Trastuzumab derived HER2-specific CARs for the treatment of trastuzumab-resistant breast cancer: CAR T cells penetrate and eradicate tumors that are not accessible to antibodies

HER2-targeted monoclonal antibodies improve the outcome for advanced breast cancer patients; however, resistance to therapy is still frequent. Epitope masking and steric hindrance to antibody binding through matrix components are thought to be the major mechanism. We asked whether tumors resistant to trastuzumab can still be eliminated by CAR T cells redirected by the same antibody domain. While saturating doses of trastuzumab in the presence of CD16.176V.NK-92 effector cells and trastuzumab derived CAR T cells equally well recognized and killed HER2-positive tumor cells in a monolayer, only CAR T cells penetrated into the core region of tumor spheroids and exhibited cytotoxic activity in vitro, whereas antibodies failed. In NSG mice treatment with trastuzumab and CD16.176V.NK-92 cells only transiently retarded tumor growth but did not induce regression of clinically trastuzumab-resistant breast cancer xenografts. In contrast, one dose of HER2-specific CAR T cells eradicated established tumors resulting in long-term survival. Data indicate that CAR T cells can successfully combat antibody resistant tumors by targeting the same epitope suggesting that CAR T cells can penetrate the tumor matrix which is a barrier for antibodies

ErbB receptorok szerepe epitél eredetű tumorokban: receptor tirozinkinázok, mint a tumor ellenes terápiák lehetséges target molekulái. = Role of ErbB receptors in epithelial tumors: receptor tyrosine kinases as targets for anticancer therapy.

Az erbB2-erbB2 és erbB2-erbB1 molekulák homo- és heteroasszociációja - akárcsak más erbB hetero-oligomereké - fontos szerepet játszhatnak a jelátvitelben így az immunoterápiában is. Kísérleteket végeztünk annak felderítésére, hogyan modulálja ezt a folyamatot más kölcsönható molekulák, pl. integrinek, CD44 (hialuronsav receptor) sejtfelszíni jelenléte. Az ErbB2 ellenes immunterápia (trastuzumab kezelés) során kialakuló trastuzumab rezisztencia lehetséges okait is vizsgáltuk. FRET eredményeink alapján elkészítettük ErbB2 dimer molekuláris modelljét. A trastuzumab rezisztens JIMT-1 ill. MKN-7 valamint a szenzitív SKBR-3 és N87 tumor sejtvonalakon végzett összehasonlító kísérleteink alapján megállapítottuk, hogy a trastuzumab kezelés korai elkezdésével még a rezisztens sejtek esetén is részleges válasz érhető el. A xenograft kísérletek rámutattak arra, hogy az antitest által közvetített citotoxicitás (ADCC) a trastuzumab kezelés kedvező hatásának elsődleges oka. A geldanamycin ill. 17AAG sejtosztódást gátló hatása hatékonyabbnak bizonyult trastuzumab rezisztens sejtekben. Ugyanakkor a CD44 leszabályozása, valamint a hialuronsav szintézis gátlása növelte a trastuzumab kezelés hatékonyságát. Mindezek az eredmények arra utalnak, hogy célzott adjuváns kezelés nagymértékben fokozhatják a trastuzumab terápia hatékonyságát. | Homo- and heteroassociations of erbB2-erbB2 and erbB2-erbB1 molecules ? similarly to that of other ErbB hetero-oligomers ? play an important role in signal transduction and immunotherapy. Our experiments aimed to reveal whether the cell surface presence of interacting molecules such as integrin and CD44 (receptor for hyaluronic acid) molecules can modulate these processes. We have also studied the possible causes of trastuzumab resistance developing during the ErbB2 targeted immunotherapy. We have prepared a useful molecular model of ErbB2 dimer on he basis or our FRET results. By comparing the behavior of trastuzumab resistant (JIMT-1 and MKN-7) and sensitive (SKBR-3 and N87) tumor cell lines we found that early trastuzumab treatment can be partially beneficial even in resistant cell lines. It was also revealed that the primary reason of the efficacy of trastuzumab treatment in xenograft models is the antibody dependent cell citotoxicity (ADCC). The treatment using geldanamycin or its derivative 17AAG was more efficient in trastuzumab resistant cells than in sensitive cells. Downregulation of CD44 and inhibition of synthesis of hyaluronic acid increased the efficiency of the trastuzumab treatment. These results suggest that targeted adjuvant therapy can significantly enhance the efficacy of the trastuzumab therapy

Anandamide Concentration-Dependently Modulates Toll-Like Receptor 3 Agonism or UVB-Induced Inflammatory Response of Human Corneal Epithelial Cells

Photodamage-induced and viral keratitis could benefit from treatment with novel nonsteroid anti-inflammatory agents. Therefore, we determined whether human corneal epithelial cells (HCECs) express members of the endocannabinoid system (ECS), and examined how the endocannabinoid anandamide (AEA, N-arachidonoyl ethanolamine) influences the Toll-like receptor 3 (TLR3) agonism- or UVB irradiation-induced inflammatory response of these cells. Other than confirming the presence of cannabinoid receptors, we show that endocannabinoid synthesizing and catabolizing enzymes are also expressed in HCECs in vitro, as well as in the epithelial layer of the human cornea in situ, proving that they are one possible source of endocannabinoids. p(I:C) and UVB irradiation was effective in promoting the transcription and secretion of inflammatory cytokines. Surprisingly, when applied alone in 100 nM and 10 μM, AEA also resulted in increased pro-inflammatory cytokine production. Importantly, AEA further increased levels of these cytokines in the UVB model, whereas its lower concentration partially prevented the transcriptional effect of p(I:C), while not decreasing the p(I:C)-induced cytokine release. HCECs express the enzymatic machinery required to produce endocannabinoids both in vitro and in situ. Moreover, our data show that, despite earlier reports about the anti-inflammatory potential of AEA in murine cornea, its effects on the immune phenotype of human corneal epithelium may be more complex and context dependent

New Possibilities for Combined Therapy of Breast Cancer

Kísérleteink fő célkitűzése az volt, hogy az emlődaganatok kezelésében már a klinikumban is alkalmazott terápiák hatását egy másik, kipróbálási fázisban lévő ágens bevonásával fokozzuk. Eredményeinket a következőkben foglaljuk össze: • In vitro kísérleteink eredményeiből megállapíthatjuk, hogy a 17-AAG az ErbB2 homodimerizációt és foszforilációt fokozza mind trastuzumab szenzitív, mind rezisztens emlőtumor sejteken, és hosszútávon a receptor leszabályozását és a sejtproliferáció gátlását idézi elő, melynek hátterében elsősorban apoptózis, nagyobb dózisoknál részben nekrózis áll. Ennek alapján a 17-AAG alkalmas lehet a trastuzumab-rezisztens, ErbB2-pozitív tumorok kezelésére. • Az ErbB2-t nagymértékben expresszáló trastuzumab szenzitív daganatsejtek esetén a 17-AAG proliferációt gátló hatása a nagyszámú ErbB2 receptor aktivációja miatt alacsonyabb. Ilyen tumoroknál a trastuzumabbal történő kombináció hatásos lehet a 17-AAG által kiváltott ErbB2 foszforiláció gátlásában, valamint az alacsony dózisú 17-AAG növekedést gátló hatásának fokozásában. • In vivo tanulmányainkban a cisztein proteináz inhibitor (CPI) fotodinámiás terápiához (PDT) társítva szinergisztikusan gátolja a patkányokba implantált szolid emlőkarcinóma növekedését, fokozza a tumornekrózist, s ennek hátterében legalábbis részben a szinergista módon csökkentett szérum VEGF szint, ill. a következményes nagyfokú neovaszkularizáció-gátlás áll. • A PDT és CPI kombinációja a Wistar patkányokba implantált szolid emlőkarcinóma progresszióját 20 mg/kg HpD, 500 μg/állat CPI és 90 J/cm2 fotondózisú besugárzás mellett képes leghatékonyabban gátolni. A legjobb eredményeket az inokulációval egyidőben elkezdett terápia adja, ami arra utal, hogy a kombinációs kezelés különösen hatékony lehet a szóródott, ill. reziduális daganatsejtek elpusztításában. ------ The main aim of our work was to enhance the effect of therapies targeting breast cancer in the clinics with another small molecule agent. • Our in vitro experiments show that 17-AAG enhances ErbB2 homodimerization and phosphorylation on trastuzumab sensitive and resistant cells. In the long run, the drug causes down-regulation of the receptor and inhibition of cell proliferation. This phenomenon can be explained by the apoptosis and in the case of higher doses the necrosis caused by 17-AAG. Thus, 17-AAG can be used to treat also trastuzumab-resistant ErbB2-positive tumours. • In trastuzumab sensitive tumours, especially with high levels of ErbB2 expression, the proliferation-inhibiting effect of 17-AAG is lower because of activation of the numerous ErbB2 receptors. In these cases, additional trastuzumab in the treatment regime can be beneficial in terms of inhibiting 17-AAG-evoked ErbB2 phosphorylation and also in enhancing the growth inhibitory effects of low doses of 17-AAG. • In our in vivo studies the combined application of photodynamic therapy (PDT) and cysteine proteinase inhibitor (CPI) had a synergistic effect on the solid mammary adenocarcinoma transplanted into Wistar rats. CPI unequivocally enhanced the effectiveness of PDT, which can be explained by the increased tumour necrosis, decreased VEGF-level in sera and the intense inhibition of vascularization. • The combination of PDT and CPI most effectively inhibits the progression of solid mammary carcinoma transplanted into Wistar rats with the application of 20 mg/kg HpD, 500 μg/animal cystatin and photon flux of 90 J/cm2. The most promising results were achieved when the therapy was started simultaneously with tumor inoculation, which promises effectiveness of combined treatment of the scattered and residual cancer cells

Limbal and Conjunctival Epithelial Cell Cultivation on Contact Lenses: different Affixing Techniques and the Effect of Feeder Cells

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Cultivation of Human Oral Mucosal Explants on Contact Lenses

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Trastuzumab derived HER2-specific CARs for the treatment of trastuzumab-resistant breast cancer: CAR T cells penetrate and eradicate tumors that are not accessible to antibodies

HER2-targeted monoclonal antibodies improve the outcome for advanced breast cancer patients; however, resistance to therapy is still frequent. Epitope masking and steric hindrance to antibody binding through matrix components are thought to be the major mechanism. We asked whether tumors resistant to trastuzumab can still be eliminated by CAR T cells redirected by the same antibody domain. While saturating doses of trastuzumab in the presence of CD16.176V.NK-92 effector cells and trastuzumab derived CAR T cells equally well recognized and killed HER2-positive tumor cells in a monolayer, only CAR T cells penetrated into the core region of tumor spheroids and exhibited cytotoxic activity in vitro, whereas antibodies failed. In NSG mice treatment with trastuzumab and CD16.176V.NK-92 cells only transiently retarded tumor growth but did not induce regression of clinically trastuzumab-resistant breast cancer xenografts. In contrast, one dose of HER2-specific CAR T cells eradicated established tumors resulting in long-term survival. Data indicate that CAR T cells can successfully combat antibody resistant tumors by targeting the same epitope suggesting that CAR T cells can penetrate the tumor matrix which is a barrier for antibodies