Tumor-associated Ags Research Articles

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

CD206+ tumor-associated macrophages cross-present tumor antigen and drive antitumor immunity

In many solid cancers, tumor-associated macrophages (TAM) represent the predominant myeloid cell population. Antigen (Ag) cross-presentation leading to tumor Ag–directed cytotoxic CD8+ T cell responses is crucial for antitumor immunity. However, the role of recruited monocyte-derived macrophages, including TAM, as potential cross-presenting cells is not well understood. Here, we show that primary human as well as mouse CD206+ macrophages are effective in functional cross-presentation of soluble self-Ag and non–self-Ag, including tumor-associated Ag (TAA), as well as viral Ag. To confirm the presence of cross-presenting TAM in vivo, we performed phenotypic and functional analysis of TAM from B16-F10 and CT26 syngeneic tumor models and have identified CD11b+F4/80hiCD206+ TAM to effectively cross-present TAA. We show that CD11b+CD206+ TAM represent the dominant tumor-infiltrating myeloid cell population, expressing a unique cell surface repertoire, promoting Ag cross-presentation and Ag-specific CD8+ T cell activation comparable with cross-presenting CLEC9A+ DCs (cDC1). The presence of cross-presenting CD206+ TAM is associated with reduced tumor burden in mouse syngeneic tumor models and with improved overall survival in cutaneous melanoma patients. Therefore, the demonstration of effective Ag cross-presentation capabilities of CD206+ TAM, including their clinical relevance, expands our understanding of TAM phenotypic diversity and functional versatility.

Dendritic Cell Maturation Defines Immunological Responsiveness of Tumors to Radiation Therapy.

Radiation therapy is capable of directing adaptive immune responses against tumors by stimulating the release of endogenous adjuvants and tumor-associated Ags. Within the tumor, conventional type 1 dendritic cells (cDC1s) are uniquely positioned to respond to these signals, uptake exogenous tumor Ags, and migrate to the tumor draining lymph node to initiate cross-priming of tumor-reactive cytotoxic CD8+ T cells. In this study, we report that radiation therapy promotes the activation of intratumoral cDC1s in radioimmunogenic murine tumors, and this process fails to occur in poorly radioimmunogenic murine tumors. In poorly radioimmunogenic tumors, the adjuvant polyinosinic-polycytidylic acid overcomes this failure following radiation and successfully drives intratumoral cDC1 maturation, ultimately resulting in durable tumor cures. Depletion studies revealed that both cDC1 and CD8+ T cells are required for tumor regression following combination therapy. We further demonstrate that treatment with radiation and polyinosinic-polycytidylic acid significantly expands the proportion of proliferating CD8+ T cells in the tumor with enhanced cytolytic potential and requires T cell migration from lymph nodes for therapeutic efficacy. Thus, we conclude that lack of endogenous adjuvant release or active suppression following radiation therapy may limit its efficacy in poorly radioimmunogenic tumors, and coadministration of exogenous adjuvants that promote cDC1 maturation and migration can overcome this limitation to improve tumor control following radiation therapy.

Immunodominant AH1 Antigen-Deficient Necroptotic, but Not Apoptotic, Murine Cancer Cells Induce Antitumor Protection.

Immunogenic cell death (ICD) occurs when a dying cell releases cytokines and damage-associated molecular patterns, acting as adjuvants, and expresses Ags that induce a specific antitumor immune response. ICD is studied mainly in the context of regulated cell death pathways, especially caspase-mediated apoptosis marked by endoplasmic reticulum stress and calreticulin exposure and, more recently, also in relation to receptor-interacting protein kinase-driven necroptosis, whereas unregulated cell death like accidental necrosis is nonimmunogenic. Importantly, the murine cancer cell lines used in ICD studies often express virally derived peptides that are recognized by the immune system as tumor-associated Ags. However, it is unknown how different cell death pathways may affect neoepitope cross-presentation and Ag recognition of cancer cells. We used a prophylactic tumor vaccination model and observed that both apoptotic and necroptotic colon carcinoma CT26 cells efficiently immunized mice against challenge with a breast cancer cell line that expresses the same immunodominant tumor Ag, AH1, but only necroptotic CT26 cells would mount an immune response against CT26-specific neoepitopes. By CRISPR/Cas9 genome editing, we knocked out AH1 and saw that only necroptotic CT26 cells were still able to protect mice against tumor challenge. Hence, in this study, we show that endogenous AH1 tumor Ag expression can mask the strength of immunogenicity induced by different cell death pathways and that upon knockout of AH1, necroptosis was more immunogenic than apoptosis in a prophylactic tumor vaccination model. This work highlights necroptosis as a possible preferred ICD form over apoptosis in the treatment of cancer.

Correction: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody-Induced Tumor Cell Killing by Effector CD8+ T Cells.

Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule--mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7- CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule-induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors.

LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody-Induced Tumor Cell Killing by Effector CD8+ T Cells.

Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule--mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7- CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule-induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors.

Abstract B125: Targeting human CD141+ DC using CLEC9A antibodies for cancer immunotherapy

Abstract Dendritic cells (DC) are a heterogeneous cell population, with specialist subtypes driving specific immune responses. In mice, the cDC1 subset (also referred to as Batf3-dependent DC, XCR1+ DC, CD8+ DC in lymphoid tissues and CD103+ DC in peripheral tissues) is essential for the induction of tumor immune responses and for the efficacy of checkpoint inhibitor blockade and adoptive T-cell immunotherapies. Vaccines that can deliver antigens (Ag) directly to DCs in vivo are more effective than cell-based therapies in mouse models and are promising approaches to translate to humans. CD141+ DC are the human cDC1 equivalent and specifically express the C-type lectin-like receptor CLEC9A, that facilitates cross-presentation of dead cell Ag. Targeting tumor-associated Ag (TAA) to human CD141+ DC using CLEC9A antibody (Ab) is therefore an attractive strategy to induce or boost tumor immune responses. NYESO1 and WT1 are well characterised, highly immunogenic TAA expressed by a broad array of tumor types. We developed recombinant human chimeric IgG4 Ab specific for human CLEC9A genetically fused to NYESO1 or WT1. For comparison we developed TAA fusions with chimeric IgG4 Ab specific for human DEC-205, which is expressed by many human leukocytes, and β-galactosidase as an irrelevant isotype control. CLEC9A-NYESO1 and CLEC9A-WT1 Abs retained their binding specificity for CD141+ DC. Following uptake of CLEC9A-WT1, CD141+ DC cross-presented a WT-1 HLA-A24-restricted epitope for recognition by specific CD8+ cytotoxic T-cells. Likewise, a HLA-A2-restricted NYESO1 epitope was cross-presented Ag specific CD8+ T-cells by CD141+ DC following uptake of CLEC9A-NYESO1. For both TAA, the CLEC9A Abs were more efficient at delivery of Ag for cross-presentation than DEC-205 or isotype control Abs. Moreover, using a humanized mouse model in which functional human CD141+ DC and Ag-specific T-cells develop, CLEC9A-TAA Ab induced priming of Ag-specific T-cells. Our data advocate further development of human CLEC9A targeting Abs as cancer vaccines. Citation Format: Kristen Radford, Frances Pearson, Kelly-Anne Masterman, Kirsteen Tullett, Oscar Haigh, Carina Walpole, Ghazal Daraj, Ingrid Leal Rojas, Mireille Lahoud. Targeting human CD141+ DC using CLEC9A antibodies for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B125.

Prophylactic Infusion of Multi-Antigen Specific T-Cell Products to Prevent Complications after T Cell Depleted Allogeneic Stem Cell Transplantation — a Phase I/II Study

Viral infections and disease relapses are major complications between T cell depleted allogeneic stem cell transplantation (TCD alloSCT) and donor lymphocyte infusion (DLI). The prophylactic infusion of selected donor T cells may be an effective method to restore anti-viral and anti-tumor immunity early after TCD alloSCT. In this phase I/II study, we aimed to prevent these complications by the infusion of donor-derived T cell products containing CD8+ T cells directed against cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV) antigens (Ag), tumor associated Ag (TAA) and minor histocompatibility Ag (MiHA) 6-8 weeks after TCD alloSCT. The feasibility of multi-Ag specific T cell product generation and safety of early administration were investigated. Furthermore, clinical outcome and immune reconstitution were assessed.HLA-A2+ patients treated for a hematological malignancy with a ≥9/10 HLA-matched TCD graft from a CMV+ and/or EBV+ (un)related donor were eligible for inclusion. Donor T cells directed against HLA-A2-restricted peptides of CMV, EBV, AdV, and TAA NY-ESO-1, WT-1, RHAMM, PRAME and proteinase 3 were simultaneously isolated under GMP conditions in 1 day, using MHC I-Streptamers and CliniMACS selection out of the naïve and memory T cell compartment from 2*109 PBMC. Depending on patient/donor HLA-typing, additional MHC I-Streptamers targeting viral peptides in HLA-A1, -A24, -B7, or -B8 were added to the procedure, as well as the HLA-A2/HA-1h MHC I-Streptamer in case of MiHA disparity in the GVL direction. The incidence of GVHD, mortality, disease relapses and viral reactivations was monitored until 6 months after alloSCT. In addition, follow-up samples were stained with tetramers to analyze in vivo reconstitution of target-Ag specific T cells.Products were generated for 27/28 included patients that showed stable engraftment after TCD alloSCT; 1 patient died early after alloSCT. All donors were EBV+, 14/27 donors were CMV+. 26/27 products were successfully generated and contained a median of 5.2*106 cells (range 0.4 - 26.5*106) with a median purity of target-Ag specific T cells within the CD3+ compartment of 80.4% (range 46.0-99.9). Target-Ag specific T cells consisted for 99% of virus-specific memory T cells, while the remaining 1% included TAA, MiHA and naïve virus-specific T cells. 24 patients received their product (median 58 days after alloSCT; range 51-107) without infusion related complications; in 2 patients the infusion was cancelled due to acute skin GVHD on the day of infusion. During follow-up, 1 patient experienced skin GVHD requiring immunosuppressive therapy. One patient died due to complications of a nephrotic syndrome, probably unrelated to product infusion. Four patients showed disease relapse and were treated accordingly; coinciding expansion of TAA- or MiHA-specific T cells was not observed. None of the patients experienced AdV reactivations; in 2 patients AdV-specific T cells expanded after infusion. Four patients had detectable EBV loads; in one of these patients the EBV reactivation progressed to EBV-PTLD and he was treated with rituximab followed by DLI. In 2 patients, the rise in EBV load led to expansion of EBV specific T cells in vivo. 4/5 CMV+ patients with a CMV- donor had ongoing CMV reactivations at the moment of product infusion, CMV specific T cells could already be detected just before product infusion and reactivations were cleared within 7-84 days. Of the 8 CMV+ patients with a CMV+ donor, 3 patients remained free of CMV reactivations, 4 had already a reactivation at the moment of product infusion and 1 patient developed a CMV reactivation during follow up. One patient had progressed to CMV pneumonia within 2 weeks after infusion, requiring ganciclovir. In all 5 CMV reactivating patients, T cells directed against 2-3 CMV-specific epitopes expanded significantly and viral loads declined. TCR sequencing of the CDR3 region illustrated in vivo persistence and expansion of target-Ag specific T cells present in the product.In conclusion, we have shown that MHC I-Streptamer isolation based generation and adoptive transfer of donor-derived multi Ag-specific T cell products is safe and feasible. Moreover, efficacy of the prophylactic infusion is illustrated by expansion of target-Ag specific T cells in patients coinciding viral reactivations and the prevention of viral complications in the majority of patients between TCD alloSCT and DLI. DisclosuresGermeroth:Juno Therapeutics: Employment, Equity Ownership.

PD-1 Blockade Unleashes Effector Potential of Both High- and Low-Affinity Tumor-Infiltrating T Cells.

Antitumor T cell responses involve CD8+ T cells with high affinity for mutated self-antigen and low affinity for nonmutated tumor-associated Ag. Because of the highly individual nature of nonsynonymous somatic mutations in tumors, however, immunotherapy relies often on an effective engagement of low-affinity T cells. In this study, we studied the role of T cell affinity during peripheral priming with single-peptide vaccines and during the effector phase in the tumor. To that end, we compared the antitumor responses after OVA257-264 (N4) peptide vaccination of CD8+ T cells carrying TCRs with high (OT-1) and low (OT-3) avidity for the N4 peptide in B16.N4 tumor-bearing C57BL/6 mice. Additionally, we assessed the response of OT-1 cells to either high-affinity (B16.N4) or low-affinity (B16.T4) Ag-expressing tumors after high-affinity (N4) or low-affinity (T4) peptide vaccination. We noticed that although low-affinity tumor-specific T cells expand less than high-affinity T cells, they express lower levels of inhibitory receptors and produce more cytokines. Interestingly, tumor-infiltrating CD8+ T cells show similar in vivo re-expansion capacity to their counterparts in secondary lymphoid organs when transferred to tumor-free hosts, suggesting that T cells in tumors may be rekindled upon relief of tumor immunosuppression. Moreover, our results show that αPD-1 treatment enhances tumor control of high- and low-affinity ligand-expressing tumors, suggesting that combination of high-affinity peripheral priming by altered peptide ligands and checkpoint blockade may enable tumor control upon low-affinity Ag recognition in the tumor.

Abstract 719: A tumor mitochondria vaccine protects against experimental renal cell carcinoma

Abstract Mitochondria provide energy for cells via oxidative phosphorylation. Reactive oxygen species, a byproduct of this mitochondrial respiration, can damage mitochondrial DNA (mtDNA), and somatic mtDNA mutations have been found in all colorectal, ovarian, breast, urinary bladder, kidney, lung, and pancreatic tumors studied. The resulting altered mitochondrial proteins or tumor-associated mitochondrial Ags (TAMAs) are potentially immunogenic, suggesting that they may be targetable Ags for cancer immunotherapy. In this article, we show that the RENCA tumor cell line harbors TAMAs that can drive an antitumor immune response. We generated a cellular tumor vaccine by pulsing dendritic cells with enriched mitochondrial proteins from RENCA cells. Our dendritic cell-based RENCA mitochondrial lysate vaccine elicited a cytotoxic T cell response in vivo and conferred durable protection against challenge with RENCA cells when used in a prophylactic or therapeutic setting. By sequencing mtDNA from RENCA cells, we identified two mutated molecules: COX1 and ND5. Peptide vaccines generated from mitochondrial-encoded COX1 but not from ND5 had therapeutic properties similar to RENCA mitochondrial protein preparation. Thus, TAMAs can elicit effective antitumor immune responses, potentially providing a new immunotherapeutic strategy to treat cancer. Citation Format: Stefano Pierini, Andrea Facciabene, Mireia Uribe-Herranz, Stavros Rafail, Chongyun Fang, Janos Tanyi. A tumor mitochondria vaccine protects against experimental renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 719.

The Immunogenicity of a Proline-Substituted Altered Peptide Ligand toward the Cancer-Associated TEIPP Neoepitope Trh4 Is Unrelated to Complex Stability.

Human cancers frequently display defects in Ag processing and presentation allowing for immune evasion, and they therefore constitute a significant challenge for T cell-based immunotherapy. We have previously demonstrated that the antigenicity of tumor-associated Ags can be significantly enhanced through unconventional residue modifications as a novel tool for MHC class I (MHC-I)-based immunotherapy approaches. We have also previously identified a novel category of cancer neo-epitopes, that is, T cell epitopes associated with impaired peptide processing (TEIPP), that are selectively presented by MHC-I on cells lacking the peptide transporter TAP. In this study, we demonstrate that substitution of the nonanchoring position 3 into a proline residue of the first identified TEIPP peptide, the murine Trh4, results in significantly enhanced recognition by antitumor CTLs toward the wild-type epitope. Although higher immunogenicity has in most cases been associated with increased MHC/peptide complex stability, our results demonstrate that the overall stability of H-2Db in complex with the highly immunogenic altered peptide ligand Trh4-p3P is significantly reduced compared with wild-type H-2Db/Trh4. Comparison of the crystal structures of the H-2Db/Trh4-p3P and H-2Db/Trh4 complexes revealed that the conformation of the nonconventional methionine anchor residue p5M is altered, deleting its capacity to form adequate sulfur-π interactions with H-2Db residues, thus reducing the overall longevity of the complex. Collectively, our results indicate that vaccination with Thr4-p3P significantly enhances T cell recognition of targets presenting the wild-type TEIPP epitope and that higher immunogenicity is not necessarily directly related to MHC/peptide complex stability, opening for the possibility to design novel peptide vaccines with reduced MHC/peptide complex stability.

Virus-Specific CD8+ T Cells Infiltrate Melanoma Lesions and Retain Function Independently of PD-1 Expression.

It is well known that CD8+ tumor-infiltrating lymphocytes (TILs) are correlated with positive prognoses in cancer patients and are used to determine the efficacy of immune therapies. Although it is generally assumed that CD8+ TILs will be tumor-associated Ag (TAA) specific, it is unknown whether CD8+ T cells with specificity for common pathogens also infiltrate tumors. If so, the presence of these T cells could alter the interpretation of prognostic and diagnostic TIL assays. We compared TAA-specific and virus-specific CD8+ T cells in the same tumors using murine CMV, a herpesvirus that causes a persistent/latent infection, and vaccinia virus, a poxvirus that is cleared by the host. Virus-specific CD8+ TILs migrated into cutaneous melanoma lesions during acute infection with either virus, after a cleared vaccinia virus infection, and during a persistent/latent murine CMV infection. Virus-specific TILs developed independently of viral Ag in the tumor and, interestingly, expressed low or intermediate levels of full-length PD-1 in the tumor environment. Importantly, PD-1 expression could be markedly induced by Ag but did not correlate with dysfunction for virus-specific TILs, in sharp contrast to TAA-specific TILs in the same tumors. These data suggest that CD8+ TILs can reflect an individual's immune status, rather than exclusively representing TAA-specific T cells, and that PD-1 expression on CD8+ TILs is not always associated with repeated Ag encounter or dysfunction. Thus, functional virus-specific CD8+ TILs could skew the results of prognostic or diagnostic TIL assays.

PiggyBacTM-Produced CAR-T Cells Exhibit Stem-Cell Memory Phenotype

Immunotherapy using chimeric-antigen receptor (CAR)-T cells is emerging as an exciting therapeutic approach for cancer therapies. Autologous CAR-modified T cells targeting a tumor-associated antigen (Ag) can result in robust tumor killing, in some cases resulting in complete remission of CD19+ hematological malignancies. Unlike traditional biologics and chemotherapeutics, CAR-T cells possess the capacity to rapidly reproduce upon Ag recognition, thereby potentially obviating the need for repeat treatments. To achieve this, CAR-T cells must not only drive tumor destruction initially, but must also persist in the patient as a stable population of viable memory T cells to prevent potential cancer relapses. Thus, intensive efforts have been focused on the development of CAR molecules that do not cause T cell exhaustion through Ag-independent (tonic) signaling, as well as of a CAR-T product containing early memory cells, especially stem cell memory (TSCM). It is hypothesized that a stem cell-like CAR-T would exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (TCM), effector memory (TEM) and effector T cells (TE), thereby producing better tumor eradication and long-term CAR-T engraftment.We developed a novel Centyrin-based CAR, referred to as a CARTyrin, that is specific for human B cell maturation antigen (BCMA). Centyrins are alternative scaffold molecules based on human consensus tenascin FN3 domain, are smaller than scFv molecules, and can be selected for monomeric properties that favor stability and decrease the likelihood of tonic signaling in CAR molecules. We produced a plasmid DNA transposon encoding the CARTyrin that was flanked by two cis-regulatory insulator elements to help stabilize CARTyrin expression by blocking improper gene activation or silencing. The piggyBac™ (PB) Transposon System was used for stable integration of anti-BCMA CARTyrin into resting pan T cells, whereby the transposon was co-delivered along with an mRNA transposase enzyme, called Super piggyBac™ (SPB), in a single electroporation reaction. Delivery of piggyBac™ transposon into untouched, resting primary human pan T cells resulted in 20-30% of cells with stable integration and expression of PB-delivered genes. Surprisingly, we observed that a majority of these cells were positive for expression of CD62L and CD45RA, markers commonly associated with TSCM cells. To see if this phenotype was retained upon CAR-T cell stimulation and expansion, we activated the cells via stimulation of CD3 and CD28, and later show that > 60% of CARTyrin+ T cells exhibited a stem-cell memory phenotype. Furthermore, these cells were fully capable of expressing potent anti-tumor effector function.To determine whether or not the PB system directly contributed to enhancing the expression of stem-like markers, we compared the phenotype of CAR-T cells generated either by PB transposition or lentiviral (LV) transduction. To do this, we constructed a new vector by subcloning the CARTyrin transgene into a common LV construct for production of virus. Following introduction of the CARTyrin to untouched resting T cells either by PB-transposition or LV-transduction, we expanded the CARTyrin+ cells and then allowed them to return to a resting state. A variety of phenotypic and functional characteristics were measured including kinetic analysis of memory and exhaustion-associated markers, secondary proliferation in response to homeostatic cytokine or tumor-associated Ag, cytokine production, and lytic capability in response to BCMA+ tumor cells. Unlike the PB-transposed CARTyrin+ T cells, we found that the LV-transduced CARTyrin+ T cells did not exhibit an augmented memory phenotype. In addition, PB-transposed cells exhibited a comparable or greater capability for secondary proliferation and killing of BCMA+ tumor cells. Together, these data demonstrate that CAR-T cells produced by PB transposition are predominantly TSCM cells, a highly desirable product phenotype in the CAR-T field. Furthermore, these CARTyrin+ T cells exhibit strong anti-tumor activity and may give rise to cells that persist longer in vivo due to the use of a Centyrin-based CAR, which may be less prone to tonic signaling and functional exhaustion. DisclosuresBarnett:Poseida Therapeutics: Employment. Hermanson:Poseida Therapeutics: Employment. Smith:Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Martin:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

Prevention of Viral Infections after T Cell Depleted Allogeneic Stem Cell Transplantation By Infusion of Multi-Antigen Specific T Cell Products

Opportunistic viral infections and relapse are major complications in patients after T cell depleted allogeneic stem cell transplantation (TCD alloSCT). Since the application of unmodified donor lymphocyte infusion (DLI) early after alloSCT results in a high risk of graft versus host disease (GVHD), infusion of selected populations of virus-specific donor T cells can be an effective approach to safely restore anti-viral immunity early after alloSCT. As part of the EU FP7 consortium T Control, in this phase I/II study the feasibility and safety of the generation and administration of selected populations of donor-derived T cells targeting multiple antigens (Ag) is assessed. The multi Ag-specific T cell products contained T cells targeting cytomegalovirus (CMV), Epstein Bar virus (EBV) and adenovirus (AdV) as well as T cells targeting tumor associated Ag (TAA) and minor histocompatibility Ag (MiHA) to boost the graft versus leukemia (GVL) reactivity. To assess efficacy, in-vivo appearance or expansion of Ag-specific T cells, and the effect on viral reactivations and/or disease relapse was evaluated for 20 weeks after infusion until regular DLI was applied.HLA-A*02+ patients treated for a hematological malignancy with an HLA-matched TCD alloSCT from a CMV and/or EBV seropositive donor were included in this study. 6-8 weeks after alloSCT, T-cells directed against HLA-A*02-restricted peptides of CMV, EBV and AdV, and the TAA NY-eso, WT-1, RHAMM, PRAME and proteinase 3 were isolated using the reversible streptamer-nanobead technology (Juno) by cliniMACS selection out of the naïve and/or memory T cell compartment from 2*10^9 donor PBMC. Depending on the HLA-typing of the patient/donor additional streptamers targeting viral peptides in A*01, A*24, B*07 or B*08 were added to the selection procedure. In case of patient/donor MiHA disparity in the GvL direction, the HLA-A*0201/HA-1h streptamer was also added. This procedure allows purification of T cells under GMP conditions in 1 day.20 multi Ag-specific T cell products were generated of which 19 met the release criteria. These products consisted of 0.5-12*10^6 cells containing purities of 46,0-94,3% target Ag-specific CD8+ T cells within the T cell compartment. In all products CMV and/or EBVas well as AdV virus-specific memory T cells were isolated comprising 99% of the target Ag-specific CD8+ T cells, while the other 1% included the TAA and MiHA specificities or CMV specific T cells from seronegative donors. 17 products were administered without infusion-related complications or GVHD; 2 patients experienced GVHD before infusion and did not receive their product.Of the 14 patients evaluated at this stage, 13 completed the follow-up period until DLI and 1 patient died during follow-up. No product-related adverse events were reported. In all 5 CMV- patients and in 3/9 CMV+ patients no CMV reactivation and no expansion of CMV-specific T cells was observed. The other 6 CMV+ patients experienced CMV reactivations. In 2/6 patients who received the product from a CMV+ donor CMV-specific T cells were detected with tetramer analysis and CMV was cleared. From the other 4 reactivating patients with CMV- donors 3 had circulating CMV-specific T cells already at the moment of infusion, whereas in 1 patient CMV-specific T cells clearly expanded after infusion, resulting in viral clearance in all 4 patients.All 14 donors were EBV+. In 7 patients EBV reactivations were observed, which coincided in 2/7 patients with the appearance of EBV-specific T cells and subsequent clearance of the virus. In 4/7 patients EBV reactivation was cleared without clear expansion of EBV-specific T cells. However, 1 patient required treatment for an EBV-PTLD, although ultimately EBV-specific T cells appeared and EBV was cleared.In none of the patients AdV DNA loads were detected in the follow-up period, while in 1 patient expansion of AdV-specific T cells was observed.Expansion of TAA and MiHA-specific T cells could not be demonstrated in-vivo using tetramer staining. More sensitive techniques will be required to visualize these cells. 2 patients showed relapse of their malignancy before DLI infusion.In this clinical study, we have shown that the reversible streptamer technology based generation and adoptive transfer of donor-derived multi Ag-specific T cell products is feasible and safe and can be used as a strategy to prevent viral infections in the interval between TCD alloSCT and DLI. DisclosuresGermeroth:Juno Therapeutics: Employment.

A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library

When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of VH and VL genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations.

Immunotherapy Expands and Maintains the Function of High-Affinity Tumor-Infiltrating CD8 T Cells In Situ.

Cancer cells harbor high-affinity tumor-associated Ags capable of eliciting potent antitumor T cell responses, yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and programmed death-1 (PD-1) have been used. We report in this study that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor Ag-specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Coexpression of Nur77GFP and OX40 identifies a polyclonal population of high-affinity tumor-associated Ag-specific CD8(+) T cells, which produce more IFN-γ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or programmed death ligand-1. Moreover, expansion of these high-affinity CD8 T cells prolongs survival of tumor-bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired, and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor-resident CD8 T cells, thereby increasing the frequency of cytotoxic, high-affinity, tumor-associated Ag-specific cells.

Target Density, Not Affinity or Avidity of Antigen Recognition, Determines Adoptive T Cell Therapy Outcomes in a Mouse Lymphoma Model.

Adoptive T cell therapy (ACT) with antitumor CTL is a promising and tailored treatment against cancer. We investigated the role played by the affinity and avidity of the interaction between the tumor and the CTL on the outcome of ACT against a mouse non-Hodgkin B cell lymphoma that expresses OVA as a model neoantigen. ACT was assessed under conditions where antitumor CTL expressed TCR of varying affinity for OVA. We also assessed conditions where the avidity of Ag recognition varied because the lymphoma cells expressed high or low levels of OVA. Efficient eradication of small tumor burdens was achieved by high- or low-affinity CTL. Tumors expressing low levels of OVA could also be eliminated. However, ACT against large tumor burdens was unsuccessful, accompanied by CTL deletion and functional impairment. This negative outcome was not prevented by lowering the affinity of the CTL or the expression of OVA in the lymphoma. Thus, tumor burden, rather than CTL affinity or avidity, appears to be the main determinant of ACT outcomes in our lymphoma model. Insofar as our results can be extrapolated to the clinical setting, they imply that the range of CTL and tumor-associated Ag combinations that may be effectively harnessed in ACT against lymphoma may be wider than generally assumed. CTL expressing low-affinity TCR may be effective against lymphoma, and lowly expressed tumor-associated Ag should be considered as potential targets, but tumor reduction should always be implemented before infusion of the CTL.

Landscape of Tumor Antigens in T Cell Immunotherapy

Cancer immunotherapy is a rapidly evolving field that exploits T cell responses to tumor-associated Ags to induce tumor rejection. Molecular identification of tumor rejection Ags has helped define several classes of Ags, including tissue differentiation and tumor germline Ags. The ability to genetically engineer Ag-specific receptors into T cells provides an opportunity to translate these findings into therapies. New immunotherapy agents, notably checkpoint inhibitors, have demonstrated unprecedented efficacy in certain cancers. However, the nature of the Ags driving those beneficial immune responses remains unclear. New evidence suggests that tumors express immunogenic, tumor-specific epitopes generated from the same mutations that drive cancer development. Correlations between cancer types responding to immunotherapies and the frequency of somatic mutations may clarify what drives natural antitumor immune responses. This fusion of tumor immunology and genetics is leading to new ways to target this class of ideal tumor-specific Ags and could allow the application of immunotherapy to many cancers.

A Tumor Mitochondria Vaccine Protects against Experimental Renal Cell Carcinoma.

Mitochondria provide energy for cells via oxidative phosphorylation. Reactive oxygen species, a byproduct of this mitochondrial respiration, can damage mitochondrial DNA (mtDNA), and somatic mtDNA mutations have been found in all colorectal, ovarian, breast, urinary bladder, kidney, lung, and pancreatic tumors studied. The resulting altered mitochondrial proteins or tumor-associated mitochondrial Ags (TAMAs) are potentially immunogenic, suggesting that they may be targetable Ags for cancer immunotherapy. In this article, we show that the RENCA tumor cell line harbors TAMAs that can drive an antitumor immune response. We generated a cellular tumor vaccine by pulsing dendritic cells with enriched mitochondrial proteins from RENCA cells. Our dendritic cell-based RENCA mitochondrial lysate vaccine elicited a cytotoxic T cell response in vivo and conferred durable protection against challenge with RENCA cells when used in a prophylactic or therapeutic setting. By sequencing mtDNA from RENCA cells, we identified two mutated molecules: COX1 and ND5. Peptide vaccines generated from mitochondrial-encoded COX1 but not from ND5 had therapeutic properties similar to RENCA mitochondrial protein preparation. Thus, TAMAs can elicit effective antitumor immune responses, potentially providing a new immunotherapeutic strategy to treat cancer.

CD8 T cell tolerance to a tumor-associated self-antigen is reversed by CD4 T cells engineered to express the same T cell receptor.

Ag receptors used for cancer immunotherapy are often directed against tumor-associated Ags also expressed in normal tissues. Targeting of such Ags can result in unwanted autoimmune attack of normal tissues or induction of tolerance in therapeutic T cells. We used a murine model to study the phenotype and function of T cells redirected against the murine double minute protein 2 (MDM2), a tumor-associated Ag that shows low expression in many normal tissues. Transfer of MDM2-TCR–engineered T cells into bone marrow chimeric mice revealed that Ag recognition in hematopoietic tissues maintained T cell function, whereas presentation of MDM2 in nonhematopoietic tissues caused reduced effector function. TCR-engineered CD8+ T cells underwent rapid turnover, downmodulated CD8 expression, and lost cytotoxic function. We found that MDM2-TCR–engineered CD4+ T cells provided help and restored cytotoxic function of CD8+ T cells bearing the same TCR. Although the introduction of the CD8 coreceptor enhanced the ability of CD4+ T cells to recognize MDM2 in vitro, the improved self-antigen recognition abolished their ability to provide helper function in vivo. The data indicate that the same class I–restricted TCR responsible for Ag recognition and tolerance induction in CD8+ T cells can, in the absence of the CD8 coreceptor, elicit CD4 T cell help and partially reverse tolerance. Thus MHC class I–restricted CD4+ T cells may enhance the efficacy of therapeutic TCR-engineered CD8+ T cells and can be readily generated with the same TCR.