Chimeric antigen receptor T cells (CAR-T) are genetically modified T cells with a receptor containing single chain antibodies to kill target cells. In previous work, we engineered CAR-T cells against cells infected with herpesviruses. CAR-T cells killed infected cells with proteins of Epstein-Barr Virus (EBV) or Cytomegalovirus (CMV) expressed on the surface. The EBVgp350-CAR-T cell technology was licensed out to Biosyngen/ Zelltechs (Singapore) and clinical trials are in ongoing. We are currently developing gene editing strategies using CRISPR to manufacture CAR-T-cells to treat lymphomas and EBV-associated malignancies. Our goal is to make the edited CAR-T cells more potent and persistent, while allowing us to generate “off-the-shelf” products when this is needed in the clinic. These CAR-T cells are tested pre-clinically in mice implanted with human tumors or in humanized mice (see below) infected with human viruses.

Sources:
 1: Braun T, Pruene A, Darguzyte M, Vom Stein AF, Nguyen PH, Wagner DL, Kath J, Roig-Merino A, Heuser M, Riehm LL, Schneider A, Awerkiew S, Talbot SR, Bleich A, Figueiredo C, Bornhäuser M, Stripecke R. Non-viral TRAC knocked-in CD19 KI CAR-T and gp350 KI CAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency. Front Immunol. 2023 Mar 24;14:1086433. doi: 10.3389/fimmu.2023.1086433.

2: Zhang X, Wang T, Zhu X, Lu Y, Li M, Huang Z, Han D, Zhang L, Wu Y, Li L, Klawonn F, Stripecke R. GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies. Front Immunol. 2023 Feb 2;14:1103695. doi: 10.3389/fimmu.2023.1103695. 

3: Wagner DL, Koehl U, Chmielewski M, Scheid C, Stripecke R. Review: Sustainable Clinical Development of CAR-T Cells - Switching From Viral Transduction Towards CRISPR-Cas Gene Editing. Front Immunol. 2022 Jun 17;13:865424. doi: 10.3389/fimmu.2022.865424. 

4: Kath J, Du W, Pruene A, Braun T, Thommandru B, Turk R, Sturgeon ML, Kurgan GL, Amini L, Stein M, Zittel T, Martini S, Ostendorf L, Wilhelm A, Akyüz L, Rehm A, Höpken UE, Pruß A, Künkele A, Jacobi AM, Volk HD, Schmueck-Henneresse M, Stripecke R, Reinke P, Wagner DL. Pharmacological interventions enhance virus-free generation of TRAC replaced CAR T cells. Mol Ther Methods Clin Dev. 2022 Apr 12;25:311-330. doi: 10.1016/j.omtm.2022.03.018. 

5: Slabik C, Kalbarczyk M, Danisch S, Zeidler R, Klawonn F, Volk V, Krönke N, Feuerhake F, Ferreira de Figueiredo C, Blasczyk R, Olbrich H, Theobald SJ, Schneider A, Ganser A, von Kaisenberg C, Lienenklaus S, Bleich A, Hammerschmidt W, Stripecke R. CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease. Mol Ther Oncolytics. 2020 Aug 8;18:504-524. doi: 10.1016/j.omto.2020.08.005.

6: Olbrich H, Theobald SJ, Slabik C, Gerasch L, Schneider A, Mach M, Shum T, Mamonkin M, Stripecke R. Adult and Cord Blood-Derived High-Affinity gB-CAR-T Cells Effectively React Against Human Cytomegalovirus Infections. Hum Gene Ther.2020 Apr;31(7-8):423-439. doi: 
10.1089/hum.2019.149. 

Lentivirus-induced dendritic cells (iDC) is a patented immunotherapeutic cell vaccine platform for accelerating the recovery of human T and B cells after hematopoietic stem cell transplantation. Acute myeloid leukemia (AML) is the most common acute leukemia in adults and overall survival remains poor. Some patients with AML-MRD are eligible for stem cell transplantation as a salvage therapy. Novel cellular immune therapies are needed to improve de novo immunological responses so that the body can fight against MRD to prevent relapse. We produced monocytes gene modified with lentiviral vectors expressing immune stimulatory factors (GM-CSF and IFN-alpha) and the WT1 leukemia-associated antigen. Preclinical tests were performed in humanized mice. Further, using materials and procedures compatible with good manufacturing practices, we automated the manufacturing of iDCtWT1 using the CliniMACs Prodigy ® system from Miltenyi Biotec. We also established iDCs expressing the HCMV glycoprotein B (gB). Humanized mice immunized with iDCgB were protected against HCMV infection and produced high levels of human IgG and IgA. The repertoire of anti-gB IgGs was exploited for the production of specific fully human monoclonal antibodies against HCMV.

Lentivirus-induzierte dendritische Zellen (iDC) sind eine patentierte immuntherapeutische Zellimpfstoffplattform zur Beschleunigung der Regenerierung menschlicher T- und B-Zellen nach hämatopoetischer Stammzelltransplantation. Akute myeloische Leukämie (AML) ist die häufigste akute Leukämie bei Erwachsenen und das Gesamtüberleben bleibt schlecht. Einige Patienten mit AML-MRD können eine Stammzelltransplantation als Salvage-Therapie in Anspruch nehmen. Neuartige zelluläre Immuntherapien sind erforderlich, um die De-novo-Immunreaktionen zu verbessern, damit der Körper gegen MRD kämpfen kann, um eine Rezidive zu verhindern. Wir produzierten Monozyten, die mit lentiviralen Vektoren modifiziert waren, die immunstimulierende Faktoren (GM-CSF und IFN-alpha) und das WT1-Leukämie-assoziierte Antigen exprimierten. Präklinische Tests wurden an humanisierten Mäusen durchgeführt. Darüber hinaus haben wir mithilfe von Materialien und Verfahren, die mit den guten Herstellungsverfahren kompatibel sind, die Herstellung von iDCtWT1 mithilfe des CliniMACs Prodigy ® -Systems von Miltenyi Biotec automatisiert. Wir haben auch iDCs etabliert, die das HCMV-Glykoprotein B (gB) exprimieren. Mit iDCgB immunisierte humanisierte Mäuse waren gegen eine HCMV-Infektion geschützt und produzierten hohe Mengen an menschlichem IgG und IgA. Das Repertoire an Anti-gB-IgGs wurde zur Herstellung spezifischer vollständig menschlicher monoklonaler Antikörper gegen HCMV genutzt.

Sources:
1: Theobald SJ, Kreer C, Khailaie S, Bonifacius A, Eiz-Vesper B, Figueiredo C, Mach M, Backovic M, Ballmaier M, Koenig J, Olbrich H, Schneider A, Volk V, Danisch S, Gieselmann L, Ercanoglu MS, Messerle M, Kaisenberg CV, Witte T, Klawonn F, Meyer-Hermann M, Klein F, Stripecke R. Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV. PLoS Pathog. 2020 Jul 15;16(7):e1008560. doi: 10.1371/journal.ppat.1008560. Erratum in: PLoS Pathog. 2021 Mar 1;17(3):e1009385. PMID: 32667948; PMCID: PMC7363084.

2: Bialek-Waldmann JK, Heuser M, Ganser A, Stripecke R. Monocytes reprogrammed with lentiviral vectors co-expressing GM-CSF, IFN-α2 and antigens for personalized immune therapy of acute leukemia pre- or post-stem cell transplantation. Cancer Immunol Immunother. 2019 Nov;68(11):1891-1899. doi: 10.1007/s00262-019-02406-9. Epub 2019 Oct 18. PMID: 31628525; PMCID: PMC6851032.

3: Bialek-Waldmann JK, Stripecke R. et al, in press Molecular Therapy Methods and Clinical Development.

Hematopoietic stem cell transplantation (HCT) is a routine clinical procedure performed to treat aggressive leukemia or to treat patients with inborne genetic defects. Although the number of HCT procedures performed continue to increase, the slow immune reconstitution of the recipient with the immune system of the donor is still a major clinical issue. The main problems affecting the immune-compromised patients are leukemia relapses, infections and graft-versus host diseases. An improved adjustment of the human leukocyte antigens (HLAs, expressed by antigen presenting cells) with the T cell receptors (TCR, expressed by T lymphocytes) could ameliorate all these hurdles. In previous work we have developed cellular donor-derived cellular vaccines to allow a faster “jumpstart” for accelerating the development of human T and B cells after HCT. We patented a translational technology based on monocytes gene modified with lentiviral vectors expressing cytokines and antigens. We are currently developing new cell-free strategies to speed-up the immune reconstitution. As a model system to study HCT and test novel therapies, we use immune deficient mice engrafted with human hematopoietic stem cells. These “humanized” mice develop mature human T and B cells. Recently, we showed that “humanized” mice can be explored for generation of fully human monoclonal antibodies. We are currently elucidating how “humanized” mice without a human thymus can develop functional human T cells and how to further harness the human immune system.

Sources:
1: Kumar S, Koenig J, Schneider A, Wermeling F, Boddul S, Theobald SJ, Vollmer M, Kloos D, Lachmann N, Klawonn F, Lienenklaus S, Talbot SR, Bleich A, Wenzel N, von Kaisenberg C, Keck J, Stripecke R. In Vivo Lentiviral Gene Delivery of HLA-DR and Vaccination of Humanized Mice for Improving the Human T and B Cell Immune Reconstitution. Biomedicines. 2021 Aug 5;9(8):961. doi: 10.3390/biomedicines9080961. 

2: Bialek-Waldmann JK, Domning S, Esser R, Glienke W, Mertens M, Aleksandrova K, Arseniev L, Kumar S, Schneider A, Koenig J, Theobald SJ, Tsay HC, Cornelius ADA, Bonifacius A, Eiz-Vesper B, Figueiredo C, Schaudien D, Talbot SR, Bleich A, Spineli LM, von Kaisenberg C, Clark C, Blasczyk R, Heuser M, Ganser A, Köhl U, Farzaneh F, Stripecke R. Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL. Mol Ther Methods Clin Dev. 2021 Apr 9;21:621-641. doi: 10.1016/j.omtm.2021.04.004. 

3: Volk V, Theobald SJ, Danisch S, Khailaie S, Kalbarczyk M, Schneider A, Bialek-Waldmann J, Krönke N, Deng Y, Eiz-Vesper B, Dragon AC, von Kaisenberg C, Lienenklaus S, Bleich A, Keck J, Meyer-Hermann M, Klawonn F, Hammerschmidt W, Delecluse HJ, Münz C, Feuerhake F, Stripecke R. PD-1 Blockade Aggravates Epstein-Barr Virus+ Post-Transplant Lymphoproliferative Disorder in Humanized Mice Resulting in Central Nervous System Involvement and CD4+ T Cell Dysregulations. Front Oncol. 2021 Jan 12;10:614876. doi:10.3389/fonc.2020.614876. 

4: Theobald SJ, Kreer C, Khailaie S, Bonifacius A, Eiz-Vesper B, Figueiredo C, Mach M, Backovic M, Ballmaier M, Koenig J, Olbrich H, Schneider A, Volk V, Danisch S, Gieselmann L, Ercanoglu MS, Messerle M, Kaisenberg CV, Witte T, Klawonn F, Meyer-Hermann M, Klein F, Stripecke R. Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV. PLoS Pathog. 2020 Jul 15;16(7):e1008560. doi: 10.1371/journal.ppat.1008560. 

5: Stripecke R, Münz C, Schuringa JJ, Bissig KD, Soper B, Meeham T, Yao LC, Di Santo JP, Brehm M, Rodriguez E, Wege AK, Bonnet D, Guionaud S, Howard KE, Kitchen S, Klein F, Saeb-Parsy K, Sam J, Sharma AD, Trumpp A, Trusolino L, Bult C, Shultz L. Innovations, challenges, and minimal information for standardization of humanized mice. EMBO Mol Med. 2020 Jul 7;12(7):e8662. doi:10.15252/emmm.201708662. 

6: Koenig J, Theobald SJ, Stripecke R. Modeling Human Cytomegalovirus in Humanized Mice for Vaccine Testing. Vaccines (Basel). 2020 Feb 17;8(1):89. doi:10.3390/vaccines8010089. 

7: Bialek-Waldmann JK, Heuser M, Ganser A, Stripecke R. Monocytes reprogrammed with lentiviral vectors co-expressing GM-CSF, IFN-α2 and antigens for personalized immune therapy of acute leukemia pre- or post-stem cell transplantation. Cancer Immunol Immunother. 2019 Nov;68(11):1891-1899. doi: 10.1007/s00262-019-02406-9. 

8: Theobald SJ, Khailaie S, Meyer-Hermann M, Volk V, Olbrich H, Danisch S, Gerasch L, Schneider A, Sinzger C, Schaudien D, Lienenklaus S, Riese P, Guzman CA, Figueiredo C, von Kaisenberg C, Spineli LM, Glaesener S, Meyer-Bahlburg A, Ganser A, Schmitt M, Mach M, Messerle M, Stripecke R. Signatures of T and B Cell Development, Functional Responses and PD-1 Upregulation After HCMV Latent Infections and Reactivations in Nod.Rag.Gamma Mice Humanized With Cord Blood CD34+ Cells. Front Immunol. 2018 Nov 22;9:2734. doi:10.3389/fimmu.2018.02734.