Serological Changes of Monoclonal Antibody CAMA3C8 against Human Breast Cancer Cell Line

Background: Tumor associated antigen are glycoproteins and glycolipids expressed on the surface or in the cytoplasm of tumor cells. The antigenic components are shed from the tumor cells into the tissue culture medium or blood or other human body fluids like human milk. Objective: In the present study, investigate the serological assay of the antigen recognized by CAMA3C8 in patients with carcinoma of the breast. Methods: The study is desgriptive cross section study.The study was divited into four groups based on the expression of tumor associated antigen. Results: In breast cancer CAMA3C8 levels were significantly increased in stage 4 when compared with stage 1 breast cancer cell line. Conclusion: In the present study, we conclude that significantly recognized CAMA3C8 defined antigens in breast cancer.

Labyrinthin: A distinct pan-adenocarcinoma diagnostic and immunotherapeutic tumor specific antigen

Structural analysis and detection of optimal cell surface localization of labyrinthin, a pan-adenocarcinoma target, was studied with respect to adenocarcinoma specificity vs. normal and non-adenocarcinoma cells. Patient-derived tissue microarray immunohistochemistry (IHC) was performed on 729 commercially prepared tissue blocks of lung, colon, breast, pancreas, prostate, and ovary cancers combined, plus a National Cancer Institute (NCI) tissue microarray derived from another 236 cases. The results confirmed that anti-labyrinthin mouse monoclonal MCA 44-3A6 antibody recognized adenocarcinomas, but not normal or non-adenocarcinoma cancer cells. The consensus of multiple topology analysis programs on labyrinthin (255 amino acids) estimate a type II cell membrane associated protein with an N-terminus signal peptide. However, because the labyrinthin sequence is enveloped within the 758 amino acids of the intracellular aspartyl/asparaginyl beta-hydroxylase (ASPH), a purported tumor associated antigen, standard IHC methods that permeabilize cells can expose common epitopes. To circumvent antibody cross-reactivity, cell surface labyrinthin was distinguished from intracellular ASPH by FACS analysis of permeabilized vs non-permeabilized cells. All permeabilized normal, adeno-and non-adenocarcinoma cells produced a strong MCA 44-3A6 binding signal, likely reflecting co-recognition of intracellular ASPH proteins along with internalized labyrinthin, but in non-permeabilized cells only adenocarcinoma cells were positive for labyrinthin. Confocal microscopy confirmed the FACS results. Labyrinthin as a functional cell-surface marker was suggested when: 1) WI-38 normal lung fibroblasts transfected with labyrinthin sense cDNA displayed a cancerous phenotype; 2) antisense transfection of A549 human lung adenocarcinoma cells appeared more normal; and 3) MCA44-3A6 suppressed A549 cell proliferation. Collectively, the data indicate that labyrinthin is a unique, promising adenocarcinoma tumor-specific antigen and therapeutic target. The study also raises a controversial issue on the extent, specificity, and usefulness of ASPH as an adenocarcinoma tumor-associated antigen.

Variable Expression of the Disialoganglioside GD2 in Breast Cancer Molecular Subtypes

The disialoganglioside GD2 is a tumor-associated antigen that may allow for the application of targeted immunotherapies (anti-GD2 antibodies, GD2 CAR T cells) in patients with neuroblastoma and other solid tumors. We retrospectively investigated GD2 expression in a breast cancer cohort, using immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays (TMAs), and its impact on survival. GD2 expression on IHC (n = 568) and IF (n = 503) was investigated in relation to subtypes and patient outcome. Overall, 50.2% of the 568 IHC-assessed samples and 69.8% of the 503 IF-assessed samples were GD2-positive. The highest proportion of GD2-positive tumors was observed in luminal tumors. Significantly fewer GD2-positive cases were detected in triple-negative breast cancer (TNBC) compared with other subtypes. The proportion of GD2-expressing tumors were significantly lower in HER2-positive breast cancer in comparison with luminal tumors on IF staining (but not IHC). GD2 expression of IHC or IF was not significantly associated with disease-free or overall survival, in either the overall cohort or in individual subtypes. However, GD2 expression can be seen in more than 50% of breast cancer cases, with the highest frequency in hormone receptor-positive tumors. With this high expression frequency, patients with GD2-positive advanced breast cancer of all subtypes may benefit from GD2-targeting immunotherapies, which are currently subject to clinical testing.

Bispecific antibodies in colorectal cancer therapy: recent insights and emerging concepts

Colorectal cancer (CRC) is identified as a life-threatening malignancy. Despite several efforts and proceedings available for CRC therapy, it is still a health concern. Among a vast array of novel therapeutic procedures, employing bispecific antibodies (BsAbs) is currently considered to be a promising approach for cancer therapy. BsAbs, as a large family of molecules designed to realize two distinct epitopes or antigens, can be beneficial microgadgets to target the tumor-associated antigen pairs. On the other hand, applying the immune system's capabilities to attack malignant cells has been proven as a tremendous development in cancer therapeutic projects. The current study has attempted to overview some of the approved BsAbs in CRC therapy and those under clinical trials. For this purpose, reputable scientific search engines and databases, such as PubMed, ScienceDirect, Google Scholar, Scopus, etc., were explored using the keywords ‘bispecific antibodies’, ‘colorectal cancer’, ‘immunotherapy’ and ‘tumor markers’.

P06.06 Enhancing trafficking and resistance to immunosuppression of synthetic agonistic receptor-transduced T cells in solid tumor models

BackgroundChimeric antigen receptor therapy – although very efficacious in B cell malignancies – is facing many challenges which limit its success in solid tumors, e.g. on-target off-tumor toxicities, antigen heterogeneity, lack of T cell migration into tumors and an immunosuppressive tumor microenvironment. To better control on-target off-tumor effects and address antigen heterogeneity we developed a modular approach where we equipped T cells with a synthetic agonistic receptor (SAR). The SAR is only activated in the presence of a bispecific antibody (BiAb) cross-linking the receptor with a tumor-associated antigen. While we could show efficacy of the SAR platform in different models, limited infiltration and immune suppression still hamper its function. We could previously demonstrate that T cell infiltration can be enhanced by transduction with carefully chosen chemokine receptors like CXCR6, CCR4 and CCR8. At the same time, gene silencing of checkpoint molecules like PD-1 can make T cells more resistant to immunosuppression, thus we assumed that combining these approaches might generate a desired T cell product.Materials and MethodsAll constructs had been generated previously by overlap-extension cloning. The EGFRvIII (E3) SAR consists of extracellular EGFRvIII, transmembrane CD28 and intracellular CD28 and CD3ζ. Human CXCR6-GFP, CCR4-GFP and CCR8-GFP are composed of the chemokine receptors fused to GFP via a 2A sequence. Primary human T cells were retrovirally transduced to stably express the SAR and chemokine receptors. We analyzed migration, cytotoxicity and activation of the single and double (E3 SAR and chemokine receptor) transduced T cells. In addition, PD-1 was knocked out using CRISPR-Cas9 and killing kinetics of target cells and T cell activation were assessed.ResultsCo-transduction with chemokine receptors significantly increased migration of E3 SAR T cells to their respective ligand while lysis of target-expressing tumor cell and T cell activation in the presence of BiAb were not affected in vitro. Additionally knocking out PD-1 enhanced killing kinetics and activation of E3 SAR and E3 SAR + CXCR6-GFP transduced T cells compared to corresponding mock electroporated T cells.ConclusionsUsing the controllable and modular SAR – BiAb platform SAR T cell activation can be limited by stopping BiAb dosing if adverse events occur. In addition, SAR T cells can be redirected to an alternative tumor-associated antigen by exchanging the BiAb in the case of antigen escape. Here we present add-ons to this approach for increased tumor infiltration and resistance to immunosuppression. Since migration is enhanced upon co-transduction with chemokine receptors and target cell lysis is accelerated upon PD-1 knockout in vitro these two additional modifications seem very promising options to further improve tumor control in vivo.Disclosure InformationM. Schwerdtfeger: None. M. Benmebarek: None. F. Märkl: None. C.H. Karches: A. Employment (full or part-time); Significant; Daiichi Sankyo Deutschland GmbH. A. Öner: None. M. Geiger: A. Employment (full or part-time); Significant; Roche. B. Cadilha: None. S. Endres: None. V. Desiderio: None. C. Klein: A. Employment (full or part-time); Significant; Roche. S. Kobold: None.

Tumor associated antigen specific T cells with nivolumab are safe and persist in vivo in rel/ref Hodgkin Lymphoma

Hodgkin Lymphoma (HL) Reed Sternberg cells express tumor associated antigens (TAA) that are potential targets for cellular therapies. We recently demonstrated that TAA specific T cells (TAA-T) targeting WT1, PRAME and Survivin were safe and associated with prolonged time to progression in solid tumors. Hence, we evaluated whether TAA-T when given alone or with nivolumab were safe and could elicit anti-tumor effects in vivo in patients with relapsed/refractory (r/r) HL. Ten patients were infused TAA-T (8 autologous; 2 allogeneic) for active HL(n=8) or as adjuvant therapy after hematopoietic stem cell transplant (n=2) at cumulative doses ranging from 0.5 X107 to 4 X107cells/m2. Six patients received nivolumab priming before TAA-T and continued until disease progression or unacceptable toxicity. All 10 products recognized 1 or more TAAs, were polyfunctional and did not demonstrated autoreactivity. Patients were monitored for safety for six weeks following the TAA-T and for response until disease progression. The infusions were safe with no clear dose limiting toxicities. Patients receiving TAA-T as adjuvant therapy remain in continued remission at 2+ years. Of the 8 patients with active disease,1 patient had a complete response and 7 had stable disease at 3 months, 3 of whom remain with stable disease at 1 year. Antigen spreading and long-term persistence of TAA-T in vivo were observed in responding patients. Nivolumab priming impacted the TAA-T recognition and persistence. In conclusion, treatment of r/r HL patients with TAA-T alone or in combination with nivolumab was safe and produced promising results (clinicaltrials.gov NCT022039303 and NCT03843294).

Colloidal Assemblies Composed of Polymeric Micellar/Emulsified Systems Integrate Cancer Therapy Combining a Tumor-Associated Antigen Vaccine and Chemotherapeutic Regimens

Integrative medicine comprising a tumor-associated antigen vaccine and chemotherapeutic regimens has provided new insights into cancer therapy. In this study, the AB-type diblock copolymers poly(ethylene glycol)–polylactide (PEG–PLA) were subjected to the dispersion of poorly water-soluble molecules in aqueous solutions. The physicochemical behavior of the chemotherapeutic agent DBPR114 in the PEG–PLA-polymeric aqueous solution was investigated by dynamic light scattering (DLS) technology. In vitro cell culture indicated that replacing the organic solvent DMSO with PEG–PLA polymeric micelles could maintain the anti-proliferative effect of DBPR114 on leukemia cell lines. A murine tumor-associated antigen vaccine model was established in tumor-bearing mice to determine the effectiveness of these formulas in inducing tumor regression. The results demonstrated that the therapeutic treatments effectively reinforced each other via co-delivery of antitumor drug/antigen agents to synergistically integrate the efficacy of cancer therapy. Our findings support the potential use of polymeric micellar systems for aqueous solubilization and expansion of antitumor activity intrinsic to DBPR114 and tumor-associated antigen therapy.

First-in-Human Trial of EphA2-Redirected CAR T-Cells in Patients With Recurrent Glioblastoma: A Preliminary Report of Three Cases at the Starting Dose

Glioblastoma is the most common primary brain malignancy with limited treatment options. EphA2 is a tumor-associated-antigen overexpressed in glioblastoma. Pre-clinical studies have demonstrated the promise of EphA2-redirected CAR T-cells against glioblastoma. We conduct the first-in-human trial of EphA2-redirected CAR T-cells in patients with EphA2-positive recurrent glioblastoma and report the results of three patients enrolled as the first cohort receiving the starting dosage (1×106 cells/kg). A single infusion of EphA2-redirected CAR T-cells was administrated intravenously, with the lymphodepletion regimen consisting of fludarabine and Cyclophosphamide. In two patients, there was grade 2 cytokine release syndrome accompanied by pulmonary edema, which resolved completely with dexamethasone medication. Except that, there was no other organ toxicity including neurotoxicity. In both the peripheral blood and cerebral-spinal-fluid, we observed the expansion of CAR T-cells which persisted for more than four weeks. In one patient, there was a transit diminishment of the tumor. Among these three patients, one patient reported SD and two patients reported PD, with overall survival ranging from 86 to 181 days. At the tested dose level (1×106 cells/kg), intravenously infusion of EphA2-rediretected CAR T-cells were preliminary tolerable with transient clinical efficacy. Future study with adjusted dose and infusion frequency of CAR T-cells is warranted.Trial Registration NumbersNCT 03423992

Innovative and Promising Strategies to Enhance Effectiveness of Immunotherapy for CNS Tumors: Where Are We?

Although there are several immunotherapy approaches for the treatment of Central Nervous System (CNS) tumors under evaluation, currently none of these approaches have received approval from the regulatory agencies. CNS tumors, especially glioblastomas, are tumors characterized by highly immunosuppressive tumor microenvironment, limiting the possibility of effectively eliciting an immune response. Moreover, the peculiar anatomic location of these tumors poses relevant challenges in terms of safety, since uncontrolled hyper inflammation could lead to cerebral edema and cranial hypertension. The most promising strategies of immunotherapy in neuro-oncology consist of the use of autologous T cells redirected against tumor cells through chimeric antigen receptor (CAR) constructs or genetically modified T-cell receptors. Trials based on native or genetically engineered oncolytic viruses and on vaccination with tumor-associated antigen peptides are also under evaluation. Despite some sporadic complete remissions achieved in clinical trials, the outcome of patients with CNS tumors treated with different immunotherapeutic approaches remains poor. Based on the lessons learned from these unsatisfactory experiences, novel immune-therapy approaches aimed at overcoming the profound immunosuppressive microenvironment of these diseases are bringing new hope to reach the cure for CNS tumors.