Abstract 2761: Antibody and T cell response profiling in pancreatic cancer patients before and after chemotherapy identify tumor associated antigens suitable for immunotherapy

Abstract CD4+ T cells are crucial for the induction and maintenance of cytotoxic T cell responses, but can also mediate direct tumor rejection. The therapeutic efficacy of peptide-based cancer vaccines may thus be improved by including HLA class II epitopes to stimulate T helper cells. In contrast to HLA class I ligands, only a small number of class II ligands of TAA has been described so far. We recently reported on the overexpression of HLA class II in AML cells as compared to autologous monocytes and granulocytes as well as on the first HLA class I leukemia associated antigens identified directly on the cell surface of primary AML cells (Stickel et. al. abstract in Blood 2012). In this study we characterized the HLA class II ligandome in AML to identify additional ligands for a peptide-based immunotherapy approach. HLA class II ligands from primary AML cells as well as bone marrow and peripheral blood mononuclear cell (BMNCs/PBMCs) of healthy donors were analyzed using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry (LC-MS/MS). LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived tumor associated antigens (LiTAAs) and to select peptide vaccine candidates. Most abundantly detected peptides were functionally characterized with regard to their ability to induce a specific CD4+ T-cell response in healthy donors and in tumor patients using ELISpot. Samples from 10 AML patients (5 FLT3-ITD mutated) and 18 healthy donors were analyzed. We identified more than 2,100 AML-derived HLA class II ligands representing >1,000 different source proteins, of which 315 were exclusively represented in AML, but not in healthy PBMC/BMNC. Data mining for broadly represented LiTAAs pinpointed 26 HLA class II ligands from 8 source proteins that were presented exclusively on more than 40% of all analyzed AML samples as most promising targets. Amongst them were already described TAAs (e.g., RAB5A) as well as several so far understated proteins (e.g. calsyntenin 1, glycophorin A, mannose-binding lectin 2). Subset analysis revealed 58 LiTAAs presented exclusively on FLT3-ITD mutated AML cells. Additional screening for HLA class II ligands from described leukemia associated antigens showed positive results for NPM1 (1 peptide sequence) and MPO (13 peptide sequences). Peptides from calsyntenin 1 and RAB5A were able to elicit CD4+-T-cell response in 25% of tested AML patients (n=16). Thus, our study identified, for the first time, HLA class II tumor associated antigens directly obtained from the HLA ligandomes of AML patients and thereby represents a further step to our goal of developing a multipeptide vaccine for immunotherapy of AML. Disclosures: No relevant conflicts of interest to declare.

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VXM01, an oral T-cell vaccine targeting the tumor vasculature: Results from a randomized, controlled, first-in-man study in pancreatic cancer patients.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.3090 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 3090-3090 ◽

Cited By ~ 2

Author(s):

Friedrich Hubertus Schmitz-Winnenthal ◽

Lars Grenacher ◽

Tobias Friedrich ◽

Heinz Lubenau ◽

Marco Springer ◽

...

Keyword(s):

Pancreatic Cancer ◽

Placebo Group ◽

T Cell ◽

Cell Response ◽

T Cell Responses ◽

T Cell Response ◽

Adverse Event Rate ◽

Tumor Perfusion ◽

Dce Mri ◽

Cell Responses

3090 Background: VXM01 is an orally available, bacterially transmitted DNA vaccine targeting VEGFR-2. Pre-clinically, VXM01 showed anti-tumor activity in multiple tumor types. This first-in-human study was designed to evaluate the safety and tolerability of VXM01. Secondary endpoints included VEGFR-2 specific T-cell responses, tumor perfusion changes, and related biomarkers. Methods: A randomized, double-blinded, placebo-controlled, dose-escalation study was conducted in 45 patients with advanced pancreatic cancer. VXM01 or placebo was given on days 1, 3, 5, and 7. Doses were escalated from 10(6) CFU to 10(10) CFU over 5 dose groups, each including 6 VXM01 and 3 placebo patients. VEGFR-2 specific T-cell activity was monitored by ELISpot and T(reg) specificity assays before, during and after the vaccination course. Tumor perfusion was assessed by DCE-MRI on days 0 and 38. Biomarkers included CA19-9, VEGF-A and collagen IV. Results: Patients were enrolled from 12/2011 to 10/2012. Most commonly observed AEs were leukopenia, abdominal pain, and diarrhea, which were all equally distributed between treatment and placebo group. While a mild elevation in average blood pressure was observed in the VXM01 group over the placebo group, the hypertension adverse event rate did not differ between both groups. No DLTs were observed. VEGFR-2 specific effector T-cell response was increased in 57% of evaluable VXM01 treated patients, during and after the vaccination course. In 25% of the VXM01 group, the T-cell response score post-vaccination was higher than maximum placebo levels. In contrast, VEGFR-2 specific T(reg) responses were overall reduced in vaccinated patients. DCE-MRI data indicated a >33% drop in K(trans)/tumor perfusion in 35% of evaluable VXM01 treated patients vs. 10% in the placebo group. Mean changes were -4% (VXM01) and +15% (placebo). Reduced tumor perfusion correlated with VEGFR-2 specific T-cell responses and biomarker responses. Conclusions: VXM01 appeared safe and was well tolerated without DLTs across 5 tested dose levels. The data suggest further that VXM01 induces and enhances a VEGFR-2 specific T-cell response and impacts tumor perfusion. Clinical trial information: ISRCTN68809279.

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Live attenuated oral Salmonella platform for effective T- and B-cell targeting of PD-L1.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.74 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. 74-74

Author(s):

Sébastien Wieckowski ◽

Heiko Smetak ◽

Iris Kobl ◽

Lilli Podola ◽

Anne-Lucie Nugues ◽

...

Keyword(s):

T Cell ◽

Tumor Immunity ◽

Cell Response ◽

Regulatory Protein ◽

T Cell Response ◽

Eukaryotic Expression ◽

Peripheral Tolerance ◽

Tumor Associated Antigens ◽

T Cell Vaccination

74 Background: Significant progresses have been recently achieved in cancer vaccines, yet novel immunization solutions to deliver efficiently tumor-associated antigens to professional antigen-presenting cells, and to overcome the peripheral tolerance and the immunosuppressive tumor microenvironment that prevent the eradication of cancer in most of patients, are urgently needed. VAXIMM is developing a unique and versatile oral T-cell vaccination platform based on the FDA-approved live-attenuated Salmonella Typhi strain Ty21a vaccine Vivotif, capable of delivering tumor-associated antigens encoded in DNA expression construct to the gut-associated lymphoid tissue, breaking immune tolerance and inducing effective anti-tumor immunity. Methods: This study summarizes the immunogenicity and antileukemia efficacy of VXM10 vaccines based on the live-attenuated Salmonella Typhimurium strain SL7207, transformed with a eukaryotic expression plasmid encoding different variants of the murine PD-L1 protein. Results: The antileukemia activity of VXM10 was evaluated in the FBL-3 disseminated model of leukemia, in which the tumor cells strongly express PD-L1. Multiple oral administrations of VXM10 vaccines produced a strong anti-tumor effect, with 100% of surviving animals 80 days after challenge with FBL3 leukemia in the highest dose groups. Moreover, 100% of long-term surviving mice resisted re-challenge with FBL-3 cells, demonstrating that vaccination with VXM10 generated a potent memory T cell response against the leukemia. Importantly, full leukemia control was achieved in both prophylactic and therapeutic settings. Upon immunization with VXM10 vaccines, T cell response was raised against PD-L1 epitopes after in vitro restimulation of the splenocytes, and anti-PD-L1 antibodies were detected in the serum. The precise mechanism of action of VXM10 vaccines is currently being investigated. Conclusions: This study provides further evidence that VAXIMM’s oral T-cell vaccination platform can be employed to stimulate anti-tumor immunity against antigens of the immune checkpoint regulatory protein PD-L1. These data paved the way for advancing the development of VXM10 into clinical development.

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Gastrin vaccine and immune checkpoint antibody therapy for pancreatic cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.4_suppl.259 ◽

2019 ◽

Vol 37 (4_suppl) ◽

pp. 259-259

Author(s):

Jill P Smith ◽

Nicholas Osborne ◽

Rebecca Sundseth ◽

Julian Burks ◽

Hong Cao ◽

...

Keyword(s):

Pancreatic Cancer ◽

T Cell ◽

Tumor Microenvironment ◽

Immune Checkpoint ◽

Cell Response ◽

Combined Therapy ◽

T Cell Response ◽

Effector Memory ◽

Antibody Treatment ◽

Pancreatic Cancer Cells

259 Background: Pancreatic cancer is poorly responsive to therapy due to fibrosis in the tumor microenvironment and nonspecificity of treatments. The peptide gastrin stimulates growth of pancreatic cancer in an autocrine fashion. Polyclonal Antibody Stimulator (PAS) is a gastrin vaccine that in clinical trials prolongs survival of patients who develop neutralizing gastrin antibodies. We hypothesized that PAS also elicits a memory and T-cell response that would improve effectiveness of immune checkpoint antibodies. Methods: C57BL/6 mice were injected sc with syngeneic mT3 murine pancreatic cancer cells. After 1 week (tumors evident), groups were treated with PBS (control); PAS (100 μg or 250 μg); PD-1 antibody (150 μg); or combined therapy with PAS100/PD-1. PAS was given ip at weeks 0, 1 and 3. Anti-PD-1 was given on days 0, 4, 8, 15 and 21. On day 31 spleens were collected for T-cell surface analysis and cytokine activation by flow cytometry. Tumors were stained for fibrosis and with immunohistochemical stains for CD8+ and FoxP3+ tumor infiltrating lymphocytes (TILs). Results: PAS resulted in T-cell activation; however, the portion of terminally differentiated effector memory double negative T-cells (CD4- CD8-) in CD3+cells from mice treated with the combination PAS100/PD-1 was two-fold higher than those portions from mice treated with PBS, PD-1 or PAS100. Lymphocytes from PAS-treated mice elicited cytokine release (Interferon-γ, granzyme, perforin, and TNF-α) upon re-stimulation with gastrin in vitro, whereas PAS100/PD-1-treated mice show the highest endogenous cytokines levels. PAS250 monotherapy decreased tumor growth. Neither PAS100 nor anti-PD-1 monotherapy decreased tumor size, but combination PAS100/PD-1 antibody tumors were smaller, had less fibrosis, fewer T-regulatory lymphocytes, and increased CD8+ TILs. Conclusions: In addition to eliciting a B-cell response, PAS activates T-lymphocytes rendering tumors susceptible to immune checkpoint antibody treatment. Smaller tumors with combined therapy may be due to the anti-fibrotic effect of PAS on the tumor microenvironment and changes in CD8+ TILs. This study supports that PAS is an important immunotherapy that elicits both B- and T-cell anti-cancer responses.

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Rapid T Cell Response to Vaccination Can Occur without Antibody Response in Children Post HSCT.

Blood ◽

10.1182/blood.v104.11.2235.2235 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2235-2235

Author(s):

W. Nicholas Haining ◽

J. Evans ◽

N. Seth ◽

G. Callaway ◽

K. Wucherpfennig ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Cell Response ◽

Influenza A ◽

T Cell Response ◽

T Cell Memory ◽

Cell Immunity ◽

Before And After ◽

B Cell Response

Abstract Vaccination is widely used to improve pathogen-specific immunity in patients post HSCT, but it is not known whether patients can mount an effective T cell response to vaccine antigens (vAg). Moreover the relationship between T and B cell response to vAg has not been studied. We hypothesized that a sufficiently sensitive assay of T cell response to vAg would allow vaccination to be used as a tool to measure immune recovery post HSCT and improve vaccine design. We therefore: (1) developed a flow-cytometry-based approach to quantify and characterize T cells specific for vAg; (2) validated it by measuring T cell immunity to influenza A in normal donors; and (3) characterized the T and B cell response to influenza vaccination in pediatric HSCT patients. PBMC were labeled with CFSE and stimulated in vitro with whole influenza Ag. Ag-specific T cells were sensitively detected by their proliferation (loss of CFSE fluorescence) and simultaneous expression of the activation marker HLA-DR. Proliferating/active T cells could be readily detected after stimulation with influenza A Ag in healthy adult (n=4) and pediatric (n=19) donors but were absent in control conditions. Both CD4+ and CD8+ T cell proliferation was detected in all donors but one, and in children as young as 6mo. Staining with MHC I- and MHC II-tetramers confirmed that the proliferating/active population contained T cells specific for immunodominant CD8+ and CD4+ epitopes, demonstrating that vAg were processed and presented to epitope-specific T cells. To characterize the phenotype of influenza-specific T cell memory, we separated memory and naive CD4+ cells prior to antigen-stimulation. Antigen-experienced (CD45RA−/CCR7−) but not naive (CD45RA+/CCR7+) T cells proliferated to vAg confirming that the assay detected pre-existing influenza-A-specific T cell memory. We next assessed Influenza-A-specific T cell immunity before and after influenza vaccination in five pediatric HSCT recipients (mean age 10.6y, range 5–15y; mean time from transplant 13m, range 3–21m). Prior to vaccination the CD4 proliferation to influenza-A was a mean of 3.3% (range 0.04–11%). Following vaccination CD4 proliferation increased significantly in all patients (mean 19.0%, range 6.9%–31.8%, p=0.02). This increase was specific as proliferation to control Ag was unchanged. Influenza-A CD8+ proliferation also increased in 3 of 5 patients but was not statistically significant for the group consistent with the limited efficacy of soluble vAg in inducing CD8+ T cell response. All patients had detectable influenza-A-specific IgG levels prior to vaccination but despite a T cell response to vaccination in all patients, none had a significant increase in IgG level following vaccination. Only one patient had an IgM response; this patient also had the highest influenza-A-specific CD4 proliferation before and after immunization suggesting that there may be a threshold of T cell response required for a B cell response. Using a novel assay we demonstrate that a T cell response to vaccination can occur without an accompanying B cell response. This assay provides a more sensitive measure of immunity to vaccination and allows vaccine response to be used as a benchmark of strategies to accelerate post-HSCT T cell reconstitution.

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