IND-enabling studies for a clinical trial to genetically program a persistent cancer-targeted immune system

Purpose: To improve persistence of adoptively transferred T-cell receptor (TCR)–engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. Experimental Design: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/K^b mice within a formal Good Laboratory Practice (GLP)–compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)–compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. Results: TCR genetically modified and ex vivo–cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. Conclusions: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471

IND-enabling studies for a clinical trial to genetically program a persistent cancer-targeted immune system

Purpose: To improve persistence of adoptively transferred T-cell receptor (TCR)–engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. Experimental Design: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/K^b mice within a formal Good Laboratory Practice (GLP)–compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)–compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. Results: TCR genetically modified and ex vivo–cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. Conclusions: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471

Adeno-associated Virus Enhances Wild-type And Oncolytic Adenovirus Spread

The contamination of adenovirus (Ad) stocks with adeno-associated viruses (AAV) is usually unnoticed, and it has been associated with lower Ad yields upon large-scale production. During Ad propagation, AAV contamination needs to be detected routinely by polymerase chain reaction without symptomatic suspicion. In this study, we describe that the coinfection of either Ad wild type 5 or oncolytic Ad with AAV results in a large-plaque phenotype associated with an accelerated release of Ad from coinfected cells. This accelerated release was accompanied with the expected decrease in Ad yields in two out of three cell lines tested. Despite this lower Ad yield, coinfection with AAV accelerated cell death and enhanced the cytotoxicity mediated by Ad propagation. Intratumoral coinjection of Ad and AAV in two xenograft tumor models improved antitumor activity and mouse survival. Therefore, we conclude that accidental or intentional AAV coinfection has important implications for Ad-mediated virotherapy

A pRb-responsive, RGD-modified, and hyaluronidase-armed canine oncolytic adenovirus for application in veterinary oncology

Human and canine cancer share similarities such as genetic and molecular aspects, biological complexity, tumor epidemiology, and targeted therapeutic treatment. Lack of good animal models for human adenovirotherapy has spurred the use of canine adenovirus 2-based oncolytic viruses. We have constructed a canine oncolytic virus that mimics the characteristics of our previously published human adenovirus ICOVIR17: expression of E1a controlled by E2F sites, deletion of the pRb-binding site of E1a, insertion of an RGD integrin-binding motif at the fiber Knob, and expression of hyaluronidase under the major late promoter/IIIa protein splicing acceptor control. Preclinical studies showed selectivity, increased cytotoxicity, and strong hyaluronidase activity. Intratumoral treatment of canine osteosarcoma and melanoma xenografts in mice resulted in inhibition of tumor growth and prolonged survival. Moreover, we treated six dogs with different tumor types, including one adenoma, two osteosarcomas, one mastocitoma, one fibrosarcoma, and one neuroendocrine hepatic carcinoma. No virusassociated adverse effects were observed, but toxicity associated to tumor lysis, including disseminated intravascular coagulation and systemic failure, was found in one case. Two partial responses and two stable diseases warrant additional clinical testing

IND-Enabling Studies for a Clinical Trial to Genetically Program a Persistent Cancer-Targeted Immune System

PURPOSE: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. EXPERIMENTAL DESIGN: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. RESULTS: TCR genetically modified and ex vivo-cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. CONCLUSIONS: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells

Isolation and characterization of NY-ESO-1–specific T cell receptors restricted on various MHC molecules

Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2–restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2

Estratègies de millora de la potència antitumoral dels adenovirus oncolítics basades en l’increment de l’alliberament viral i l’extravasació vascular

La viroteràpia amb adenovirus oncolítics es basa en la lisi selectiva de la cèl•lula tumoral a través de la replicació viral. Les principals limitacions d’aquesta teràpia són: la limitada arribada del virus al tumor després de l’administració sistèmica, la baixa dispersió intratumoral i la resposta immune antiviral. En aquesta tesi hem presentat estratègies per millorar la disseminació intratumoral basades en l’augment de l’alliberament viral i en la millora de l’extravasació i penetració del virus al parènquima tumoral, augmentant en els dos casos l’eficàcia antitumoral. Una de les principals barreres a la dispersió intratumoral són els fibroblasts que infiltren el tumor, i que reben el nom de fibroblasts associats a tumor o CAFs. Els CAFs presenten un fenotip tumorigènic i a més no són permissius a la replicació viral. Amb l’objectiu de superar aquesta barrera vam dur a terme un procés de bioselecció a partir d’un estoc d’adenovirus salvatge mutagenitzat. Després de vàries rondes de replicació en CAFs vam aïllar varis mutants que presentaven un alliberament viral incrementat en aquestes cèl•lules. Vam escollir el mutant que presentava el millor fenotip i vam identificar la mutació responsable del seu fenotip a partir de la seqüenciació del genoma complet i del mapatge funcional. La mutació responsable del fenotip és la mutació iLG397T que provoca la truncació de l’extrem C-terminal de la proteïna i-leader de l’adenovirus. Aquest mutant presenta un alliberament viral i una citotoxicitat incrementades en CAFs, fibroblasts normals i un panell de línies cel•lulars tumorals. La truncació de la proteïna i-leader augmenta l’eficàcia antitumoral de l’adenovirus en models de tumors subcutanis en ratolins immunodeficients establerts a partir de cèl•lules tumorals i de mescles de cèl•lules tumorals i fibroblasts, sense afectar a la toxicitat hepàtica i hematològica. Amb l’objectiu d’augmentar encara més la potència oncolítica de l’adenovirus vam combinar la mutació iLG397T amb la mutació E3/19K-445A aïllada prèviament pel nostre grup i que també augmenta l’alliberament viral. El virus amb les dues mutacions presentava un alliberament incrementat però perdia producció de la progènie viral. Per augmentar la potència antitumoral de l’adenovirus oncolític ICOVIR15, vam introduir la mutació iLG397T al seu genoma, generant l’ICOVIR-15i. Aquest virus també presenta alliberament viral, citotoxicitat i eficàcia antitumoral incrementades en un model de carcinoma de pulmó humà. En el model de hàmster siri, un model immunocompetent i semipermissiu a la replicació de l’adenovirus, aquest virus presentava eficàcia antitumoral només al combinar-lo amb gemcitabina. Aquesta combinació augmentava la proliferació de limfòcits després de l’estimulació amb cèl•lules tumorals, i a més el seu efecte no s’observà en el model murí immunodeficient amb tumors subcutanis de hàmster siri. Aquests resultats ens permeten suggerir que l’efecte antitumoral de la combinació de l’ICOVIR-15i amb gemcitabina es deu a una resposta immune antitumoral. També vam demostrar que la combinació amb verapamil, un inhibidor dels canals de calci, augmentava l’alliberament viral, la citotoxicitat i l’eficàcia antitumoral en varis models tumorals. Finalment amb l’objectiu d’augmentar l’extravasació del virus cap a la massa tumoral vam introduir el pèptid de penetració tumoral iRGD a la càpsida del l’adenovirus oncolític ICOVIR-15K. Aquest pèptid permet la internalització de les macromolècules a les que està unit a través de la interacció amb integrines i neuropilina-1 sobreexpressades a la vasculatura i a les cèl•lules tumorals, i de la proteòlisi per part una proteasa tumoral. La inserció d’aquest pèptid a la càpsida de l’adenovirus millorà la transducció tumoral sense afectar a la resta d’òrgans, la disseminació intratumoral i l’eficàcia antitumoral del virus. Aquestes diferents estratègies milloren la disseminació intratumoral del virus i l’eficàcia antitumoral i poden ser combinades amb altres estratègies com la degradació de la matriu extracel•lular per millorar encara més l’eficàcia de la teràpia.Virotherapy with oncolytic adenoviruses holds promise as a new cancer treatment. The main barriers to virotherapy are the poor tumor targeting after systemic administration, the limited intratumoral dissemination hampered by the fibroblasts, and the extracellular matrix present in the tumors and the antiviral immune response. In this thesis we present different strategies to enhance adenovirus intratumoral dissemination based on optimizing adenovirus release and enhancing its extravasation and penetration into the tumor. After multiple rounds of replication in cancer associated fibroblasts (or CAFs) of a mutagenized stock of adenovirus wild type, we isolated an enhanced-release mutant. The mutation responsible for this phenotype, the iLG397T mutation, was identified by sequencing and functional mapping. This mutation enhances adenovirus release and cytotoxicity in CAFs, normal fibroblasts, and a panel of tumor cells. Moreover this mutation increases the antitumor efficacy of the virus in immunodeficient mice with tumors established from tumor cells and mixtures of tumor cells and CAFs. To increase the potency of the oncolytic adenovirus ICOVIR-15, we introduced the iLG397T mutation into its genome, generating the ICOVIR-15i. This virus also displayed enhanced release, cytotoxicity, and antitumor efficacy in a lung carcinoma model. In Syrian hamsters, an immunocompetent semi-permissive model, only the combination of ICOVIR-15i with gemcitabine shows antitumor activity. This antitumor activity led to a lymphocyte proliferation increase after stimulation with tumor cells, and was not observed when the same experiment was performed in immunodeficient mice. These results suggest that the antitumor efficacy of the combination of ICOVIR-15i and gemcitabine is associated to an immune response against tumor antigens. We also described the combination of the virus with verapamil, a calcium channel blocker, to enhance adenovirus release, cytotoxicity and antitumor efficacy. Finally to improve adenovirus extravasation and penetration into the tumor mass, we inserted the iRGD tumor penetrating peptide into ICOVIR15K capsid. The iRGD peptide enhanced internalization into cells by the interaction with integrins and neuropilin-1, after a proteolytic cleavage by a tumor protease. Integrins and neuropilin-1 are overexpressed in the tumor cells and vasculature. The insertion of the iRGD peptide enhanced tumor transduction, intratumoral dissemination of the virus and antitumor efficacy

Estratègies de millora de la potència antitumoral dels adenovirus oncolítics basades en l’increment de l’alliberament viral i l’extravasació vascular

[cat]La viroteràpia amb adenovirus oncolítics es basa en la lisi selectiva de la cèl•lula tumoral a través de la replicació viral. Les principals limitacions d’aquesta teràpia són: la limitada arribada del virus al tumor després de l’administració sistèmica, la baixa dispersió intratumoral i la resposta immune antiviral. En aquesta tesi hem presentat estratègies per millorar la disseminació intratumoral basades en l’augment de l’alliberament viral i en la millora de l’extravasació i penetració del virus al parènquima tumoral, augmentant en els dos casos l’eficàcia antitumoral. Una de les principals barreres a la dispersió intratumoral són els fibroblasts que infiltren el tumor, i que reben el nom de fibroblasts associats a tumor o CAFs. Els CAFs presenten un fenotip tumorigènic i a més no són permissius a la replicació viral. Amb l’objectiu de superar aquesta barrera vam dur a terme un procés de bioselecció a partir d’un estoc d’adenovirus salvatge mutagenitzat. Després de vàries rondes de replicació en CAFs vam aïllar varis mutants que presentaven un alliberament viral incrementat en aquestes cèl•lules. Vam escollir el mutant que presentava el millor fenotip i vam identificar la mutació responsable del seu fenotip a partir de la seqüenciació del genoma complet i del mapatge funcional. La mutació responsable del fenotip és la mutació iLG397T que provoca la truncació de l’extrem C-terminal de la proteïna i-leader de l’adenovirus. Aquest mutant presenta un alliberament viral i una citotoxicitat incrementades en CAFs, fibroblasts normals i un panell de línies cel•lulars tumorals. La truncació de la proteïna i-leader augmenta l’eficàcia antitumoral de l’adenovirus en models de tumors subcutanis en ratolins immunodeficients establerts a partir de cèl•lules tumorals i de mescles de cèl•lules tumorals i fibroblasts, sense afectar a la toxicitat hepàtica i hematològica. Amb l’objectiu d’augmentar encara més la potència oncolítica de l’adenovirus vam combinar la mutació iLG397T amb la mutació E3/19K-445A aïllada prèviament pel nostre grup i que també augmenta l’alliberament viral. El virus amb les dues mutacions presentava un alliberament incrementat però perdia producció de la progènie viral. Per augmentar la potència antitumoral de l’adenovirus oncolític ICOVIR15, vam introduir la mutació iLG397T al seu genoma, generant l’ICOVIR-15i. Aquest virus també presenta alliberament viral, citotoxicitat i eficàcia antitumoral incrementades en un model de carcinoma de pulmó humà. En el model de hàmster siri, un model immunocompetent i semipermissiu a la replicació de l’adenovirus, aquest virus presentava eficàcia antitumoral només al combinar-lo amb gemcitabina. Aquesta combinació augmentava la proliferació de limfòcits després de l’estimulació amb cèl•lules tumorals, i a més el seu efecte no s’observà en el model murí immunodeficient amb tumors subcutanis de hàmster siri. Aquests resultats ens permeten suggerir que l’efecte antitumoral de la combinació de l’ICOVIR-15i amb gemcitabina es deu a una resposta immune antitumoral. També vam demostrar que la combinació amb verapamil, un inhibidor dels canals de calci, augmentava l’alliberament viral, la citotoxicitat i l’eficàcia antitumoral en varis models tumorals. Finalment amb l’objectiu d’augmentar l’extravasació del virus cap a la massa tumoral vam introduir el pèptid de penetració tumoral iRGD a la càpsida del l’adenovirus oncolític ICOVIR-15K. Aquest pèptid permet la internalització de les macromolècules a les que està unit a través de la interacció amb integrines i neuropilina-1 sobreexpressades a la vasculatura i a les cèl•lules tumorals, i de la proteòlisi per part una proteasa tumoral. La inserció d’aquest pèptid a la càpsida de l’adenovirus millorà la transducció tumoral sense afectar a la resta d’òrgans, la disseminació intratumoral i l’eficàcia antitumoral del virus. Aquestes diferents estratègies milloren la disseminació intratumoral del virus i l’eficàcia antitumoral i poden ser combinades amb altres estratègies com la degradació de la matriu extracel•lular per millorar encara més l’eficàcia de la teràpia.[eng]Virotherapy with oncolytic adenoviruses holds promise as a new cancer treatment. The main barriers to virotherapy are the poor tumor targeting after systemic administration, the limited intratumoral dissemination hampered by the fibroblasts, and the extracellular matrix present in the tumors and the antiviral immune response. In this thesis we present different strategies to enhance adenovirus intratumoral dissemination based on optimizing adenovirus release and enhancing its extravasation and penetration into the tumor. After multiple rounds of replication in cancer associated fibroblasts (or CAFs) of a mutagenized stock of adenovirus wild type, we isolated an enhanced-release mutant. The mutation responsible for this phenotype, the iLG397T mutation, was identified by sequencing and functional mapping. This mutation enhances adenovirus release and cytotoxicity in CAFs, normal fibroblasts, and a panel of tumor cells. Moreover this mutation increases the antitumor efficacy of the virus in immunodeficient mice with tumors established from tumor cells and mixtures of tumor cells and CAFs. To increase the potency of the oncolytic adenovirus ICOVIR-15, we introduced the iLG397T mutation into its genome, generating the ICOVIR-15i. This virus also displayed enhanced release, cytotoxicity, and antitumor efficacy in a lung carcinoma model. In Syrian hamsters, an immunocompetent semi-permissive model, only the combination of ICOVIR-15i with gemcitabine shows antitumor activity. This antitumor activity led to a lymphocyte proliferation increase after stimulation with tumor cells, and was not observed when the same experiment was performed in immunodeficient mice. These results suggest that the antitumor efficacy of the combination of ICOVIR-15i and gemcitabine is associated to an immune response against tumor antigens. We also described the combination of the virus with verapamil, a calcium channel blocker, to enhance adenovirus release, cytotoxicity and antitumor efficacy. Finally to improve adenovirus extravasation and penetration into the tumor mass, we inserted the iRGD tumor penetrating peptide into ICOVIR15K capsid. The iRGD peptide enhanced internalization into cells by the interaction with integrins and neuropilin-1, after a proteolytic cleavage by a tumor protease. Integrins and neuropilin-1 are overexpressed in the tumor cells and vasculature. The insertion of the iRGD peptide enhanced tumor transduction, intratumoral dissemination of the virus and antitumor efficacy