Abstract:While anti-CEA antibodies have no direct effect on CEA-positive tumors, they can be used to direct potent antitumor effects as an antibody-IL-2 fusion protein (immunocytokine, ICK), and at the same time reduce the toxicity of IL-2 as a single agent. Using a fusion protein of humanized anti-CEA with human IL-2 (M5A-IL-2) in a transgenic murine model expressing human CEA, we show high tumor uptake of the ICK to CEA-positive tumors with additional lymph node targeting. ICK treated CEA-positive tumors exhibit sign… Show more
“…[81][82][83] found that the addition of CY to ICK therapy depletes immunosuppressive regulatory T cells (Treg) cells. 34 Chmielewski et. al.…”
Section: Discussionmentioning
confidence: 99%
“…The immunocytokine (ICK) was produced as described by Kujawski et al . 34 Figure 1. Anti-CEA CAR T cells specifically target CEA-positive mouse adenocarcinoma cells in vitro.…”
Section: Methodsmentioning
confidence: 99%
“…The constructs are shown in (Figure 1a). The immunocytokine (ICK) was produced as described by Kujawski et al 34…”
Section: Dna Constructsmentioning
confidence: 99%
“…34,[60][61][62] Specifically, combined stereotactic radiation therapy (SRT) with anti-CEA-IL2 in the MC38/CEA and E0771/CEA tumor models in CEATg mice was found to not only stimulate a T cellmediated antitumor response but also to lower regulatory T cell infiltration and stimulate a memory antitumor effect. 34 To determine if the addition of ICK to anti-CEA CAR T cell therapy would also improve the antitumor response, anti-CEA CAR or mock transduced T cells were co-cultured with MC38/GFP vs. MC38/CEA/GFP cells in in the presence or absence of anti-CEA -IL2 ICK. The anti-CEA CAR T cells with ICK exhibited a dosedependent cytotoxic response to CEA + vs. CEA − cells and secreted more IFNγ than anti-CEA CAR T cells alone or mock transduced T cells (Figure 5a-d).…”
Section: Immunocytokine Anti-cea-il2 Increases the Cytotoxic Activitymentioning
confidence: 99%
“…We have previously shown that the ICK, humanized anti-CEA antibody (M5A) fused to IL2, was effective against orthotopic CEA transfected breast carcinoma E0771 tumors in a CEATg mouse model. 34 Furthermore, combination of ICK with fractionated stereotactic radiation therapy (SRT) eradicated tumors and established tumor immunity. These studies prompted us to combine two antigen-targeted therapies, namely anti-CEA CAR T cells plus ICK therapy to determine their therapeutic efficacy.…”
Targeted immunotherapy of solid cancers with chimeric antigen receptor (CAR) T cells and immunocytokines are attractive options in that they both rely on the specificity of tumor-targeted antibodies. Since carcinoembryonic antigen (CEA) expression in both colon and breast cancers is correlated with poor prognosis, it was chosen as a model tumor target in immunocompetent CEA transgenic (CEATg) mice. A second-generation anti-CEA CAR derived from CEA-specific antibody T84.66 was used to treat murine MC38 colon or E0771 breast carcinomas transfected with CEA. Anti-CEA CAR vs. mock transduced T cells exhibited a CEA-specific cytotoxic and IFNγ dose response to both CEA transfected cell lines vs. their CEAnegative controls. Anti-CEA CAR vs. mock transduced T cells delayed the median survival of CEA transfected s.c. MC38 or orthotopic E0771 tumor-bearing CEATg mice by 2 days. With the addition of oneday prior cyclophosphamide (CY) lymphodepletion, anti-CEA CAR T cell treatment delayed the median survival of MC38/CEA and E0771/CEA tumor-bearing CEATg mice by ten and 3 days, respectively. Since CAR T cells require IL2 for survival and expansion, anti-CEA-IL2 immunocytokine (ICK) treatment was performed post CAR T cell therapy. Single ICK treatment 1 day after CY plus anti-CEA CAR T cell therapy in the MC38/CEA model, and two ICK treatments every 3 days after CY plus anti-CEA CAR T cell therapy in the E0771/CEA model were ineffective, while four ICK treatments every 3 days after CY plus anti-CEA CAR T cell therapy completely eradicated MC38/CEA tumor growth and induced tumor immunity when the mice were re-challenged with tumor. These studies show the therapeutic potential of anti-CEA CAR T cells combined with ICK to treat CEA-positive tumors.
“…[81][82][83] found that the addition of CY to ICK therapy depletes immunosuppressive regulatory T cells (Treg) cells. 34 Chmielewski et. al.…”
Section: Discussionmentioning
confidence: 99%
“…The immunocytokine (ICK) was produced as described by Kujawski et al . 34 Figure 1. Anti-CEA CAR T cells specifically target CEA-positive mouse adenocarcinoma cells in vitro.…”
Section: Methodsmentioning
confidence: 99%
“…The constructs are shown in (Figure 1a). The immunocytokine (ICK) was produced as described by Kujawski et al 34…”
Section: Dna Constructsmentioning
confidence: 99%
“…34,[60][61][62] Specifically, combined stereotactic radiation therapy (SRT) with anti-CEA-IL2 in the MC38/CEA and E0771/CEA tumor models in CEATg mice was found to not only stimulate a T cellmediated antitumor response but also to lower regulatory T cell infiltration and stimulate a memory antitumor effect. 34 To determine if the addition of ICK to anti-CEA CAR T cell therapy would also improve the antitumor response, anti-CEA CAR or mock transduced T cells were co-cultured with MC38/GFP vs. MC38/CEA/GFP cells in in the presence or absence of anti-CEA -IL2 ICK. The anti-CEA CAR T cells with ICK exhibited a dosedependent cytotoxic response to CEA + vs. CEA − cells and secreted more IFNγ than anti-CEA CAR T cells alone or mock transduced T cells (Figure 5a-d).…”
Section: Immunocytokine Anti-cea-il2 Increases the Cytotoxic Activitymentioning
confidence: 99%
“…We have previously shown that the ICK, humanized anti-CEA antibody (M5A) fused to IL2, was effective against orthotopic CEA transfected breast carcinoma E0771 tumors in a CEATg mouse model. 34 Furthermore, combination of ICK with fractionated stereotactic radiation therapy (SRT) eradicated tumors and established tumor immunity. These studies prompted us to combine two antigen-targeted therapies, namely anti-CEA CAR T cells plus ICK therapy to determine their therapeutic efficacy.…”
Targeted immunotherapy of solid cancers with chimeric antigen receptor (CAR) T cells and immunocytokines are attractive options in that they both rely on the specificity of tumor-targeted antibodies. Since carcinoembryonic antigen (CEA) expression in both colon and breast cancers is correlated with poor prognosis, it was chosen as a model tumor target in immunocompetent CEA transgenic (CEATg) mice. A second-generation anti-CEA CAR derived from CEA-specific antibody T84.66 was used to treat murine MC38 colon or E0771 breast carcinomas transfected with CEA. Anti-CEA CAR vs. mock transduced T cells exhibited a CEA-specific cytotoxic and IFNγ dose response to both CEA transfected cell lines vs. their CEAnegative controls. Anti-CEA CAR vs. mock transduced T cells delayed the median survival of CEA transfected s.c. MC38 or orthotopic E0771 tumor-bearing CEATg mice by 2 days. With the addition of oneday prior cyclophosphamide (CY) lymphodepletion, anti-CEA CAR T cell treatment delayed the median survival of MC38/CEA and E0771/CEA tumor-bearing CEATg mice by ten and 3 days, respectively. Since CAR T cells require IL2 for survival and expansion, anti-CEA-IL2 immunocytokine (ICK) treatment was performed post CAR T cell therapy. Single ICK treatment 1 day after CY plus anti-CEA CAR T cell therapy in the MC38/CEA model, and two ICK treatments every 3 days after CY plus anti-CEA CAR T cell therapy in the E0771/CEA model were ineffective, while four ICK treatments every 3 days after CY plus anti-CEA CAR T cell therapy completely eradicated MC38/CEA tumor growth and induced tumor immunity when the mice were re-challenged with tumor. These studies show the therapeutic potential of anti-CEA CAR T cells combined with ICK to treat CEA-positive tumors.
BackgroundImmunocytokines (ICKs) are antibody directed cytokines produced by genetic fusion of an antibody to a cytokine.MethodsWe now show that antibodies conjugated by click chemistry to interleukin‐2 (IL‐2)‐Fc form fully active conjugates, and in one example, equivalent activity to a genetically produced ICK.ResultsAn IL‐2‐Fc fusion protein was optimized for click chemistry at hinge cysteines using protein stabilizing IL‐2 mutations at Lys35 and Cys125 and Fc hinge mutations at Cys142 and Cys148. The IL‐2‐Fc fusion protein with K35E and C125S mutations with 3 intact hinge cysteines, designated as IL‐2‐Fc Par, was selected based on its minimal tendency to aggregate. IL‐2‐Fc‐antibody clicked conjugates retained high IL‐2 activity and bound target antigens comparable to parent antibodies. An IL‐2‐Fc‐anti‐CEA click conjugate showed comparable anti‐tumor activity to an anti‐CEA‐IL‐2 ICK in immunocompetent CEA transgenic mice bearing CEA positive orthotopic breast tumors. Significant increases in IFNγ+/CD8+ and decreases in FoxP3+/CD4+ T‐cells were found for the clicked conjugate and ICK therapies, suggesting a common mechanism of tumor reduction.ConclusionThe production of antibody targeted IL‐2 therapy via a click chemistry approach is feasible with comparable activity to genetically produced ICKs with the added advantage of multiplexing with other monoclonal antibodies.
BackgroundThe potent immune effects of interleukin‐2 (IL‐2) for cancer therapy can be increased by genetic fusion of IL‐2 to the Fc domain of an antibody (IL‐2‐Fc) or tumor targeted by genetic fusion to a whole antibody known as an immunocytokine (ICK).MethodsAn anti‐CEA ICK (M5A‐IL‐2) was compared to an IL‐2‐Fc fusion protein using tumor therapy and PET imaging in CEA transgenic immunocompetent mice bearing CEA positive colon or breast tumors. Combination with stereotactic radiation therapy (SRT) was performed with either ICK or IL‐2‐Fc.ResultsICK and IL‐2‐Fc had comparable antitumor effects in both tumor models, although ICK had higher tumor uptake and slower blood clearance than an IL‐2‐Fc. Analysis of IFNγ+/CD8+ and FoxP3+/CD4+ T cells revealed higher levels of IFNγ‐producing CD8+ T cells in ICK treated mice versus more efficient Treg elimination in IL‐2‐Fc treated mice. No significant or lasting toxicity was detected for either agent. Combination therapies with SRT revealed comparable efficacy and induction of immune memory for both ICK and IL‐2‐Fc when mice were rechallenged post‐therapy.ConclusionsIL‐2‐Fc had comparable antitumor efficacy to CEA‐targeted M5A‐IL‐2 ICK, while both fusion proteins induced immune memory when combined with SRT. Differences in the therapeutic mechanisms of both agents were observed.
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