Murine Monoclonal Antibody Research Articles

Occurrence of DEA1.1 in blood donor dogs in Cuiabá, Mato Grosso, Brazil

This study aimed to assess Dog Erythrocyte Antigen (DEA) 1.1 in donor dogs at the Federal University of Mato Grosso, Cuiabá, Brazil, and review the relevant literature. The blood (60 samples; 1.5 mL volume, each) was collected in separate vacutainer tubes containing ethylenediaminetetraacetic acid and submitted for complete blood count; in addition, the samples were typed by RapidVet® based on agglutination due to specific interaction between DEA 1 antigen at the membrane surface of the erythrocyte and lyophilised murine monoclonal antibody on the test card. DEA1.1 positivity was observed in 81.6% (49 of 60) of test samples, while negative results were obtained in the remaining 18.3% (11 of 60). DEA 1.1 positive samples were comprised of 42.8% of purebred dogs and 38.3% of mixed breed dogs. With regard to sex in the DEA 1.1 positive group, 48.3% were male dogs and 33.3% were female dogs. The blood donor canine population showed high prevalence of DEA 1.1, which confirms that blood typing should be performed prior to blood transfusion in previously sensitised dogs.

Abstract A85: The role of LAP positive immune cells in cancer

Abstract Immunotherapy that acts by counteracting the suppressive environment established by the tumor became a very promising approach for the treatment of cancer. Regulatory immune cells promote cancer by suppressing antitumor immune responses, but there are few cell surface molecules that can specifically be targeted to neutralize their function. We characterized a regulatory T-cell population that expresses surface latency-associated peptide (LAP). LAP is an adaptor and regulator of TGF-beta, a potent immunosuppressive cytokine. An increase in LAP+ CD4 T cells has been reported in human cancer, including head and neck and colorectal cancer. To study the regulatory role of LAP+ cells in cancer, we developed a murine monoclonal anti-LAP antibody. We found that treatment with anti-LAP antibody reduces tumor growth and increases survival in various cancer models, including colon carcinoma, glioblastoma and melanoma. Anti-LAP antibody is able to block TGF-beta release from cells expressing LAP and also reduces both the number and suppressive abilities of tumor-associated LAP+ regulatory cells. Anti-LAP antibody treatment triggers a profound peripheral immune response by acting on both innate and adaptive arms of the immune system. A synergistic antitumor effect was observed by combining anti-LAP antibody with dendritic cell vaccination. To study different LAP+ immune cells on a single-cell level, we developed a 19-panel flow cytometry approach. Based on the TCGA data analysis, the expression of LAP-associated genes correlates inversely with patient survival in a number of cancers. In summary, LAP+ immune cells contribute to cancer progression, and targeting these cells for cancer treatment represents a promising immunotherapeutic approach against cancer. Citation Format: Galina Gabriely, Andre da Cunha, Rafael Rezende, Nathaniel Skillin, Brendan Kenyon, Lena Walton, Murugaiyan Gopal, Howard Weiner. The role of LAP positive immune cells in cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A85.

Antibodies that conformationally activate ADAMTS13 allosterically enhance metalloprotease domain function

AbstractPlasma ADAMTS13 circulates in a folded conformation that is stabilized by an interaction between the central Spacer domain and the C-terminal CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. Binding of ADAMTS13 to the VWF D4(-CK) domains or to certain activating murine monoclonal antibodies (mAbs) induces a structural change that extends ADAMTS13 into an open conformation that enhances its function. The objective was to characterize the mechanism by which conformational activation enhances ADAMTS13-mediated proteolysis of VWF. The activating effects of a novel anti-Spacer (3E4) and the anti-CUB1 (17G2) mAbs on the kinetics of proteolysis of VWF A2 domain fragments by ADAMTS13 were analyzed. mAb-induced conformational changes in ADAMTS13 were investigated by enzyme-linked immunosorbent assay. Both mAbs enhanced ADAMTS13 catalytic efficiency (kcat/Km) by ∼twofold (3E4: 2.0-fold; 17G2: 1.8-fold). Contrary to previous hypotheses, ADAMTS13 activation was not mediated through exposure of the Spacer or cysteine-rich domain exosites. Kinetic analyses revealed that mAb-induced conformational extension of ADAMTS13 enhances the proteolytic function of the metalloprotease domain (kcat), rather than augmenting substrate binding (Km). A conformational effect on the metalloprotease domain was further corroborated by the finding that incubation of ADAMTS13 with either mAb exposed a cryptic epitope in the metalloprotease domain that is normally concealed when ADAMTS13 is in a closed conformation. We show for the first time that the primary mechanism of mAb-induced conformational activation of ADAMTS13 is not a consequence of functional exosite exposure. Rather, our data are consistent with an allosteric activation mechanism on the metalloprotease domain that augments active site function.

Development and Characterization of Mouse Monoclonal Antibodies Specific for Clostridiodes (Clostridium) difficile Lipoteichoic Acid.

Clostridiodes (Clostridium) difficile is an anaerobic Gram-positive, spore-forming nosocomial, gastrointestinal pathogen causing C. difficile-associated disease with symptoms ranging from mild cases of antibiotic-associated diarrhea to fatal pseudomembranous colitis. We developed murine monoclonal antibodies (mAbs) specific for a conserved cell surface antigen, lipoteichoic acid (LTA)of C. difficile. The mAbs were characterized in terms of their thermal stability, solubility, and their binding to LTA by surface plasmon resonance and competitive ELISA. Synthetic LTA molecules were prepared in order to better define the minimum epitope required to mimic the natural antigen, and three repeat units of the polymer were required for optimal recognition. One of the murine mAbs was chimerized with human constant region domains and was found to recognize the target antigen identically to the mouse version. These mAbs may be useful as therapeutics (standalone, in conjunction with known antitoxin approaches, or as delivery vehicles for antibody drug conjugates targeting the bacterium), as diagnostic agents, and in infection control applications.

SPECT-CT Imaging of Dog Spontaneous Diffuse Large B-Cell Lymphoma Targeting CD22 for the Implementation of a Relevant Preclinical Model for Human.

Antibodies directed against CD22 have been used in radioimmunotherapy (RIT) clinical trials to treat patients with diffuse large B-cell lymphoma (DLBCL) with promising results. However, relevant preclinical models are needed to facilitate the evaluation and optimization of new protocols. Spontaneous DLBCL in dogs is a tumor model that may help accelerate the development of new methodologies and therapeutic strategies for RIT targeting CD22. Seven murine monoclonal antibodies specific for canine CD22 were produced by the hybridoma method and characterized. The antibodies' affinity and epitopic maps, their internalization capability and usefulness for diagnosis in immunohistochemistry were determined. Biodistribution and PET imaging on a mouse xenogeneic model of dog DLBCL was used to choose the most promising antibody for our purposes. PET-CT results confirmed biodistribution study observations and allowed tumor localization. The selected antibody, 10C6, was successfully used on a dog with spontaneous DLBCL for SPECT-CT imaging in the context of disease staging, validating its efficacy for diagnosis and the feasibility of future RIT assays. This first attempt at phenotypic imaging on dogs paves the way to implementing quantitative imaging methodologies that would be transposable to humans in a theranostic approach. Taking into account the feedback of existing human radioimmunotherapy clinical trials targeting CD22, animal trials are planned to investigate protocol improvements that are difficult to consider in humans due to ethical concerns.

Functional characterization of a broad and potent neutralizing monoclonal antibody directed against outer membrane protein (OMP) of Salmonella typhimurium.

In the present study, we have generated a murine monoclonal antibody (mAb) named Sal-06 by using the crude outer membrane protein preparation of Salmonella enteric subsp. enterica serovar Typhimurium ATCC 14028 strain as antigen. Sal-06mAb belonging to IgG1 isotype demonstrated broad cross-reactivity to standard and isolated strains of genus Salmonella and others such as Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. Cross-reactivity across several bacterial genera indicated that the epitopes reactive to Sal-06mAb are conserved among these members. Neutralizing effects of Sal-06mAb on Salmonella growth and survival was evaluated in vitro using bacteriostatic and bactericidal activity with and without complement and bacterial invasion inhibition assay. Sal-06mAb demonstrated a bacteriostatic effect on the growth of S. typhimurium ATCC 14028 strain which is both time and concentration (of mAb) dependent. It was also found that the bacterial growth inhibition was complement independent. When the bacterial cells were preincubated with Sal-06mAb, it reduced the adherence and invasion of bacterial cells into A549 epithelial cell line. This was confirmed by CFU count analysis, phase contrast, and fluorescence microscopy. Scanning electron microscope (SEM) imaging confirmed the antimicrobial effects of Sal-06mAb on S. typhimurium ATCC 14028. The development of broadly reactive and cross protective Sal-06mAb opens new possibilities for immunotherapy of sepsis caused by Gram-negative Enterobacteriaceae members.

Impact of Peri-Transplant Rituximab and Host/Donor Fc Receptor Polymorphisms for Patients with Relapsed or Refractory CD20+ B-Cell Malignancies

We previously reported results of allogeneic hematopoietic cell transplantation (HCT) in 93 older or medically unfit patients with non-Hodgkin's lymphoma (NHL) or chronic lymphocytic lymphoma (CLL) after conditioning with fludarabine (FLU) and 2-3 Gy total body irradiation (TBI). Three-year survival ranged from 45% to 52% depending on the underlying histology and disease stage. The major complication in those studies was disease relapse (24% at 1 year). The current prospective study in 63 NHL patients tested the hypothesis that disease relapse could be reduced, and overall survival (OS) improved, by peri-transplant rituximab (RTX). Treatment (Figure 1) consisted of RTX (375 mg/m²/day) on days -3, +10, +24 and +38 in addition to 2-3 Gy TBI ± FLU (30 mg/m² × 3 days). As pharmacokinetics (PK) and optimal dosing of RTX in allogeneic HCT are undefined, we collected PK data to optimize RTX dosing. RTX levels were assayed with murine anti-idiotype monoclonal antibody (18C9) binding to RTX. Median therapeutic RTX levels of >25 μg/mL were achieved through day 84 after HCT, with peak median level of 200.5 μg/mL at day 64. RTX levels were not correlated with relapse or graft-vs-host disease (GVHD) rates. NHL patients had significantly higher median RTX levels than CLL patients (day 84, p=0.003, Figure 2). We collected data on donor and recipient FCγRIIIa receptor polymorphisms expressed predominantly on natural killer cells, thought to affect responsiveness to RTX. This was done to identify patients or donor/recipient pairs most likely to benefit from peri-transplant RTX. FCγRIIIa polymorphisms were determined by PCR and single-strand confirmation polymorphism (SSCP) from DNA isolated from patient cells. Recipients with F/F and V/F FCγRIIIa polymorphisms experienced higher relapse rates compared to recipients with V/V polymorphism (p=0.15). Overall, almost a third of pre-HCT F/F patients relapsed. Probably due to small numbers, no difference in outcome was found with V/V donor pairing for these patients. Results are shown in Figure 3. Relapse rates at 5 years were similar among RTX-treated patients and historical controls (32% vs, 28%; P=0.94). RTX-treated patients experienced higher 5-year OS and progression-free survival; (47% vs, 38%; P=0.13) (41 vs, 32%; P=0.12), respectively, but none of the differences was statistically significant. Non-relapse mortality was lower among RTX-treated patients versus controls (27% vs, 40; P=0.05), respectively; incidences of grade 2-4 acute GVHD and grade 3-4 acute GVHD were similar (60% vs, 57%, 13% vs. 17%); and 5-year chronic GVHD was higher among RTX-treated patients (62% vs, 47%). These findings suggest that for patients with relapsed or refractory NHL, peri-transplant RTX neither reduced relapse nor improved OS or other post-HCT parameters. The role of donor-recipient pairing by FCγRIIIa polymorphisms in outcome remains to be determined.

Cryo-Electron Microscopy Structure of the αIIbβ3-Abciximab Complex.

The αIIbβ3 antagonist antiplatelet drug abciximab is the chimeric antigen-binding fragment comprising the variable regions of murine monoclonal antibody 7E3 and the constant domains of human IgG1 and light chain κ. Previous mutagenesis studies suggested that abciximab binds to the β3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent β1-α1 helix. These studies could not, however, assess whether 7E3 or abciximab prevents fibrinogen binding by steric interference, disruption of either the αIIbβ3-binding pocket for fibrinogen or the β3 SDL (which is not part of the binding pocket but affects fibrinogen binding), or some combination of these effects. To address this gap, we used cryo-electron microscopy to determine the structure of the αIIbβ3-abciximab complex at 2.8 Å resolution. Approach and Results: The interacting surface of abciximab is comprised of residues from all 3 complementarity-determining regions of both the light and heavy chains, with high representation of aromatic residues. Binding is primarily to the β3 SDL and neighboring residues, the β1-α1 helix, and β3 residues Ser211, Val212 and Met335. Unexpectedly, the structure also indicated several interactions with αIIb. As judged by the cryo-electron microscopy model, molecular-dynamics simulations, and mutagenesis, the binding of abciximab does not appear to rely on the interaction with the αIIb residues and does not result in disruption of the fibrinogen-binding pocket; it does, however, compress and reduce the flexibility of the SDL. We deduce that abciximab prevents ligand binding by steric interference, with a potential contribution via displacement of the SDL and limitation of the flexibility of the SDL residues.

Contribution of Fc-dependent cell-mediated activity of a vestigial esterase-targeting antibody against H5N6 virus infection

ABSTRACT The highly pathogenic avian influenza A (H5N6) virus has caused sporadic human infections with a high case fatality rate. Due to the continuous evolution of this virus subtype and its ability to transmit to humans, there is an urgent need to develop effective antiviral therapeutics. In this study, a murine monoclonal antibody 9F4 was shown to display broad binding affinity against H5Nx viruses. Furthermore, 9F4 can neutralize H5N6 pseudotyped particles and prevent entry into host cells. Additionally, ADCC/ADCP deficient L234A, L235A (LALA) and CDC deficient K322A mutants were generated and displayed comparable binding affinity and neutralizing activity as wild type 9F4 (9F4-WT). Notably, 9F4-WT, 9F4-LALA and 9F4-K322A exhibit in vivo protective efficacies against H5N6 infections in that they were able to reduce viral loads in mice. However, only 9F4-WT and 9F4-K322A but not 9F4-LALA were able to reduce viral pathogenesis in H5N6 challenged mice. Furthermore, depletion of phagocytic cells in mice lungs nullifies 9F4-WT's protection against H5N6 infections, suggesting a crucial role of the host's immune cells in 9F4 antiviral activity. Collectively, these findings reveal the importance of ADCC/ADCP function for 9F4-WT protection against HPAIV H5N6 and demonstrate the potential of 9F4 to confer protection against the reassortant H5-subtype HPAIVs.

Generation and characterization of murine monoclonal antibodies against immunoreactive trypsinogen for newborn screening of cystic fibrosis

Cystic fibrosis (CF) is a multisystem disorder that reduces quality of life and survival in affected individuals. In newborns, the release of pancreatic enzymes into the blood raises the levels of immunoreactive trypsinogen (IRT), the main marker for CF screening, which is detected in dried blood samples on filter paper by immunoenzymatic assays. In Cuba, CF has an estimated incidence of 1/9862 live births and should be included in the national basic newborn screening (NBS) panel given its benefits in terms of nutrition, lung function and survival. The Immunoassay Center develops and produces diagnostic kits allowing the establishment of large-scale NBS programs for inherited metabolic disorders in Cuba and other Latin American countries. IRT-specific monoclonal antibodies (MAbs) obtained at the Immunoassay Center are essential for developing an affordable immunoassay for IRT to support CF NBS in our low-income country. An immunization scheme with trypsinogen-1 originated two IgG1-producing murine hybridomas. 4C9C9 and 4C9E11 MAbs recognized different determinants on both trypsin-1 and trypsin-2 molecules. Both antibodies identified conformational epitopes on the molecule of trypsin-1 and of its zymogen. As 4C9E11 MAb cross-reacted with proteins structurally and functionally related to trypsinogen, it was used as revealing antibody in a sandwich-type UMELISA® assay for IRT determination with 4C9C9 MAb for capture. This combination, aside from detecting several commercially available trypsins, adequately quantified IRT from dried blood samples on filter paper of newborns. The evaluation of the assay's accuracy yielded percentage recoveries ranging 93.3–109.2% for commercial controls. The properties of the studied MAbs demonstrate their suitability for being used in a sandwich-type UMELISA® assay for the CF NBS in Cuba.

A close relationship between HIF-1α expression and bone metastases in advanced NSCLC, a retrospective analysis.

Background: Hypoxia-inducible factor (HIF-1) is a transcription factor produced in hypoxia condition, it is closely associated with tumor angiogenesis and metastasis. Aim: To investigate the expression of HIF-1α in relation with the presence or absence of bone metastasis. Methods A retrospective analysis was carried out on samples deriving from bronchial biopsy and CT-guided trans-thoracic needle biopsy. Detection of HIF-1 expression was performed on tissue sample by a monoclonal murine antibody, comparing patients with or without bone metastases (BM+). Findings: In the total population the main histotype was adenocarcinoma (71.5%), COPD the prevalent comorbidity (73.6%), the mean pack-year was 36.4. Ninety-five histology samples were considered for analysis and comparison. Subdividing the population according to the presence or not of bone metastases, significant differences were found in pack-years (p = 0.02), time to progression (TTP) (p = 0.001) and COPD comorbidity (p = 0.04). The survival comparison between the two subgroups obtained by Kaplan–Meier method showed a longer TTP in patients with visceral metastases with a HR of 1.3 though the comparison by this method was not significant (p = 0.1). A higher intensity and percentage of expression of HIF-1α was recorded in the group with bone metastases (p = 0.02). The main variable affecting HIF expression in a multivariate analysis was the presence of bone metastases (p = 0.01). Interpretation: Patients affected by NSCLC IV stage with bone metastasis have lower survival. There is a very close link between bone metastasis and HIF-1α expression level. The latter could be considered a predictive factor of bone spread and poor prognosis.

A novel approach to immunoapheresis of C3a/C3 and proteomic identification of associates.

BackgroundComplement factor C3 represents the central component of the complement cascade and its activation split product C3a plays an important role in inflammation and disease. Many human disorders are linked to dysregulation of the complement system and alteration in interaction molecules. Therefore, various therapeutic approaches to act on the complement system have been initiated.Methods and ResultsAiming to develop a tool to eliminate C3a/C3 from the circulation, in a first step a high affine murine monoclonal antibody (mAb) (3F7E2-mAb) was generated against complement factor C3 and selected for binding to the C3a region to serve as immunoaffinity reagent. Functional testing of the 3F7E2-mAb revealed an inhibition of Zymosan-induced cleavage of C3a from C3. Subsequently, a C3a/C3 specific 3F7E2-immunoaffinity column was developed and apheresis of C3a/C3 and associates was performed. Finally, a proteomic analysis was carried out for identification of apheresis products. C3a/C3 was liberated from the 3F7E2-column together with 278 proteins. C3a/C3 interaction specificity was validated by using a haptoglobin immunoaffinity column as control and biostatistic analysis revealed 39 true C3a/C3 interactants.ConclusionA novel and functionally active mAb was developed against complement factor C3a/C3 and used in a specific immunoaffinity column that allows apheresis of C3a/C3 and associates and their identification by proteomic analysis. This methodological approach of developing specific antibodies that can be used as immunoaffinity reagents to design immunoaffinity columns for elimination and further identification of associated proteins could open new avenues for the development of tailored immunotherapy in various complement-mediated or autoimmune diseases.

A Murine Monoclonal Antibody With Potent Neutralization Ability Against Human Adenovirus 7

B1-type human adenoviruses (HAdVs) HAdV-3, HAdV-7, and HAdV-55 have caused epidemics in North America, Asia, and Europe. However, to date, no adenovirus vaccines or antiviral drugs have been approved for general use. In the present work, a scFv-phage immune library was constructed and mouse monoclonal antibody (MMAb) 10G12 was obtained through selection. 10G12 is specific for HAdV-7 and binds the hexon loop1 and loop2 (LP12), resulting in strong neutralization activity against HAdV-7. Additionally, it is stable in serum and buffer at various pH values. The findings provide insight into adenovirus and antibody responses and may facilitate the design and development of adenovirus vaccines and antiviral drugs.

Therapeutic monoclonal antibody treatment protects nonhuman primates from severe Venezuelan equine encephalitis virus disease after aerosol exposure.

There are no FDA licensed vaccines or therapeutics for Venezuelan equine encephalitis virus (VEEV) which causes a debilitating acute febrile illness in humans that can progress to encephalitis. Previous studies demonstrated that murine and macaque monoclonal antibodies (mAbs) provide prophylactic and therapeutic efficacy against VEEV peripheral and aerosol challenge in mice. Additionally, humanized versions of two neutralizing mAbs specific for the E2 glycoprotein, 1A3B-7 and 1A4A-1, administered singly protected mice against aerosolized VEEV. However, no studies have demonstrated protection in nonhuman primate (NHP) models of VEEV infection. Here, we evaluated a chimeric antibody 1A3B-7 (c1A3B-7) containing mouse variable regions on a human IgG framework and a humanized antibody 1A4A-1 containing a serum half-life extension modification (Hu-1A4A-1-YTE) for their post-exposure efficacy in NHPs exposed to aerosolized VEEV. Approximately 24 hours after exposure, NHPs were administered a single bolus intravenous mAb. Control NHPs had typical biomarkers of VEEV infection including measurable viremia, fever, and lymphopenia. In contrast, c1A3B-7 treated NHPs had significant reductions in viremia and lymphopenia and on average approximately 50% reduction in fever. Although not statistically significant, Hu-1A4A-1-YTE administration did result in reductions in viremia and fever duration. Delay of treatment with c1A3B-7 to 48 hours post-exposure still provided NHPs protection from severe VEE disease through reductions in viremia and fever. These results demonstrate that post-exposure administration of c1A3B-7 protected macaques from development of severe VEE disease even when administered 48 hours following aerosol exposure and describe the first evaluations of VEEV-specific mAbs for post-exposure prophylactic use in NHPs. Viral mutations were identified in one NHP after c1A3B-7 treatment administered 24 hrs after virus exposure. This suggests that a cocktail-based therapy, or an alternative mAb against an epitope that cannot mutate without resulting in loss of viral fitness may be necessary for a highly effective therapeutic.

Monoclonal antibody anti-PBP2a protects mice against MRSA (methicillin-resistant Staphylococcus aureus) infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium responsible for serious nosocomial and community-acquired infections worldwide. Since few antibiotics are effective for treating MRSA infections, the development of new therapies is of great importance. Previous studies demonstrated that PBP2a is a target that generates protective antibodies against MRSA. A murine monoclonal antibody (MAb) that recognizes PBP2a from MRSA strains was previously isolated and characterized. In this report, we evaluated the biodistribution of this MAb in blood and tissues, as well as the extent of protection conferred using prophylactic and therapeutic assays compared to vancomycin treatment. Biodistribution was evaluated 12–96 h after MAb administration. It predominantly remained in the serum, but it was also detectable in the kidneys, lungs, and spleen at low concentrations (about 4.5% in the kidneys, 1.9% in the lungs, and 0.7% the spleen) at all observed timepoints. Prophylactic studies in a murine model demonstrated a significant bacterial load reduction in the kidneys of the groups treated with either with IgG (greater than 3 logs) or F(ab’)2 (98%) when compared to that of the control groups (untreated). Mice were challenged with a lethal dose, and the survival rate was higher in the treated mice. Treatment with the MAb resulted in a bacterial load reduction in the kidneys similar to that of mice treated with vancomycin, and a MAb/vancomycin combination therapy was also effective. These results demonstrate that an anti-PBP2a MAb may be a promising therapeutic for treating MRSA infections.

A dendritic cell-targeted chimeric hepatitis B virus immunotherapeutic vaccine induces both cellular and humoral immune responses in vivo

ABSTRACT Chimigen® HBV Immunotherapeutic Vaccine (C-HBV), a recombinant chimeric fusion protein comprising hepatitis B virus (HBV) S1 and S2 surface antigen fragments, Core antigen and a murine monoclonal antibody heavy chain fragment (Fc), was designed and produced in Sf9 insect cells. C-HBV targets the host immune system through specific receptors present on dendritic cells (DCs) which facilitates antigen internalization, processing, and presentation on MHC class I and II to induce both cellular and humoral immune responses against HBV antigens. T cell responses, previously assessed by ex vivo antigen presentation assays using human peripheral blood mononuclear cell (PBMC)-derived DCs and T cells from uninfected and HBV chronic-infected donors, demonstrated that C-HBV was highly immunogenic. A vaccine dose response study was performed in sheep to analyze the immunogenicity of C-HBV in vivo. Sheep (n = 8/group) received three consecutive subcutaneous injections of each dose of C-HBV at four-week intervals. Analysis of serum antibody levels confirmed C-HBV induced a dose-dependent antibody response to C-HBV and S1/S2-Core. Kinetics of the S1/S2-Core specific antibody response was similar to hepatitis B surface antigen (HBsAg)-specific antibody responses induced by ENGERIX-B. Analysis of cell-mediated immune responses (CMI) confirmed C-HBV induced both dose-dependent S1/S2-Core-specific lymphocyte proliferative responses and IFN-γ secretion. These responses were stronger with blood lymphocytes than with cells isolated from the lymph node draining the vaccination site. No correlation was seen between antibody titers and CMI. The results confirm C-HBV is an effective delivery vehicle for the induction of T cell responses and may be an appropriate candidate for immunotherapy for chronic HBV infections.

Targeting the FMS-like Tyrosin Kinase 3 with the Unicar System: Preclinical Comparison of Murine and Humanized Single-Chain Variable Fragment-Based Targeting Modules

Clinical translation of chimeric antigen receptor (CAR) T cell therapy in myeloid malignancies is progressing slowly compared to its success in treatment of B cell malignancies. Clinical experiences with CAR T cell therapies against the currently investigated tumor-associated antigens (TAA) (e.g. CD33, CD123 and FMS-like tyrosine kinase 3 (FLT3)) were discouraging and severe side effects occurred (cytokine release syndrome, neurotoxicity and myeloid aplasia) (Hoffmann et al. Journal of Clinical Medicine 2019). Probably targeting a single TAA is insufficient to treat high risk myeloid malignancies with CAR T cell therapies. Therefore, combined targeting of two or even more TAAs seems to be a promising approach. In order to implement such a multiple tumor targeting strategy, we developed a modular CAR T cell system termed UniCAR. The system consists of a universal CAR (UniCAR) directed against the La peptide epitope E5B9 combined with single-chain variable fragment (scFv) -based target modules (TM). In contrast to conventional CARs, anti-tumor activity of UniCAR T cells is only turned on in the presence of the TMs. Thus, this approach will allow UniCAR T cell control due to the short half-life of the TM and therefore has a favorable safety profile. Furthermore, different TMs against several TAAs can be administered both sequentially or in parallel to increase the anti-tumor efficacy or face disease relapse due to antigen escape mechanisms. In the field of myeloid malignancies our group developed retargeting strategies against the TAAs CD33 and CD123 (Cartellieri et al. Blood Cancer Journal 2016). In addition, we have developed a new TM for the UniCAR system that is directed against the TAA FLT3. FLT3 is highly expressed on acute myeloid leukemia (AML) cells and also present on CD123low AML samples (Riccioni et al. British Journal of Haematology 2011). The novel FLT3 TM was constructed by fusion of the variable domain of the heavy and the light chain of the murine anti-FLT3 monoclonal antibody (4G8) to the E5B9 UniCAR epitope. In light of a potential clinical application, we in parallel generated a humanized FLT3 TM to further decrease its immunogenicity. Both FLT3 TMs were tested in vitro against different AML cell lines, by using flow cytometry based killing assays as described elsewhere (Fasslrinner et al. British Journal of Haematology 2019). The functionality of the FLT3 TMs in vitro was highly effective. Both FLT3 TMs were able to redirect UniCAR T cells for AML cell lysis already in the picomolar range and were moreover comparable effective than the previously developed CD123 TM. Thus, humanization of the FLT3 TM did not lead to a decrease in anti-tumor efficacy. In summary, we could show that both the novel murine FLT3 TM and the humanized counterpart redirected UniCAR T cells and induced highly effective elimination of AML cells in vitro. Thus, the flexible application of the FLT3-based UniCAR system seems to be a promising tool for cell-based AML therapy alone or even in combination with other AML-specific TMs (e.g. CD33, CD123). Disclosures Koristka: Intellia Therapeutics: Employment. Jung:Synimmune: Other: shareholder interest. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

Treatment of Testicular Relapse of B-cell Acute Lymphoblastic Leukemia With CD19-specific Chimeric Antigen Receptor T Cells

BackgroundIrradiation has been a standard treatment for testicular relapse but is associated with severe hypogonadism. Because CD19-specific chimeric antigen receptor T (CAR-T) cells can eradicate leukemic blasts in cerebrospinal fluid, a pharmacologic sanctuary site, we tested the efficacy of this therapy in 7 boys with isolated testicular relapse of B-cell acute lymphoblastic leukemia. Patients and MethodsCD19-specific CAR-T cells were generated with the use of autologous T cells transduced with a lentiviral vector to express a CAR molecule containing anti-CD19 scFv derived from the HI19α murine monoclonal antibody, human CD8α hinge, and human 4-1BB (CD137) and CD3ζ costimulatory signaling transmembrane domains. After the conditioning regimen, which consisted of intravenous fludarabine and intravenous cyclophosphamide, 7 patients with a median age of 9 years (range, 2-10 years) with isolated testicular relapse received a single infusion of CD19 CAR-T cells at a total dose of 5 × 106 all T cells per kilogram. ResultsAll 7 patients achieved complete remission with normal testes. Six patients remained in second remission for 5 to 23 months (median, 14 months), and 1 patient subsequently relapsed in the bone marrow. The probability of event-free survival for all patients at 12 months of follow-up was 83.3% ± 15.2% (standard error). The treatment was well-tolerated, with grade 1 cytokine-release syndrome developing in 5 patients. ConclusionThese results suggest that CAR-T cell therapy is a treatment option for patients with testicular relapse.

Antibody-mediated targeting of TNFR2 activates CD8+ T cells in mice and promotes antitumor immunity.

Tumor necrosis factor receptor 2 (TNFR2) is the alternate receptor for TNF and can mediate both pro- and anti-inflammatory activities of T cells. Although TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in autoimmune diseases, the viability of TNFR2 as a target for cancer immunotherapy has been underappreciated. Here, we show that new murine monoclonal anti-TNFR2 antibodies yield robust antitumor activity and durable protective memory in multiple mouse cancer cell line models. The antibodies mediate potent Fc-dependent T cell costimulation and do not result in significant depletion of Tregs Corresponding human agonistic monoclonal anti-TNFR2 antibodies were identified and also had antitumor effects in humanized mouse models. Anti-TNFR2 antibodies could be developed as a novel treatment option for patients with cancer.

EP1.04-37 Biomarkers in Non-Small Cell Lung Cancer: Expression of PD-L1 Protein and p16 in Squamous Cell Carcinoma Histologic Subtype

Squamous cell carcinoma (SCC), 30% of Non-Small Cell Lung Cancer (NSCLC), cigarette is its mayor etiology. >50% NSCLC are diagnosed in advanced/stage, 10–15%EIIIB, 40%E-IV. In terms of survival, NSCLC is heterogeneous and variable. Survival at 5 years is <15%, treatment is not curative. In cancer, the evasion of the immune system and the uncontrolled tumor proliferation is important. The programmed-cell-death-ligand (PD-L1) is a lymphocyteT protein. The union PD-1/PD-L1 inhibits the lymphocyteT’s activity. Overexpression of PD/L1 in CD8+lymphocytes inhibits apoptosis, reduces survival. In NSCLC, PD-L1 is a predictor of successful immunotherapy. Inhibition PD:PD-L1 is an effective antitumoral therapy. The p16INK4a intervenes in pulmonary carcinogenesis, localized in chromosome 9p21(locus CDKN2A), its codes suppressor/tumoral proteins:p16INK4a-p14ARF. P16 inhibits D1-dependent quinases4-6(CDK4/6,ciclinaD1) which regulate the retinoblastoma protein through phosphorylation. Dysfunctional p16 inactivates Rb through hyperphosphorilation and progression of the cell cycle. Its expression contributes to the therapeutic response and to survival in NSCLC. We describe the clinical, pathological and survival characteristics in SCC based in expression of PD-L1/p16. Descriptive study, 24 patients with SCC, 2009–2013. PD–L1 studied with antibody 22C3 pharmDxkit(Agilent,Santa-Clara,CA,USA) in Autostainer Link/48DAKO®with murine monoclonal antibody(Clone/E6H4™) in BenchMark/Autostainer(Ventana®). PD-L1 was classified with TPS(Tumor/Proportion/Score), high expression TPS>50%, low expression TPS1-49%, or negative TPS=0%. P16(+) with expression >70% in nuclei, tumoral membranes. STATAv.14®, ShapiroWilk, Chi squared, Fisher, t-Student or U/Mann/Whitney were used. Survival with Kaplan-Meier. Age 67±14, 63%men. 54% of smokers, 7% had COPD. E-IV54%, E-IIIA25%, E-IIIB13%, E-IIB y E-IIA4%. PD-L1(+) more in smokers (85%) p=0.001. Treatment: palliative/care (25%), chemotherapy/surgery (17%); chemotherapy/radiotherapy/surgery (12%); radiotherapy/surgery(8%), only surgery(4%). PD-L1, high-expression survival 33m, low expression/negative 66m (log-rank test p=0.0041), Figure1: p16(+) in 10(41.6%), survival 36.2m, p16(-) survival 66.8m, p16/PD-L1(-) survival 66.8m, p16/PD-L1(+) survival 36.2m. SCC with PD-L1 TPS≥50%, p16(+), smokers with high/tumor/burden had lower survival rates. Immunotherapy against programmed cell death (PD-1) is a promising alternative impacting survival in advanced/metastatic NSCLC.