173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

Download Full-text

A Novel Approach for the Treatment of T Cell Malignancies: Targeting T Cell Receptor Vβ Families

Vaccines ◽

10.3390/vaccines8040631 ◽

2020 ◽

Vol 8 (4) ◽

pp. 631

Author(s):

Jie Wang ◽

Katarzyna Urbanska ◽

Prannda Sharma ◽

Reza Nejati ◽

Lauren Shaw ◽

...

Keyword(s):

Monoclonal Antibody ◽

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

T Cell Lymphomas ◽

Novel Approach ◽

T Cell Malignancies

Peripheral T cell lymphomas (PTCLs) are generally chemotherapy resistant and have a poor prognosis. The lack of targeted immunotherapeutic approaches for T cell malignancies results in part from potential risks associated with targeting broadly expressed T cell markers, namely T cell depletion and clinically significant immune compromise. The knowledge that the T cell receptor (TCR) β chain in human α/β TCRs are grouped into Vβ families that can each be targeted by a monoclonal antibody can therefore be exploited for therapeutic purposes. Here, we develop a flexible approach for targeting TCR Vβ families by engineering T cells to express a chimeric CD64 protein that acts as a high affinity immune receptor (IR). We found that CD64 IR-modified T cells can be redirected with precision to T cell targets expressing selected Vβ families by combining CD64 IR-modified T cells with a monoclonal antibody directed toward a specific TCR Vβ family in vitro and in vivo. These findings provide proof of concept that TCR Vβ-family-specific T cell lysis can be achieved using this novel combination cell–antibody platform and illuminates a path toward high precision targeting of T cell malignancies without substantial immune compromise.

Download Full-text

Evidence for in vivo clonal proliferation of unique population of blood CD4-/CD8- T cells bearing T-cell receptor alpha and beta chains in two normal men

Blood ◽

10.1182/blood.v79.11.2965.2965 ◽

1992 ◽

Vol 79 (11) ◽

pp. 2965-2972 ◽

Cited By ~ 20

Author(s):

Y Kusunoki ◽

Y Hirai ◽

S Kyoizumi ◽

M Akiyama

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Nk Cell ◽

Cell Receptor ◽

Cell Markers ◽

Clonal Proliferation ◽

Alpha Beta

Abstract Rare T lymphocytes bearing CD3 surface antigen and T-cell receptor (TCR) alpha and beta chains, but lacking both CD4 and CD8 antigens, viz, TCR alpha beta+CD4–8- cells, appear at a frequency of 0.1% to 2% in peripheral blood TCR alpha beta+ cells of normal donors. Here we report two unusual cases, found among 100 healthy individuals studied, who showed an abnormally elevated frequency of these T cells, ie, 5% to 10% and 14% to 19%. Southern blot analyses of the TCR alpha beta+CD4–8- clones all showed the identical rearrangement patterns for each individual, demonstrating that these are derivatives of a single T cell. The same rearrangement patterns were also observed for the freshly isolated lymphocytes of TCR alpha beta+CD4-CD8- fraction, which excludes the possible bias in the processes of in vitro cloning. These TCR alpha beta+CD4–8- T cells were found to express other mature T-cell markers such as CD2, CD3, and CD5 antigens, as well as natural killer (NK) cell markers (CD11b, CD16, CD56, and CD57 antigens) for both individuals. Further, although lectin-dependent or redirected antibody- dependent cell-mediated cytotoxicities were observed for both freshly sorted lymphocytes of TCR alpha beta+CD4–8- fraction and in vitro established clones, NK-like activity was not detected.

Download Full-text

The Caspase 8 Inhibitor c-FLIPL Modulates T-Cell Receptor-Induced Proliferation but Not Activation-Induced Cell Death of Lymphocytes

Molecular and Cellular Biology ◽

10.1128/mcb.22.15.5419-5433.2002 ◽

2002 ◽

Vol 22 (15) ◽

pp. 5419-5433 ◽

Cited By ~ 109

Author(s):

Susanne M. A. Lens ◽

Takao Kataoka ◽

Karen A. Fortner ◽

Antoine Tinel ◽

Isabel Ferrero ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

T Cell Receptor ◽

Death Receptor ◽

Cell Receptor ◽

Caspase 8 ◽

Activation Induced Cell Death

ABSTRACT The caspase 8 inhibitor c-FLIPL can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIPL in the T-cell compartment (c-FLIPL Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIPL Tg mice. In contrast, activation-induced cell death of T cells in c-FLIPL Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIPL Tg mice differed from Fas-deficient mice by showing no accumulation of B220+ CD4− CD8− T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIPL Tg mice. Thus, a major role of c-FLIPL in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.

Download Full-text

T Cells Genetically Modified to Be CD19-Specific with Improved Potential for Trafficking and Activation.

Blood ◽

10.1182/blood.v104.11.1748.1748 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1748-1748

Author(s):

Zaid Al-Kadhimi ◽

Lisa Marie Serrano ◽

Simon Olivares ◽

Sergio Gonzalez ◽

Timothy Pfeiffer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cytokine Production ◽

Genetically Modified ◽

Cell Receptor ◽

Effector T Cells ◽

Concentration Gradients ◽

Chimeric Immunoreceptor

Abstract The safety and feasibility of adoptive immunotherapy using ex vivo-expanded differentiated human effector T cells that express tumor-specific chimeric receptors are being evaluated in clinical trials. Typically, these T cells are CCR7neg and bear a T-cell receptor of unknown specificity. To improve the therapeutic potential of genetically engineered T cells in general, and CD19-specific T cells in particular, strategies are needed to improve their ability traffic to sites of residual/macroscopic disease where infused T cells can be specifically activated for proliferation, cytokine secretion, and tumor-lysis. To accomplish these goals we have generated a selection process that uses genetically modified T cells, expressing influenza A matrix protein 1 (MP1) or CMV pp65, to act as antigen presenting cells (T-APC) in order to expand autologous viral-specific T cells in vitro and in vivo. The viral-specific effector T cells can then be genetically modified with a CD19-specific chimeric immunoreceptor (CD19R), which recognizes CD19 on malignant B cells, independent of MHC. By using these viral-specific T cells as a platform for the introduction of CD19R, we now demonstrate that bi-specific T cells express the chemokine receptor CCR7, which is implicated in the trafficking of T cells to lymph nodes. We demonstrate that this chemokine receptor functions to directionally chemotax the genetically modified bi-specific T-cells along concentration gradients of CCL19 or CCL21. We further demonstrate that both the endogenous and introduced chimeric immunoreceptor continue to function in CCR7+ bi-specific T cells. Indeed, the bi-specific T cells are capable of augmented cytokine production and proliferation upon docking with both CD19 and MP1 antigens, compared with these same T cells interacting with either CD19 or MP1 alone. This enhanced activation is an explanation for the enhanced in vivo anti-tumor activity demonstrated by bi-specific T-cells when stimulated with MP1+ T-APC in treating CD19+ lymphoma in NOD/scid mice. An advantage of this methodology is that the CCR7+ bi-specific T cells and T-APC can be genetically modified and expanded in compliance with current good manufacturing practice (cGMP) for 2nd generation Phase I/II clinical trials to test their ability to traffic to sites of lymphoma providing potent regional/local T-cell activation. Legend: (A) CCR7+ viral- and CD19-bi-specific T cells migrate along recombinant CCL19 and CCL21 concentration gradients, whereas CCR7neg CD19-specific T cells do not. (B) Stimulation of both introduced chimeric immunoreceptor and endogenous T-cell receptor on CD19- and MP1- bi-specific T-cells, using artificial APC, results in augmented cytokine production. Figure Figure

Download Full-text

Increased Activation and Oligoclonality of Peripheral CD8+ T Cells in the Chronic Human Helminth Infection Alveolar Echinococcosis

Infection and Immunity ◽

10.1128/iai.70.3.1168-1174.2002 ◽

2002 ◽

Vol 70 (3) ◽

pp. 1168-1174 ◽

Cited By ~ 21

Author(s):

Burkhard J. Manfras ◽

Stefan Reuter ◽

Thomas Wendland ◽

Peter Kern

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Alveolar Echinococcosis ◽

Ex Vivo ◽

Active Role ◽

Cell Receptor ◽

T Cell Subsets ◽

And Function

ABSTRACT Alveolar echinococcosis (AE) in humans is a chronic disease characterized by slowly expanding liver lesions. Cellular immunity restricts the spreading of the extracellular pathogen, but functional contributions of CD4+ and CD8+ T cells are not defined. Here we studied ex vivo the phenotype and function of circulating T-cell subsets in AE patients by means of flow cytometry, T-cell receptor spectratyping, and lymphocyte proliferation. AE patients with parasitic lesions displayed a significant increase of activation of predominantly CD8+ T cells compared to healthy controls and AE patients without lesions. In vitro, proliferative T-cell responses to polyclonal stimulation with recall antigens and Echinococcus multilocularis vesicular fluid antigen were sustained during chronic persisting infection in all AE patients. Only in AE patients with parasitic lesions did T-cell receptor spectratyping reveal increased oligoclonality of CD8+ but not CD4+ T cells, suggesting a persistent antigenic drive for CD8+ T cells with subsequent proliferation of selected clonotypes. Thus, our data provide strong evidence for an active role of CD8+ T cells in AE.

Download Full-text

T Cells Engineered with a Novel Chimeric Receptor Demonstrate Durable In Vivo Efficacy Against Disseminated Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-118569 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 962-962 ◽

Cited By ~ 2

Author(s):

Ksenia Bezverbnaya ◽

Vivian Lau ◽

Craig Aarts ◽

Galina Denisova ◽

Arya Afsahi ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Inhibitory Effect ◽

In Vivo Efficacy ◽

Engineered T Cells

Abstract Despite recent therapeutic developments, multiple myeloma remains an incurable plasma cell malignancy. Poor prognosis for myeloma patients relapsing post-transplant calls for the need for novel treatment options. Immunotherapy with engineered T cells has proven highly efficacious against B-cell cancers, and early-phase clinical trials suggest that multiple myeloma is susceptible to this form of therapy. We designed a new chimeric T cell receptor, T cell antigen coupler (TAC), which relies upon activation through endogenous T cell receptor complex, thus allowing engineered T cells to auto-regulate their activity (Helsen et al, Nat. Comm., 2018). Using published single-chain antibody fragments (scFvs) C11D5.3 and J22.9-xi, we generated B cell maturation antigen (BCMA)-specific TAC receptors for targeting multiple myeloma. Primary human T cells were transduced with lentiviral vectors carrying different BCMA TAC constructs and assessed for in vitro functionality via cytokine production, cytotoxicity, and proliferation assays. In vivo efficacy and T cell tracking were performed in an established orthotopic xenograft mouse model based on a BCMA-positive KMS-11 cell line. C11D5.3 and J22.9-xi TAC T cells demonstrated comparable in vitro performance with both types of cultures efficiently killing BCMA-expressing targets, producing IFN-γ, TNF-α, and IL-2 cytokines, and undergoing multiple rounds of proliferation. In vivo, TAC T cells carrying either scFv were capable of curing mice bearing disseminated myeloma; however, the TAC T cells carrying J22.9-xi scFv were more potent on a per-cell basis (Figure 1A, top panel). Mice in remission 3 months post-treatment with a single dose of 106 TAC-positive T cells showed evidence of sustained anti-tumor protection upon rechallenge with a fresh dose of 106 KMS-11 tumor cells (Figure 1B). Mice treated with low-dose J22.9-xi T cells were more resistant to rechallenge than mice treated with a comparable dose of C11D5.3 TAC T cells. Tracking of the TAC T cells in vivo revealed that the J22.9-xi TAC T cells expanded to a much larger extent than the C11D5.3 TAC T cells (Figure 1A, bottom panel), indicating that there were likely more J22.9-xi TAC T cells present at the time of tumor rechallenge. To understand whether biological aspects of BCMA may influence the proliferative response of the TAC T cells, we explored the influence of APRIL, the soluble ligand for BCMA, on TAC T cell proliferation in vitro. Strikingly, despite comparable proliferation of both TAC T cell populations following stimulation with KMS-11 tumor cells in the absence of APRIL in vitro, the presence of APRIL had a strong inhibitory effect on proliferation of C11D5.3 TAC T cells and only a modest inhibitory effect on J22.9-xi TAC T cells. Our preclinical findings support further development of TAC T cells for the treatment of multiple myeloma and underscore the importance of T cell expansion in determining the therapeutic activity of engineered T cells. This work further reveals a novel observation that the natural ligand of BCMA can impair the therapeutic impact of T cells engineered with chimeric receptors directed against BCMA and provide a basis for advancing BCMA-specific TAC T cells into the clinic. Disclosures Denisova: Triumvira Immunologics: Patents & Royalties. Afsahi:Triumvira Immunologics: Patents & Royalties. Helsen:Triumvira Immunologics: Employment, Patents & Royalties. Bramson:Triumvira Immunologics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Download Full-text

Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection

Nature Communications ◽

10.1038/s41467-020-18256-4 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Ioana Sandu ◽

Dario Cerletti ◽

Manfred Claassen ◽

Annette Oxenius

Keyword(s):

T Cells ◽

T Cell ◽

Cell Function ◽

Viral Infections ◽

Cell Receptor ◽

Target Cells ◽

Tcr Signaling ◽

And Function

Abstract Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.

Download Full-text

The Avidity Spectrum of T Cell Receptor Interactions Accounts for T Cell Anergy in a Double Transgenic Model

Journal of Experimental Medicine ◽

10.1084/jem.189.2.265 ◽

1999 ◽

Vol 189 (2) ◽

pp. 265-278 ◽

Cited By ~ 38

Author(s):

Laila Girgis ◽

Mark M. Davis ◽

Barbara Fazekas de St. Groth

Keyword(s):

T Cells ◽

Cell Division ◽

T Cell ◽

Cytochrome C ◽

T Cell Receptor ◽

Cytokine Production ◽

Cell Receptor ◽

T Cell Anergy ◽

Cell Anergy

The mechanism of self-tolerance in the CD4+ T cell compartment was examined in a double transgenic (Tg) model in which T cell receptor (TCR)-α/β Tg mice with specificity for the COOH-terminal peptide of moth cytochrome c in association with I-Ek were crossed with antigen Tg mice. Partial deletion of cytochrome-reactive T cells in the thymus allowed some self-specific CD4+ T cells to be selected into the peripheral T cell pool. Upon restimulation with peptide in vitro, these cells upregulated interleukin (IL)-2 receptor but showed substantially lower cytokine production and proliferation than cells from TCR Tg controls. Proliferation and cytokine production were restored to control levels by addition of saturating concentrations of IL-2, consistent with the original in vitro definition of T cell anergy. However, the response of double Tg cells to superantigen stimulation in the absence of exogenous IL-2 was indistinguishable from that of TCR Tg controls, indicating that these self-reactive cells were not intrinsically hyporesponsive. Measurement of surface expression of Tg-encoded TCR α and β chains revealed that cells from double Tg mice expressed the same amount of TCR-β as cells from TCR Tg controls, but only 50% of TCR-α, implying expression of more than one α chain. Naive CD4+ T cells expressing both Tg-encoded and endogenous α chains also manifested an anergic phenotype upon primary stimulation with cytochrome c in vitro, suggesting that low avidity for antigen can produce an anergic phenotype in naive cells. The carboxyfluorescein diacetate succinimidyl ester cell division profiles in response to titered peptide ± IL-2 indicated that expression of IL-2 receptor correlated with peptide concentration but not TCR level, whereas IL-2 production was profoundly affected by the twofold decrease in specific TCR expression. Addition of exogenous IL-2 recruited double Tg cells into division, resulting in a pattern of cell division indistinguishable from that of controls. Thus, in this experimental model, cells expressing more than one α chain escaped negative selection to a soluble self-protein in the thymus and had an anergic phenotype indistinguishable from that of low avidity naive cells. The data are consistent with the notion that avidity-mediated selection for self-reactivity in the thymus may lead to the appearance of anergy within the peripheral, self-reactive T cell repertoire, without invoking the induction of hyporesponsiveness to TCR-mediated signals.

Download Full-text

Antigen-dependent Proliferation of CD4+ CD25+ Regulatory T Cells In Vivo

Journal of Experimental Medicine ◽

10.1084/jem.20030315 ◽

2003 ◽

Vol 198 (2) ◽

pp. 249-258 ◽

Cited By ~ 461

Author(s):

Lucy S.K. Walker ◽

Anna Chodos ◽

Mark Eggena ◽

Hans Dooms ◽

Abul K. Abbas

Keyword(s):

T Cells ◽

Population Dynamics ◽

T Cell ◽

Regulatory T Cells ◽

T Cell Receptor ◽

Cell Receptor ◽

Tissue Antigen ◽

Treg Population

The failure of CD25+ regulatory T cells (Tregs) to proliferate after T cell receptor (TCR) stimulation in vitro has lead to their classification as naturally anergic. Here we use Tregs expressing a transgenic TCR to show that despite anergy in vitro, Tregs proliferate in response to immunization in vivo. Tregs also proliferate and accumulate locally in response to transgenically expressed tissue antigen whereas their CD25− counterparts are depleted at such sites. Collectively, these data suggest that the anergic state that characterizes CD25+ Tregs in vitro may not accurately reflect their responsiveness in vivo. These observations support a model in which Treg population dynamics are shaped by the local antigenic environment.

Download Full-text