An Lu

Enhanced tumor trafficking of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b.

For adoptive T-cell therapy to be effective against solid tumors, tumor-specific T cells must be able to migrate to the tumor site. One requirement for efficient migration is that the effector cells express chemokine receptors that match the chemokines produced either by tumor or tumor-associated cells. In this study, we investigated whether the tumor trafficking of activated T cells (ATCs) bearing a chimeric antigen receptor specific for the tumor antigen GD2 (GD2-CAR) could be enhanced by forced coexpression of the chemokine receptor CCR2b, as this receptor directs migration toward CCL2, a chemokine produced by many tumors, including neuroblastoma. Neuroblastoma cell lines (SK-N-SH and SK-N-AS) and primary tumor cells isolated from 6 patients all secreted high levels of CCL2, but GD2-CAR transduced ATCs lacked expression of CCR2 (<5%) and migrated poorly to recombinant CCL2 or tumor supernatants. After retroviral transduction, however, ATCs expressed high levels of CCR2b (>60%) and migrated well in vitro. We expressed firefly luciferase in CCR2b-expressing ATCs and observed improved homing (>10-fold) to CCL2-secreting neuroblastoma compared with CCR2-negative ATCs. As a result, ATCs co-modified with both CCR2b and GD2-CAR had greater antitumor activity in vivo.

Antitumor Activity of EBV-specific T Lymphocytes Transduced With a Dominant Negative TGF-β Receptor

Transforming growth factor (TGF)-β is produced in most human tumors and markedly inhibits tumor antigen-specific cellular immunity, representing a major obstacle to the success of tumor immunotherapy. TGF-β is produced in Epstein-Barr virus (EBV)-positive Hodgkin disease and non-Hodgkin lymphoma both by the tumor cells and by infiltrating T-regulatory cells and may contribute the escape of these tumors from infused EBV-specific T cells. To determine whether tumor antigen-specific cytotoxic T lymphocytes (CTLs) can be shielded from the inhibitory effects of tumor-derived TGF-β, we previously used a hemagglutinin-tagged dominant negative TGF-βRII expressed from a retrovirus vector to provide CTLs with resistance to the inhibitory effects of TGF-β in vitro. We now show that human tumor antigen-specific CTLs can be engineered to resist the inhibitory effects of tumor-derived TGF-β both in vitro and in vivo using a clinical grade retrovirus vector in which the dominant negative TGF-β type II receptor (DNRII) was modified to remove the immunogenic hemagglutinin tag. TGF-β–resistant CTL had a functional advantage over unmodified CTL in the presence of TGF-β–secreting EBV-positive lymphoma, and had enhanced antitumor activity, supporting the potential value of this countermeasure.

Antigen-specific cytotoxic T lymphocytes can target chemoresistant side-population tumor cells in Hodgkin lymphoma

Side-population (SP) analysis has been used to identify progenitor cells from normal and malignant tissues as well as revealing tumor cells with increased resistance to radiation and chemotherapy. Despite enhanced chemoresistance, tumor SP cells may still express tumor-associated antigens (TAAs), which may render them susceptible to elimination by the immune system. In this study, we show that both Hodgkin lymphoma (HL) cell lines and primary HL tumor samples contain a distinct SP phenotype. Importantly, while these cells showed increased resistance to gemcitabine, a commonly used drug for the treatment of refractory HL, HL SP cells also expressed higher levels of the TAAs MAGEA4, SSX2, survivin, and NY-ESO-1, which allowed them to be specifically recognized and killed by TAA-specific cytotoxic T lymphocytes. This study suggests that chemoresistant HL SP cells can be targeted by the immune system, providing a rationale for combined chemotherapy and immunotherapy for the treatment of HL.

Genetic modification of T cells with IL-21 enhances antigen presentation and generation of central memory tumor-specific cytotoxic T-lymphocytes.

An optimized antigen-presenting cell for tumor immunotherapy should produce a robust antigen specific cytotoxic T lymphocytes (CTL) response to tumor-associated antigens, which can persist in vivo and expand on antigen reencounter. Interleukin (IL)-21 synergizes with other γ-chain cytokines to enhance the frequency and cytotoxicity of antigen-specific CTL. As T cells themselves may serve as effective antigen-presenting cells (T antigen-presenting cells; TAPC) and may be useful in vivo as cellular vaccines, we examined whether CD8+ T cells genetically modified to produce IL-21 could induce immune responses to tumor associated antigen peptides in healthy human leukocyte antigen-A2+ donors. We found that IL-21 modified TAPC enhanced both the proliferation and survival of MART-1 specific CD8+ T cells, which were enriched by >8-fold over cultures with control nontransgenic TAPC. MART-1-specific CTL produced interferon-γ in response to cognate peptide antigen and killed primary tumor cells expressing MART-1 in a major histocompatibility complex restricted manner. IL-21 modified TAPC similarly enhanced generation of functional CTL against melanoma antigen gp100 and the B-cell chronic lymphocytic leukemia associated RHAMM antigen. Antigen-specific CTL generated using IL-21 gene-modified TAPC had a central memory phenotype characterized by CD45RA–, CD44high, CD27high, CD28high, CD62Lhigh, and IL-7 receptor-αhigh, contrasting with the terminal effector phenotype of CTL generated in the absence of IL-21. Thus, TAPC stimulation in the presences of IL-21 enhances proliferation of tumor antigen-specific T cells and favors induction of a central memory phenotype, which may improve proliferation, survival, and efficacy of T-cell based therapies for the treatment of cancer.

Autologous designer antigen-presenting cells by gene modification of T lymphocyte blasts with IL-7 and IL-12.

An effective immune response to antigen requires professional antigen-presenting cell (APC), which not only present antigen, but also provide costimulation and cytokines (eg, IL-12) that drive T cell differentiation down the appropriate effector pathway (Tc1/TH1). For T cell-based immunotherapy protocols, the availability of large numbers of autologous professional APC is a major limitation because professional APC do not proliferate in vitro. T cells themselves can proliferate exponentially in vitro and have the ability to present antigen. They can also express costimulatory molecules after activation. Therefore, we hypothesized that if activated T cells were genetically modified to express proinflammatory cytokines required to polarize T cells toward a Tc1 response, they could fulfill the requirements for an abundant, autologous APC. To test this potential, T cells were activated by CD3/CD28 antibodies and pulsed with model HLA-A2+ peptides derived from CMVpp65, MAGE-3, and MART-1. Activated T-APC readily reactivated CD8+ pp65 memory T cells from healthy CMV seropositive donors; however, the activation of MAGE-3 and MART-1-specific CD8+ T cells required both IL-7 and IL-12, which could be provided either exogenously or by genetic modification of the T-APC. Responder T cells could be expanded to large numbers with subsequent stimulations using activated, peptide-pulsed T-APC and IL-2. Tumor antigen-specific T cell lines killed both peptide-pulsed target cells and tumor cell lines. Thus, T cells provide a platform for the generation of autologous APC that can be customized to express both antigens and therapeutic molecules for the induction of antigen-specific T cell immunity.

Selective elimination of a chemoresistant side population of B-CLL cells by cytotoxic T lymphocytes in subjects receiving an autologous hCD40L/IL-2 tumor vaccine

Side-population (SP) analysis identifies precursor cells in normal and malignant tissues. Cells with this phenotype have increased resistance to many cytotoxic agents, and may represent a primary drug-resistant population in malignant diseases. To discover whether drug-resistant malignant SP cells are nonetheless sensitive to immune-mediated killing, we first established the presence of a malignant CD5+CD19+ SP subset in the blood of 18/21 subjects with B-cell chronic lymphocytic leukemia (B-CLL). We examined the fate of these cells in six of these individuals who received autologous human CD40 ligand and interleukin-2 (hCD40L/IL-2) gene-modified tumor cells as part of a tumor vaccine study. Vaccinated patients showed an increase in B-CLL-reactive T cells followed by a corresponding decline in circulating CD5+CD19+ SP cells. T-cell lines and clones generated from vaccinated patients specifically recognized B-CLL SP tumor cells. Elimination of SP cells is likely triggered by their increased expression of target antigens, such as receptor for hyaluronan-mediated motility (RHAMM), after stimulation of the malignant cells by hCD40L, as CD8+ RHAMM-specific T cells could be detected in the peripheral blood of immunized patients and were associated with the decline in B-CLL SP cells. Hence, malignant B cells with a primary drug-resistant phenotype can be targeted by T- cell-mediated effector activity after immunization of human subjects.

In vivo fluorescent optical imaging of cytotoxic T lymphocyte migration using IRDye800CW near-infrared dye.

We describe a method to measure in vivo migration of human T cells by using the near-infrared (NIR) dye IRDye800CW. Labeling of Epstein-Barr virus-specific T cells with IRDye800CW did not affect viability, proliferation, or T cell function. Following tail vein injection into mice bearing subcutaneous tumors, the NIR signal could be measured in vivo at the tumor site. Analysis of tumors revealed T cell infiltration and an increased NIR signal, confirming T cell migration. To test specific migration with IRDye800CW, tumors were modified to express CCL5 to measure site-specific migration. The NIR signal was increased at CCL5-secreting tumors compared with control tumors. Together, these data suggest that IRDye800CW may be used to study the trafficking of T cells in a small animal model and may have potential as a short-term reporter molecule for human immunotherapy studies.

Selective depletion of a minor subpopulation of B-chronic lymphocytic leukemia cells is followed by a delayed but progressive loss of bulk tumor cells and disease regression

Cancer precursor/progenitor cells may initiate and sustain the growth of tumors, but evidence for their existence in human disease is indirect, relying on their in vitro properties and animal models. More directly, specific elimination of these rare cells from cancer patients should produce a delayed but progressive disappearance of differentiated malignant progeny. Here, we describe selective eradication of a putative precursor population in a patient with B-cell chronic lymphocytic leukemia, followed 6 months later by a progressive loss of mature tumor cells without further treatment. This outcome supports the presence of a rare population of precursor/progenitor cells in human malignancies, and suggests benefit from their removal.

746. Go-TCR: Inducible MyD88/CD40 (iMC) Enhances Proliferation and Survival of Tumor-Specific TCR-Modified T Cells, Increasing Anti-Tumor Efficacy

Introduction: Use of tumor antigen-specific T cell receptors (TCRs) to refocus T cell killing has shown tantalizing clinical efficacy; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Also, MHC class I downregulation in tumors further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a small molecule dimerizer (rimiducid)-dependent “activation switch”, called inducible MyD88/CD40 (iMC), along with tumor-targeted TCRs to regulate T cell expansion and activation, while affecting upregulation of MHC class I on tumors.Methods: Human T cells were CD3/CD28-activated and transduced with αβTCR-encoding γ-retroviruses recognizing either the CT antigen, PRAME (HLA-A*0201/SLLQHLIGL), or the B-cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*0702/APAPTAVVL). Parallel “Go-TCR” vectors co-expressed iMC, comprising MyD88 and CD40 signaling domains along with rimiducid-binding FKBP12-V36. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed T2 cells (PRAME only) or against PRAME+/Bob1+, HLA-A2+ -B7+ U266 myeloma cells +/- 10 nM rimiducid. MHC class I induction was measured using transwell assays and flow cytometry. In vitro tumor killing was analyzed by T cell and tumor coculture assays at various effector to target ratios over a 7-day period. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 1×107 transduced T cells. iMC was activated in vivo by weekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo BLI imaging and flow cytometry.Results: All vectors efficiently (~85%) transduced activated T cells and showed antigen-specific IFN-γ production and cytotoxicity against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 cells. However, both iMC signaling and TCR ligation of PRAME peptide-pulsed T2 Cells were required for IL-2 production. Coculture assays with U266 cells showed that tumor elimination, IL-2 secretion and robust (~ 50-fold) T cell proliferation (vs TCR signaling alone) was optimized with concurrent rimiducid-driven iMC activation in both “Go-PRAME” and “Go-Bob1” constructs. Further, iMC activation produced TCR-independent IFN-γ that increased (~100-fold) MHC class I expression on tumor cells. In NSG mice engrafted with U266 tumors, iMc-PRAME TCR-modified T cells persisted for at least 81 days post-injection and prevented tumor growth, unlike other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ~1000-fold on day 81 post-injection vs T cells expressing only the PRAME TCR (p ~ 0.001).Summary: The novel rimiducid-regulated “Go” switch, iMC, greatly augments activation and expansion of TCR-engineered T cells while sensitizing tumors to T cells via cytokine-induced MHC class I upregulation. iMC-enhanced TCRs are prototypes of novel “Go-TCR” engineered T cell therapies that increase efficacy, safety and durability of adoptive T cell therapies.

Abstract B078: GoTCR: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells and improves antitumor efficacy in myeloma

Introduction: Use of T cells engineered to express antigen-specific T cell receptors (TCRs) has shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor T cell persistence and expansion in vivo . Additionally, MHC class I downregulation on tumor cells further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a novel, small molecule dimerizer (rimiducid)-dependent T cell “activation switch”, called inducible MyD88/CD40 (iMC), along with tumor antigen-specific TCRs to regulate T cell activation and expansion, while upregulating MHC class I expression on tumor cells. Methods: Human T cells were activated with anti-CD3/CD28 and transduced with γ-retroviruses encoding TCR α and β chains recognizing either the cancer-testes antigen PRAME (HLA-A*201-restricted SLLQHLIGL) or the B cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*702-restricted APAPTAVVL). Parallel “GoTCR” vectors co-expressed the αβ TCR and iMC, comprising signaling domains from MyD88 and CD40 fused in frame with tandem rimiducid-binding FKBP12v36 domains. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed EGFPluc-expressing T2 cells (PRAME only) or PRAME + /Bob1 + , HLA-A2 + HLA-B7 + EGFPluc-expressing U266 myeloma cells ± rimiducid (10 nM). MHC class I upregulation on tumor cells was measured using transwell assays and flow cytometry. In vitro tumor killing and T cell proliferation were analyzed using T cell and tumor coculture assays by either measuring loss of luciferase activity overnight or by flow cytometry over a period of 4-7 days. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 5×10 6 -1×10 7 transduced T cells. iMC was activated in vivo by weekly or biweekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo bioluminescence imaging and flow cytometry, respectively. Results: All vectors efficiently (∼85%) transduced activated T cells and showed antigen-specific IFN-γ production and cytolytic function against peptide-pulsed T2 cells and/or PRAME + Bob1 + U266 myeloma cells. However, both TCR ligation and rimiducid-dependent iMC costimulation were required for IL-2 production against PRAME peptide-pulsed T2 cells. Coculture assays against U266 cells showed that tumor elimination was optimized with concurrent rimiducid-driven iMC activation in both “GoPRAME” and “GoBob1” constructs, and this was accompanied by greatly increased IL-2 secretion and robust T cell proliferation (∼ 50-fold vs PRAME or Bob1-specific TCRs alone). Further, iMC activation produced IFN-γ independently of TCR ligation, which significantly increased MHC class I expression on tumor cells (∼ 7-fold) relative to PRAME TCR-transduced T cells. In NSG mice engrafted with PRAME + U266 myeloma tumors, GoPRAME TCR-modified T cells persisted for 81 days post-injection and prevented tumor growth, unlike any of the other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ∼1000-fold on day 81 post-injection compared to T cells expressing only the PRAME TCR (p Summary: iMC is a novel “Go” switch that utilizes rimiducid, a small molecule dimerizer, to provide costimulation to PRAME and Bob1-specific TCR-engineered T cells while sensitizing tumors to TCR-mediated recognition via cytokine-induced MHC I upregulation. These iMC-enhanced TCRs are prototypes of novel “GoTCR” engineered T cell therapies that may increase efficacy, safety and durability of adoptive T cell therapies. Citation Format: Tsvetelina Pentcheva-Hoang, David Torres, Tania Rodriguez, Ana Korngold, An Lu, Jeannette Crisostomo, Annemarie Moseley, Lorenz Jahn, Mirjam H.M. Heemskerk, Kevin Slawin, David Spencer, Aaron Foster. GoTCR: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells and improves antitumor efficacy in myeloma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B078.