Impact of prophylactic TNF blockade in the dual PD-1 and CTLA-4 immunotherapy efficacy and toxicity

The TNF blockade therapy is currently a well-established treatment option for a variety of autoimmune diseases such as rheumatoid arthritis (RA), psoriasis or Crohn's disease, given the proinflammatory role of TNF in the course of these diseases. Importantly, TNF neutralization is also used for the treatment of corticosteroid-refractory immune-related adverse events (irAEs) induced by the combined anti-PD-1 and anti-CTLA-4 immunotherapy. The manifestation of these toxicities is an important limiting factor for the successful implementation of the inhibitory checkpoint blockade therapy (ICB), restraining its anti-tumor efficacy. In our recent study (Perez-Ruiz et al., Nature 569(7756): 428-432.), we analyzed the potential impact of prophylactic TNF neutralization therapy in the anti-PD1/CTLA-4 efficacy. Through several mouse models, we demonstrated that TNF neutralization ameliorated ICB-exacerbated colitis in addition to improving ICB-dependent anti-tumor efficacy. Similar results were obtained after prophylactic TNF blockade in graft vs host xenografted mouse models with human immune cells, which showed a reduction in colitis and hepatitis. Importantly, there was a preservation of the immunotherapeutic control of xenografted tumors after ICB treatment. Moreover, TNF and TNF-dependent gene expression is upregulated in the colon mucosa from patients affected by colitis as a side effect of ipilimumab and nivolumab. Our results, thus, provide evidence of the successful combination of prophylactic TNF neutralization with ICB therapy strategy to ameliorate toxicities, while keeping or even ameliorating anti-tumor efficacy. The prophylactic TNF blockade strategy is clinically feasible since excellent TNF inhibitors have been approved for the treatment of autoimmunity and are used for the immune-related serious adverse events in immunotherapy

Enhancement of antibody-dependent cellular cytotoxicity of cetuximab by a chimeric protein encompassing interleukin-15

Enhancement of antibody-dependent cellular cytotoxicity (ADCC) may potentiate the antitumor efficacy of tumor-targeted monoclonal antibodies. Increasing the numbers and antitumor activity of NK cells is a promising strategy to maximize the ADCC of standard-of-care tumor-targeted antibodies. For this purpose, we have preclinically tested a recombinant chimeric protein encompassing the sushi domain of the IL15Rα, IL-15, and apolipoprotein A-I (Sushi-IL15-Apo) as produced in CHO cells. The size-exclusion purified monomeric fraction of this chimeric protein was stable and retained the IL-15 and the sushi domain bioactivity as measured by CTLL-2 and Mo-7e cell proliferation and STAT5 phosphorylation in freshly isolated human NK and CD8+ T cells. On cell cultures, Sushi-IL15-Apo increases NK cell proliferation and survival as well as spontaneous and antibody-mediated cytotoxicity. Scavenger receptor class B type I (SR-B1) is the receptor for ApoA-I and is expressed on the surface of tumor cells. SR-B1 can adsorb the chimeric protein on tumor cells and can transpresent IL-15 to NK and CD8+ T cells. A transient NK-humanized murine model was developed to test the increase of ADCC attained by the chimeric protein in vivo. The EGFR+ human colon cancer cell line HT-29 was intraperitoneally inoculated in immune-deficient Rag2-/-γc-/- mice that were reconstituted with freshly isolated PBMCs and treated with the anti-EGFR mAb cetuximab. The combination of the Sushi-IL15-Apo protein and cetuximab reduced the number of remaining tumor cells in the peritoneal cavity and delayed tumor engraftment in the peritoneum. Furthermore, Sushi-IL15-Apo increased the anti-tumor effect of a murine anti-EGFR mAb in Rag1-/- mice bearing subcutaneous MC38 colon cancer transfected to express EGFR. Thus, Sushi-IL15-Apo is a potent tool to increase the number and the activation of NK cells to promote the ADCC activity of antibodies targeting tumor antigens

Daratumumab in combination with urelumab to potentiate anti-myeloma activity in lymphocytedeficient mice reconstituted with human NK cells

Daratumumab is an anti-CD38 fully human IgG1 mAb approved for multiple myeloma treatment. One of the proposed mechanisms of action is the induction of antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells. NK cells acquire surface CD137 expression in the presence of solid-phase-attached daratumumab and when encountering a daratumumab-coated CD38+ tumor cell line. In this setting, addition of the agonist anti-CD137 mAb urelumab enhances NK-cell activation increasing CD25 expression and IFNɣ production. However, in vitro ADCC is not increased by the addition of urelumab both in 4h or 24h lasting experiments. To study urelumab-increased daratumumab-mediated ADCC activity in vivo, we set up a mouse model based on the intravenous administration of a luciferase-transfected multiple myeloma cell line of human origin, human NK cells and daratumumab to immuno-deficient NSG mice. In this model, intravenous administration of urelumab 24h after daratumumab delayed tumor growth and prolonged mice survival

On mechanisms underlying efficacy and safety in cancer immunotherapy

La inmunoterapia del cáncer basada en el bloqueo de puntos de control ha supuesto una revolución en el tratamiento del cáncer. Sin embargo, es necesario profundizar en los factores que afectan a su eficacia y seguridad para poder conseguir mejorar las estrategias terapéuticas actuales. Para contribuir al estudio de los factores que afectan a la eficacia de la inmunoterapia, nos hemos centrados en estudiar si la muerte celular mediada por células citotóxicas es inmunogénica. La respuesta inmune frente al cáncer se conceptualiza con el ciclo de inmunidad y cáncer. Un paso esencial en esta interpretación es que los antígenos liberados por los tumores son presentados por las células dendríticas a las células T naïve o de memoria en los ganglios linfáticos que drenan el tumor. Actualmente se desconoce si la muerte de las células tumorales resultante de la citotoxicidad, mediada por las células T o los linfocitos NK, es realmente inmunogénica. Observamos en cocultivos de células tumorales y células citotóxicas efectoras, la presencia de marcadores de muerte celular inmunogénica como la exposición de calreticulina y la proteína soluble HMGB1. Las células de cáncer de colon MC38 transfectadas con OVA, pulsadas exógenamente para presentar el epítopo gp100, son destruidas en cultivo por células T CD8 con TCR transgénico específicas de gp100 de ratón. La inmunización de ratones con las células destruidas resultantes induce la propagación del epítopo como se observa mediante la detección de células T específicas de OVA y rechazo de células de linfoma EG7. Se observaron resultados similares en ratones inmunizados con restos celulares generados por citotoxicidad mediada por células NK. Los ratones deficientes en células dendríticas dependientes de BATF3 (cDC1) no logran desarrollar una respuesta anti-OVA cuando se inmunizan con células tumorales destruidas por linfocitos citotóxicos. En línea con esto, las células dendríticas cDC1 cultivadas pueden presentar el antígeno de las células tumorales destruidas por citotoxicidad con linfocitos T CD8. Tomados en conjunto, estos resultados respaldan que una respuesta inmune antitumoral citotóxica en curso puede conducir a la muerte inmunogénica de las células tumorales. Por otra parte, se estudiaron factores que afectan a la seguridad del tratamiento. La inmunoterapia dirigida a PD-1 y CTLA-4 con Nivolumab e Ipilimumab logra una notable eficacia contra el melanoma, el carcinoma de células renales y el cáncer de pulmón de células no pequeñas. Sin embargo, se asocia a eventos adversos inmunes frecuentes, graves y limitantes de dosis, que requieren reducciones de la dosis de Ipilimumab. En ratones tratados conjuntamente con anticuerpos monoclonales anti-PD-1 y anti-CTLA-4, se exacerba la colitis autoinmune concomitante a la eficacia terapéutica en modelos de cáncer trasplantables. El tratamiento previo con inhibidores de TNF mejora la colitis y mejora inesperadamente la eficacia antitumoral. Curiosamente, el eje inflamatorio de TNF está hiperactivado en el intestino de pacientes que sufren colitis después del tratamiento con Ipilimumab más Nivolumab. El bloqueo profiláctico del TNF humano mejora la colitis y la hepatitis inducida por el PBMCs humanas exacerbadas por el tratamiento conjunto con Ipilimumab más Nivolumab, al tiempo que preserva el control inmunoterapéutico de los tumores xenoinjertos. Estos resultados presentan estrategias clínicamente viables para disociar la eficacia y la toxicidad en el uso del bloqueo combinado de puntos de control inmunitario para la inmunoterapia del cáncer

On mechanisms underlying efficacy and safety in cancer immunotherapy

La inmunoterapia del cáncer basada en el bloqueo de puntos de control ha supuesto una revolución en el tratamiento del cáncer. Sin embargo, es necesario profundizar en los factores que afectan a su eficacia y seguridad para poder conseguir mejorar las estrategias terapéuticas actuales. Para contribuir al estudio de los factores que afectan a la eficacia de la inmunoterapia, nos hemos centrados en estudiar si la muerte celular mediada por células citotóxicas es inmunogénica. La respuesta inmune frente al cáncer se conceptualiza con el ciclo de inmunidad y cáncer. Un paso esencial en esta interpretación es que los antígenos liberados por los tumores son presentados por las células dendríticas a las células T naïve o de memoria en los ganglios linfáticos que drenan el tumor. Actualmente se desconoce si la muerte de las células tumorales resultante de la citotoxicidad, mediada por las células T o los linfocitos NK, es realmente inmunogénica. Observamos en cocultivos de células tumorales y células citotóxicas efectoras, la presencia de marcadores de muerte celular inmunogénica como la exposición de calreticulina y la proteína soluble HMGB1. Las células de cáncer de colon MC38 transfectadas con OVA, pulsadas exógenamente para presentar el epítopo gp100, son destruidas en cultivo por células T CD8 con TCR transgénico específicas de gp100 de ratón. La inmunización de ratones con las células destruidas resultantes induce la propagación del epítopo como se observa mediante la detección de células T específicas de OVA y rechazo de células de linfoma EG7. Se observaron resultados similares en ratones inmunizados con restos celulares generados por citotoxicidad mediada por células NK. Los ratones deficientes en células dendríticas dependientes de BATF3 (cDC1) no logran desarrollar una respuesta anti-OVA cuando se inmunizan con células tumorales destruidas por linfocitos citotóxicos. En línea con esto, las células dendríticas cDC1 cultivadas pueden presentar el antígeno de las células tumorales destruidas por citotoxicidad con linfocitos T CD8. Tomados en conjunto, estos resultados respaldan que una respuesta inmune antitumoral citotóxica en curso puede conducir a la muerte inmunogénica de las células tumorales. Por otra parte, se estudiaron factores que afectan a la seguridad del tratamiento. La inmunoterapia dirigida a PD-1 y CTLA-4 con Nivolumab e Ipilimumab logra una notable eficacia contra el melanoma, el carcinoma de células renales y el cáncer de pulmón de células no pequeñas. Sin embargo, se asocia a eventos adversos inmunes frecuentes, graves y limitantes de dosis, que requieren reducciones de la dosis de Ipilimumab. En ratones tratados conjuntamente con anticuerpos monoclonales anti-PD-1 y anti-CTLA-4, se exacerba la colitis autoinmune concomitante a la eficacia terapéutica en modelos de cáncer trasplantables. El tratamiento previo con inhibidores de TNF mejora la colitis y mejora inesperadamente la eficacia antitumoral. Curiosamente, el eje inflamatorio de TNF está hiperactivado en el intestino de pacientes que sufren colitis después del tratamiento con Ipilimumab más Nivolumab. El bloqueo profiláctico del TNF humano mejora la colitis y la hepatitis inducida por el PBMCs humanas exacerbadas por el tratamiento conjunto con Ipilimumab más Nivolumab, al tiempo que preserva el control inmunoterapéutico de los tumores xenoinjertos. Estos resultados presentan estrategias clínicamente viables para disociar la eficacia y la toxicidad en el uso del bloqueo combinado de puntos de control inmunitario para la inmunoterapia del cáncer

Impact of prophylactic TNF blockade in the dual PD-1 and CTLA-4 immunotherapy efficacy and toxicity

The TNF blockade therapy is currently a well-established treatment option for a variety of autoimmune diseases such as rheumatoid arthritis (RA), psoriasis or Crohn's disease, given the proinflammatory role of TNF in the course of these diseases. Importantly, TNF neutralization is also used for the treatment of corticosteroid-refractory immune-related adverse events (irAEs) induced by the combined anti-PD-1 and anti-CTLA-4 immunotherapy. The manifestation of these toxicities is an important limiting factor for the successful implementation of the inhibitory checkpoint blockade therapy (ICB), restraining its anti-tumor efficacy. In our recent study (Perez-Ruiz et al., Nature 569(7756): 428-432.), we analyzed the potential impact of prophylactic TNF neutralization therapy in the anti-PD1/CTLA-4 efficacy. Through several mouse models, we demonstrated that TNF neutralization ameliorated ICB-exacerbated colitis in addition to improving ICB-dependent anti-tumor efficacy. Similar results were obtained after prophylactic TNF blockade in graft vs host xenografted mouse models with human immune cells, which showed a reduction in colitis and hepatitis. Importantly, there was a preservation of the immunotherapeutic control of xenografted tumors after ICB treatment. Moreover, TNF and TNF-dependent gene expression is upregulated in the colon mucosa from patients affected by colitis as a side effect of ipilimumab and nivolumab. Our results, thus, provide evidence of the successful combination of prophylactic TNF neutralization with ICB therapy strategy to ameliorate toxicities, while keeping or even ameliorating anti-tumor efficacy. The prophylactic TNF blockade strategy is clinically feasible since excellent TNF inhibitors have been approved for the treatment of autoimmunity and are used for the immune-related serious adverse events in immunotherapy

Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy

Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy

Enhancement of antibody-dependent cellular cytotoxicity of cetuximab by a chimeric protein encompassing interleukin-15

Enhancement of antibody-dependent cellular cytotoxicity (ADCC) may potentiate the antitumor efficacy of tumor-targeted monoclonal antibodies. Increasing the numbers and antitumor activity of NK cells is a promising strategy to maximize the ADCC of standard-of-care tumor-targeted antibodies. For this purpose, we have preclinically tested a recombinant chimeric protein encompassing the sushi domain of the IL15Rα, IL-15, and apolipoprotein A-I (Sushi-IL15-Apo) as produced in CHO cells. The size-exclusion purified monomeric fraction of this chimeric protein was stable and retained the IL-15 and the sushi domain bioactivity as measured by CTLL-2 and Mo-7e cell proliferation and STAT5 phosphorylation in freshly isolated human NK and CD8+ T cells. On cell cultures, Sushi-IL15-Apo increases NK cell proliferation and survival as well as spontaneous and antibody-mediated cytotoxicity. Scavenger receptor class B type I (SR-B1) is the receptor for ApoA-I and is expressed on the surface of tumor cells. SR-B1 can adsorb the chimeric protein on tumor cells and can transpresent IL-15 to NK and CD8+ T cells. A transient NK-humanized murine model was developed to test the increase of ADCC attained by the chimeric protein in vivo. The EGFR+ human colon cancer cell line HT-29 was intraperitoneally inoculated in immune-deficient Rag2-/-γc-/- mice that were reconstituted with freshly isolated PBMCs and treated with the anti-EGFR mAb cetuximab. The combination of the Sushi-IL15-Apo protein and cetuximab reduced the number of remaining tumor cells in the peritoneal cavity and delayed tumor engraftment in the peritoneum. Furthermore, Sushi-IL15-Apo increased the anti-tumor effect of a murine anti-EGFR mAb in Rag1-/- mice bearing subcutaneous MC38 colon cancer transfected to express EGFR. Thus, Sushi-IL15-Apo is a potent tool to increase the number and the activation of NK cells to promote the ADCC activity of antibodies targeting tumor antigens

Human CD8 T cells are susceptible to TNF-mediated activation-induced cell death

Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role of TNF in AICD of CD8 T lymphocytes. Methods: Human peripheral mononuclear cells were isolated from healthy donors and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinicalgrade TNF blocking agents. Results: A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals which damage DNA. Conclusion: The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in immunotherapy strategies

Human CD8 T cells are susceptible to TNF-mediated activation-induced cell death

Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role of TNF in AICD of CD8 T lymphocytes. Methods: Human peripheral mononuclear cells were isolated from healthy donors and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinicalgrade TNF blocking agents. Results: A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals which damage DNA. Conclusion: The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in immunotherapy strategies