Immune ◽

A375 Melanoma

BackgroundAdaptive immunity to cancer cells forms a crucial part of cancer immunotherapy. Recently, the importance of tumor B-cell signatures were shown to correlate with melanoma survival. We investigated whether tumor-targeting antibodies could be isolated from a patient that cured (now 13 years tumor-free) metastatic melanoma following adoptive transfer of ex vivo expanded autologous T cells.MethodsPatient‘s peripheral blood B cells were isolated and tested for the presence of tumor-reactive B cells using AIMM’s immmortalisation technology. Antibody AT1412 was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes. AT1412 binds the tetraspanin CD9, a broadly expressed protein involved in multiple cellular activities in cancer and induces ADCC and ADCP by effector cells.ResultsSpontaneous immune rejection of tumors was observed in human immune system (HIS) mouse models implanted with CD9 genetically-disrupted A375 melanoma (A375-CD9KO) tumor cells, while A375wt cells were not cleared. Most notably, no tumor rejection of A375-CD9KO tumors was observed in NSG mice, indicating that blockade of CD9 makes tumor cells susceptible to immune rejection.CD9 has been described to regulate integrin signaling, e.g. LFA-1, VLA-4, VCAM-1 and ICAM-1. AT1412 was shown to modulate CD9 function by enhancing adhesion and transmigration of T cells to endothelial (HUVEC) cells. AT1412 was most potently enhancing transendothelial T-cell migration, in contrast to a high affinity version of AT1412 or other high affinity anti-CD9 reference antibodies (e.g. ALB6). Enhanced immune cell infiltration is also observed in immunodeficient mice harbouring a human immune system (HIS). AT1412 strongly enhanced CD8 T-cell and macrophage infiltration resulting in tumor rejection (A375 melanoma). PD-1 checkpoint blockade is further sustaining this effect. In a second melanoma model carrying a PD-1 resistant and highly aggressive tumor (SK-MEL5) AT1412 together with nivolumab was inducing full tumor rejection, while either one of the antibodies alone did not.ConclusionsThe safety of AT1412 has been assessed in preclinical development and is well tolerated up to 10 mg/kg (highest dose tested) by non human primates. AT1412 demonstrated a half-life of 8.5 days, supporting 2–3 weekly administration in humans. Besides transient thrombocytopenia no other pathological deviations were observed. No effect on coagulation parameters, bruising or bleeding were observed macro- or microscopically. The thrombocytopenia is reversible, and its recovery accelerated in those animals developing anti-drug antibodies. First in Human clinical study is planned to start early 2021.Ethics ApprovalStudy protocols were approved by the Medical Ethical Committee of the Leiden University Medical Center (Leiden, Netherlands).ConsentBlood was obtained after written informed consent by the patient.

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A Modified Tyrosinase-Related Protein 2 Epitope Generates High-Affinity Tumor-Specific T Cells but Does Not Mediate Therapeutic Efficacy in an Intradermal Tumor Model

The Journal of Immunology ◽

10.4049/jimmunol.177.1.155 ◽

2006 ◽

Vol 177 (1) ◽

pp. 155-161 ◽

Cited By ~ 27

Author(s):

Jennifer A. McWilliams ◽

Sean M. McGurran ◽

Steven W. Dow ◽

Jill E. Slansky ◽

Ross M. Kedl

Keyword(s):

T Cells ◽

Therapeutic Efficacy ◽

Tumor Model ◽

Related Protein ◽

High Affinity ◽

Tyrosinase Related Protein

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Interleukin 2 receptor-targeted cytotoxicity. Interleukin 2 receptor-mediated action of a diphtheria toxin-related interleukin 2 fusion protein.

Journal of Experimental Medicine ◽

10.1084/jem.167.2.612 ◽

1988 ◽

Vol 167 (2) ◽

pp. 612-622 ◽

Cited By ~ 163

Author(s):

P Bacha ◽

D P Williams ◽

C Waters ◽

J M Williams ◽

J R Murphy ◽

...

Keyword(s):

T Cells ◽

Protein Synthesis ◽

T Cell ◽

Diphtheria Toxin ◽

Interleukin 2 ◽

Elongation Factor ◽

High Affinity ◽

Interleukin 2 Receptor ◽

Human T Cell ◽

Inhibition Of Protein Synthesis

The IL-2 toxin-mediated inhibition of protein synthesis in high affinity IL-2-R-positive murine and human T cell lines has been examined. Both excess free IL-2 and mAb to the Tac epitope of the p55 subunit of IL-2-R are shown to block the action of IL-2 toxin; whereas, agents that interact with other receptors or antigens on the T cell surface have no effect. We show that IL-2 toxin, like diphtheria toxin, must pass through an acidic vesicle in order to intoxicate target T cells. Finally, we demonstrate that the IL-2 toxin-mediated inhibition of protein synthesis in both human and murine T cells that bear the high affinity IL-2-R is due to the classic diphtheria toxin fragment A-catalyzed ADP ribosylation of elongation factor 2.

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Corrigendum to ‘High-affinity human programmed death-1 ligand-1 variant promotes redirected T cells to kill tumor cells’ [Cancer Lett. 447 (2019) 164–173]

Cancer Letters ◽

10.1016/j.canlet.2019.03.026 ◽

2019 ◽

Vol 452 ◽

pp. 268-269

Author(s):

Zhaoduan Liang ◽

Yanyan Li ◽

Ye Tian ◽

Huanling Zhang ◽

Wenxuan Cai ◽

...

Keyword(s):

T Cells ◽

Tumor Cells ◽

High Affinity ◽

Programmed Death ◽

Programmed Death 1

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IL-21 from high-affinity CD4 T cells drives differentiation of brain-resident CD8 T cells during persistent viral infection

Science Immunology ◽

10.1126/sciimmunol.abb5590 ◽

2020 ◽

Vol 5 (51) ◽

pp. eabb5590 ◽

Cited By ~ 3

Author(s):

Heather M. Ren ◽

Elizabeth M. Kolawole ◽

Mingqiang Ren ◽

Ge Jin ◽

Colleen S. Netherby-Winslow ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Viral Infection ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Central Nervous System Infection ◽

Cell Receptor ◽

Cd4 T Cell ◽

Persistent Viral Infection ◽

High Affinity

Development of tissue-resident memory (TRM) CD8 T cells depends on CD4 T cells. In polyomavirus central nervous system infection, brain CXCR5hi PD-1hi CD4 T cells produce interleukin-21 (IL-21), and CD8 T cells lacking IL-21 receptors (IL21R−/−) fail to become bTRM. IL-21+ CD4 T cells exhibit elevated T cell receptor (TCR) affinity and higher TCR density. IL21R−/− brain CD8 T cells do not express CD103, depend on vascular CD8 T cells for maintenance, are antigen recall defective, and lack TRM core signature genes. CD4 T cell–deficient and IL21R−/− brain CD8 T cells show similar deficiencies in expression of genes for oxidative metabolism, and intrathecal delivery of IL-21 to CD4 T cell–depleted mice restores expression of electron transport genes in CD8 T cells to wild-type levels. Thus, high-affinity CXCR5hi PD-1hi CD4 T cells in the brain produce IL-21, which drives CD8 bTRM differentiation in response to a persistent viral infection.

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