Chimeric carrier proteins for targeted delivery of tumor antigens to professional antigen presenting cells

Tumor-specific T lymphocytes can be regarded as a highly effective mechanism for tumor rejection. A substantial number of T-cell defined tumor antigens including mutated oncoproteins and differentiation antigens have been identified. However, while most spontaneous tumors appear to be antigenic, few are immunogenic. Activation of tumor-specific cytotoxic T cells (CTL) requires presentation of tumor antigens by professional antigen presenting cells (APCs) via MHC I molecules. Due to their crucial role in T-cell activation, APCs are being exploited for active cancer immunotherapy. Present experimental strategies include the incubation of dendritic cells with synthetic, tumor specific peptides to achieve uptake of tumor antigens and presentation in the context of MHC molecules. Alternatively, gene therapeutic approaches are aimed at the endogenous expression of tumor antigens in APCs upon transfer of suitable vector constructs. Our strategy for the presentation of tumor antigens by APCs is based on the intracellular delivery of tumor antigens as part of a fusion protein specifically targeted to APC cell surface receptors. We have constructed prototype molecules that contain a soluble fragment of CTLA-4 for cell binding via interaction with B7 molecules, genetically fused to a protein fragment derived from the tumor-associated antigen ErbB2. To improve uptake and direct the antigenic determinant preferentially to the MHC class I pathway, in one of these protein vaccines also the translocation domain of the bacterial Pseudomonas exotoxin A has been included. In the parental toxin this protein domain facilitates escape from the endosomal compartment to the cytosol upon receptor mediated endocytosis. Here we have investigated the in vitro cell binding activity of such reagents and their antitumoral activity in immunocompetent murine model systems. Specific binding to B7 molecules and uptake of bacterially expressed protein vaccines could be demonstrated. Ex vivo restimulation with an ErbB2-derived peptide of splenocytes from Balb/c mice injected with the fusion proteins resulted in enhanced IFN-gamma production by T cells. Protective and therapeutic effects of ErbB2 protein vaccines were also investigated. Vaccinated animals were protected against subsequent challenge with syngeneic ErbB2 expressing tumor cells. Likewise, s.c. injection of ErbB2 protein vaccines in the vicinity of established tumors resulted in tumor rejection and long lasting protection indicating that immunological memory was induced. Our results suggest that chimeric proteins combining a tumor antigen and specific recognition of APCs in a single molecule are suitable for targeted delivery of antigens to professional APCs and might become valuable tools for cancer immunotherapy

The Science Performance of JWST as Characterized in Commissioning

This paper characterizes the actual science performance of the James Webb Space Telescope (JWST), as determined from the six month commissioning period. We summarize the performance of the spacecraft, telescope, science instruments, and ground system, with an emphasis on differences from pre-launch expectations. Commissioning has made clear that JWST is fully capable of achieving the discoveries for which it was built. Moreover, almost across the board, the science performance of JWST is better than expected; in most cases, JWST will go deeper faster than expected. The telescope and instrument suite have demonstrated the sensitivity, stability, image quality, and spectral range that are necessary to transform our understanding of the cosmos through observations spanning from near-earth asteroids to the most distant galaxies.Comment: 5th version as accepted to PASP; 31 pages, 18 figures; https://iopscience.iop.org/article/10.1088/1538-3873/acb29

The Science Performance of JWST as Characterized in Commissioning

This paper characterizes the actual science performance of the James Webb Space Telescope (JWST), as determined from the six month commissioning period. We summarize the performance of the spacecraft, telescope, science instruments, and ground system, with an emphasis on differences from pre-launch expectations. Commissioning has made clear that JWST is fully capable of achieving the discoveries for which it was built. Moreover, almost across the board, the science performance of JWST is better than expected; in most cases, JWST will go deeper faster than expected. The telescope and instrument suite have demonstrated the sensitivity, stability, image quality, and spectral range that are necessary to transform our understanding of the cosmos through observations spanning from near-earth asteroids to the most distant galaxies

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Induction of anti-tumor immunity by targeted delivery of ErbB2 cancer vaccines to antigenpresenting cells

Tumor-specific T lymphocytes are being regarded as a highly effective mechanism for tumor rejection. Activation of tumor-specific cytotoxic CD8+ T cells (Tc or CTLs) requires presentation of tumor antigens by professional antigen presenting cells (APCs) via MHC class I molecules. Due to this crucial role in T-cell activation, APCs are being exploited for active cancer immunotherapy. Independent of CTLs, also CD4+ helper T cells (Th) can mediate a number of anti-tumor effector pathways. Activation of CD4+ T cells requires the presentation of antigen via MHC class II. So far only a limited number of MHC class II-presented, tumorspecific Th-epitopes have been identified. Therefore most current vaccination strategies are focusing on the use of defined Tc epitopes able to elicit tumor-antigen specific CTL responses. Such strategies include vaccines based on tumor-specific peptides, proteins or protein fragments, or DNA constructs encoding such tumor antigens. Since these reagents on their own are usually poorly immunogenic, they need to be administered together with an adjuvant to stimulate sufficiently strong immune responses. Dendritic cells (DCs), due to their ability to initiate primary immune responses, are the most efficient APCs and can be regarded as "nature’s adjuvant". Vaccine development therefore focuses more and more on exploiting the unique capabilities of DCs. Present experimental studies include loading of DCs with synthetic, tumor-specific peptides or tumor lysates, to achieve uptake of tumor antigens and presentation in the context of MHC molecules. However, while such vaccination approaches in some cases yielded promising results, they are associated with laborious and cost-intensive ex vivo processing of primary cells and, in the case of peptide antigens, are restricted to well-defined T-cell epitopes known to match the patient’s MHC haplotype. Therefore constructs that directly target vaccines to DCs in vivo and/or deliver a number of different T-cell epitopes, are highly desirable and might overcome some of the limitations of present approaches. The optimization of vaccine delivery can be expected to improve efficacy, reduce doses and the risk of side effects, and improve control of immunological outcome. In this thesis, construction and functional characterization of two novel types of cancer vaccines are described. These vaccines were designed to be applicable for in vivo vaccination and induction of anti-tumor immunity specific for the tumor-associated antigen ErbB2 (HER2/neu). ErbB2 is overexpressed in a variety of human malignancies including breast and ovarian carcinomas, and adenocarcinomas of the lung. Other types of carcinomas that show variable ErbB2 overexpression are those of the colon, kidney, bladder and salivary gland, prostate and pancreas. The first type of cancer vaccines intended to improve uptake and presentation of tumor antigen by APCs is based on the intracellular delivery of an antigenic determinant as part of a fusion protein specifically targeted to cell surface molecules exclusively expressed on APCs. As a potential cell targeting domain, a fragment derived from the extracellular part of the B7 counter receptor CTLA-4 (CTLA-4125) was chosen and expressed as a recombinant protein in E. coli. By FACS analysis, soluble human CTLA-4125 purified from bacterial lysates could be shown to bind specifically to murine and human B7 molecules selectively expressed on APCs, suggesting that CTLA-4125 could serve as an effective targeting domain for the delivery of tumor antigens to APCs. As chimeric protein vaccines for targeted delivery to APCs, two fusion proteins termed C-ErbB2(N) and C-E-ErbB2(N) were constructed, that comprise CTLA-4125 at the N-terminus for binding to B7, and a fragment from the extracellular part of the human tumor-associated ErbB2 antigen (ErbB2(N), amino acid residues 1-222) at the C-terminus. In the C-E-ErbB2(N) protein, between cell targeting domain and antigenic determinant the translocation domain of Pseudomonas exotoxin A (ETA II) was included to enable endosome escape upon receptor-mediated uptake of fusion protein by APC, possibly resulting in preferential delivery to the MHC class I pathway. Chimeric CTLA-4-ErbB2 fusion proteins were expressed in E. coli and purified from bacterial lysates by metal chelate affinity chromatography. Specific binding to B7 molecules on the surface of cells and uptake of the recombinant antigen-carrier proteins by B7 expressing human Raji cells could be demonstrated by FACS analysis and laser scanning microscopy. Immune responses induced by CTLA-4-ErbB2 fusion proteins were analyzed in Balb/c mice. Ex vivo restimulation with an ErbB2 derived peptide of splenocytes from animals vaccinated with the chimeric proteins led to enhanced IFN-g production by T cells. Subcutaneous challenge of Balb/c mice with syngeneic ErbB2+ renal carcinoma cells (Renca-lacZ/ErbB2) resulted in rejection of tumors by the majority of mice vaccinated with CTLA-4-ErbB2 fusion proteins indicating the induction of protective immunity. While both fusion proteins were effective, C-ErbB2(N) protein which lacks the bacterial translocation domain appeared to be more potent than C-E-ErbB2(N) in several independent experiments. Mice vaccinated with CTLA-4-ErbB2 fusion proteins were not protected against challenge with ErbB2 negative Renca-lacZ cells demonstrating specificity of the immune responses induced by the vaccines. T-cell depletion experiments showed that the protective effects were strictly dependent on the presence of CD8+ CTLs. Intravenous rechallenge of vaccinated, tumor-free animals two months after the first, subcutaneous tumor challenge did not result in the formation of lung metastasis suggesting that long-lasting immune responses had been induced. In therapeutic vaccination experiments more closely resembling a possible clinical situation, CTLA-4-ErbB2 fusion proteins were injected into the periphery of established, subcutaneously growing Renca-lacZ/ErbB2 tumors. In two independent experiments therapeutic vaccination with C-ErbB2(N) resulted in the cure of 7 out of 8 mice, whereas CE-ErbB2(N) also in a therapeutic setting appeared to be slightly less effective curing 6 out of 8 animals. Cured animals were also protected against intravenous re-challenge with RencalacZ/ErbB2 cells confirming the induction of long-lasting anti-tumor immunity. As an alternative to production of recombinant protein vaccines in bacteria, also direct injection of DNA constructs for in vivo expression and secretion of CTLA-4-ErbB2 fusion proteins was tested. Based on plasmid pSecTag2 mammalian expression vectors were constructed, which encode under the control of a CMV promoter the C-ErbB2(N) and C-EErbB2(N) proteins tagged for secretion with an Igk leader sequence. Upon transfection of COS-7 cells in tissue culture, secretion of CTLA-4-ErbB2 fusion proteins into the culture supernatant and B7 binding activity of such proteins could be confirmed by immunoblot and FACS analysis. In protective vaccination experiments, intramuscular injection of CTLA-4-ErbB2 DNA constructs in Balb/c mice resulted in complete protection from subcutaneous challenge with Renca-lacZ/ErbB2 tumor cells, whereas all animals vaccinated with pSecTag2 control DNA developed rapidly growing tumors. In conclusion, the data obtained in this first part of the thesis project demonstrate that chimeric protein vaccines combining in a single polypeptide chain a cell recognition domain for specific targeting to APCs with an antigenic protein fragment derived from human ErbB2 are effective in inducing ErbB2-specific, T-cell mediated anti-tumor immunity in vivo. Thereby both, injection of recombinant proteins produced in bacteria, and vaccination with mammalian expression plasmids for in vivo production of chimeric protein vaccines was effective resulting in the rejection of syngeneic, ErbB2 expressing tumors. The chimeric protein vaccines described above contain a large protein fragment derived from a human tumor antigen for delivery to and uptake by APCs. This approach is not necessarily dependent on a given MHC type and might be particularly useful to induce immune responses against antigens for which peptides suitable for presentation by most prevalent MHC alleles have not been defined. For tumor antigens where individual peptides have already been characterized as potential rejection antigens, in a limited number of cases synthetic derivatives of such peptides have successfully been used to induce T-cell responses of single specificity. However, also in this case inclusion of appropriate antigen carriers could facilitate improved uptake and presentation by APCs and enhance immunogenicity. Therefore in an alternative approach to induce ErbB2-specific anti-tumor immunity, in vivo application of synthetic vaccine complexes was investigated which are based on a liposomal carrier as a vector for peptide antigens linked to the liposomal surface via a special adjuvant lipopeptide anchor (Pam3CysSerSer). This part of the thesis project was carried out in close collaboration with Audrey Roth, Dr. Benoit Frisch and Dr. Francis Schuber, Laboratoire de Chimie Bioorganique, Faculté de Pharmacie, Université Louis Pasteur (ULP), Strasbourg, who prepared and provided all liposomal vaccine constructs tested in this study. Two liposomal formulations were synthesized. One single-epitope construct, referred to as Tc-ErbB2-liposomes, carried a Tc-epitope (TYLPTNASL) derived from the human ErbB2 amino acid sequence. This peptide has previously been shown to bind to murine H-2Kd MHC class I with high affinity and to elicit peptide-specific CTL responses in Balb/c mice. The second liposomal formulation, termed Tc-ErbB2/Th-liposomes, contained in addition to the ErbB2 peptide a universal Th-epitope derived from influenza virus hemagglutinin (HA 307-319) attached to the adjuvant anchor. Inclusion of the Th-epitope was intended to increase CTL responses against the Tc-epitope through additional recruitment of CD4+ T cell help. Both liposomal vaccines were able to induce activation of Tc-specific CTLs in vivo, as determined by ex vivo restimulation with the respective ErbB2 peptide of splenocytes from Balb/c mice vaccinated with the liposomal constructs. Using the immunocompetent Balb/c mouse model already employed for characterization of CTLA-4-ErbB2 protein vaccines, antitumoral activity of the liposomal complexes was investigated. Vaccination of mice with both constructs, Tc-ErbB2-liposomes and Tc-ErbB2/Th-liposomes resulted in protection of mice from subsequent subcutaneous challenge with ErbB2 expressing Renca-lacZ/ErbB2 cells. Therapeutic vaccination of mice bearing established ErbB2+ tumors with Tc-ErbB2-liposomes or Tc-ErbB2/Th-liposomes led to delayed tumor growth in some and complete tumor rejection in the remaining animals. In control animals treated with liposomal carrier no signs of regression were noted. While both liposomal formulations were effective in these protective and therapeutic vaccination experiments, addition of the Th-epitope to the Tc-ErbB2 liposomes enhanced both, ErbB2-specific CTL responses and anti-tumor immunity. This underscores the beneficial role of synergistic CD4+ T cell activation as part of anti-tumor immune responses. While different in their mode of action and chemical composition, both types of cancer vaccines investigated in this thesis project were effective and, upon in vivo application resulted in ErbB2-specific, T-cell mediated anti-tumor immunity. Chimeric protein vaccines containing a large antigenic protein fragment facilitate targeted delivery to and uptake by APCs, followed by in vivo processing of the proteins into small antigenic peptides for MHC presentation and activation of tumor-specific T cells. This approach might be particularly useful to induce immune responses against antigens for which peptides suitable for presentation by most prevalent MHC alleles have not yet been defined, or to apply as a single vaccine construct composite antigens consisting of different antigenic determinants. For tumor antigens where individual peptides have already been defined as potential rejection antigens, novel liposomal carriers containing in addition to the Tc-epitope an immunostimulatory lipopeptide anchor and a strong Th-epitope might provide a means to increase immunogenicity and enhance antitumoral activity of synthetic peptide vaccines.Tumorspezifische T-Zellen stellen den im Prinzip wirkungsvollsten Mechanismus zur Abstoßung von Tumoren dar. Voraussetzung für die Aktivierung tumorspezifischer zytotoxischer CD8+ T-Zellen (Tc oder CTLs) ist die Präsentation von Tumorantigenen auf MHC-Klasse I Molekülen durch professionelle Antigen-präsentierende Zellen (APCs). Wegen ihrer Schlüsselrolle bei der Aktivierung von T-Zellen werden APCs in der aktiven Krebs-Immuntherapie eingesetzt. Unabhängig von CTLs können auch CD4+ Helfer T-Zellen (Th) eine Reihe effektiver Anti-Tumor Mechanismen einleiten. Die Aktivierung von CD4+ erfordert die Präsentation des Antigens via MHC-Klasse II Molekülen. Bis jetzt konnten jedoch nur einige wenige tumor-spezifische Th-Epitope identifiziert werden, welche auf MHC-Klasse II Molekülen präsentiert werden. Aus diesem Grund benutzen die meisten derzeitigen Vakzinierungsstrategien definierte Tc-Epitope, die in der Lage sind, spezifische Immunantworten gegen das Tumorantigen auszulösen. Diese Vakzine basieren auf tumorspezifischen Peptiden, Proteinen oder Proteinfragmenten, aber auch auf DNA-Konstrukten, welche für ein bestimmtes Tumorantigen kodieren. Um allerdings eine ausreichend starke Immunreaktion auszulösen, ist es notwendig diese normalerweise nur schwach immunogenen Substanzen zusammen mit Adjuvanzien zu verabreichen. Dendritische Zellen (DCs) sind hoch-effiziente APCs. Weil sie in der Lage sind, die primäre Immunantwort einzuleiten werden sie auch als „natürliches Adjuvanz“ bezeichnet. Nicht zuletzt deswegen werden bei der Entwicklung von Vakzinen immer mehr die einzigartigen Eigenschaften von DCs ausgenutzt. Gegenwärtige Studien untersuchen daher die Beladung von DCs mit synthetischen, tumorspezifischen Peptiden oder Tumor-Lysaten, um eine Aufnahme des Tumorantigens sowie dessen anschließende Präsentation auf MHCKlasse I Molekülen zu erreichen. Obgleich einige dieser Vakzinierungs-Ansätze zu vielversprechenden Ergebnissen führten, sind solche Strategien mit der aufwendigen und kostenintensiven ex vivo Aufbereitung primärer Dendritischer Zellen verbunden. Zudem muss gewährleistet sein, dass die ausgewählten T-Zell Epitope auch vom jeweiligen MHC-Haplotyp des Patienten präsentiert werden. Um einige dieser Nachteile auszugleichen, wären „Konstrukte“ wünschenswert, welche in vivo einen zielgerichteten Transport eines oder besser, mehrerer T-Zell Epitope zu den Dendritischen Zellen ermöglicht. Mit der Entwicklung und Optimierung von Systemen, die Tumorantigene gezielt zu und in APCs transportieren, könnte die Effizienz herkömmlicher Vakzinierungen gesteigert und deren Dosierung verringert werden. In dieser Arbeit wird die Herstellung sowie die funktionelle Charakterisierung zweier neuartiger Vakzine beschrieben. Diese Vakzine wurden für die in vivo Vakzinierung gegen das tumorassoziierte Antigen ErbB2 (HER2/neu) entwickelt. ErbB2 wird in einer Reihe von Tumoren wie Brust- und Ovarialkarzinomen oder Adenokarzinomen der Lunge überexprimiert. Andere Tumorenentitäten wie Kolon-, Nieren-, Blasen-, Speicheldrüsen-, Prostata- und Pankreaskarzinome zeigen ebenfalls eine variable Überexpression von ErbB2. Ziel des ersten der beiden Vakzinierungsansätze war eine verbesserte Aufnahme und Präsentation von Tumorantigenen durch APCs zu erreichen. Als Teil eines Fusionsproteins sollte hier die zielgerichtete Bindung einer antigenen Determinante an APC-spezifische Oberflächenmoleküle und deren anschließende Aufnahme und Prozessierung innerhalb von APCs ermöglicht werden. Als mögliche APC-Bindedomäne wurde ein Fragment gewählt, welches sich vom extrazellulären Anteil des B7-Rezeptors CTLA-4 (CTLA-4125) ableitet. B7 ist ein Oberflächenprotein, das speziell auf APCs exprimiert wird. Nach der bakteriellen Expression und Reinigung wurde durch FACS-Analyse gezeigt, dass lösliches humanes CTLA-4125 spezifisch sowohl an murine als auch humane B7 Moleküle bindet und daher als effektive APC-Bindungsdomäne für den zielgerichteten Transport von Tumorantigenen zu APCs verwendet werden kann. Als chimäre Proteinvakzine zur spezifischen Bindung an APCs wurden zwei mit C-ErbB2(N) bzw. C-E-ErbB2(N) bezeichnete Fusionsproteine konstruiert. Diese setzen sich aus CTLA-4125 am N-Terminus für die Bindung an APCs und einem Fragment des extrazellulären Teils des humanen tumorassoziierten ErbB2 Antigens (ErbB2(N), Aminosäuren 1-222) am C-Terminus zusammen. Beim C-E-ErbB2(N) Protein wurde zusätzlich die Translokationsdomäne von Pseudomonas exotoxin A (ETA II) zwischen der APC-Bindungsdomäne und der antigenen Determinante eingefügt. Diese Domäne könnte nach Rezeptor-vermittelter Aufnahme des Fusionsproteins die Freisetzung des Tumorantigens aus dem Endosom ins Zytoplasma verbessern und so die Prozessierung und Präsentation des Antigens via MHC-Klasse I verstärken. Beide chimären CTLA-4-ErbB2 Fusionsproteine wurden in E. coli exprimiert und mittels Metallchelat-Chromatographie aus den bakteriellen Lysaten gereinigt. Die spezifische Bindung der rekombinanten Fusionsproteine an B7 Moleküle auf der Zelloberfläche sowie deren Aufnahme durch B7-exprimierende humane Raji Zellen wurde mittels FACS Analyse und konfokaler Mikroskopie nachgewiesen. In Balb/c Mäusen wurde untersucht, inwieweit die Fusionsproteine eine Immunantwort gegen das Tumorantigen auslösen können. Die Vakzinierung von Balb/c Mäusen mit den CTLA-4 Fusionsproteinen bewirkte die Abstoßung anschließend subkutan injizierter syngener ErbB2-positiver Nierenkarzinomzellen (Renca-lacZ/ErbB2) nach subkutaner Injektion. Dies deutet auf die Erzeugung protektiver Immunität hin. Obgleich beide Fusionsproteine effektiv waren, zeigte das Fusionsprotein C-ErbB2(N), dem die bakterielle Translokationsdomäne fehlt, in mehreren unabhängigen Experimenten eine höhere Wirksamkeit als C-E-ErbB2(N). Vakzinierung mit den CTLA-4 Fusionsproteinen erzeugte keinen Schutz gegen ErbB2-negative Kontroll-Tumorzellen. Dies Experimente zeigt die Antigen-Spezifität der induzierten Immunantwort nach Vakzinierung mit CTLA-4 Fusionsproteinen. T-Zell Depletionsexperimente ergaben, dass die protektiven Effekte eindeutig von CD8+ CTLs abhängig waren. Eine erneute, diesmal intravenöse Applikation von ErbB2+ Tumorzellen in vakzinierte, tumorfreie Mäuse zwei Monate nach der ersten, subkutanen Injektion („Re-challenge“) führte nicht zur Ausbildung von Lungenmetastasen. Dies deutet darauf hin, dass durch die Vakzinierung eine langanhaltende Immunreaktion induziert wurde. In therapeutischen Vakzinierungsexperimenten, die wohl am ehesten eine klinische Situation in Tumorpatienten widerspiegeln, wurden die CTLA-4 Fusionsproteine in die Umgebung bereits etablierter subkutaner Renca-lacZ/ErbB2 Tumoren injiziert. In zwei unabhängigen Experimenten konnte gezeigt werden, dass durch die therapeutische Vakzinierung mit CErbB2(N) 7 von 8 Mäusen geheilt werden konnten, wohingegen die Wirkung von C-EErbB2(N) – von 8 Mäusen wurden 6 geheilt – auch im therapeutischen Einsatz etwas schwächer zu sein schien. Nach einem intravenösen „Re-challenge“ mit Renca-lacZ/ErbB2 Zellen waren bereits geheilte Tiere auch vor der Ausbildung von Lungenmetastasen geschützt. Dies bestätigt eine durch die Vakzinierung hervorgerufene lang-anhaltende Anti-Tumor Immunität. Alternativ zur Produktion rekombinanter Proteinvakzine in Bakterien wurde auch die direkte Injektion von DNA-Konstrukten zur in vivo Expression und Sekretion der CTLA-4 Fusionsproteine getestet (DNA-Vakzinierung). Basierend auf dem Plasmid pSecTag2 wurden Säugerzell-Expressionsvektoren hergestellt, welche unter der Kontrolle eines CMVPromotors die beiden Fusionsproteine C-ErbB2(N) bzw. C-E-ErbB2(N) kodieren. Ein Igk “leader” Signalpeptid ermöglicht hierbei die Sekretion der produzierten Proteine. Nach Transfektion der Plasmide in COS-7 Zellen konnte Sekretion der CTLA-4-Fusionsproteine in den Zellkulturüberstand sowie deren Zellbindungsspezifität für B7, mit Hilfe von Western-Blot und FACS-Analysen nachgewiesen werden. Protektionsexperimente ergaben, dass alle Balb/c Mäuse, die vorher durch intramuskuläre Injektion der CTLA-4-ErbB2 DNA-Vakzine vakziniert wurden, vor dem Anwachsen subkutan injizierter Renca-lacZ/ErbB2 Tumorzellen geschützt waren. Mäuse, die als Kontrolle mit dem leeren pSecTag2 Vektor vakziniert wurden, entwickelten dagegen schnell wachsende Tumoren. In diesem ersten Teil dieser Arbeit konnte gezeigt werden, dass chimäre Proteinvakzine, die in einer einzigen Polypeptidkette eine Zellerkennungsdomäne zur spezifischen Bindung an APCs und ein antigenes Proteinfragment aus dem humanen ErbB2 enthalten, in vivo eine äußerst wirkungsvolle ErbB2-spezifische T-Zell vermittelte Anti-Tumor Immunantwort auslösen. Hierbei konnte sowohl durch die Injektion von rekombinanten, in Bakterien hergestellten Proteinen, wie auch durch DNA-Vakzinierung zur in vivo Produktion dieser chimären Proteinvakzine erreicht werden, dass syngene, ErbB2 exprimierende Tumoren abgestoßen wurden. Die oben beschriebenen chimären Proteinvakzine enthalten ein großes Proteinfragment, abgeleitet aus einem humanen Tumorantigen, welches zu APCs befördert und von ihnen aufgenommen wird. Dieser Ansatz ist nicht unbedingt abhängig von einem bestimmten MHCTyp und möglicherweise besonders dann wirkungsvoll, wenn Immunantworten gegen Antigene erzeugt werden sollen, für die noch keine von MHC-Molekü

Induction d'une immunité anti-tumorale par le ciblage de constructions vaccinales anti-ErbB2 vers des cellules présentatrices d'antigènes

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceGermanyFRD

Light-sheet microscopy in thick media using scanned Bessel beams and two-photon fluorescence excitation

In this study we show that it is possible to successfully combine the benefits of light-sheet microscopy, self-reconstructing Bessel beams and two-photon fluorescence excitation to improve imaging in large, scattering media such as cancer cell clusters. We achieved a nearly two-fold increase in axial image resolution and 5-10 fold increase in contrast relative to linear excitation with Bessel beams. The light-sheet penetration depth could be increased by a factor of 3-5 relative to linear excitation with Gaussian beams. These findings arise from both experiments and computer simulations. In addition, we provide a theoretical description of how these results are composed. We investigated the change of image quality along the propagation direction of the illumination beams both for clusters of spheres and tumor multicellular spheroids. The results reveal that light-sheets generated by pulsed near-infrared Bessel beams and two photon excitation provide the highest image resolution, contrast and light-sheet penetration at a minimum amount of artifacts

Self-reconstructing sectioned Bessel beams offer submicron optical sectioning for large fields of view in light-sheet microscopy

One of main challenges in light-sheet microscopy is to design the light-sheet as extended and thin as possible - extended to cover a large field of view, thin to optimize resolution and contrast. However, a decrease of the beam's waist also decreases the illumination beam's depth of field. Here, we introduce a new kind of beam that we call sectioned Bessel beam. These beams can be generated by blocking opposite sections of the beam's angular spectrum. In combination with confocal-line detection the optical sectioning performance of the light-sheet can be decoupled from the depth of field of the illumination beam. By simulations and experiments we demonstrate that these beams exhibit self-reconstruction capabilities and penetration depths into thick scattering media equal to those of conventional Bessel beams. We applied sectioned Bessel beams to illuminate tumor multicellular spheroids and prove the increase in contrast. Sectioned Bessel beams turn out to be highly advantageous for the investigation of large strongly scattering samples in a light-sheet microscope

Extent, impact, and predictors of diagnostic delay in Pompe disease: A combined survey approach to unveil the diagnostic odyssey

Background Early diagnosis is of substantial benefit for patients with Pompe disease. Yet underdiagnosing and substantial diagnostic delay are still frequent and the determinants of this are unknown. This study is the first to systematically investigate the diagnostic odyssey in Pompe disease from patients', parents', and physicians' perspectives. Methods Patients with infantile or late onset Pompe disease, their parents as well as their metabolic experts were invited to fill in respective surveys. The survey addressed perceived disease symptoms at onset and during the course of the disease, specialties of involved physicians, activities of patient-initiated search for diagnosis and the perceived impact of time to diagnosis on outcome. Results of experts' and patients'/parents' surveys were compared and expressed by descriptive statistics. Results and Discussion We collected data on 15 males and 17 females including 9 infantile and 23 late onset Pompe patients. All received the correct diagnosis at a metabolic or musculoskeletal expert center. Patients with direct referral to the expert center had the lowest diagnostic delay, while patients who were seen by several physicians, received the correct diagnosis after 44%-200% longer delay. The proportion of direct referral varied strongly between pediatricians (57%) and other disciplines (18%-36%). Conclusion Our study highlights a substantially larger diagnostic delay in Pompe patients that are not directly referred to expert centers for diagnostic work. Our findings may be used to develop more successful strategies for early diagnosis. Synopsis Diagnostic delay in Pompe disease is substantial particularly in patients that are not directly referred to expert centers for diagnostic workup, so facilitating direct referral may be a new strategy for early diagnosis