Purification of bacteriophage M13 by anion exchange chromatography

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast™ Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious

Detection of hepatitis B virus core antigen by phage display mediated TaqMan real-time immuno-PCR

The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10 ng of HBcAg with 108 pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples

Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia

Acute myeloid leukemia (AML) is an attractive entity for the development of chimeric antigen receptor (CAR) T-cell immunotherapy because AML blasts are susceptible to T-cell–mediated elimination. Here, we introduce sialic acid–binding immunoglobulin-like lectin 6 (Siglec-6) as a novel target for CAR T cells in AML. We designed a Siglec-6–specific CAR with a targeting domain derived from the human monoclonal antibody JML-1. We found that Siglec-6 is commonly expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6 CAR T cells confers specific antileukemia reactivity that correlates with Siglec-6 expression in preclinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6 expression on transformed B cells in chronic lymphocytic leukemia (CLL), and specific anti-CLL reactivity of Siglec-6 CAR T cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSPCs) and that treatment with Siglec-6 CAR T cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6 CAR T-cell therapy may be used to effectively treat AML without the need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lack of expression of Siglec-6 on normal HSPCs is a key to differentiating it from other Siglec family members (eg, Siglec-3 [CD33]) and other CAR target antigens (eg, CD123) that are under investigation in AML, and it warrants the clinical investigation of Siglec-6 CAR T-cell therapy

Entwicklung chimärer Antigenrezeptor T-Zellen mit verbessertem Therapeutischen Index in der Krebsimmuntherapie durch die Verwendung von nicht-viralen Gentransfer und Genomeditierung

The advances in genetic engineering have enabled us to confer T cells new desired functions or delete their specific undesired endogenous properties for improving their antitumor function. Due to their efficient gene delivery, viral vectors have been successfully used in T-cell engineering to provide gene transfer medicinal products for the treatment of human disease. One example is adoptive cell therapy with T cells that were genetically modified with gamma-retroviral and lentiviral (LV) delivery vectors to express a CD19-specific chimeric antigen receptor (CAR) for cancer treatment. This therapeutic approach has shown remarkable results against B-cell malignancies in pilot clinical trials. Consequently, there is a strong desire to make CAR T cell therapy scalable and globally available to patients. However, there are persistent concerns and limitations with the use of viral vectors for CAR T cell generation with regard to safety, cost and scale of vector production. In order to address these concerns, we aimed to improve non-viral gene transfer and genome editing tools as an effective, safe and broadly applicable alternative to viral delivery methods for T-cell engineering. In the first part of the study, we engineered CAR T cells through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles rather than conventional SB plasmids. This novel approach dramatically increased stable gene transfer rate and cell viability and resulted in higher yield of CAR+ T cells without the need of long ex vivo expansion to generate therapeutic doses of CAR+ T cells. Importantly, CD19-CAR T cells modified by MC-based SB transposition were equally effective as LV transduced CD19-CAR T cells in vitro and in a murine xenograft model (NSG/Raji-ffLuc), where a single administration of CD8+ and CD4+ CAR T cells led to complete eradication of lymphoma and memory formation of CAR T cells after lymphoma clearance. To characterize the biosafety profile of the CAR T cell products, we did the most comprehensive genomic insertion site analysis performed so far in T cells modified with SB. The data showed a close-to-random integration profile of the SB transposon with a higher number of insertions in genomic safe harbors compared to LV integrants. We developed a droplet digital PCR assay that enables rapid determination of CAR copy numbers for clinical applications. In the second part of the study, we ablated expression of PD-1, a checkpoint and negative regulator of T cell function to improve the therapeutic index of CAR T cells. This was accomplished using non-viral CRISPR/Cas9 via pre-assemble Cas9 protein and in vitro-transcribed sgRNA (Cas9 RNP). Finally, we combined our developed Cas9 RNP tool with CAR transposition from MC vectors into a single-step protocol and successfully generated PD-1 knockout CAR+ T cells. Based on the promising results achieved from antibody-mediated PD-1 blockade in the treatment of hematological and solid tumors, we are confident that PD-1 knockout CAR T cells enhance the potency of CAR T cell therapies for treatment of cancers without the side effects of antibody-based therapies. In conclusion, we provide a novel platform for virus-free genetic engineering of CAR T cells that can be broadly applied in T-cell cancer therapy. The high level of gene transfer rate and efficient genome editing, superior safety profile as well as ease-of-handling and production of non-viral MC vectors and Cas9 RNP position our developed non-viral strategies to become preferred approaches in advanced cellular and gene-therapy.Die Fortschritte des genetischen Engineerings erlauben uns, T-Zellen neue, erwünschte Funktionen zu verleihen oder ihnen bestimmte, unerwünschte endogenen Eigenschaften zu nehmen, um ihre Antitumorfunktion zu verbessern. Aufgrund ihrer Effizienz im Gentransport, werden virale Vektoren für das TZellengineering verwendet, um gentransferierte, medizinische Produkte zur Behandlung humaner Krankheiten herzustellen. Ein Beispiel hierfür ist die adoptive Zelltherapie mit T-Zellen, die mit gamma-retroviralen und lentiviralen (LV) Vektoren genetisch modifiziert wurden, so dass sie einen CD19-spezifischen chimären Antigenrezeptor (CAR) exprimieren. In klinischen Pilotstudien zu B-Zellerkrankungen zeigte dieser therapeutische Ansatz bereits beachtliche Erfolge. Hieraus resultiert das Bestreben, die CAR-T-Zelltherapie für Patienten skalierbar und weltweit zugänglich zu machen. Aufgrund gesundheitlicher Risiken, finanzieller Kosten und dem Umfang der Vektorenproduktion bestehen jedoch anhaltende Bedenken und Grenzen bezüglich der Verwendung viraler Vektoren für die Herstellung von CAR-T-Zellen. Um diese Problematiken zu umgehen, beabsichtigten wir, den nicht-viralen Gentransfer sowie genomverändernde Techniken soweit zu verbessern, dass sie als eine effiziente, sichere und umfassend einsetzbare Alternative zum virusbasierten T-Zellengineering verwendet werden können. Im ersten Teil dieser Arbeit stellten wir durch die Sleeping Beauty (SB) Transposition von CAR-Genen auf minimalistischen DNA Vektoren (sogenannten Minicircles) CART-Zellen her. Die Minicircles wurden anstelle von konventionellen SB Plasmiden verwendet. Mithilfe dieser neuen Vorgehensweise wurden die Rate des stabilen Gentransfers sowie das Überleben der Zellen drastisch erhöht und führte zu einer gesteigerten Rate an CAR+ T-Zellen, ohne dass eine langwierige ex vivo Expansion zur Herstellung therapeutisch relevanter CAR-T-Zelldosen nötig wurde. CD19-CART-Zellen, die mit MC-basierter SB-Transposition modifiziert wurden, zeigten in vitro und in einem murinen Xenograftmodell (NSG/Raji-ffLuc) eine vergleichbar hohe Effizienz, wie LV-transduzierte CD19-CAR-T-Zellen. Hierbei genügte eine einzige Verabreichung von CD4+ und CD8+ CAR-T-Zellen für eine komplette Eliminierung des Lymphoms und der anschließenden Gedächtnisbildung von CAR-T-Zellen. Um die Biosicherheit der CAR-T-Zellprodukte zu charakterisieren, führten wir die bislang umfassendste vergleichende Analyse von Genominsertionsstellen nach SB-basierter Modifikation von T-Zellen durch. Im Vergleich zur LV Integration zeigten diese Daten ein beinahe zufälliges Integrationsmuster des SB Transposons mit höheren Integrationsraten in genomisch „sicheren Häfen“. Wir entwickelten eine Analyse basierend auf digitaler Tröpfchen-PCR, um eine rasche Ermittlung der Anzahl an CAR-Genkopien in klinischen Anwendungen zu ermöglichen. Im zweiten Teil der Arbeit verminderten wir die Expression von PD-1, einer Prüfstelle und negativen Regulator der T-Zellfunktion, um den therapeutischen Index der CART- Zellen zu verbessern. Dies wurde durch die Verwendung eines nicht-viralen CRISPR/Cas9, durch das Zusammensetzen von Cas9 Protein und in vitrotranskribierter sgRNA (Cas9 RNP), erzielt. Schließlich verwendeten wir unsere entwickelte Cas9 RNP-Technik in Kombination mit CAR-Transposition von MCVektoren, um PD-1-knock out, CAR-positive T-Zellen herzustellen. Da die antikörperbasierte PD-1-Blockade in der Behandlung hämatologischer und solider Tumore vielversprechende Ergebnisse zeigt, sind wir zuversichtlich, dass PD-1-knock out CAR-T-Zellen die Effizienz von CAR-T-Zelltherapien verschiedener Krebsarten verbessern können und dabei die Nebenwirkungen der antikörperbasierten Therapien umgehen. Wir zeigen in der vorliegenden Arbeit Möglichkeiten mit virusfreien, gentechnischen Methoden CAR-T-Zellen herzustellen, die in der T-Zellkrebstherapie umfassend Anwendung finden können. Das hohe Level der Gentransferraten und der effizienten Genomeditierung, ein zu bevorzugendes Sicherheitsprofil sowie die einfache Handhabung und Produktion nichtviraler MC-Vektoren und Cas9 RNP machen es möglich, dass unser neuentwickelter, nichtviraler Ansatz zu einer bevorzugten Herangehensweise in der künftigen Zell- und Gentherapie werden kann

Detection of Hepatitis B Core Antigen by Phage Display Mediated Immuno-Taqman Real-Time Polymerase Chain Reaction

Hepatitis B virus (HBV) causes acute and chronic viral hepatitis and it is closely related to the development of liver cirrhosis and hepatocellular carcinoma. One of the HBV serological markers is hepatitis B core antigen (HBcAg) and it is closely related to the viral DNA load. Phage display mediated immuno-PCR (PD-IPCR) is a new strategy that combines the advantages of phage display technology and immuno-PCR (IPCR). IPCR combines the versatility of enzyme-linked immunosorbent assay (ELISA) with the amplification and sensitivity of PCR. As a consequence, IPCR leads to about 1,000- to 10,000-fold improvement in sensitivity as compared to a conventional ELISA.Previously, by the aid of phage display technology, a fusion bacteriophage bearing the sequence WSFFSNI, which interacts tightly with HBcAg was isolated. This fusion phage has the potential to be further developed as a diagnostic reagent for IPCR. Traditionally, the fusion phage required in this study has been purified by cesium chloride (CsCl) density gradient ultracentrifugation. This conventional method of phage purification is highly labourious and time consuming. In order to overcome the disadvantages of the conventional purification method, a simple, rapid and efficient method for purification of M13 phage based on anion exchange chromatography was established. A pre-packed SepFast™ Super Q column connected to a fast protein liquid chromatography system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. The purified fusion phage was used for developing a phage display mediated TaqMan real-time immuno-PCR for detecting HBcAg. The initial conditions for real-time PCR like probe concentration and annealing temperature were optimised. The best threshold cycle (Ct) was obtained when 100 nM of probe with an annealing temperature of 60 ºC were used. The efficiency of the real-time PCR reaction based on the slope of a standard curve was 96.7%. This method detected as low as 5 ng of HBcAg by using 108 pfu/ml of the fusion phage. The specificity of the assay was assessed by testing the reactivity of the fusion phage with healthy human sera. Moreover, the developed assay was also used to detect HBcAg in the sera of HBV positive patients. Based on the cutoff value which was obtained from healthy serum reactivity with the phage, the assay managed to detect the HBcAg in the sera of HBV positive patients. In conclusion, the phage displayed peptide which interacts tightly with HBcAg and its encoding DNA can replace monoclonal antibody and chemically bond DNA, respectively

Inhibition of TGF-β-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer

BackgroundImmunotherapy with chimeric antigen receptor (CAR)-engineered T-cells is effective in some hematologic tumors. In solid tumors, however, sustained antitumor responses after CAR T-cell therapy remain to be demonstrated both in the pre-clinical and clinical setting. A perceived barrier to the efficacy of CAR T-cell therapy in solid tumors is the hostile tumor microenvironment where immunosuppressive soluble factors like transforming growth factor (TGF)-β are thought to inhibit the cellular immune response. Here, we analyzed whether CAR T-cells specific for the receptor tyrosine kinase-like orphan receptor 1 (ROR1) antigen, that is frequently expressed in triple-negative breast cancer (TNBC), are susceptible to inhibition by TGF-β and evaluated TGF-β-receptor signaling blockade as a way of neutralizing the inhibitory effect of this cytokine.MethodsCD8+ and CD4+ ROR1-CAR T-cells were prepared from healthy donors and their antitumor function analyzed using the TNBC cell line MDA-MB-231 in vitro and in a microphysiologic 3D tumor model. Analyses were performed in co-culture assays of ROR1-CAR T-cells and MDA-MB-231 cells with addition of exogenous TGF-β.ResultsThe data show that exposure to TGF-β engages TGF-β-receptor signaling in CD8+ and CD4+ ROR1-CAR T-cells as evidenced by phosphorylation of small mothers against decapentaplegic homolog 2. In the presence of TGF-β, the cytolytic activity, cytokine production and proliferation of ROR1-CAR T-cells in co-culture with MDA-MB-231 TNBC cells were markedly impaired, and the viability of ROR1-CAR T-cells reduced. Blockade of TGF-β-receptor signaling with the specific kinase inhibitor SD-208 was able to protect CD8+ and CD4+ ROR1-CAR T-cells from the inhibitory effect of TGF-β, and sustained their antitumor function in vitro and in the microphysiologic 3D tumor model. Combination treatment with SD-208 also led to increased viability and lower expression of PD-1 on ROR1-CAR T-cells at the end of the antitumor response.ConclusionWe demonstrate the TGF-β suppresses the antitumor function of ROR1-CAR T-cells against TNBC in preclinical models. Our study supports the continued preclinical development and the clinical evaluation of combination treatments that shield CAR T-cells from TGF-β, as exemplified by the TGF-β-receptor kinase inhibitor SD-208 in this study

Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia

Acute myeloid leukemia (AML) is an attractive entity for the development of chimeric antigen receptor (CAR) T-cell immunotherapy because AML blasts are susceptible to T-cell-mediated elimination. Here, we introduce sialic acid-binding immunoglobulin-like lectin 6 (Siglec-6) as a novel target for CAR T cells in AML. We designed a Siglec-6-specific CAR with a targeting domain derived from the human monoclonal antibody JML-1. We found that Siglec-6 is commonly expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6 CAR T cells confers specific antileukemia reactivity that correlates with Siglec-6 expression in preclinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6 expression on transformed B cells in chronic lymphocytic leukemia (CLL), and specific anti-CLL reactivity of Siglec-6 CART cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSPCs) and that treatment with Siglec-6 CAR T cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6 CAR T-cell therapy may be used to effectively treat AML without the need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naive) B cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lack of expression of Siglec-6 on normal HSPCs is a key to differentiating it from other Siglec family members (eg, Siglec-3 [CD33]) and other CAR target antigens (eg, CD123) that are under investigation in AML, and it warrants the clinical investigation of Siglec-6 CAR T-cell therapy