Target Antigen For Immunotherapy Research Articles

A T-cell receptor as a promising immunotherapeutic target in acute myeloid leukemia: expression of T cell immunoglobulin and mucin-containing-3 on bone marrow hematopoietic cells

Background The emergence of immunotherapy has changed the treatment landscape of various types of cancers. However, limited target antigen remains to be a challenge for its application in acute myeloid leukemia (AML). T cell immunoglobulin and mucin-containing-3 (Tim-3) is an immune checkpoint receptor that plays a major role in the escape of host immune surveillance in AML. Thus, its role in cancer treatment needs to be thoroughly investigated. Methods Tim-3 expression on blasts, lymphocytes, and monocytes was analyzed by flow cytometry in bone marrow samples of 60 newly diagnosed AML patients and 30 matched non-neoplastic controls. Its association with different prognostic clinicopathological parameters was also evaluated. ROC curve confirmed the value of Tim-3 expression in discriminating AML patients from healthy controls and the role of Tim-3 in disease aggressiveness was also investigated. Results The median percentage of myeloblasts positive for Tim-3% and MFI were significantly higher in AML than normal controls (64.27% and 1.34 in patients vs. 41.89% and 1.12 in controls) and it was markedly associated with poor overall survival of the patients. Besides, the upregulation of Tim-3 on AML blasts was significantly higher than on lymphocytes and monocytes in denovo AML patients (P <0.001). Conclusion This study indicates Tim-3 as a poor prognostic marker in AML and highlights Tim-3 as a new therapeutic target antigen for immunotherapy with low off-target toxicity.

The combined HPV16-E2/E6/E7 T cell response in oropharyngeal cancer predicts superior survival

Tumor-infiltrating HPV16-E2-specific CD8+ Tcells have been detected in HPV16-induced oropharyngeal squamous cell carcinoma (OPSCC). Whether intratumoral CD4+ Tcells target HPV16 E2 and if HPV16-E2-specific immunity contributes to better clinical outcome is unknown. In a prospective HPV16+ OPSCC cohort, we regularly detect HPV16-E2-specific CD4+ and CD8+ intratumoral Tcells, albeit at lower frequencies than the co-infiltrating HPV16-E6/E7-specific Tcells. These HPV16-reactive Tcells produce multiple cytokines when activated, indicating their polyfunctionality. Importantly, their combined intratumoral presence predicts superior survival, emphasizing the value of HPV16-E2-specific Tcells in anti-tumor immunity and suggests its use as a target antigen for immunotherapy.

B7-H3-targeting Fc-optimized antibody for induction of NK cell reactivity against sarcoma.

Natural killer (NK) cells largely contribute to antibody-dependent cellular cytotoxicity (ADCC), a central factor for success of monoclonal antibodies (mAbs) treatment of cancer. The B7 family member B7-H3 (CD276) recently receives intense interest as a novel promising target antigen for immunotherapy. B7-H3 is highly expressed in many tumor entities, whereas expression on healthy tissues is rather limited. We here studied expression of B7-H3 in sarcoma, and found substantial levels to be expressed in various bone and soft-tissue sarcoma subtypes. To date, only few immunotherapeutic options for treatment of sarcomas that are limited to a minority of patients are available. We here used a B7-H3 mAb to generate chimeric mAbs containing either a wildtype Fc-part (8H8_WT) or a variant Fc part with amino-acid substitutions (S239D/I332E) to increase affinity for CD16 expressing NK cells (8H8_SDIE). In comparative studies we found that 8H8_SDIE triggers profound NK cell functions such as activation, degranulation, secretion of IFNγ and release of NK effector molecules, resulting in potent lysis of different sarcoma cells and primary sarcoma cells derived from patients. Our findings emphasize the potential of 8H8_SDIE as novel compound for treatment of sarcomas, particularly since B7-H3 is expressed in bone and soft-tissue sarcoma independent of their subtype.

Research Progress in Alpha-fetoprotein-induced Immunosuppression of Liver Cancer.

Liver cancer is one of the most common malignant tumors, with limited treatment and 8.2% mortality. Liver cancer is the fourth leading cause of cancer-related deaths, which seriously endangers human life and health. Approximately 70% of liver cancer patients show increased serum Alpha- Fetoprotein (AFP) levels. AFP is the main diagnostic and prognostic indicator of liver cancer. AFP, a key marker of liver cancer, plays a crucial role in regulating the proliferation of tumor cells, apoptosis, and induction of cellular immune escape. High levels of AFP during embryonic development protect the embryos from maternal immune attack. AFP also promotes immune escape of liver cancer cells by inhibiting Tumor-Infiltrating Lymphocytes (TILs), Natural Killer cells (NK), Dendritic Cells (DC), and macrophages; thus, it is also used as a target antigen in immunotherapy for liver cancer. AFP is highly expressed in liver cancer cells. In addition to being used in the diagnosis of liver cancer, it has become a target of immunotherapy for liver cancer as a tumor-associated antigen. In immunotherapy, it was also confirmed that early AFP response was positively correlated with the efficacy of immunotherapy. Early AFP responders had longer PFS and OS than non-responders. At present, the methods of immunotherapy for liver cancer mainly include Adoptive Cell Transfer Therapy (ACT), tumor vaccine therapy, immune checkpoint inhibitors (ICIs) therapy, etc. A large number of studies have shown that AFP mainly plays a role in ACT and liver cancer vaccines. This review presents the research progress of AFP and immunosuppression of liver cancer.

Abstract 2078: CLN-049 is a bispecific T cell engaging IgG-like antibody targeting FLT3 on AML cells

Abstract Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, with few options for curative treatment. In lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable success. However, such immunotherapeutic strategies are not yet established for AML, likely due to a dearth of appropriate targets. The fms like tyrosine kinase 3 (FLT3)/CD135 surface receptor is expressed on AML cells in the large majority of AML patients and constitutes a highly selective target antigen for immunotherapy, as expression on healthy tissues is limited to low levels on dendritic cells, monocytes and hematopoietic progenitor cells. We report here on the development of CLN-049, a bispecific humanized antibody that binds to FLT3 on leukemic cells and the CD3 epsilon subunit of the T cell receptor complex on T cells. CLN-049 recognizes the membrane proximal domain of FLT3 and has a silenced Fc gamma 1 domain, with anti-CD3 single chain variable fragments (scFv) fused to the C-termini of the heavy chains. While FLT3 kinase inhibitors are limited to treatment of patients with specific intracellular FLT3 mutations, CLN-049 recognizes the extracellular domain of FLT3 and would therefore be more broadly applicable in AML therapy. CLN-049 showed low-nanomolar binding to both FLT3-expressing and CD3-expressing cells. The antibody induced target-dependent activation of CD4+ and CD8+ T cells as well as corresponding cytokine production by T cells only when co-cultured with FLT3+ AML cells. Human AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed upon treatment with sub-nanomolar concentrations of CLN-049 when co-cultured with heterologous PBMCs, regardless of the expression level or mutational status of FLT3. Intriguingly, FLT3-expressing hematopoietic progenitor cells and dendritic cells were not found to be sensitive to CLN-049 killing. The in vitro lysis of leukemic cells by CLN-049 was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft studies, CLN-049 was highly active against the MOLM-13 human AML model. In these studies, CLN-049 demonstrated significant dose-dependent activity as measured by survival as well as reduction of AML cells in the blood. In summary, CLN-049 is a promising FLT3-targeted T cell engaging antibody construct expected to have robust anti-tumor activity in the clinic against AML. CLN-049 is currently in a phase 1 clinical trial for the treatment of patients with relapsed/refractory AML. Citation Format: Kristan Meetze, Naveen K. Mehta, Martin Pfluegler, Bochong Li, Patrick A. Baeuerle, Jennifer S. Michaelson, Gundram Jung, Helmut Salih. CLN-049 is a bispecific T cell engaging IgG-like antibody targeting FLT3 on AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2078.

AFM26 - Targeting B Cell Maturation Antigen (BCMA) for NK Cell-Mediated Immunotherapy of Multiple Myeloma

Significant progress in multiple myeloma (MM) treatment has been made in recent years and achieving sustained minimal residual disease (MRD) negativity now constitutes a major goal of treatment. High dose melphalan combined with autologous stem cell transplantation (ASCT) is currently regarded the standard of care in 1st line treatment of MM in eligible patients, but fails to eradicate MRD in the majority of patients ultimately leading to disease relapse. Consequently, additional therapies are needed to target residual disease to delay or potentially prevent relapse.The cell surface antigen B cell maturation antigen (BCMA, CD269) constitutes a highly attractive target antigen for immunotherapy because of its restricted expression in non-malignant tissue but almost universal expression on MM cells.AFM26 is a BCMA-targeted tetravalent bispecific antibody that selectively engages natural killer (NK) cells to induce myeloma cell lysis. Through its high-affinity bivalent binding to CD16A (FcγRIIIa), AFM26 possesses prolonged cell retention times, and has been shown to be less prone to interference by high levels of circulating IgG than antibody Fc-domains. Thus, AFM26 can be used to “arm” NK cells for effective killing of tumor cells. AFM26 also induces markedly lower levels of pro-inflammatory cytokines in peripheral blood mononuclear cell (PBMC) cultures in the presence of target cells compared to BCMA-directed T cell activating approaches. In contrast to other monoclonal antibodies (mAbs) such as daratumumab and elotuzumab, AFM26 does not confer target-independent NK cell activation or NK cell depletion.NK cells are promising effectors to target MRD. Due to their early reconstitution following ASCT and their ability to rapidly destroy malignant cells through direct cytotoxicity. Redirecting NK cells through AFM26 may therefore offer an effective treatment of MM patients early after ASCT.Employing a wide range of functional assays, we demonstrate here that AFM26 exhibits potent cytotoxic activity against primary myeloma cells using patient (autologous) and donor-derived (allogeneic) NK cells as well as against a panel of high and low BCMA expressing cell lines. In particular, we show that AFM26 induces NK cell cytotoxicity towards cells expressing almost undetectable surface levels of BCMA, suggesting the potential for broad and deep anti-MM activity. In summary, AFM26 is well-differentiated from other mAbs and a promising therapeutic candidate to address the unmet medical need of MRD positivity in MM. DisclosuresGantke:Affimed: Employment. Reusch:Affimed: Employment. Kellner:Affimed: Research Funding. Klausz:Affimed: Research Funding. Haneke:Affimed: Employment. Knackmuss:Affimed: Employment. Müller:Affimed: Employment. Ellwanger:Affimed: Employment. Fucek:Affimed: Employment. Schniegler-Mattox:Affimed: Employment. Koch:Affimed: Employment. Kerber:Affimed: Employment. Peipp:Affimed: Research Funding. Treder:Affimed: Employment.

First-in-Human Phase I Dose Escalation and Expansion Study Evaluating the Fc Optimized FLT3 Antibody Flysyn in Acute Myeloid Leukemia Patients with Minimal Residual Disease

Even when achieving morphological complete remission (CR) after induction therapy, roughly half of acute myeloid leukemia (AML) patients display measurable residual disease (MRD) and eventually relapse. The surface receptor FLT3/CD135 is expressed on AML cells in almost all patients and constitutes a highly selective target antigen for immunotherapy, as expression on healthy tissues is limited to low levels on dendritic cells, monocytes and hematopoietic progenitor cells. FLYSYN is a chimeric Fc-optimized IgG1 antibody that binds specifically and with high avidity to human FLT3 (CD135). Here we report updated results of an open-label, single-arm, first in man multicenter trial (recruitment March 2017 to March 2020) evaluating safety/tolerability and preliminary efficacy of FLYSYN in patients with AML (NCT02789254). Morphological CR with stable or increasing MRD in two sequential measurements using central RT-qPCR and/or next generation sequencing (NGS) constituted the main inclusion criterion. FLYSYN was administered i.v. over 3 h as single application in cohorts 1-5 (0.5 mg/m², 1.5 mg/m², 5 mg/m², 15 mg/m², 45 mg/m²); in cohort 6, 15 mg/m² were applied on day 1, 15 and 29. Three patients were treated per cohort except for cohorts 4 and 6, which were expanded to comprise 9 and 10 patients, respectively. Molecular response was defined as >1 log MRD reduction or negativity in bone marrow (BM). In total, 31 patients (median age 58 years; range, 21-80 years; male:female ratio: 1:1.8) were enrolled, of which 27, 3 and 1 were MRD-positive for mutated NPM1, mutated IDH2 and RUNX1-RUNX1T1, respectively. Based on pharmacokinetic analysis, the half-life of FLYSYN was estimated to be 6.5 days. In 8 patients (26%), a transient decrease of neutrophil count (2 adverse events (AEs) grade 3, others ≤ grade 2) was observed. No relevant effect on stem cell reserve as assessed by colony forming unit assays was detected in the so far analyzed 26 patients. No other AE > grade 2 or dose-limiting toxicity were observed. The most frequent AEs were unspecific and comprised fatigue and flu like symptoms (12%), musculoskeletal symptoms (8%) and laboratory abnormalities (42%). With regard to efficacy, molecular response to treatment was achieved in 11/31 patients (35%), with so far two patients achieving MRD negativity documented one year after treatment. Among the patients receiving 45 mg/m2 FLYSYN (in total, upon single or repetitive dosing), objective responses were achieved in 46% (6/13) cases, whereas 28% (5/18) responded to treatment with lower doses. Together, the results of our phase I trial demonstrate that FLYSYN is safe and very well tolerated as monotherapy in AML patients with molecular MRD. Early efficacy data are promising and warrant further evaluation in an up-coming phase II clinical trial. Disclosures Heuser: PriME Oncology: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Stemline Therapeutics: Consultancy; Janssen: Consultancy; Astellas: Research Funding; Roche: Research Funding; BerGenBio ASA: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Amgen: Research Funding; Karyopharm: Research Funding; Abbvie: Consultancy. Thol:Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kapp-Schwoerer:Jazz Pharmaceuticals: Honoraria, Research Funding. Grosse-Hovest:Synimmune: Current Employment. Steiner:Synimmune: Current Employment. Schlenk:Roche: Research Funding; Novartis: Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accomodations, Expenses, Research Funding, Speakers Bureau; PharmaMar: Research Funding; AstraZeneca: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Salih:Pfizer: Consultancy; Medigene: Consultancy; Novartis: Consultancy; Philogen: Consultancy; Synimmune: Consultancy, Research Funding.

MUC16 affects the biological functions of ovarian cancer cells and induces an antitumor immune response by activating dendritic cells.

BackgroundOvarian cancer is the 5th most common lethal gynecological malignancy with a 5-year survival rate of about 47% and a localized stage diagnosis of 15%, leading to about 125,000 global deaths each year. Therefore, it is urgent to explore novel and effective strategies for radical cure.MethodsShort hairpin RNA targeting the Mucin16 (MUC16) gene was used to establish MUC16 knockdown in ovarian cancer cells. RT-PCR was performed to quantify the expression of MUC16 mRNA, and western blotting was performed to detect the expression of MUC16 and epithelial-mesenchymal transition-related proteins. Cell counting kit 8 (CCK8) wound healing and transwell assays were performed to assess cell proliferation and cell invasion. Flow cytometry was used to detect CD80-, CD83-, and CD86-expressing dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs) activated by MUC16-pulsed DCs.ResultsIn this study, we identified MUC16 as a novel target antigen for immunotherapy against ovarian cancer, which was significantly up regulated in ovarian cancer cells and high-grade ovarian serous adenocarcinoma tissues. MUC16 knockdown in Ovcar3 cells using short hairpin RNA targeting the MUC16 gene suppressed the proliferation of migration, invasion, epithelial-mesenchymal transition (EMT), and PI3K/Akt signaling pathway in Ovcar3 cells markedly. MUC16 significantly up-regulated CD80, CD83, and CD86 (mature makers) expression in DCs and T-cell transformation into CD8+ T-cells detected by Flow cytometry.ConclusionsFor malignant ovarian cancer, MUC16 overexpression promoted cell proliferation, migration, and invasion via the PI3K/AKT signaling pathway. MUC16 pulsing mediated DC maturation and activated CTL response in vitro. Our study offers promising DC-based immunotherapy of considerable clinical value for patients with ovarian cancer.

GD2-targeted chimeric antigen receptor T cells prevent metastasis formation by elimination of breast cancer stem-like cells

ABSTRACT Expression of the disialoganglioside GD2 has been identified as a marker antigen associated with a breast cancer stem-like cell (BCSC) phenotype. Here, we report on the evaluation of GD2 as a BCSC-specific target antigen for immunotherapy. GD2 expression was confirmed at variable degree in a set of breast cancer cell lines, predominantly in triple-negative breast cancer (TNBC). To target GD2, we have generated novel anti-GD2 chimeric antigen receptors (GD2-CAR), based on single-chain variable fragments (scFv) derived from the monoclonal antibody (mAb) ch14.18, also known as dinutuximab beta. Expressed on T cells, GD2-CARs mediated specific GD2-dependent T-cell activation and target cell lysis. In contrast to previously described GD2-CARs, no signs of exhaustion by tonic signaling were found. Importantly, application of GD2-CAR expressing T cells (GD2-CAR-T) in an orthotopic xenograft model of TNBC (MDA-MB-231) halted local tumor progression and completely prevented lung metastasis formation. In line with the BCSC model, GD2 expression was only found in a subpopulation (4-6%) of MDA-MB-231 cells before injection. Significant expansion of GD2-CAR-T in tumor-bearing mice as well as T-cell infiltrates in the primary tumor and the lungs were found, indicating site-specific activation of GD2-CAR-T. Our data strongly support previous findings of GD2 as a BCSC-associated antigen. GD2-targeted immunotherapies have been extensively studied in human. In conclusion, GD2-CAR-T should be considered a promising novel approach for GD2-positive breast cancer, especially to eliminate disseminated tumor cells and prevent metastasis formation.

Prediction and identification of human leukocyte antigen-A2-restricted cytotoxic T lymphocyte epitope peptides from the human papillomavirus 58 E7 protein.

Persistent infection with high-risk human papilloma virus (HPV) is the primary cause of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. HPV58 is the third most common HPV genotype in China after HPV16 and HPV18. HPV E6 and E7 are oncoproteins and are constitutively expressed in HPV-associated cancer cells, therefore they are considered to be ideal target antigens for immunotherapy, including HPV therapeutic vaccine. In the present study, human leukocyte antigen (HLA)-A2-restricted cytotoxic T lymphocyte (CTL) epitope peptides were predicted and screened from HPV58 E7 antigen and their immunogenicity was subsequently determined. A total of 6 HLA-A2-binding peptides derived from HPV58 E7 were predicted and selected using 3 different prediction programs. A negative control peptide and PBS were used as two negative controls. Peripheral blood mononuclear cells (PBMCs) with HLA-A2(+) allele were used to detect the specific cellular immune response among the 6 predicted peptides by enzyme-linked immunospot assay (ELISOPT). Following preliminary screening for the predicted peptides, the antigenicity of the peptide HPV58 E772-80 was further assessed by an immunoassay to a vaccine contained HPV58 E7 antigen. Specific humoral and cellular immunity were detected using the peptide HPV58 E772-80 as the specific antigen. A total of 6 peptides from HPV58 E7 protein were predicted and subsequently named P1 (E77-15: TLREYILDL), P2 (E714-22: DLHPEPTDL), P3 (E769-77: CINSTTTDV), and P4 (E772-80: STTTDVRTL), P5 (E779-87: TLQQLLMGT) and P6 (E783-91: LLMGTCTIV). In the ELISPOT assay on HLA-A2 (+) human PBMCs, interferon (IFN)-γ-production was evident in the P2 and P4 groups. The average numbers of IFN-γ associated spots in the P2 and P4 groups was 50.61±5.37 spot-forming cells (SFC)/1×105 and 266±34.42 SFC/1×105, respectively. The numbers of spots in the two peptides were significantly increased compared with the other 4 peptides and the control groups (P<0.05). In the further antigenicity verification of P4 (HPV58 E772-80), the peptide only stimulated the humoral immune response of the AD-HPV16/18/58 mE6E7 vaccine containing HPV58 E7 antigen. Compared with the 2 negative control groups (1:400), the antibody titers of the vaccine group (1:25,600) were significantly increased (P<0.05). In cellular immunoassays the average number of IFN-γ associated spots was 143.3±32.13 SFC/1×105 in the vaccine group, which was significantly enhanced compared with the PBS group (8±5.29 SFC/1×105; P<0.01) and the AD-NC group (28±5.13 SFC/1×105; P<0.01). The peptide HPV58 E772-80 (STTTDVRTL) displayed sufficient antigenicity to a vaccine contained HPV58 E7 antigen. Therefore, HPV58 E772-80 peptide may be considered as a candidate epitope peptide for the construction of HPV58 peptide vaccines.

Back Cover: Immunopeptidomic Profiling of HLA‐A2‐Positive Triple Negative Breast Cancer Identifies Potential Immunotherapy Target Antigens

DOI: 10.1002/pmic.201700465 Immunopeptidomic profiling of biopsies from HLA-A2-positive triple negative breast cancer patients identifies a high number of shared tumor-associated HLA-A2 peptide antigens. Calculation of the average protein tumor-associated cohort coverage (aTaCC) shortlists antigens that are presented with higher abundance in tumor tissue than in normal tissue and may prioritize the selection of target antigens for immunotherapy. This is reported in article number 1700465 by Ternette et al.

Vaccination with NY-ESO-1 in Combination with Decitabine for Patients with MDS

Background: The identification of suitable target antigens for immunotherapy in myeloid cancer has been a challenge. NY-ESO-1 is a recognized target in solid tumors, but in myeloid cancers expression is silenced by dense promoter hypermethylation. We have demonstrated standard dose decitabine induced expression of NY-ESO-1 in the circulating blasts of AML patients. The level of expression was sufficient to trigger a cytotoxic response in NY-ESO-1 specific CD8+ T cells. We have previously reported our preliminary data demonstrating that vaccination against NY-ESO-1 in combination with decitabine is safe and resulted in NY-ESO-1 specific cellular immune responses in the first six treatment-na•ve myelodysplatic syndrome (MDS) patients on study. We now update our results following complete study accrual.Methods: We performed a phase I trial of NY-ESO-1 vaccine (anti-DEC-205-NY-ESO-1 fusion protein (CDX-1401) with poly-ICLC adjuvant; Celldex Therapeutics) in combination with decitabine (20 mg/m2/day x 5 days). Patients were enrolled on an IRB-approved protocol. All patients signed informed consent and were treated in accordance with the Declaration of Helsinki. Enrolled patients had intermediate/high-risk MDS by IPSS, were > 18 years old, had ECOG performance status < 2, and adequate hepatic and renal function. Patients with uncontrolled medical illness, HIV-positivity, auto-immunity or recent corticosteroid/radiation therapy were excluded. Patients were vaccinated on day -14, received decitabine on day 1 and were re-vaccinated on day 14 of each cycle. Four cycles of therapy were planned. Peripheral blood was obtained pre-treatment, twice weekly, and at end of treatment (EOT). CD11b+ myeloid cells were isolated from each sample. Immune monitoring was performed at baseline and end of treatment.Results: Nine patients (median age 64y, range 57-71) have been enrolled and treated on study; all have completed therapy. As previously reported, adverse events were decitabine and disease related. We quantified LINE-1 (surrogate for global methylation) and NY-ESO-1 promoter methylation in the CD11b+ myeloid population throughout treatment. The methylation nadir for LINE-1 and NY-ESO-1 occurred between days 8 and 18 of each decitabine cycle. Changes in LINE-1 and NY-ESO-1 methylation were correlated for eight patients (R2 = 0.91-0.99, p < 0.0001). We observed that 7/9 patients demonstrated induced expression NY-ESO-1 mRNA (by nested RT-PCR) in CD11b+ cells. There was no baseline humoral immunity to NY-ESO-1. Seroconversion was observed in two subjects, both of whom demonstrated induced NY-ESO-1 expression. 7/9 and 5/9 patients demonstrated NY-ESO-1 specific CD4+ and CD8+ T-cell responses respectively following vaccination. There were no significant changes in the frequency of CD25+ FOXP3 positive T-regulatory cells from baseline to the end of therapy. NY-ESO-1 specific T-cell recognition of myeloid cells with induced antigen expression will be analyzed.Conclusion: Circulating myeloid cells exhibit decreased global and NY-ESO-1 promoter methylation. A majority of treated patients show induction of NY-ESO-1 mRNA in the myeloid compartment with vaccination induced CD4+ and CD8+ T-cell responses in a majority of patients. Patients without evidence of induced expression in circulating myeloid cells had poor cellular immune response to vaccination. These data indicate that MDS patients receiving standard decitabine may benefit from immunological targeting of reactivated NY-ESO-1. [Display omitted] DisclosuresKarpf:Astex Pharmaceuticals: Research Funding. Wang:Incyte: Speakers Bureau; Immunogen: Research Funding. Griffiths:Astex Pharmaceuticals: Consultancy, Research Funding; Celgene Inc: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Honoraria.

A novel approach for targeted elimination of CSPG4-positive triple-negative breast cancer cells using a MAP tau-based fusion protein.

Chondroitin sulfate proteoglycan 4 (CSPG4) has been identified as a highly promising target antigen for immunotherapy of triple-negative breast cancer (TNBC). TNBC represents a highly aggressive heterogeneous group of tumors lacking expression of estrogen, progesterone and human epidermal growth factor receptor 2. TNBC is particularly prevalent among young premenopausal women. No suitable targeted therapies are currently available and therefore, novel agents for the targeted elimination of TNBC are urgently needed. Here, we present a novel cytolytic fusion protein (CFP), designated αCSPG4(scFv)-MAP, that consists of a high affinity CSPG4-specific single-chain antibody fragment (scFv) genetically fused to a functionally enhanced form of the human microtubule-associated protein (MAP) tau. Our data indicate that αCSPG4(scFv)-MAP efficiently targets CSPG4(+) TNBC-derived cell lines MDA-MB-231 and Hs 578T and potently inhibits their growth with IC50 values of ∼200 nM. Treatment with αCSPG(scFv)-MAP resulted in induction of the mitochondrial stress pathway by activation of caspase-9 as well as endonuclease G translocation to the nucleus, while induction of the caspase-3 apoptosis pathway was not detectable. Importantly, in vivo studies in mice bearing human breast cancer xenografts revealed efficient targeting to and accumulation of αCSPG4(scFv)-MAP at tumor sites resulting in prominent tumor regression. Taken together, this preclinical proof of concept study confirms the potential clinical value of αCSPG4(scFv)-MAP as a novel targeted approach for the elimination of CSPG4-positive TNBC.

NY-ESO-1 Vaccination in Combination with Decitabine for Patients with MDS Induces CD4+ and CD8+ T-Cell Responses

Background: Identification of suitable target antigens for immunotherapy has been a challenge in patients with myeloid malignancies. NY-ESO-1 has been identified as an immunotherapeutic target in solid tumors. Its use in myeloid cancer is limited due to silencing by dense promoter hypermethylation. We and others have demonstrated that decitabine can induce expression of NY-ESO-1 in leukemia cell lines. We hypothesized that vaccination against NY-ESO-1 in combination with decitabine would be safe and result in NY-ESO-1 expression sufficient to induce NY-ESO-1 specific humoral and cellular immune responses in treatment-na•ve myelodysplastic syndrome (MDS) patients.Methods: We performed a phase I trial of NY-ESO-1 vaccine (anti-DEC-205-NY-ESO-1 fusion protein (CDX-1401) with poly-ICLC adjuvant; Celldex Therapeutics) in combination with decitabine (20 mg/m2/day x 5 days). Patients with intermediate/high-risk MDS by IPSS, > 18 years old, ECOG performance status < 2, and adequate hepatic and renal function were enrolled on an IRB-approved protocol (median age 64y). Patients with uncontrolled medical illness, HIV-positivity, auto-immunity or recent corticosteroid/radiation therapy were excluded. All patients signed informed consent and were treated in accordance with the Declaration of Helsinki. Patients were vaccinated on day -14, received decitabine on day 1 and were re-vaccinated on day 14 of each cycle. Four cycles of therapy were planned. Peripheral blood was obtained pre-treatment, twice weekly, and at end of treatment (EOT). CD11b+ myeloid cells were isolated from each sample. Immune monitoring was performed at baseline and at EOT. An interim analysis, pre-specified in the protocol for the first 6 patients, was planned for safety and immunology endpoints. Three additional patients enrolled to an expansion cohort to ensure sufficient power for correlative studies remain on treatment.Results: Analysis of the initial safety cohort showed no unexpected toxicities. The most frequent adverse events were related to decitabine and included cytopenias (predominantly grades 3/4), elevated liver enzymes (grade 3), fatigue (grade 2), edema (grade 2/3) and diarrhea (grade 1/2). Two patients did not complete four cycles of therapy due to serious adverse events; 1 patient with a history of myocardial infarction (MI) developed in-stent restenosis and recurrent MI; a second patient suffered a terminal intracranial hemorrhage due to thrombocytopenia (deemed decitabine related). LINE-1 (surrogate for global methylation) and NY-ESO-1 promoter methylation in the CD11b+ myeloid population were serially quantified for the first 2 patients by bisulfite pyrosequencing. The methylation nadir for LINE-1 and NY-ESO-1 occurred between days 8 and 15 of each decitabine cycle. Changes in LINE-1 and NY-ESO-1 methylation were correlated for each patient (R2 = 0.95, p < 0.001). Expression of NY-ESO-1 mRNA (by nested RT-PCR) was performed on CD11b+ cells from days 0, 8, 15, and 22 of the first cycle for these two patients. Patient 1 exhibited NY-ESO-1 mRNA on days 8 and 15. Patient 2 did not show any NY-ESO-1 expression. Of the first 6 patients analyzed, none showed baseline humoral immunity to NY-ESO-1 and seroconversion was observed in one subject (Table 1). Five patients had induced NY-ESO-1 specific CD4+ T-cell responses and 4 patients had NY-ESO-1 specific CD8+ T-cell responses following vaccination.Table 1Response to Vaccination. T cell response assessed by ELISPOT for IFN-g and scored after subtracting background. Numbers in parentheses indicate number of epitopes recognized by T cells. Bold type indicates responses induced or enhanced by vaccination.Patient IDAntibody responseCD4+ T cell responseCD8+ T cell responsePrePostPrePostPrePost1-+++ (2)+++ (3)- (0)+ (1)2--- (0)+ (2)- (0)- (0)3--- (0)+ (2)- (0)+ (1)4--- (0)+ (1)- (0)- (0)5--- (0)+ (1)- (0)+ (2)6--+ (1)- (0)- (0)++ (3)Conclusion: Vaccination against NY-ESO-1 is safe in combination with decitabine. Circulating myeloid cells exhibited decreased NY-ESO-1 promoter methylation. 1 of 2 sampled patients demonstrated induction of NY-ESO-1 mRNA in the myeloid compartment. Vaccination successfully induced CD4+ and CD8+ T-cell responses in a majority of patients. These data indicate that vaccination against NY-ESO-1 in combination with decitabine is feasible, opening the door for future studies targeting this induced antigen in MDS. DisclosuresWang:Immunogen: Research Funding. Griffiths:Celgene: Honoraria; Alexion Pharmaceuticals: Honoraria; Astex: Research Funding.

The Fc-alpha receptor is a new target antigen for immunotherapy of myeloid leukemia.

Antibody-based immunotherapy of leukemia requires the targeting of specific antigens on the surface of blasts. The Fc gamma receptor (CD64) has been investigated in detail, and CD64-targeting immunotherapy has shown promising efficacy in the targeted ablation of acute myeloid leukemia (AML), acute myelomonocytic leukemia (AMML) and chronic myeloid leukemia cells (CML). Here we investigate for the first time the potential of FcαRI (CD89) as a new target antigen expressed by different myeloid leukemic cell populations. For specific targeting and killing, we generated a recombinant fusion protein comprising an anti-human CD89 single-chain Fragment variable and the well-characterized truncated version of the potent Pseudomonas aeruginosa exotoxin A (ETA'). Our novel therapeutic approach achieved in vitro EC50 values in range 0.2-3 nM depending on the applied stimuli, that is, interferon gamma or tumor necrosis factor alpha. We also observed a dose-dependent apoptosis-mediated cytotoxicity, which resulted in the elimination of up to 90% of the target cells within 72 hr. These findings were also confirmed ex vivo using leukemic primary cells from peripheral blood samples of three previously untreated patients. We conclude that CD89-specific targeting of leukemia cell lines can be achieved in vitro and that the efficient elimination of leukemic primary cells supports the potential of CD89-ETA' as a potent, novel immunotherapeutic agent.

Rapid monitoring of reconstituted Wilms' tumor suppressor gene-specific t lymphocytes after allogeneic stem cell transplantation using quantitative real time Polymerase chain reaction

Meeting abstracts Wilms' tumor suppressor gene (WT1) protein is over-expressed in some leukemias and various types of solid tumors, and it is considered to be an attractive target antigen for immunotherapy. After ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) from 12 patients

WT1-Specific T Cells Exhibiting a Non-Exhausted, Functional Phenotype Can Be Generated From The Natural Repertoire Of Healthy Donors For Clinical Use

Background The Wilms tumor antigen 1 (WT1) is a self-antigen expressed at high levels in leukemic cells, but not in healthy tissue. As WT1 expression in leukemic cells drives leukemogenesis, it is a favorable target antigen for immunotherapy, e.g. adoptive transfer of allogeneic T cells, to prevent or treat leukemic relapse after stem cell transplantation (Cheever et al., Clin Cancer Res 2009;15(17)). WT1-specific CD8+ T cells have been detected in healthy individuals at low frequencies (Rezvani et al., Blood 2003;102). However, a comprehensive characterization of CD4+ and CD8+WT1-specific T cells is missing and the efficient expansion of a polyclonal WT1-reactive T cell population for clinical use has remained a major challenge. In this study we aim to directly ex vivo characterize WT1-specific T cells present in the blood of healthy donors at high-resolution and to develop a rapid method for the generation of functionally potent, polyclonal CD4+ and CD8+WT1-specific T cells for clinical use. Methods For direct ex vivo analysis of CD4+ WT1-specific T cells peripheral blood mononuclear cells (PBMC) of healthy blood donors were in vitro stimulated with a pool of overlapping peptides spanning the WT1 protein for 7 hours. Subsequently CD154 (CD40L)-expressing cells were magnetically enriched and flow cytometrically examined for expression of effector cytokines and their differentiation status. Presence and phenotype of CD8+ WT1-specific T cells have been studied after stimulation of presorted naïve and memory T cell populations with WT-1 peptide pool for 30 hours, magnetic enrichment of CD137+ (4-1BB) cells and subsequent staining using pMHCI-Tetramers. For the generation of polyclonal WT1-specific CD4+ and CD8+ T cells PBMC were in vitro activated with WT-1 peptide pool for 30 hours. CD137+cells were magnetically selected and expanded for 9 days in the presence of the cytokines IL-7, IL-15 and IL-21 at low doses. Expanded T cells were analyzed for their phenotype, the expression of co-stimulatory and exhaustion markers and were tested for their functionality and cytotoxicity by restimulation experiments with antigen-loaded target cells. Results Ex vivo frequencies of WT1-specific T cells are low, 1 to 10 WT1-specific CD154+ CD4+ T cells can be detected within 1x106 CD4+ T cells. In about 80% of healthy donors (n=15) a CD4+ memory response, accompanied by production of effector cytokines like IFNγ, TNFα and IL-2, against WT1 peptides is present. Additionally, in all donors naïve WT1-specific CD4+ T cells can be detected. In contrast, detected CD137+CD8+ WT1-reactive T cells exhibit a naïve phenotype (CD45RA+CCR7+) in all donors (n=5), no WT1-reactive CD8+T cells could be enriched from presorted memory T cells. To evaluate the usefulness of our improved short-term expansion protocol to generate potent WT1-specific T cell cultures for clinical use, we characterized CD137 enriched and expanded T cells. Notably, a high frequency of CD4+ and CD8+ T cells show specific reactivity against WT1-presenting autologous cells as detected by production of effector cytokines like IFNγ, TNFα and IL-2 after antigen-specific restimulation. Cytotoxic activity against antigen-loaded target cells could be shown by direct flow-cytometry-based cytotoxicity assays and antigen-specific upregulation of the degranulation marker CD107a. Stainings using multiple WT1-MHCI-tetramers furthermore confirmed antigen-specificity and suggested polyclonality within the CD8+T cell population. In contrast to previous expansion protocols our polyclonally expanded T cells exhibit a favourable, unexhausted memory phenotype, express co-stimulatory markers CD27 and CD28 and the IL7R-a chain (CD127) which has been shown to mark cells with stem T cell like properties. Furthermore exhaustion markers like CD279 (PD-1), CD178 (FasL) and CD57 are scarcely expressed. Conclusions Functional, polyclonal, CD4+ and CD8+ WT1-specific, reactive T cells can be efficiently enriched directly ex vivo from the natural repertoire by magnetic separation of T cells after antigen-specific stimulation. Phenotypic and functional characterization revealed a non-exhausted phenotype of expanded WT1-specific T cells, thereby suggesting good persistence and functionality of the obtained T cell product in vivo. Thus, our approach holds great potential for the GMP-compliant generation of WT1-specific T cells for future clinical use. Disclosures: Schmied: Miltenyi Biotec GmbH: Employment. Richter:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec: Employment.

Abstract 3966: Development of a fully-human chimeric antigen receptor against PSCA for the treatment of pancreatic cancer.

Abstract Pancreatic cancer represents the fourth leading cause of cancer-related death in developed countries. Despite remarkable advances in the understanding of its molecular pathophysiology, treatment options remain scarce, highlighting the need for novel therapies. Adoptive cell transfer (ACT) of lymphocytes engineered ex vivo to express chimeric antigen receptors (CAR) has come of age as a safe and clinically efficacious treatment for advanced cancers. We sought to develop a CAR specific for prostate stem cell antigen (PSCA), a glycoprotein with restricted expression in normal tissues that is over-expressed in pancreatic cancer cells starting at early stages of malignant transformation. In order to establish the optimal CAR design, we generated constructs containing antigen-recognition domains derived from mouse or human antibodies, and intracellular domains containing one or two co-stimulatory domains, in addition to CD3zeta. We compared multiple constructs and found that a CAR based on human monoclonal antibody Ha1-4.117 had greater reactivity in vitro. Using a murine model of ACT we observed that CAR-engineered human lymphocytes induced an antitumor effect on HPAC xenografts. Interestingly, unlike what has been described for other CARs, a second generation PSCA CAR (containing CD28 signaling domain) induced a more potent antitumor effect than its third generation counterpart (containing CD28 and 41BB domains). Our results provide evidence to support PSCA as a target antigen for immunotherapy of pancreatic cancer, using adoptively transferred autologous lymphocytes engineered to express an Ha1-4.117-based CAR. Citation Format: Daniel Abate-Daga, Kiran H. Lagisetty, Luca Gattinoni, Zhiya Yu, Nicholas P. Restifo, Steven A. Rosenberg, Richard A. Morgan. Development of a fully-human chimeric antigen receptor against PSCA for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3966. doi:10.1158/1538-7445.AM2013-3966

A recombinant adenovirus vector encoding the light chain of human coagulation factor VII and IgG1 Fc fragment to targeting tissue factor for colorectal cancer immunotherapy in the mouse model

Over expression of tissue factor (TF) occurs in more than 50 % of colorectal cancer (CRC). Therefore, TF represents an attractive target antigen for immunotherapy in CRC. Here, we assessed the safety and efficacy of a recombinant adenovirus vector encoding the light chain of human coagulation factor VII (hfVII-LC) and human IgG1 Fc fragment (hIgG1-Fc), termed "benc vector," by targeting TF in the mouse model with colon cancer. Benc vector was administered intravenously or intratumorally in SCID mice with TF over-expressing HT-29 colon cancer. The safety and efficacy of benc vector were observed during animal experiments. Complete inhibition of tumor growth (5/5) was observed not only in the intravenously injection of benc vector group but also in the intratumorally of benc vector group. We also observed a precautionary effect on lung metastases of HT-29 cells by intratumoral injection of benc vector. In the control group of animals given empty control vector, all animals (5/5) developed lung tumors and exhibited a higher number of nodules after injection with HT-29 cells via the tail vein. In contrast, only three animals (3/5) in the treatment group receiving benc vector had any observable lung metastases and a lower number of nodules. No animals died and no bleeding was observed both in treatment groups and control groups. Moreover, only moderate liver damage was detected in mice receiving benc vector by intravenous injections. Benc vector encoding hfVII-LC and hIgG1-Fc can effectively inhibit tumor growth and metastases in SCID mice with TF over-expressing colon cancer and shows promise as an agent for CRC immunotherapy.

Limited expression of cancer-testis antigens in renal cell carcinoma patients

The aim of this study was to evaluate the frequency of expression of the cancer-testis antigens (CTAs) NY-ESO-1, MAGE-A4 and SAGE, in renal cell carcinoma (RCC) patients compared to that in head and neck cancer (HNC) patients, which represent a positive control with a high incidence of CTA expression, to identify novel target antigens for immunotherapy. We prospectively examined frozen tissue samples collected from surgery or biopsy from 35 RCC and 40 HNC patients. Total RNA was extracted, and real-time reverse transcription-polymerase chain reaction (RT)-PCR was performed to determine the expression of MAGE-A4, NY-ESO-1 and SAGE. MAGE-A4 was not detected in any of the RCC samples, although a low incidence of NY-ESO-1 (5.7%; 2/35) and SAGE (2.9%; 1/35) expression was observed. No samples demonstrated co-expression of the three CTAs. By contrast, a comparatively high incidence of CTA expression was detected in squamous cell carcinoma (SCC) specimens of HNC patients. The actual incidence was 42.5% (17/40) for MAGE-A4, 20% (8/40) for NY-ESO-1 and 15% (6/40) for SAGE. The incidence of co-expression was 7.5% (3/40) for MAGE-A4 and NY-ESO-1, 7.5% (3/40) for MAGE-A4 and SAGE, 7.5% (3/40) for NY-ESO-1 and SAGE, and 2.5% (1/40) for the CTAs. The number of HNC samples positive for MAGE-A4 was significantly higher compared to that of RCC samples. The remaining two antigens, NY-ESO-1 and SAGE, were expressed at high levels in HNC compared to RCC samples. Limited frequency of CTA (NY-ESO-1, MAGE-A4 and SAGE) expression was demonstrated in RCC compared to HNC samples.