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.

P06.03 Bispecific antibody-driven synthetic agonistic receptor engineered T cells lead to specific and conditional therapy in melanoma cancer models

BackgroundImmunotherapeutic approaches, including immune checkpoint blockade and adoptive T cell therapy (ACT) in the form of tumor-infiltrating lymphocytes (TIL), have had marked success in the treatment of melanoma. Despite these successes, many patients are refractory to treatment or relapse with therapy-resistant disease. To overcome said limitations, we propose a controlled ACT approach, where T cells are armed with synthetic agonistic receptors (SAR) that are conditionally activated only in the presence of a target melanoma-associated antigen, and a cross-linking bispecific antibody (BiAb) specific for both SAR T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans- membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap- extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. We validated our approach in two murine as well as two human cancer models expressing our melanoma-associated target antigens TYRP (murine) and MCSP (human). We confirmed conditional and specific stimulation and proliferation of our T cells, as well as their tumour-antigen-directed cytotoxicity, in vitro and in vivo.ResultsCrosslinking TYRP-EGFRvIII (murine) and MCSP-EGFRvIII (human) BiAb, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation of SAR-T cells and directed tumour cell lysis with specificity towards two TYRP-expressing murine melanoma and two MCSP-expressing human melanoma cancer models. In vivo, anti-tumoural activity was mediated by the co-administration of SAR-T cells and BiAb, in A375 and MV3 melanoma xenograft models. Further, we could show that SAR T cells exhibited resistance to MDSC-induced suppression of activation and proliferation.ConclusionsHere we apply the SAR x BiAb approach in efforts to deliver specific and conditional activation of SAR transduced T cells, and targeted tumour cell lysis. The modularity of our platform is key for a targeting approach in a tumor entity with a high mutational load such as melanoma and is fundamental in our drive towards personalised immunotherapies. Further, the SAR approach has demonstrated resistance to MDSC-induced suppression, an interesting axis that requires further investigation.Disclosure InformationM. Benmebarek: None. F. Märkl: None. J. Keyl: None. B. Loureiro Cadilha: None. M. Geiger: None. C. Karches: None. S. Endres: None. C. Klein: None. S. Kobold: None. A. Klüver: None. M. Schwerdtfeger: None.

Novel Cold-Adapted Recombinant Laccase KbLcc1 from Kabatiella bupleuri G3 IBMiP as a Green Catalyst in Biotransformation

Cold-adapted enzymes are useful tools in the organic syntheses conducted in mixed aqueous-organic or non-aqueous solvents due to their molecular flexibility that stabilizes the proteins in low water activity environments. A novel psychrophilic laccase gene from Kabatiella bupleuri, G3 IBMiP, was spliced by Overlap‑Extension PCR (OE-PCR) and expressed in Pichia pastoris. Purified recombinant KbLcc1 laccase has an optimal temperature of 30 °C and pH of 3.5, 5.5, 6.0, and 7.0 in the reaction with 2,2′‑azino‑bis(3‑ethylbenzothiazoline‑6‑sulfonic acid) (ABTS), guaiacol, sinapic acid, and syringaldazine, respectively. Moreover, laccase KbLcc1 is highly thermolabile, as it loses 40% of activity after 30 min at 40 °C and is inactivated at 50 °C after the same period of incubation. The new enzyme remained active with 1 mM of Ni2+, Cu2+, Mn2+, and Zn2+ and with 2 mM of Co2+, Ca2+, and Mg2+, but Fe2+ greatly inhibited the laccase activity. Moreover, 1% ethanol had no impact on KbLcc1, although acetone and ethyl acetate decreased the laccase activity. The presence of hexane (40%, v/v) caused a 58% increase in activity. Laccase KbLcc1 could be applied in the decolorization of synthetic dyes and in the biotransformation of ferulic acid to vanillin. After 5 days of reaction at 20 °C, pH 3.5, with 1 mM ABTS as a mediator, the vanillin concentration was 21.9 mg/L and the molar yield of transformation reached 14.39%.

Construction of recombinant Escherichia coli producing nitrogenase-related proteins from Azotobacter vinelandii

Biological nitrogen fixation by nitrogenase has attracted attention as an alternative method to chemical nitrogen fixation, which requires large amounts of fossil fuels. Azotobacter vinelandii, which produces an oxygen-sensitive nitrogenase, can fix nitrogen even under aerobic conditions; therefore, the heterologous expression of nif-related genes from A. vinelandii is a promising strategy for developing a biological nitrogen fixation method. We assembled 17 nif-related genes, which are scattered throughout the genome of A. vinelandii, into synthetic gene clusters by overlap-extension-PCR and seamless cloning and expressed them in Escherichia coli. The transcription and translation of the 17 nif-related genes were evaluated by RT-qPCR and LC-MS/MS, respectively. The constructed E. coli showed nitrogenase activity under anaerobic and microaerobic conditions. This strain would be a useful model for examining the effect of other genes from A. vinelandii on nitrogen fixation by expressing them in addition to the minimal set of nif-related genes.

Molecular and Biochemical Characterization of nifHDK Genes in Klebsiella pneumoniae

Nitrogen fixation is carried by an enzyme complex called nitrogenase which consists of two main components, a dinitrogenase that is encoded by nifD and nifK and an iron containing reductase, also called Fe protein which is encoded by nifH. Nitrogen-free medium was used to detect the ability of nitrogen fixation by Klebsiella pneumonia, then DNA was extracted and overlap extension polymerase chain reaction of nifH, nifD and nifK. To obtain nucleotide sequences of these genes, sequencing of the PCR products was one. The reverse sequence of nifH and the forward sequences of nifD and nifK were converted into amino acids using online translation tool. Homology modeling was carried out using SWISS-MODEL. The modeled amino acids sequences was validated using ERRAT and PROCHECK. The modeled sequences were reliable and of quality higher than 90%. The two subunits of Fe protein were constructed and tertiary structure was predicted together with the binding sites for prosthetic group and ADP molecule in Fe protein. The following amino acids Asp11, Lys13, Asn157, Ser158, Val183, Pro184, Arg185, Asp186, Val189, Gln190 and Glu193 seem to participate in the ADP binding. The complexity of this enzyme makes it difficult to be cloned in plants.

Bioinformatic Analysis in Designing Mega-primer in Overlap Extension PCR Cloning (OEPC) Technique

Bioinformatics has developed into an application tool for basic and applied research in the biomedical and biotechnology field. Polymerase Chain Reaction (PCR) is a common technique in the molecular area that has always involved bioinformatics science. PCR cloning techniques such as TA cloning and PCR-mediated cloning exhibit complex processes with low success rates. One easy, effective, and practical solution is to use a mega-primer with the Overlap Extension PCR Cloning (OEPC) technique. The success of PCR cloning using the mega-primer design in the OEPC technique is strongly influenced by the characteristics of the mega-primer used. Knowledge of mega-primer characteristics is one of the important factors in the success of PCR cloning. The design process for the mega-primer str promoter was characterized based on the principle of a genetic algorithm using the web-based bioinformatics tools such as ClustalW, NetPrimer, and BLAST. The success of the mega-primer construction in producing recombinant pSB1C3 vector has been confirmed by the sequencing method and the function of the reporting protein (AmilCP). DNA analysis shows a 100 % homologous sequence on the str promoter, while E. coli colonies successfully express the purplish-blue color. Mega-primer characters can save costs and time of the research by maintaining the primer parameters that provide optimal values and increase the success value of PCR cloning via bioinformatics software. Hence, implications on biological problems, especially using DNA and amino acid sequences, could solve rapidly.

Construction and Characterization of 1F5 Chimeric Anti-CD20 Monoclonal Antibodies: an Efficient Splicing by Overlap Extension-polymerase Chain Reaction Technique

Despite the unparalleled success of anti-CD20-targeted immunotherapy, the currently available mAbs are not sufficiently efficacious in the treatment of lymphoma. 1F5 is one of a panel of anti-CD20 mAbs that was used in the B-cell lymphoma serotherapy. Despite the efficacy of murine 1F5 mAbs in lymphoma patients, the 1F5 chimeric antibodies with human effector functionality are yet to be approved and widely used in the treatment of lymphoma. In this study, the conversion of 1F5 mAb from mouse IgG2a to human-mouse chimeric IgG1 was achieved and the chimeric antibody was partially characterized. We constructed the 1F5 chimeric mouse-human anti-CD20 antibody genes using an efficient Splicing by overlap extension-polymerase chain reaction (SOE-PCR) technique and cloned the chimeric heavy and light genes in pBudCE4.1 mammalian expression vector, followed by purification of the expressed chimeric 1F5 mAbs using affinity chromatography. Our investigation also included the biological properties of purified chimeric antibodies. The generated 1F5 chimeric mAbs mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against Raji and Daudi Burkitt's lymphoma cell lines, which were comparable with rituximab and exhibit superior reduction in cell viability in vitro, compared to rituximab. The current study indicated that the generated chimeric 1F5 mAbs has potential CDC and ADCC activity which was comparable with rituximab whereas it exhibits a superior reduction in cell viability, compared to rituximab. Our work contributes to future studies involving in vivo biological functions and the application of the 1F5 chimeric antibody.

Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells

Background KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins. Methods This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells. Results The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells. Discussion These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.

Multiplexed Competition in a Synthetic Squid Light Organ Microbiome Using Barcode-Tagged Gene Deletions

ABSTRACTBeneficial symbioses between microbes and their eukaryotic hosts are ubiquitous and have widespread impacts on host health and development. The binary symbiosis between the bioluminescent bacterium Vibrio fischeri and its squid host Euprymna scolopes serves as a model system to study molecular mechanisms at the microbe-animal interface. To identify colonization factors in this system, our lab previously conducted a global transposon insertion sequencing (INSeq) screen and identified over 300 putative novel squid colonization factors in V. fischeri. To pursue mechanistic studies on these candidate genes, we present an approach to quickly generate barcode-tagged gene deletions and perform high-throughput squid competition experiments with detection of the proportion of each strain in the mixture by barcode sequencing (BarSeq). Our deletion approach improves on previous techniques based on splicing by overlap extension PCR (SOE-PCR) and tfoX-based natural transformation by incorporating a randomized barcode that results in unique DNA sequences within each deletion scar. Amplicon sequencing of the pool of barcoded strains before and after colonization faithfully reports on known colonization factors and provides increased sensitivity over colony counting methods. BarSeq enables rapid and sensitive characterization of the molecular factors involved in establishing the Vibrio-squid symbiosis and provides a valuable tool to interrogate the molecular dialogue at microbe-animal host interfaces.IMPORTANCE Beneficial microbes play essential roles in the health and development of their hosts. However, the complexity of animal microbiomes and general genetic intractability of their symbionts have made it difficult to study the coevolved mechanisms for establishing and maintaining specificity at the microbe-animal host interface. Model symbioses are therefore invaluable for studying the mechanisms of beneficial microbe-host interactions. Here, we present a combined barcode-tagged deletion and BarSeq approach to interrogate the molecular dialogue that ensures specific and reproducible colonization of the Hawaiian bobtail squid by Vibrio fischeri. The ability to precisely manipulate the bacterial genome, combined with multiplex colonization assays, will accelerate the use of this valuable model system for mechanistic studies of how environmental microbes—both beneficial and pathogenic—colonize specific animal hosts.