Enhanced Antitumor Efficacy of MUC-16 Targeted T Cells Further Modified to Constitutively Express the IL-12 Cytokine in a Syngeneic Model of Ovarian Cancer,

Abstract Abstract 4176 T cells may be genetically modified to recognize tumor associated antigens (TAAs) through the introduction of genes encoding artificial T cell receptors termed chimeric antigen receptors (CARs). We have constructed SFG retroviral vectors encoding first (4H11mz) and second (4H11m28mz) generation CARs as well as IL-12 modified CAR (4H11m28mzIRESmIL12) targeted to the retained extra-cellular domain of MUC16, termed MUC-CD. This antigen is over-expressed on most ovarian carcinoma tumor cells. IL-12 is a potent inducer of a Th1 CD4+ T cell response and serves as a “signal 3” in concert with TCR activation (signal 1) and CD28 co-stimulation (signal 2) to CD8+ T cells, resulting in optimized clonal expansion and effector function. In order to mimic the clinical setting, we generated a syngeneic tumor model using the C57BL6 (B6) mice intraperitoneally (i.p.) injected with ID8(MUC-CD) cells. In our studies treatment of mice bearing established ID8(MUC-CD) ovarian tumor with MUC-CD specific T cells expressing IL-12 gene, in contrast to T cells targeted to MUC-CD alone, fully eradicate advanced intraperitoneal ovarian tumors. The mechanism of IL-12 expressing MUC-CD targeted T cell antitumor efficacy was mediated through enhanced persistence and engraftment of modified T cells, as well as the ability of IL-12 secreting T cells to recruit endogenous T cells to the tumor site. Furthermore, we observed elevated secretion of the pro-inflammatory cytokines (IFN-g and TNF-a) in the serum of IL-12 treated mice, compared to 4H11m28mz and control CD19 targeted T cell treated groups. Treatment of B6 mice with MUC-CD targeted T cells expressing IL-12 gene was dependent upon recruitment of the NK and NKT cells to the tumor site. Finally, we demonstrated the ability of IL-12 secreting T cells to overcome the immunosuppressive tumor microenvironment by switching the phenotype of the tumor-associated macrophages (TAMs) from a predominately immunosuppressive M2 to an immunostimulatory M1 phenotype. These data, obtained in the context of a clinically relevant syngeneic tumor model supports the application of this approach in the treatment of the patients with relapsed ovarian carcinomas. Disclosures: No relevant conflicts of interest to declare.

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Effect of modulation of the hostile tumor microenvironment through adoptive transfer of IL-12 expressing MUC-16 targeted T cells on ovarian tumors in vivo.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.2586 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 2586-2586

Author(s):

Alena A. Chekmasova ◽

Samith Sandadi ◽

David R. Spriggs ◽

Renier J. Brentjens

Keyword(s):

Ovarian Cancer ◽

T Cells ◽

T Cell ◽

Tumor Model ◽

Retroviral Vectors ◽

Ovarian Tumors ◽

Adoptive T Cell Therapy ◽

Antitumor Efficacy ◽

Ovarian Carcinomas ◽

T Cell Therapy

2586 Background: T cells may be genetically modified to recognize tumor associated antigens (TAAs) through the introduction of genes encoding artificial T cell receptors termed chimeric antigen receptors (CARs). MUC16 (CA125) is an antigen over-expressed on ovarian carcinomas and a serum marker for the diagnosis of ovarian cancer. We have previously demonstrated enhanced antitumor efficacy of CAR+ T cells further modified to secrete IL-12. We therefore tested whether MUC-16 targeted T cells further modified to express IL-12 would exhibit an enhanced antitumor efficacy in a syngeneic immunocompetent tumor model of ovarian cancer. Methods: We have constructed SFG retroviral vectors encoding the second (4H11m28mz) generation CARs as well as IL-12 modified CAR (4H11m28mz/mIL12) targeted to the retained extra-cellular domain of MUC16, termed MUC-CD. We demonstrated an antitumor efficacy of these T cells in a syngeneic tumor model using the C57BL6 (B6) mice intraperitoneally (i.p.) injected with ID8(MUC-CD) tumor cells. Results: In our studies treatment of mice bearing established ID8(MUC-CD) ovarian tumor with MUC-CD specific T cells expressing IL-12 gene, in contrast to T cells targeted to MUC-CD alone, fully eradicate highly advanced intraperitoneal ovarian tumors. Significantly, we found that mice treated with 4H11m28mz/mIL12 T cells had increased number of modified T cells in the peritoneum at day 4 and 7 with increased recruitment of endogenous T cells to the site of the tumor when compared to controls and mice treated with 4H11m28mz T cells. The observed antitumor effect did not required prior lymphodepletion and was well tolerated in treated mice. Conclusions: CAR modified T cells targeted to the MUC-16 antigen efficiently eradicate orthotopic ovarian cancer in syngeneic immunocompetent mice with markedly enhanced antitumor efficacy seen in those mice treated with CAR+ T cells further modified to secrete IL-12. These data support future clinical trials utilizing adoptive T cell therapy in patients with relapsed ovarian cancer.

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Enhancing Cytotoxicity of Autologous T Cells in AML By Blockade of CTLA-4 and PD-1

Blood ◽

10.1182/blood.v128.22.4057.4057 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4057-4057 ◽

Cited By ~ 1

Author(s):

Kirsten Marie Boughan ◽

Xiaohua Chen ◽

Paul Szabolcs

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Response ◽

Conflicts Of Interest ◽

Expression Profiles ◽

T Cell Response ◽

Inhibitory Receptors

Abstract Background: AML remains a disease diagnosed in the aging population with chemotherapy followed by bone marrow transplant in some cases being the standard of care. Although response rates remain around 50-60%, treatment related mortality and disease relapse remain high. Adoptive immunotherapy, especially those targeting T cell co-inhibitory receptors, has proven successful in solid malignancies however, AML remains less explored. Our laboratory has previously demonstrated the feasibility to generate autologous AML reactive T cells in vitro (Mehta/Szabolcs; Immunotherapy 2016). It was noted that "resistant" AML blasts over expressed a number of genes associated with immunosuppressive characteristics. Over expression of these genes may induce T cell functional exhaustion. Therefore, we hypothesized that blocking PD-1 and/or CTLA-4 during co-culture with IFNg activated AML blasts, may enhance T cell activation and cytotoxicity. To test this hypothesis, we tested CTL responses against AML blasts and IFNg ELISpot formation after blocking with PD-1, CTLA-4 or both receptors, and compared the response in untreated T cells. Gene expression profiles of co-stimulatory/co-inhibitory receptors were also monitored to test for correlation. Methods: We evaluated 12 patients with newly diagnosed AML under an IRB approved protocol with written informed consent of patients. Mononuclear cell preparation was generated from fresh marrow samples or drawn from a biorepository of previously cryopreserved leukophereses. T cells were then purified using immunomagnetic CD3/CD28 beads (Life technologies) and cultivated in media with IL-2 and IL-7 for 2 weeks. AML blasts were cultured over a supporting layer of mesenchymal stromal cells (MSCs) derived from healthy BM donors for 1 week and then cryopreserved. T cells were then co-cultured with restored and irradiated autologous AML cells at an effector: target (E: T) ratio of 5:1 to 40:1. AML and T cells were co-cultured in the presence of Ipilimumab (anti-CTLA-4), or Nivolumab (anti-PD-1), or a combination of both drugs. T cells and AML were re stimulated in X-vivo 15 with IL-12, IL-15 and IL-2 weekly x 3weeks. T cell response to AML was quantitated by IFNg ELISpot assay and Europium TDA (EuTDA) CTL assays independently. Co-stimulatory/co-inhibitory expression on T cells was examined with RT-q PCR assay. Paired-sample student t test was used for statistical analysis with p<0.05. Results and Discussion: Out of 12 samples, 10 (83%) yielded viable AML cells available for cytotoxicity assay. One third (33%) of co-cultures exhibited a positive T cell response in CTL assays ("killers"). There was no difference in CTL activity by blockade of either PD-1 or CTL-4 (Fig 1). IFN-ɣ spot formation in ELISpot was observed in 4/10 samples (40%) with statistical significance noted in cells blocked with PD-1 as compared to all other blockade types (Fig 2). The results indicated that in vitro priming with autologous AML blasts or together with blocking PD-1 can enhance T cell response in 33-40%. By gene expression analysis, the ratio of co-stimulatory to co-inhibitory genes was calculated. In PD-1 blocked cells, the ratio of activation/inhibition was not impacted in T cells from "killers" (0.9; p=0.1), however, T cells from "non-killer cells" had a diminished ratio due to higher expression of co-inhibitory molecules (0.4; p=0.04) (Fig 3). This trend was also present in CTLA-4 blocked cells (0.85; p=0.4 in killers vs 0.54; p=0.03 in non-killers) (data not shown). Interestingly, dual blockage failed to influence gene expression ratio, data not shown. Conclusion: The above studies demonstrate that cytotoxicity can be achieved in T cells when primed against autologous AML. PD-1 blockade can enhance IFNg production and cytotoxic responses, but CTLA-4 and dual blockade failed to enhance T cell function. The upregulation of an inhibitory pattern of genes in T cells that did not express cytotoxicity (non-killers) could allude to an "inhibitory phenotype" that may be resistant to immunotherapy drug blockade and requires further study. Disclosures No relevant conflicts of interest to declare.

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Targeting FVIII-Specific B Cells Using BAR-Transduced Regulatory T Cells

Blood ◽

10.1182/blood.v128.22.329.329 ◽

2016 ◽

Vol 128 (22) ◽

pp. 329-329 ◽

Cited By ~ 7

Author(s):

Ai-Hong Zhang ◽

Jeong Heon Yoon ◽

Yong Chan Kim ◽

David W. Scott

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Neutralizing Antibodies ◽

Conflicts Of Interest ◽

Rational Solution ◽

T Cell Response ◽

Treg Suppression ◽

Risk Patients

Abstract Up to a third of hemophilia A (HA) patients receiving therapeutic FVIII develop neutralizing antibodies termed "inhibitors". Once inhibitors develop, clinical management of HA patients becomes extremely difficult. Thus, a rational solution would be to establish FVIII-specific immune tolerance to FVIII in high risk patients. To address this issue in a mouse model of human HA, we elected to use an antigen-specific regulatory T cell (Treg) approach. Analogous to the chimeric antigen receptor (CAR) strategy successfully used in cancer immunotherapy, we have created a chimeric receptor comprising a protein antigen or its domain, linked with the transmembrane and signal transduction domains, CD28-CD3ζ. We termed this receptor "BAR" for B-cell-targeting antibody receptor. Human Tregs (CD4+CD25hiCD127low) were retrovirally transduced with a BAR containing FVIII C2 domain (C2-BAR) or FVIII A2 domain (A2-BAR) and expanded successfully in vitro. These cells stained positively with anti-C2 and anti-A2 monoclonal antibodies, respectively, and maintained Treg phenotypic markers in terms of co-expression of Foxp3 and Helios. Control human Tregs were transduced with a BAR containing chicken ovalbumin (OVA-BAR). To test the hypothesis that BAR-transduced Tregs could directly and effectively suppress the activity of specific B cells, a xenogeneic model was employed. On day 0, FVIII-/- HA mice were injected intravenously with 106 transduced human Tregs. The mice were then immunized subcutaneously on day 1 with FVIII in incomplete Freund's adjuvant, and anti-FVIII antibody development was followed. By two weeks after immunization, anti-FVIII antibodies could be detected in the control mice (n = 4). However, in the experimental group (n = 5) that received a mixture of equal number of C2-and A2-BAR Tregs, anti-FVIII antibody development was reproducibly completely blocked for at least 8 weeks. To examine the possible mechanism of BAR Treg suppression, purified B cells and T cells from "tolerized" (A2+C2-BAR) or "control" (OVA-BAR) recipients were mixed and tested for recall responses to FVIII in vitro. The results suggested that the FVIII-specific B cells were directly tolerized while the T-cell response remained intact. Taken together, we report here a successful approach utilizing FVIII-specific BAR-Tregs to directly target FVIII-specific B cells, an approach which could be adapted to address other adverse immune response as well. (Supported in part by a NIH grant HL127495) Disclosures No relevant conflicts of interest to declare.

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Immunisation against Human c-MYC Elicits a T-Cell Response and Influences Growth of High Grade B-Cell Lymphoma in Mice.

Blood ◽

10.1182/blood.v114.22.5120.5120 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5120-5120

Author(s):

Florian Helm ◽

Andrea Wilke ◽

Thomas Kammertoens ◽

Christian Friese ◽

Josef Mautner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Response ◽

Conflicts Of Interest ◽

Disease Free Survival ◽

B Cell Lymphoma ◽

T Cell Response ◽

Transfer Model ◽

High Grade ◽

Myc Gene

Abstract Abstract 5120 Overexpression of the proto-oncogene c-myc due to chromosomal translocation is the hallmark of Burkitt-lymphoma. The evolving high grade lymphoma is dependent on the overexpression of c-myc, which provides the necessary signal to drive uncontrolled proliferation. Therefore loss of function or recognition of c-myc overexpressing cells by c-MYC specific T-cells should result in killing of the target and a halt to lymphoma progression. C-myc is also expressed in a variety of other human malignancies. Peptide prediction reveals several potential foreign epitopes in the context of murine H2b due to 87% homology between human and mouse c-MYC. In this study we explored whether the human c-myc gene product can be a target for T-cell therapy. Wildtype C57BL/6 mice were immunized with recombinant human c-MYC protein in combination with incomplete Freund′s adjuvans and CpG, and were boosted at various time points thereafter using either c-MYC protein or 40mer peptides encompassing the non homologous regions. Control animals were vaccinated with recombinant GFP or OVA protein. C-MYC vaccinated animals displayed a higher IFNg release upon re-stimulation with c-MYC pulsed dendritic cells compared to control vaccinated animals. In ELISPOT assays we observed a higher number of IFNg positive cells (299±17 vs. 122±8.5 (GFP vaccinated) vs. 66±8.5 (OVA vaccinated)). Vaccination using single peptides revealed that peptides spanning the region from amino acid 87-123, 216-255 and 334-376 produced similar results. In addition, using a human c-MYC specific ELISA we were able to detect c-MYC specific antibodies in serum from immunized mice in a concentration up to 40mg/l. Using established cell lines from l-hu-c-myc transgenic mice, where the human c-myc gene is overexpressed due to the juxtaposition of elements of the immunoglobuline lambda locus as found in t(8;22) of Burkitt's lymphoma, we investigated whether vaccination with human c-MYC protein would influence lymphoma growth in a lymphoma transfer model. Animals were s.c. challenged with 0.1 Mio 291cells overexpressing human c-MYC and were monitored for lymphoma growth. C-MYC vaccinated animals (n=15) displayed a delay in tumor onset and a significantly better disease free survival (28 vs. 22 days, p=0.012) compared to control (OVA) vaccinated animals (n=10). This delayed growth was associated with an increased number of infiltrating CD3+/Perforin+ cells. However, all mice eventually succumbed to lymphoma growth, indicating that the T-cell response was not sufficient to control lymphoma growth in the long term. From these data we conclude that the human c-MYC is a possible target antigen for T-cells, but responses are weak and presumably low in frequency. Disclosures No relevant conflicts of interest to declare.

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IL7-IL12 Engineered Mesenchymal Stem Cells (MSCs) Improve A CAR T Cell Attack Against Colorectal Cancer Cells

Cells ◽

10.3390/cells9040873 ◽

2020 ◽

Vol 9 (4) ◽

pp. 873 ◽

Cited By ~ 3

Author(s):

Andreas A. Hombach ◽

Ulf Geumann ◽

Christine Günther ◽

Felix G. Hermann ◽

Hinrich Abken

Keyword(s):

Stem Cells ◽

T Cells ◽

Mesenchymal Stem Cells ◽

T Cell ◽

Tumor Model ◽

T Cell Response ◽

Car T Cells ◽

Car T Cell ◽

Activation Induced Cell Death ◽

Car T

Chimeric antigen receptor (CAR) redirected T cells are efficacious in the treatment of leukemia/lymphoma, however, showed less capacities in eliminating solid tumors which is thought to be partly due to the lack of cytokine support in the tumor lesion. In order to deliver supportive cytokines, we took advantage of the inherent ability of mesenchymal stem cells (MSCs) to actively migrate to tumor sites and engineered MSCs to release both IL7 and IL12 to promote homeostatic expansion and Th1 polarization. There is a mutual interaction between engineered MSCs and CAR T cells; in presence of CAR T cell released IFN-γ and TNF-α, chronic inflammatory Th2 MSCs shifted towards a Th17/Th1 pattern with IL2 and IL15 release that mutually activated CAR T cells with extended persistence, amplification, killing and protection from activation induced cell death. MSCs releasing IL7 and IL12 were superior over non-modified MSCs in supporting the CAR T cell response and improved the anti-tumor attack in a transplant tumor model. Data demonstrate the first use of genetically modified MSCs as vehicles to deliver immuno-modulatory proteins to the tumor tissue in order to improve the efficacy of CAR T cells in the treatment of solid malignancies.

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T Cells From CLL Patients Recognize Spontaneously Peptides Derived of the Receptor Tyrosine Kinase Ror1

Blood ◽

10.1182/blood.v116.21.3603.3603 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3603-3603

Author(s):

Mohammad Hojjat -Farsangi ◽

Amir Hossein Daneshmanesh ◽

Mahmood Jeddi-Tehrani ◽

Anders Osterborg ◽

Fazel Shokri ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Cell Response ◽

Progressive Disease ◽

Conflicts Of Interest ◽

T Cell Response ◽

Potential Implication ◽

Ifn Γ

Abstract Abstract 3603 Background: The receptor tyrosine kinase Ror1 is a tumor-associated molecule over-expressed in chronic lymphocytic leukemia (CLL) and a variety of other malignancies with potential implication for targeted immunotherapy. Little is known about the functional role of Ror1 in the leucomogenesis of CLL. Aims: To assess spontaneous T cell response against Ror1 in CLL patients. Methods: Autologous T cell response against Ror1 was studied in 9 CLL patients and 6 healthy subjects expressing the HLA-A2 allele. Four synthetic 9-mer HLA-A2 restricted peptides selected from different parts of the Ror1 molecule were loaded on dendritic cells and co-cultured with enriched autologous T cells. T cell response was determined by enumeration of the frequency of IFN-γ, IL-5 and IL-17A secreting T cells by ELISPOT. Cell proliferation was determined by 3H-thymidine incorporation. Results: Our results demonstrated a significantly higher frequencies of IFN-γ producing T cells (p<0.05-0.0001) and to a lesser extent IL-17A secreting autologous T cells (p<0.05) in response to Ror1 peptides in CLL patients compared to healthy controls. No IL-5 secreting T cells were noted. The frequency of IFN-γ secreting T cells was significantly higher in non-progressive as compared to progressive CLL patients (P<0.05). No differences were observed between patients with mutated and unmutated immunoglobulin heavy chain variable region (IGHV) genes. Conclusion: Ror1 may spontaneously induce mainly a type 1 T cell response in CLL patients, particularly in those with non-progressive disease as compared to progressive disease. Ror1 may be an immunodominant antigen in CLL which, has also been suggested by Fakuda et al. PNAS, 105, 3047, 2008. Active immunotherapy using Ror1 as a target might be an interesting approach to test in the clinic. Disclosures: No relevant conflicts of interest to declare.

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Caveolin-1 Is Required For T Cell-Mediated Acute GvHD

Blood ◽

10.1182/blood.v122.21.4461.4461 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4461-4461

Author(s):

Sebastian Wohlfeil ◽

Hechinger Anne-Kathrin ◽

Franziska Leonhardt ◽

Gabriele Prinz ◽

Annette Schmitt-Gräff ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Intracellular Signaling ◽

Acute Gvhd ◽

Conflicts Of Interest ◽

Serum Levels ◽

Membrane Dynamics ◽

T Cell Response ◽

Gamma Chain ◽

Caveolin 1

Caveolin-1 (Cav-1) is a key organizer of membrane specializations that coordinate membrane and protein traffic in different cells including T cells. Cav-1 promotes the formation and stability of plasma membrane raft microdomains that serve as platforms for signal transduction and binds a wide array of signal transducers through interactions with its phosphorylated tyrosine 14 or the so-called scaffolding domain. Several of the proteins identified as Cav-1 binding partners have been suggested to play a role in TCR-regulated membrane dynamics and intracellular signaling. Therefore, we aimed to determine the requirement of Cav-1 for T cells during acute graft-versus-host disease (GvHD). We observed upregulation of Cav-1 in the T cells of mice developing acute GvHD which was dependent on both tissue damage and homeostatic signals as investigated in conditioned WT recipients or RAG-/-C gamma chain-/- non-conditioned mice. By using gene targeted mice we found that Cav-1 deficiency of the donor led to improved survival (p=0.02), lower GvHD histopathology scores (p=0.004) and reduced serum levels of IL-6 (p=0.02) while Treg numbers increased when Cav-1 deficient donors were used. Comparative microarray analysis of WT compared to Cav-1 deficient T cells indicated a reduced IL-17A production in Cav-1 T cells exposed to allogeneic dendritic cells. These observations were confirmed on the protein level. In summary we show a role for Cav-1 in T cells during GvHD as an alloantigen driven T cell response. Regarding the mechanism we describe the relevance of Cav-1 for Th-17 lineage commitment of alloreactive T cells during GvHD. Disclosures: No relevant conflicts of interest to declare.

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MLL Histone Methyltransferase Restrains Effector Differentiation and PD-1 Expression in Human CD8+ T Cells While Promoting Their Proliferation

Blood ◽

10.1182/blood.v128.22.863.863 ◽

2016 ◽

Vol 128 (22) ◽

pp. 863-863

Author(s):

Janaki Purushe ◽

Hongxin Sun ◽

Shan He ◽

Yali Dou ◽

Yi Zhang

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Specific Effect ◽

Cd8 T Cell ◽

T Cell Response ◽

Ifn Γ ◽

The Impact

Abstract Adoptive cellular therapy (ACT) for cancer requires amplification and persistence of tumor-specific T cells. To completely eliminate malignant tumor cells, infused tumor-reactive T cells must retain the capacity to expand over weeks and months in order to produce sufficient effector cells. However, T-cell potency can be blunted by excessive differentiation and upregulation of PD-1, which mediates exhaustion and impairs their proliferative capacity. Histone methylation is thought to be central in directing transcriptional programs important for effector proliferation, survival and differentiation; however, the epigenetic regulator(s) of this process are not well characterized. We report that the histone methyltransferases Mixed Lineage Leukemia 1 (MLL1) and MLL4, which catalyze trimethylation of histone H3 at lysine 4 (H3K4me3), play important roles in restraining effector differentiation and promoting proliferation of activated human CD8+ T cells. Upon T cell receptor (TCR) activation, human CD8+ T cells upregulated MLL1 and MLL4, however, down-regulated the global level of H3K4me3. To assess the specific effect of MLL1 in CD8+ T cell differentiation, we produced lentivirus encoding short hairpin RNA (shRNA) specific to MLL1 or MLL4. Knockdown of either protein increased the frequency of IFN-γ-producing cells by 50% to 100%, with silencing of MLL1 having the more potent effect. Pharmacological inhibition with MI-2-2, which simultaneously inhibits the menin-MLL1 and menin-MLL4 interactions, increased the frequency of IFN-γ + CD8+ T cells three-fold and reduced cell proliferation from 94% to 64%. Using a second MLL1 inhibitor MM-401, which affects MLL1 specifically, we confirmed that inhibiting MLL1 resulted in a two-to-four-fold increase in the expression of numerous effector molecule transcripts, including IFNG, TNFA, PRF1, FASL and GZMB. Our results suggest that while both MLL1 and MLL4 are important for proliferation of activated CD8+ T cells, MLL1 potently restrains effector differentiation. T-BET and EOMES are two transcription factors critical for mediating effector differentiation. We found that inhibition of MLL1 in cultured, TCR-activated CD8+ T cells using either MI-2-2 or MM-401 led to a significant increase in expression of EOMES, but had no significant effect on T-BET expression. Flow cytometric analysis showed that silencing MLL1 also increased the expression of EOMES protein in activated CD8+ T cells. Interestingly, MLL1 knockdown impaired subsequent persistence of ex vivo expanding CD8+ T cells, which was associated with a substantial increase of CD45RO+CCR7- short-lived effector CD8+ T cells. Remarkably, knockdown of MLL1 in proliferating CD8+ T cells led to their upregulation of PD-1. Taken together, these data suggest that MLL1 may play an important role in restraining precocious effector differentiation and exhuastion in CD8+ T cells. Future studies will investigating the impact of both MLL1 and MLL4 in regulating the CAR CD8+ T cell response in vivo and in vitro. Results from these experiments will allow us to identify epigenetic mechanisms that regulate the generation and persistence of antitumor effector and memory CD8+ T cells. Disclosures No relevant conflicts of interest to declare.

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A BCMAxCD3 bispecific T cell–engaging antibody demonstrates robust antitumor efficacy similar to that of anti-BCMA CAR T cells

Blood Advances ◽

10.1182/bloodadvances.2020002736 ◽

2021 ◽

Vol 5 (5) ◽

pp. 1291-1304

Author(s):

David J. DiLillo ◽

Kara Olson ◽

Katja Mohrs ◽

Thomas Craig Meagher ◽

Kevin Bray ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Antitumor Efficacy ◽

Therapeutic Effects ◽

Tumor Site ◽

Car T Cells ◽

Car T

Abstract CD3-engaging bispecific antibodies (bsAbs) and chimeric antigen receptor (CAR) T cells are potent therapeutic approaches for redirecting patient T cells to recognize and kill tumors. Here we describe a fully human bsAb (REGN5458) that binds to B-cell maturation antigen (BCMA) and CD3, and compare its antitumor activities vs those of anti-BCMA CAR T cells to identify differences in efficacy and mechanism of action. In vitro, BCMAxCD3 bsAb efficiently induced polyclonal T-cell killing of primary human plasma cells and multiple myeloma (MM) cell lines expressing a range of BCMA cell surface densities. In vivo, BCMAxCD3 bsAb suppressed the growth of human MM tumors in murine xenogeneic models and showed potent combinatorial efficacy with programmed cell death protein 1 blockade. BCMAxCD3 bsAb administration to cynomolgus monkeys was well tolerated, resulting in the depletion of BCMA+ cells and mild inflammatory responses characterized by transient increases in C-reactive protein and serum cytokines. The antitumor efficacy of BCMAxCD3 bsAb was compared with BCMA-specific CAR T cells containing a BCMA-binding single-chain variable fragment derived from REGN5458. Both BCMAxCD3 bsAb and anti-BCMA CAR T cells showed similar targeted cytotoxicity of MM cell lines and primary MM cells in vitro. In head-to-head in vivo studies, BCMAxCD3 bsAb rapidly cleared established systemic MM tumors, whereas CAR T cells cleared tumors with slower kinetics. Thus, using the same BCMA-binding domain, these results suggest that BCMAxCD3 bsAb rapidly exerts its therapeutic effects by engaging T cells already in place at the tumor site, whereas anti-BCMA CAR T cells require time to traffic to the tumor site, activate, and numerically expand before exerting antitumor effects.

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Tuning Of mTOR In Therapeutic T Cells As A Strategy To Manufacture Effector and Memory T Cells

Blood ◽

10.1182/blood.v122.21.4488.4488 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4488-4488

Author(s):

Velica Pedro ◽

Mathias Zech ◽

Hans Stauss ◽

Ronjon Chakraverty

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Conflicts Of Interest ◽

Genetic Manipulation ◽

Ex Vivo ◽

T Cell Response ◽

Mtor Pathway ◽

Retroviral Gene Transfer

Retroviral gene transfer of T cell receptors (TCRs) or chimeric-antigen receptors has been successfully used to redirect T cell specificity to tumor antigens and has shown promising results in clinical trials for patients with melanoma and B cell malignancies. Successful therapy relies not only on the generation of an efficient effector response but also in the ability of T cells to engraft and persist during and after cancer rejection. Yet, both aspects can be compromised. On the one hand, effector T cells can be disarmed in vivo by the immunosuppressive environment of the tumor, where the depletion of key nutrients (glucose, arginine, etc.) and the predominance of inhibitory signals (TGF-β, PD-L1, etc.) dampen their functions. On the other hand, current retroviral gene transfer protocols require T cells to be activated and expanded ex vivo,driving terminal effector differentiation at the expense of the capacity for self-renewal (“stemness”). This may prevent the generation of long-term protective memory and compromise therapeutic success. The mechanistic target of rapamycin (mTOR) pathway has recently emerged as a driver of effector differentiation in CD8 T cells by acting as a signalling bridge between extracellular stimuli and effector differentiation. Whilst high doses of rapamycin are known to be immunosuppressive, a low dose regimen increases memory output after a viral challenge without impairing viral clearance. These and other studies suggest that mTOR can be used as a rheostat to regulate T cell differentiation to clinical advantage. Using a mouse model of TCR gene therapy we aimed to produce T cells in which the mTOR pathway is either hyperactive, in order to generate ‘super-effectors’ that can function in the tumor microenvironment, or hypoactive in order to preserve “stemness”, increase engraftment and produce better recall immunity. To this purpose we transferred genes into therapeutic T cells encoding either (1) an mTOR activator, RHEB (Ras homolog enriched in brain) or (2) an mTOR inhibitor, PRAS40 (proline-rich Akt substrate 40 kDa). Upon stimulation in vitro,the phosphorylation of ribosomal protein S6, an mTOR downstream target, was increased in RHEB-expressing cells and decreased in PRAS40-expressing cells. RHEB T cells demonstrated a greater propensity than controls for blast formation, lower expression of L-selectin, increased IFN-g production and resistance to TGF-b. In contrast, PRAS40 T cells were smaller in size, expressed higher surface levels of L-selectin and had reduced but not ablated production of IFN-g, IL-2 and TNF-a production. In vivo, RHEB T cells co-transduced with a tumor-specific TCR showed increased initial expansion that was associated with increased tumor protection. However, this was followed by a steep decrease in T cell numbers compared to control cells, with preliminary experiments indicating that RHEB cells produced a less robust re-call response upon antigenic re-challenge. In contrast, PRAS40 cells failed to expand in vivo or to infiltrate tumors, resulting in complete lack of protection. Yet, the few PRAS40 cells remaining in circulation were predominately high for L-selectin, IL-7Ra and Sca-1 (markers associated with ‘stemness’). These results demonstrated that constitutive suppression of mTOR was of no therapeutic advantage. To circumvent this, we developed a doxycycline (DOX)-inducible PRAS40 vector in which mTOR suppression in T cells can be temporally controlled in vivo. Unlike the constitutive vector used previously, with the DOX-ON system the level of mTOR suppression was lower and furthermore, could be applied only during the period of antigen exposure. Thus, when DOX was added during tumor challenge, PRAS40-expressing T cells could reject tumor as well as controls. However, after DOX was withdrawn and transgene expression turned off, increased numbers of therapeutic T cells were found in peripheral blood. When re-challenged, these T cells produced a stronger recall response relative to cells in which mTOR had not been suppressed initially. Thus, our results show that genetic manipulation of mTOR can be used to alter the intrinsic properties of T cells and direct either their effector or memory differentiation. This approach maybe useful in designing better therapeutic immunotherapeutic strategies according to the type of T cell response required in vivo. Disclosures: No relevant conflicts of interest to declare.

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