752 Novel, orally administered HPK1 inhibitors demonstrate anti-tumor efficacy and enhanced immune response

BackgroundHematopoietic progenitor kinase 1 (HPK1, MAP4K1) is emerging as a well-renowned, druggable target for T cell-based immunotherapies. HPK1 is a member of the serine/threonine MAP4K family, predominantly expressed in hematopoietic cell lineages and shown to be a negative regulator of the T cell receptor (TCR) signaling pathway. Upon TCR activation, HPK1 is recruited to the proximity of the cell membrane and phosphorylates an adaptor protein SLP-76 at the Ser376 residue which, in turn, abrogates TCR signaling. Other studies point to a potential role of HPK1 in T cell exhaustion as well as in functional re-programming of regulatory T cells. Moreover, mounting evidence suggest that HPK1 kinase activity suppresses the immune functions of a wide range of other immune cell subsets like B cells and dendritic cells. Taken together, these observations support small-molecule HPK1 inhibitors as an attractive modality in cancer immunotherapy either as single agents or in combination with immune checkpoint inhibitors.MethodsActivity of compounds against HPK1 and selected off- and anti-targets was assessed in biochemical assays. Phosphorylation of SLP-76 was measured either by flow cytometry or TR-FRET. Jurkat and primary T cells were activated and cultured in the presence of tested compounds and immunosuppressive agents. Impact on TCR selectivity and T cell function was measured by AlphaLISA and flow cytometry. Target engagement was measured in splenocytes of mice administered orally with tested compounds followed by IP injection of aCD3 antibody. Anti-tumor efficacy of HPK1 inhibitors was assessed in a syngeneic tumor model.ResultsRyvu's proprietary small molecule HPK1 inhibitors exhibit sub-nanomolar activity against human and mouse HPK1 proteins and good selectivity against other TCR pathway kinases. Tested compounds efficiently block phosphorylation of SLP-76 upon TCR engagement. TCR selectivity of Ryvu's inhibitors, measured as a ratio between CD69 and pSer376 SLP-76 inhibition, is on par or superior to reference molecules. Tested compounds are not only able to overcome PGE-2 induced resistance following TCR activation in human PBMCs, inducing elevated IL-2 release but also affect T cell function in co-culture assay. Developed molecules have favorable PK profiles, allowing for sustained target coverage in proposed dosing regimens and demonstrate efficacy in a mammary carcinoma syngeneic model.ConclusionsRyvu has developed potent and selective HPK1 inhibitors with favorable PK and PD profiles, whose activity in vitro translates to in vivo efficacy. Further preclinical work is warranted to select a lead candidate for IND-enabling studies and subsequently clinical studies across a variety of solid tumors.

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Histone Deacetylase 11 (HDAC11) Regulates Cytotoxic T-Cell Function and Memory Phenotype

Blood ◽

10.1182/blood.v120.21.840.840 ◽

2012 ◽

Vol 120 (21) ◽

pp. 840-840

Author(s):

David M Woods ◽

Karrune V. Woan ◽

Eva Sahakian ◽

John Powers ◽

Fengdong Cheng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cell Function ◽

Cd8 T Cell ◽

Central Memory ◽

Negative Regulator ◽

Wild Type ◽

Potential Target ◽

T Cell Function

Abstract Abstract 840 T-cells are an essential component of immune mediated tumor rejection. Adoptive transfer of T-cells results in a durable anti-tumor response in some patients with hematological malignancies. To further improve the efficacy of T-cell adoptive transfers, a better understanding of the regulatory checkpoints of these cells is needed. Here we show that HDAC11 is a negative regulator of CD8+ T-cell function, thus representing a potential target in adoptive immunotherapy. HDACs are a group of enzymes initially known for their role in deacetylating histones, thereby condensing chromatin structure and repressing gene expression. The known roles of HDACs as epigenetic regulators have recently expanded to include more complex regulatory functions including interactions with non-histone targets. HDAC11 is the most recently identified member of the HDAC family, and is highly expressed in brain, testis and T-cells. Recently, our group reported HDAC11 as a regulator of IL-10 production in antigen presenting cells. To determine the role of HDAC11 in T-cell biology, T-cells from HDAC11 knock out (HDAC11KO) mice were compared to wild-type T-cells in number, function and phenotype. HDAC11KO T-cells had no differences in absolute number or percentages of CD4+ or CD8+ lymphocytes. However CD8+ T-cells were hyper-proliferative upon CD3/CD28 stimulation and produced significantly higher levels of the pro-inflammatory, Tc1 cytokines IL-2, INF-γ, and TNF-α. However, no significant increases in the production of the Tc2 cytokines IL-4, IL-6 or IL-10 were seen. Further investigation of phenotypic differences also revealed that HDAC11KO mice have a larger percentage of central memory CD8+ T-cells. Additionally, HDAC11KO CD8+ T-cells express higher levels of the transcription factor Eomes, a known contributor to central memory cell formation as well as a controller of granzyme B and perforin production in CD8+ T-cells. This Tc1 and central memory-like phenotype translated to delayed tumor progression and survival in vivo in C1498 AML bearing mice treated with adoptively transferred HDAC11KO T-cells, as compared with wild type T-cells. Collectively, we have demonstrated HDAC11 as a negative regulator of CD8+ T-cell function, and a novel potential target to augment the efficacy of adoptive T-cell tumor immunotherapy. Disclosures: No relevant conflicts of interest to declare.

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Small molecule inhibition of Csk alters affinity recognition by T cells

eLife ◽

10.7554/elife.08088 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 26

Author(s):

Boryana N Manz ◽

Ying Xim Tan ◽

Adam H Courtney ◽

Florentine Rutaganira ◽

Ed Palmer ◽

...

Keyword(s):

T Cells ◽

Catalytic Activity ◽

T Cell ◽

Small Molecule ◽

Src Family Kinases ◽

Negative Regulator ◽

Proliferation Inhibition ◽

Tcr Signaling ◽

Cell Responses ◽

Small Molecule Inhibition

The C-terminal Src kinase (Csk), the primary negative regulator of Src-family kinases (SFK), plays a crucial role in controlling basal and inducible receptor signaling. To investigate how Csk activity regulates T cell antigen receptor (TCR) signaling, we utilized a mouse expressing mutated Csk (CskAS) whose catalytic activity is specifically and rapidly inhibited by a small molecule. Inhibition of CskAS during TCR stimulation led to stronger and more prolonged TCR signaling and to increased proliferation. Inhibition of CskAS enhanced activation by weak but strictly cognate agonists. Titration of Csk inhibition revealed that a very small increase in SFK activity was sufficient to potentiate T cell responses to weak agonists. Csk plays an important role, not only in basal signaling, but also in setting the TCR signaling threshold and affinity recognition.

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Trib1 regulates T cell differentiation during chronic infection by restraining the effector program

Journal of Experimental Medicine ◽

10.1084/jem.20190888 ◽

2020 ◽

Vol 217 (5) ◽

Author(s):

Kelly S. Rome ◽

Sarah J. Stein ◽

Makoto Kurachi ◽

Jelena Petrovic ◽

Gregory W. Schwartz ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

Cell Function ◽

T Cell Differentiation ◽

Negative Regulator ◽

Chronic Infections ◽

Tcr Signaling ◽

Viral Control ◽

Cell Immunity

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.

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562 THE EFFECT OF IONISING-RADIATION AND THE TUMOUR MICRO-ENVIRONMENT (TME) ON TREATMENT NAIVE ESOPHAGEAL ADENOCARCINOMA PATIENT T CELL FUNCTION AND ACTIVITY.

Diseases of the Esophagus ◽

10.1093/dote/doaa087.147 ◽

2020 ◽

Vol 33 (Supplement_1) ◽

Author(s):

N Donlon ◽

A Sheppard ◽

M Davern ◽

C Donohoe ◽

N Ravi ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Cell Expansion ◽

Cell Function ◽

Ionising Radiation ◽

Nutrient Deprivation ◽

T Cell Function ◽

T Cell Expansion ◽

The Impact

Abstract There is extensive literature demonstrating CD8+ T cells are essential for initial tumour control following radiation, however, effects are reduced after time due to T cell exhaustion and a lack of release Damage Associated Molecular Patterns (DAMPS) which are essential for anti-tumour immune responses. In vivo, activated T-cells migrate to the tumour site within the field of irradiation, however translational studies on the effects of radiotherapy on T-cell activation, function and activity are lacking. Methods EAC patient (n = 6) PBMCs were isolated by density centrifugation in Ficoll Paque. T cells were activated and were irradiated at 1.8Gy, 3.6Gy bolus dosing and fractionation for 72 hrs. A panel of immune checkpoints, DAMPS, activation markers, and cytokines were assessed by flow cytometry. To determine the effect of the TME on T cells, PBMCs were cultured under conditions of nutrient deprivation (No Glucose & No Glutamine) under conditions of normoxia and hypoxia. We then ran the aforementioned panel by flow cytometry. We also activated PBMCs with immune checkpoint blockers to determine its effects on T cell expansion and survival. Results 3.6Gy induced a significantly higher expression of DAPMS (Fig 1 p < 0.001); Calreticulin and HMGB1, most notably under conditions of nutrient deprivation (p < 0.001). Ionising radiation also resulted in an increase in the expression of cytokines and importantly in the context of targeted therapy, IR at both the conventional 1.8Gy and 3.6Gy induced a higher expression of checkpoints PD-1, PD-L1, TIGIT, and TIM-3 (p < 0.001). Interestingly, when T cells are activated in the presence of ICB (Atezolizumab, Pembrolizumab, Nivolumab), it increases the rate of T cell expansion, and enhances their survival compared to T cell activated only. (p < 0.001). Conclusion This work demonstrates the impact of clinically utilised fractions of radiation, and conditions of the TME on T cell function and activity, with improved T cell expansion and survival in the presence of ICB’s suggesting it may be a feasible combination therapy as an adjunct to radiotherapy.

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MicroRNA miR-181—A Rheostat for TCR Signaling in Thymic Selection and Peripheral T-Cell Function

International Journal of Molecular Sciences ◽

10.3390/ijms21176200 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6200

Author(s):

Zoe Grewers ◽

Andreas Krueger

Keyword(s):

T Cells ◽

T Cell ◽

Cell Function ◽

Signal Strength ◽

Cell Receptor ◽

Tcr Signaling ◽

Cell Repertoire ◽

T Cell Function ◽

Complex Process ◽

Peripheral T Cells

The selection of T cells during intra-thymic d evelopment is crucial to obtain a functional and simultaneously not self-reactive peripheral T cell repertoire. However, selection is a complex process dependent on T cell receptor (TCR) thresholds that remain incompletely understood. In peripheral T cells, activation, clonal expansion, and contraction of the active T cell pool, as well as other processes depend on TCR signal strength. Members of the microRNA (miRNA) miR-181 family have been shown to be dynamically regulated during T cell development as well as dependent on the activation stage of T cells. Indeed, it has been shown that expression of miR-181a leads to the downregulation of multiple phosphatases, implicating miR-181a as ‘‘rheostat’’ of TCR signaling. Consistently, genetic models have revealed an essential role of miR-181a/b-1 for the generation of unconventional T cells as well as a function in tuning TCR sensitivity in peripheral T cells during aging. Here, we review these broad roles of miR-181 family members in T cell function via modulating TCR signal strength.

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Src-like Adaptor Protein (Slap) Is a Negative Regulator of T Cell Receptor Signaling

Journal of Experimental Medicine ◽

10.1084/jem.191.3.463 ◽

2000 ◽

Vol 191 (3) ◽

pp. 463-474 ◽

Cited By ~ 87

Author(s):

Tomasz Sosinowski ◽

Akhilesh Pandey ◽

Vishva M. Dixit ◽

Arthur Weiss

Keyword(s):

T Cells ◽

T Cell ◽

Interleukin 2 ◽

Adaptor Protein ◽

Sh2 Domain ◽

Jurkat Cells ◽

Negative Regulator ◽

Tcr Signaling ◽

Activated T Cells ◽

Sh2 Domains

Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)–, and interleukin 2–dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3ζ, ZAP-70, SH2 domain–containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

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Ligand-Independent Exhaustion of Killer Immunoglobulin-Like Receptor-Positive CD8+ T Cells in Human Immunodeficiency Virus Type 1 Infection

Journal of Virology ◽

10.1128/jvi.00341-08 ◽

2008 ◽

Vol 82 (19) ◽

pp. 9668-9677 ◽

Cited By ~ 19

Author(s):

Galit Alter ◽

Suzannah Rihn ◽

Hendrik Streeck ◽

Nickolas Teigen ◽

Alicja Piechocka-Trocha ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

T Cell ◽

Human Immunodeficiency Virus Type ◽

Cell Function ◽

T Cell Function ◽

Immunodeficiency Virus ◽

Tcr Activation ◽

Hiv 1

ABSTRACT Virus-specific CD8+ T cells play a central role in the control of viral infections, including human immunodeficiency virus type 1 (HIV-1) infection. However, despite the presence of strong and broad HIV-specific CD8+ T-cell responses in chronic HIV-1 infection, these cells progressively lose critical effector functions and fail to clear the infection. Mounting evidence suggests that the upregulation of several inhibitory regulatory receptors on the surface of CD8+ T cells during HIV-1 infection may contribute directly to the impairment of T-cell function. Here, we investigated the role of killer immunoglobulin receptors (KIR), which are expressed on NK cells and on CD8+ T cells, in regulating CD8+ T-cell function in HIV-1 infection. KIR expression was progressively upregulated on CD8+ T cells during HIV-1 infection and correlated with the level of viral replication. Expression of KIR was associated with a profound inhibition of cytokine secretion, degranulation, proliferation, and activation by CD8+ T cells following stimulation with T-cell receptor (TCR)-dependent stimuli. In contrast, KIR+ CD8+ T cells responded potently to TCR-independent stimulation, demonstrating that these cells are functionally competent. KIR-associated suppression of CD8+ T-cell function was independent of ligand engagement, suggesting that these regulatory receptors may constitutively repress TCR activation. This ligand-independent repression of TCR activation of KIR+ CD8+ T cells may represent a significant barrier to therapeutic interventions aimed at improving the quality of the HIV-specific CD8+ T-cell response in infected individuals.

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