scholarly journals Mechanisms of Therapeutic Response to Tipifarnib in a Mouse Model of Angioimmunoblastic T-Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
Jose Rodriguez Cortes ◽  
Anisha Cooke ◽  
Aidan Quinn ◽  
Bobby B Shih ◽  
Ioan Filip ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Mouse Model ◽  
Tumor Cells ◽  
Cell Lymphoma ◽  
Cell Populations ◽  
Lymphoma Cells ◽  
Mouse Lymphoma

Angioimmunoblastic T-cell lymphomas (AITLs) are aggressive non-Hodgkin lymphomas arising from malignant transformation of follicular helper T-cells (Tfh) and are associated with limited response to intensified chemotherapy and poor prognosis. The genomic landscape of AITL is characterized by loss of function mutations in epigenetic regulators, including TET2, and the highly prevalent RHOA G17V mutation identified in almost 70% of AITL cases (Palomero et al., 2014). Using a combination of loss of TET2 and expression of RHOA G17V in CD4+ T-cells, our group developed a new mouse model of AITL, which accurately recapitulates the features of the human disease (Cortes et al., 2018). Our model constitutes a relevant platform for studying AITL pathogenesis and developing experimental therapeutic approaches for the treatment of this disease. Tipifarnib is a farnesyltransferase inhibitor that has been linked to reduced cell proliferation and increased apoptosis in a variety of solid tumors and leukemias (Alsina et al., 2004; Kirschbaum et al., 2011). Interestingly, a phase II study of tipifarnib in patients with relapsed/refractory lymphoma indicated that, while tipifarnib had a modest anti-lymphoma activity within the whole cohort, it elicited an overall response rate of 31% within the Peripheral T-cell Lymphoma cases, particularly AITL (Witzig et al, 2011). However, the molecular bases for the therapeutic activity of tipifarnib in AITL are still unknown. To understand the role and the mechanisms of action of tipifarnib in AITL, we have used our TET2-/-RHOA G17V mouse AITL lymphoma model. Using this model, we first identified that treatment in vitro with increasing doses of tipifarnib showed limited effect on the viability of AITL lymphoma cells; however, when mouse lymphoma cells were co-cultured in the presence of splenocytes as supporting cells, tipifarnib induced a strong decrease in cell viability and proliferation, suggesting that tipifarnib might be exerting its therapeutic effects not only on the tumor cells, but also indirectly by regulating the tumor microenvironment. Indeed, tipifarnib treatment in a lymphoma model in vivo led to significantly decreased tumor load and substantial reduction of lymphoma cell infiltration in solid organs, demonstrating a consistent and strong anti-tumor effect of tipifarnib in AITL in vivo. Mechanistic analysis of signaling pathways regulated by tipifarnib indicated that tipifarnib induced downregulation of surface expression of the CXCR4 receptor in tumor cells and increased the circulating levels of its ligand, CXCL12, resulting in an effective increase of the CXCL12/CXCR4 ratio. In addition, our data revealed a significant tipifarnib-induced decrease in the levels of IFN-G, TNF-A, IL-10 and IL-17 cytokines. We also performed gene expression profiling in sorted AITL lymphoma cells and sorted stroma splenocytes from mice that had been treated with tipifarnib or vehicle control. Interestingly, most of the genes specifically affected by tipifarnib were expressed in stromal cells, where we detected an upregulation of Fas-L and, more notably, of multiple genes associated with NK cell function and differentiation (CD226, CD244a, Klre1, Xcl1 and Eomes). CYBERSORT analysis of hematopoietic cell populations indicated that treatment with tipifarnib in our mouse lymphoma model is associated with enrichment in NK signatures and a potential decrease of monocytes and neutrophils within the tumor microenvironment. In summary, we have demonstrated that tipifarnib has a strong anti-lymphoma effect on a mouse model of AITL and that this effect might be mediated by the recruitment and/or activation of different hematopoietic cell populations in the tumor microenvironment that can alter the survival and proliferation of lymphoma cells. Disclosures Palomero: Kura Onclology: Research Funding.

Apoptotic Lymphoma Cells Evoke a Pro-Senescent Stromal Signal That Limits Lymphoma Development.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2392-2392
Author(s):  
Maurice Reimann ◽  
Soyoung Lee ◽  
Christoph Loddenkemper ◽  
Jan Dörr ◽  
Vedrana Tabor ◽  
...  
Keyword(s):  
B Cell ◽  
Tumor Cells ◽  
Transgenic Mouse ◽  
Cellular Senescence ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Anti Cancer ◽  
Mouse Lymphoma

Abstract Abstract 2392 Poster Board II-369 Introduction: Cancer entities frequently exhibiting constitutive Myc expression, such as aggressive B-cell lymphomas, typically display significant amounts of apoptotic cell death. So far, cellular senescence as another cell-autonomous oncogene-inducible safeguard program has been recognized in RAS/BRAF-driven scenarios but not as a bona fide Myc-evoked anti-cancer mechanism. Understanding how oncogenic Myc may provoke not only apoptosis but cellular senescence as a failsafe mechanism to counter tumor development has broad implications for the clinical presentation and therapeutic strategies in frequently Myc-driven lymphoma entities such as Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL). Results: Using the Burkitt's like Eμ-myc transgenic mouse lymphoma model, we show here that cellular senescence serves as another crucial anti-neoplastic barrier during Myc-driven tumorigenesis in addition to apoptosis. Eμ-myc lymphomas harbor a substantial fraction of senescent tumor cells, that stain positive for histone H3 lysine 9 (H3K9)-trimethylation. Lymphomas lacking the H3K9 methyltransferase Suv39h1 display no senescence and develop significantly faster, although apoptosis is not affected by Suv39h1 deficiency. While Myc, unlike other Ras-type oncogenes, shows rather modest pro-senescent activity in vitro, we identified the cytostatic cytokine TGF-β as the main paracrine senescence trigger in vivo. When neutralizing TGF-β action during Myc-driven lymphomagenesis utilizing a secretable TGF-β receptor II ecto-domain, senescence is completely blunted and tumor latency is significantly shortened. We identify macrophages, but not lymphoma cells, as the main source of exogenous TGF-β, that is secreted upon phagocytosis of apoptotic lymphoma cells. Lymphomas harboring a Bcl2-mediated apoptotic block presented with a much lower frequency of both infiltrating macrophages and senescent cells in vivo, suggesting that there is a functional link between cell-autonomous Myc-triggered apoptosis and non-cell-autonomous, macrophage-induced senescence. Both pharmacological suppression of TGF-β production in macrophages via the angiotensin-converting enzyme (ACE) inhibitor lisinopril and depletion of macrophages in Eμ-myc lymphoma-harboring mice by systemic exposure to clodronate resulted in a profound reduction of senescence, thereby underscoring the important role for tumor-infiltrating macrophages in TGF-β-mediated senescence in vivo. We recapitulated components of such a mechanism in human aggressive B-cell lymphomas, a frequently Myc-activated entity where TGF-β1 signaling has previously been identified as a component of the prognostically favourable “stromal-1” signature (Lenz-G et al., NEJM, 2008). A panel of 30 DLBCL samples was sub-divided based on Ki67 immunoreactivity into a very high proliferation (Ki67hi; ≥80% Ki67-positive cells) and a lower proliferation (Ki67lo; <80% Ki67-positive cells) group. Ki67lo samples exhibited a higher frequency of H3K9me3-positive cells, indicative of cellular senescence. Importantly, the Ki67lo group also presented with a higher fraction of apoptotic cells, more lymphoma-infiltrating macrophages, and a stronger reactivity for the TGF-β signaling mediator Smad3-P, thereby representing a subgroup in DLBCL that displays features highly reminiscent of the macrophage-derived mechanism of senescence induction. Conclusions: Our study expands the relevance of oncogene-induced senescence to Myc-driven cancers and demonstrates that different tumor suppressor programs - such as apoptosis and senescence - are enforced in an interdependent fashion between the tumor- and non-malignant stroma cells during lymphomagenesis. Utilizing the Eμ-myc transgenic mouse lymphoma model and furthermore supported by evidence from human aggressive B-cell lymphoma samples, this study establishes a novel network of heterotypic cell-cell interactions within a tumor in which apoptotic tumor cells induce a paracrine response in non-malignant bystander cells that limits lymphomagenesis by cellular senescence. Given the anti-cancer relevance of senescence and the demonstrated inducibility of senescence by a non-DNA damaging cytokine, such as TGF-β, these findings open the exciting perspective to utilize Suv39h1/H3K9me3-mimicking approaches for future cancer therapies. Disclosures: No relevant conflicts of interest to declare.

Targeting DKK1 for the Immunotherapy of B-Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 465-465
Author(s):  
Jianfei Qian ◽  
Sungyoul Hong ◽  
Liang Zhang ◽  
Yuhuan Zheng ◽  
Haiyan Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Specific Ctls

Abstract Abstract 465 Immunotherapy may complement the current treatments for lymphomas. The lack of suitable shared lymphoma-associated antigens limits its applicability. Therefore, identification and utilization of novel and more potent tumor-associated antigens, particularly those shared among patients, are urgently needed to improve the efficacy of immunotherapy in the diseases. Recent studies have shown that Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by myeloma and other tumor cells, and is absent from normal tissues and organs except placenta and prostate. In the present study we demonstrated that DKK1 is also overexpressed in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated HLA-A*0201+ DKK1-specific CTL lines and clones in vitro. These CTLs effectively lysed DKK1+/HLA-A*0201+ lymphoma cell lines Jeko-1 and Granta 519 cells, but not DKK1-/HLA-A*0201+ BJAB, RL and Mino cells nor DKK1+/HLA-A*020- CA46 and Daudi cells. Furthermore, the T-cell clones efficiently killed DKK1+/HLA-A*0201+ primary B-cell lymphoma cells from patients but not lymphoma cells from DKK1–/HLA-A*0201+ patients. HLA-ABC or HLA-A*0201 blocking mAbs significantly inhibited T cell-mediated cytotoxicity against peptide-pulsed T2 cells (P < .01, compared with medium control). No inhibitory effect was observed with mAb against HLA-DR and isotype control IgG. The results indicate that the cytotoxicity was attributed to MHC class I and more specifically, HLA-A*0201-restricted CD8+ CTLs. The CTLs did not kill DKK1–/HLA-A*0201+ DCs, B cells, or PBMCs, These results suggest that the CTLs recognized DKK1 peptides that are naturally processed and presented in the context of HLA-A*0201 molecules on lymphoma cells. To determine the in vivo antitumor activity, NOD-SCID and SCID-hu mice were used for lymphoma cell lines and primary lymphoma cells, respectively. Mice were treated with DKK1-specific CTLs after tumor established in NOD-SCID and SCID-hu mice. Control mice were treated with naïve CD8+ T cells or PBS alone. Tumor burden was measured according to levels of circulating human B2M, and survival rates were determined. Low levels (< 50 ng/ml) of circulating human B2M were detected in group treated DKK1-specific CTLs, while high levels (≥ 150 ng/ml) of circulating human B2M were detected in control mice. In SCID-hu model, X-ray examination showed that established tumors were eradicated in 60% mice treated with DKK1-specific CTLs, while large tumor burdens were found in all control mice. In NOD-SCID model, 40% of mice survived with the treatment of DKK1-specific CTLs. TUNEL assay further confirmed that tumor cells were lysed by DKK1-specific CTLs not naïve CD8+ T cells. These results indicate that DKK1-specific CTLs are able to eradicate established, patient-derived primary B- cell lymphoma in the hosts and adoptive transfer of DKK1-specific CTLs may be used for B-cell lymphoma therapy. Disclosures: No relevant conflicts of interest to declare.

Preclinical analysis of an autologous CD4-targeted chimeric antigen receptor T-cell (CAR-T) immunotherapy for relapsed or refractory peripheral T-cell lymphoma (PTCL) or cutaneous T-cell lymphoma (CTCL).

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14510-e14510
Author(s):  
Eric Zeng ◽  
Tailan Zhan ◽  
Yang Wu ◽  
Meili Chen ◽  
Junzheng Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lymphoma ◽  
Masking Effect ◽  
Car T ◽  
Target Binding ◽  
Anti Tumor Activity

e14510 Background: CD4 is highly and uniformly expressed T-cell lymphomas (TCL) including PTCL and CTCL, suggesting its potential as a surface target for CAR-T therapy. However, there is a significant risk of potential antigen masking by CAR introduced into tumor cells, which thereby leads to escape of tumor cells from recognition by CAR-T cells. For example, Ruella M et al ( Nat Med, 2018) reported that contaminating malignant B cells transduced with anti-CD19 CAR (called CAR-B cells) in the manufacturing process led to the CD19 antigen masking by CAR molecules. Hence, CAR-B tumor cells could not be recognized by CAR-T cells, which resulted in relapse of CAR expressing B lymphoma cells. A similar antigen masking effect might occur for the anti-CD4 CAR-T product. Methods: To identify single-chain variable fragment (scFv) without antigen-masking effect, large panels of fully human monoclonal antibodies were converted to format of CAR modality with the scFv-4-1BB-CD3z structure and introduced into CD4+ and CD8+ T cells. After lentiviral transduction, residual CD4+ T cells were quantified and the CARs with complete elimination of CD4+ T cells were selected for further validation. The selected CAR constructs were then introduced into CD4+ TCL cells HH (CAR-HH cells) to mimic the potential risk of introducing CAR into contaminating malignant T cells in the manufacturing process. Thereafter, CAR-HH cells were subject to in vitro killing assay by LB1901. Last, identified CAR constructs were further tested for in vitro and in vivo anti-tumor efficacy and off-target binding and killing. Results: Introduction of LB1901 CAR into CD4+ and CD8+ T cells led to complete elimination of CD4+ T cells, suggesting no masking effect of CAR on CD4 antigen. Furthermore, the introduction of CAR into CD4+ HH cells did not protect HH cells from being recognized and eliminated by LB1901, further confirming that the CAR modality of LB1901 does not mask CD4 antigen. An in vivo anti-tumor efficacy study showed that LB1901 exhibited dose-dependent anti-tumor activity without significant adverse effect. As low as 0.3 million CAR+ cells completely suppressed tumor growth, suggesting the potent anti-tumor activity by LB1901. Immunohistochemical analysis of normal tissues with LB1901 scFV binder showed no off-target binding. Furthermore, no killing toward CD4- cell lines and primary cells derived from vital organs or antigen-independent cytokine release was observed in vitro. Conclusions: Altogether, the in vitro and in vivo studies showed that LB1901 did not “mask” the CD4 antigen but exhibited potent anti-tumor activity without off-target effects. A phase 1 study of LB1901 CAR-T in patients with relapsed or refractory PTCL or CTCL is ongoing in the US to assess the safety and tolerability of LB1901 CAR-T (NCT04712864).

CMVpp65-Specific T cells generated from naïve T cell populations recognize atypical but not canonical epitopes and may be protective in Vivo

Cytotherapy ◽  
2015 ◽  
Vol 17 (6) ◽  
pp. S9-S10
Author(s):  
Patrick Hanley ◽  
Joseph Melenhorst ◽  
Russell Cruz ◽  
Caridad Martinez ◽  
Helen Heslop ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Populations ◽  
Naïve T Cell ◽  
Naive T Cell

scholarly journals P09.05 Plasma CD27, a surrogate of intratumoral CD27-CD70 interaction, correlates with immunotherapy resistance in renal cancer

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A30.1-A30
Author(s):  
N Benhamouda ◽  
I Sam ◽  
N Epaillard ◽  
A Gey ◽  
A Saldmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Principal Investigator ◽  
Cell Phenotype ◽  
Metastatic Rcc ◽  
Research Grant

BackgroundCD70, a costimulatory molecule on antigen presenting cells, is known to activate CD27-expressing T cells. CD27-CD70 interaction leads to the release of soluble CD27 (sCD27). However, persistent interaction of CD27 and CD70 such as in chronic infection may exhaust the T cell pool and promote apoptosis. Surprisingly, our analysis based on TCGA database show that clear cell renal cell carcinoma (ccRCC) expresses the highest levels of CD70 among all solid tumors. Despite the important clinical efficacy of immunotherapy by anti-PD-1 in RCC patients, the overall response to anti-PD1 remains modest. The relationship between the CD27-CD70 interaction in the RCC and the response to immunotherapy is still unclear.Materials and MethodsTo study the CD27 and CD70 expression in the tumor microenvironment (TME), FFPE tumor tissues from 25 RCC patients were analysed using multiplex in situ immunofluorescence. 10 fresh RCC tumor samples were collected to analyse the phenotype of CD27+ T cells by flow cytometry and 4 samples were proceeded for single-cell RNA-seq analysis. A cohort of metastatic RCC patients (n = 35) treated by anti-PD-1 were enrolled for the measurement of plasma sCD27 by ELISA and the survival analysis is also realized.ResultsIn the TME, we demonstrated that CD27+ T cells interact with CD70-expressing tumor cells. In fresh tumors from RCC patients, CD27+ T cells express higher levels of cleaved caspase 3 (a classical marker of apoptosis) than CD27- T cells. We confirmed the apoptotic signature (BAX, FASLG, BCL2L11, CYCS, FBXO32, LGALS1, PIK3R1, TERF1, TXNIP, CDKN2A) of CD27+ T cells by single-cell RNAseq analysis. CD27+T cells also had a tissue resident memory T cell phenotype with enriched gene expression of ITGAE, PRDM1, RBPJ and ZNF683. Moreover, CD27+T cells display an exhaustion phenotype with the expression of multiple inhibitory receptors gene signature (PDCD1, CTLA4, HAVCR2, LAG3, etc). Besides, intratumoral CD27-CD70 interaction significantly correlates with plasma sCD27 concentration in RCC (p = 0.0017). In metastatic RCC patients treated with anti-PD-1, higher levels of sCD27 predict poor overall survival (p = 0.037), while it did not correlate with inflammatory markers or clinical prognostic criteria.ConclusionsIn conclusion, we demonstrated that sCD27, a surrogate of T cell dysfunction in tumors likely induced by persistent interactions of CD27+T cells and CD70-expressing tumor cells, is a predictive biomarker of resistance to immunotherapy in mRCC. To our knowledge, this is the first report showing that a peripheral blood biomarker may reflect certain aspects of the tumor-host interaction in the tumor microenvironment. Given the frequent expression of CD70 and CD27 in solid tumors, our findings may be further extended to other types of tumors. CD70-CD27 interaction could thus be considered as a mechanism of tumor escape, but also a novel therapeutic target in cancers.Disclosure InformationN. Benhamouda: None. I. Sam: None. N. Epaillard: None. A. Gey: None. A. Saldmann: None. J. Pineau: None. M. Hasan: None. V. Verkarre: None. V. Libri: None. S. Mella: None. C. Granier: None. C. Broudin: None. P. Ravel: None. B. Jabla: None. N. Chaput: None. L. Albiges: None. Y. Vano: None. O. Adotevi: None. S. Oudard: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; SIRIC CARPEM, FONCER. E. Tartour: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Fondation ARC, INCA PLBio, Labex Immuno-Oncology, SIRIC CARPEM, FONCER, IDEX université de Paris, Inserm Transfert.

scholarly journals 121 ICAM-1-specific affinity tuned CAR T cells expressing SSTR2 for real-time imaging

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A130-A130
Author(s):  
Jingmei Hsu ◽  
Eric von Hofe ◽  
Michael Hsu ◽  
Koen Van Besien ◽  
Thomas Fahey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Phase I ◽  
Tumor Cells ◽  
Somatostatin Receptor ◽  
Laboratory Animals ◽  
Therapeutic Window ◽  
Car T Cells ◽  
Car T

BackgroundThe use of CAR T cells for solid tumors has a number of challenges, such as lack of tumor-specific targets, CAR T cell exhaustion, and the immunosuppressive tumor microenvironment. To address these challenges, AffyImmune has developed technologies to affinity tune and track CAR T cells in patients. The targeting moiety is affinity tuned to preferentially bind to tumor cells overexpressing the target while leaving normal cells with low basal levels untouched, thereby increasing the therapeutic window and allowing for more physiological T cell killing. The CAR T cells are designed to express SSTR2 (somatostatin receptor 2), which allows for the tracking of CAR T cells in vivo via PET/CT scan using FDA-approved DOTATATE.MethodsAIC100 was generated by affinity tuning the I-domain of LFA-1, the physiological ligand to ICAM-1. Various mutants with 106-fold difference in affinity were evaluated for affinity. This allowed structure activity relationships to be conducted using CAR T cells expressing the various affinity mutants against targets with varying antigen densities. The variant with micromolar affinity was clearly the most effective in non-clinical animal models. AIC100 is currently being evaluated to assess safety, CAR T expansion, tumor localization, and preliminary activity in patients with advanced thyroid cancer in a phase I study (NCT04420754). Our study uses a modified toxicity probability interval design with three dosage groups of 10 x 106, 100 x 106, and 500 x 106 cells.ResultsPreclinical studies demonstrated greater in vivo anti-tumor activity and safety with lower affinity CAR T cells. A single dose of AIC100 resulted in tumor elimination and significantly improved survival of animals. AIC100 activity was confirmed in other high ICAM-1 tumor models including breast, gastric, and multiple myeloma. In a Phase I patient given 10-million CAR T cells, near synchronous imaging of FDG and DOTATATE revealed preliminary evidence of transient CAR T expansion and tumor reduction at multiple tumor lesions, with the peak of CAR T density coinciding with the spike in CAR T numbers in blood.ConclusionsWe have developed affinity tuned CAR T cells designed to selectively target ICAM-1 overexpressing tumor cells and to spatiotemporally image CAR T cells. Near-synchronous FDG and DOTATATE scans will enhance patient safety by early detection of off-tumor CAR T activity and validation of tumor response. We anticipate that our ‘tune and track’ technology will be widely applicable to developing potent yet safe CAR T cells against hard-to-treat solid cancers.Trial RegistrationNCT04420754Ethics ApprovalIRB number19-12021154IACUC (animal welfare): All animal experiments were performed in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. Animal handling protocols were approved by the Institutional Laboratory Animal Use and Care Committee of Weill Cornell Medicine (Permit Number: 2012–0063).

scholarly journals Immune cell topography predicts response to PD-1 blockade in cutaneous T cell lymphoma

2020 ◽  
Author(s):  
Darci Phillips ◽  
Magdalena Matusiak ◽  
Belén Rivero Gutierrez ◽  
Salil S. Bhate ◽  
Graham L. Barlow ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clinical Response ◽  
Tumor Cells ◽  
Spatial Organization ◽  
Cell Lymphoma ◽  
Post Treatment ◽  
T Cell Lymphoma ◽  
T Cell Subsets ◽  
Cutaneous T Cell Lymphoma

Anti-PD-1 immunotherapies have transformed cancer treatment, yet the determinants of clinical response are largely unknown. We performed CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced cutaneous T cell lymphoma (CTCL) patients enrolled in a clinical trial of pembrolizumab therapy. Clinical response was not associated with the frequency of tumor-infiltrating T cell subsets, but rather with striking differences in the spatial organization and functional immune state of the tumor microenvironment (TME). After treatment, pembrolizumab responders had a localized enrichment of tumor and CD4+ T cells, which coincided with immune activation and cytotoxic PD-1+ CD4+ T cells. In contrast, non-responders had a localized enrichment of Tregs pre- and post-treatment, consistent with a persistently immunosuppressed TME and exhausted PD-1+ CD4+ T cells. Integrating these findings by computing the physical distances between PD-1+ CD4+ T cells, tumor cells, and Tregs revealed a spatial biomarker predictive of pembrolizumab response. Finally, the chemokine CXCL13 was upregulated in tumor cells in responders post-treatment, suggesting that chemoattraction of PD-1+ CD4+ T cells towards tumor cells facilitates a positive outcome. Together, these data show that T cell topography reflects the balance of effector and suppressive activity within the TME and predicts clinical response to PD-1 blockade in CTCL.

scholarly journals A Long-Acting Pharmacological Grade Interleukin-7 Molecule Logarithmically Accelerates Ucart Proliferation, Differentiation, and Tumor Killing

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2199-2199
Author(s):  
Matt L Cooper ◽  
Karl W. Staser ◽  
Julie Ritchey ◽  
Jessica Niswonger ◽  
Byung Ha Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Effector Memory ◽  
Photon Flux ◽  
Interleukin 7 ◽  
Tumor Killing ◽  
Long Acting

Abstract Background: Chimeric antigen receptor T cell (CART) therapy is revolutionizing modern cancer therapy, with two anti-CD19 CARTs FDA-approved for relapsed/refractory B cell lymphoma/leukemia and many other CARTs for solid and liquid tumors currently undergoing clinical trials. Our group recently demonstrated multiplexed CRISPR/Cas9 gene-editing of anti-CD7 CARTs to produce CD7 and T cell receptor alpha constant (TRAC)-deleted "off-the-shelf" universal (U)CART7s that effectively kill CD7+ T cell lymphoma in vivo without causing GVHD or fratricide (Cooper et al, Leukemia, 2018). However, in current clinical practice, suboptimal CART persistence and tumor killing permit tumor cell escape and, ultimately, disease relapse. Reasoning that a pro-lymphoid growth factor could promote CART efficacy, we supplemented UCART infusion with subcutaneous injections of the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7) in vivo using a CD19+ lymphoma xenograft model. Methods: To create anti-CD19 universal CARTs (UCART19), we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9 mRNA and a TRAC-targeted gRNA, and virally transduced an anti-CD19 scFv 3rd generation CAR containing a peptidase 2A-cleaved human CD34 construct for both purification and tracking in vivo. Residual TRAC+ cells were depleted using magnetic selection. For xenograft tumor modeling in vivo, we injected NOD-scid IL2Rgammanull (NSG) mice with 5x105 RamosCBR-GFP cells four days prior to UCART19 (2x106 cells). Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART19 infusion. Results:RamosCBR-GFP mice receiving NT-I7 without UCART19 (NT-I7 only group) survived marginally longer (24 day med survival) than mice receiving RamosCBR-GFP cells alone (No tx group) (21 day medium survival, p=0.018, NT-I7 only vs. No Tx). While RamosCBR-GFP mice treated with UCART19 alone (UCART19 group) survived 33 days, 100% of RamosCBR-GFP mice treated with UCART19 and NT-I7 (UCART19+NT-I7 group) were alive at 80 days (Fig 1a), with no mouse showing signs of xenogeneic GVHD (p<0.0001, UCART19+NT-I7 vs. UCART19). At three weeks post UCART19 infusion, bioluminescent imaging (BLI) revealed minimal tumor signal in UCART19+NT-I7 treated mice (108 vs. 1010 photon flux/s, p<0.05, UCART19+NT-I7 vs. UCART19) and near-undetectable photon flux/s at four weeks (107 vs 1011 photon flux/s, p<0.0001, UCART19+NT-I7 vs. UCART19). Quantitative 17-parameter flow cytometric analyses of the blood, bone marrow, and spleens revealed an up to ~8000-fold increase in UCART19 cells in NT-I7-treated mice four weeks post UCART19 infusion (Fig 1a). These UCART19 cells demonstrated a predominantly effector and effector memory phenotype. Discussion: CARTs engineered to express interleukin-7 and CCL19 showed increased migration to and killing of solid tumors (Adachi et al, Nature Biotechnology, 2018). However, genetically engineered potentiation strategies lack "off-switches" and may preclude additional genetic enhancements required for universal "off-the-shelf" CART development. Here, we demonstrate that a pharmacological grade long-acting interleukin-7 agonist can potentiate adoptive cellular therapies. Specifically, NT-I7 can dramatically enhance gene modified T cell proliferation, persistence and tumor killing in vivo, resulting in enhanced survival, providing a tunable clinic-ready adjuvant for reversing suboptimal CART activity in vivo. Disclosures Cooper: WUGEN: Consultancy, Equity Ownership. Lee:NeoImmuneTech: Employment. Park:NeoImmuneTech: Employment.

Development and characterization of a HCMV multiantigen therapeutic vaccine for GBM using the UNITE platform.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14565-e14565
Author(s):  
Amit Adhikari ◽  
Juliete Macauley ◽  
Yoshimi Johnson ◽  
Mike Connolly ◽  
Tim Coleman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Mouse Model ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
T Cell Response

e14565 Background: Glioblastoma (GBM) is an aggressive form of brain cancer with a median survival of 15 months which has remained unchanged despite technological advances in the standard of care. GBM cells specifically express human cytomegalovirus (HCMV) proteins providing a unique opportunity for targeted therapy. Methods: We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins- pp65, gB and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated protein 1 (LAMP1) with target antigens resulting in increased antigen presentation by MHC-I and II. ELISpot, flow cytometry and ELISA techniques were used to evaluate the vaccine immunogenicity and a syngeneic, orthotopic GBM mouse model that expresses HCMV proteins was used for efficacy studies. The tumor microenvironment studies were done using flow cytometry and MSD assay. Results: ITI-1001 vaccination showed a robust antigen-specific CD4 and CD8 T cell response in addition to a strong humoral response. Using GBM mouse model, therapeutic treatment of ITI-1001 vaccine resulted in ̃56% survival with subsequent long-term immunity. Investigating the tumor microenvironment showed significant CD4 T cell infiltration as well as enhanced Th1 and CD8 T cell activation. Regulatory T cells were also upregulated upon ITI-1001 vaccination and would be an attractive target to further improve this therapy. In addition, tumor burden negatively correlated with number of activated CD4 T cells (CD4 IFNγ+) reiterating the importance of CD4 activation in ITI-1001 efficacy and potentially identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+ and CD8+ T cells in responders compared with non- responders along with higher CD8 T cell activation. Conclusions: Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant anti-tumor activity that leads to enhanced survival in mice with GBM.

scholarly journals Involvement of leukocyte function-associated antigen-1 (LFA-1) in the invasion of hepatocyte cultures by lymphoma and T-cell hybridoma cells.

1987 ◽  
Vol 105 (1) ◽  
pp. 553-559 ◽  
Author(s):  
E Roos ◽  
F F Roossien
Keyword(s):  
T Cell ◽  
Tumor Cells ◽  
Cell Types ◽  
Hybridoma Cells ◽  
Lymphoma Cells ◽  
Hepatocyte Cultures ◽  
Leukocyte Function ◽  
Vitro Model

We studied the interaction of MB6A lymphoma and TAM2D2 T cell hybridoma cells with hepatocyte cultures as an in vitro model for in vivo liver invasion by these tumor cells. A monoclonal antibody against leukocyte function-associated antigen-1 (LFA-1) inhibited adhesion of the tumor cells to the surface of hepatocytes and consequently strongly reduced invasion. This effect was specific since control antibodies, directed against Thy.1 and against T200, of the same isotype, similar affinity, and comparable binding to these cells, did not inhibit adhesion. This suggests that LFA-1 is involved in the formation of liver metastases by lymphoma cells. TAM2D2 T cell hybridoma cells were agglutinated by anti-LFA-1, but not by control antibodies. Reduction of adhesion was not due to this agglutination since monovalent Fab fragments inhibited adhesion as well, inhibition was also seen under conditions where agglutination was minimal, and anti-LFA-1 similarly affected adhesion of MB6A lymphoma cells that were not agglutinated. The two cell types differed in LFA-1 surface density. TAM2D2 cells exhibited 400,000 surface LFA-1 molecules, 10 times more than MB6A cells. Nevertheless, the level of adhesion and the extent of inhibition by the anti-LFA-1 antibody were only slightly larger for the TAM2D2 cells.

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