Human/rat HER2-specific Exosome-targeted T Cell Vaccine For HER2~+ Breast Cancer Immunotherapy

Objective: To investigate the immunotherapeutic effect of heterologous human/rat HER2-specific exosome(EXO)-targeted T cell(Hu Rt-TEXO)vaccine in HER2-positive breast cancer.Methods:1)The recombinant transfer plasmid pshuttle-CMV-Hu Rt expressing human HER21-407aa(Hu)fused with rat neu408-690aa(Rt)protein(Hu Rt)was constructed by common recombinant DNA technology.The pshuttle-CMV-Hu Rt transfer plasmid was linearized by Pme I and cotransformed with Arg-Gly-Asp(RGD)-modified p Ad Easy-1 adenoviral backbone plasmid into BJ5183 competent bacteria for homologous recombination.The resultant p Ad Easy-1-pshuttle-CMV-Hu Rt(termed p Ad-Hu Rt)homologous recombinant adenoviral plasmid was then linearized by Pac I and transfected into 293 human embryonic kidney cells using Lipofectamine2000,leading to the formation of recombinant adenovirus Ad VHu Rt.Furthermore,the Ad VHu Rt was abundantly amplified in 293 cells and purified by cesium chloride(Cs Cl)density-gradient ultracentrifugation.2)Dendritic cells(DCs)were generated by culturing bone marrow(BM)cells derived from wild-type BALB/c(H-2Kd)or double transgenic(Tg)HLA-A2/HER2(HLA-A2,HLA-A2-restricted and human HER2-specific self-immune tolerance)mice.Mouse BM-DCs were then infected with Ad VHu Rt or Ad VHER2.Exosomes(EXOs)were then purified from culture supernatants of above DCs by differential ultracentrifugation,and termed EXOHu Rt(H-2K d),EXOHER2(H-2K d);and EXOHu Rt(HLA-A2),EXOHER2(HLA-A2),respectively.3)The activated T cells were generated by culturing splenocytes derived from BALB/c or HLA-A2/HER2 mice in presence of Con A.The activated CD4+ T cells were then purified by mouse CD4+ T cell isolation kit,and termed CD4+ TH-2K d or CD4+ THLA-A2.4)The CD4+ TH-2K d cells were incubated with EXOHu Rt(H-2K d)or EXOHER2(H-2K d)to form BALB/c(H-2Kd)-derived Hu Rt-TEXO or HER2-TEXO vaccine used for BALB/c mice vaccination.The CD4+ THLA-A2 cells were incubated with EXOHu Rt(HLA-A2)or EXOHER2(HLA-A2)to form HLA-A2/HER2(HLA-A2)-derived Hu Rt-TEXO or HER2-TEXO vaccine used for HLA-A2/HER2 mice vaccination.5)The BALB/c mice were intravenously(i.v.)immunized with BALB/c(H-2Kd)-derived Hu Rt-TEXO or HER2-TEXO(used as a control).Six days post immunization,the CD4+ T cell responses using double staining with FITC-anti-CD4(FITC-CD4)antibody(Ab)and PE-anti-CD44(PE-CD44)Ab,and HER2-specific CD8+ CTL responses using double staining with FITC-anti CD8(FITC-CD8)Ab and PE-Tetramer were analyzed by flow cytometry,respectively.Moreover,the splenocytes of BALB/c mice were labeled with either high or low concentration of carboxyfluorescein succinimidyl ester(CFSE).These high CFSE-labeled(CFSEhigh)cells were pulsed with H-2Kd-restricted HER2 peptide and served as HER2-specific CFSEhigh target cells.These low CFSE-labeled(CFSElow)cells were pulsed with irrelevant H-2Kd-restricted peptide and served as negative control target cells.These CFSEhigh and CFSElow target cells were co-injected i.v.at 1:1 ratio into immunized mice six days after immunization.The residual CFSEhigh and control CFSElow target cells remaining in the mouse spleens were then analyzed by flow cytometry to assess the H-2Kd-restricted and HER2-specific cytotoxic effect of Hu Rt-TEXO-or HER2-TEXO-stimulated effector CTLs.In addition,the immunized mice were i.v.boosted with Ad VHER2-infected BM-DC(DCHER2)from BALB/c mice thirty days after primary immunization,followed by flow cytometry analysis of immune recall responses using FITC-CD8 Ab and PE-Tetramer double staining four days post the boost.6)The BALB/c mice were i.v.immunized with BALB/c(H-2Kd)-derived Hu Rt-TEXO or HER2-TEXO(used as a control)twice with two weeks interval.Sera of immunized mice harvested three weeks post the second immunization.To determine HER2-specific antibody responses,the concentration of anti-HER2 Ab in sera was measured by HER2-binding enzyme-linked immunosorbent assay(ELISA).7)The BALB/c mice were i.v.or subcutaneously(s.c.)injected with Tg mouse breast cancer cells 4T1HER2 expressing human HER2.After establishment of early stage lung metastatic tumors or s.c.tumors,the tumor-bearing mice were i.v.injected with BALB/c(H-2Kd)-derived Hu Rt-TEXO or HER2-TEXO(used as a control).The therapeutic antitumor immunity of Hu Rt-TEXO or HER2-TEXO was analyzed.8)The double Tg HLA-A2/HER2 mice were i.v.immunized with HLA-A2/HER2(HLA-A2)-derived Hu Rt-TEXO or HER2-TEXO(used as a control).Six days post immunization,the mice were s.c.challenged with Tg BL6-10A2/HER2 mouse melanoma tumor cells expressing HLA-A2 and HER2.The preventive antitumor immunity was assessed to examine whether Hu Rt-TEXO or HER2-TEXO could overcome HER2-specific immune tolerance.9)The double Tg HLA-A2/HER2 mice were i.v.immunized with HLA-A2/HER2(HLA-A2)-derived Hu Rt-TEXO.CD8+ T cells were purified from mouse splenocytes six days after immunization,re-stimulated in vitro in culture by irradiated Ad VHER2-infected BM-DC(DCHER2)from HLA-A2/HER2 mice,and then purified to generate HLA-A2-restricted and HER2-specific CD8+ CTLs.The in vitro cytotoxicity of these amplified HER2-specific CD8+ CTLs to trastuzumab-resistant HLA-A2+HER2+ BT474A2(HLA-A2 Tg)human breast cancer cells was evaluated.10)Athymic BALB/c nude mice were s.c.injected with trastuzumab-resistant HLA-A2+HER2+ BT474A2 human breast cancer cells to establish human breast cancer s.c.tumors.The re-stimulated and amplified HER2-specific CD8+ CTLs were adoptively transferred i.v.to tumor-bearing mice.The in vivo therapeutic effect of these CTLs on trastuzumab-resistant HLA-A2+HER2+ BT474A2 human breast cancer s.c.tumors in athymic nude mice was then assessed.Results:1)The adenovirus expressing human HER21-407aa(Hu)fused with rat neu408-690aa(Rt)protein(Hu Rt)was successfully constructed.The heterologous human/rat HER2-specific exosome-targeted T cell vaccine Hu Rt-TEXO was also successfully prepared.2)In BALB/c wild-type mice,Hu Rt-TEXO vaccine more efficiently stimulated CD4+ T cell responses than HER2-TEXO did.The percentage of active CD4+CD44+ T cells in total CD4+ T cell population(29.4%)in mouse peripheral blood in Hu Rt-TEXO vaccination group was higher than that(23.1%)in HER2-TEXO control group(P<0.05).Hu Rt-TEXO also induced more potent HER2-specific humoral responses.The concentration of anti-HER2 antibody in the sera of Hu Rt-TEXO-immunized mice was estimated to be 70 ?g/ml,which is significantly higher than that(40 ?g/ml)in HER2-TEXO-immunized mice(P<0.05).3)In BALB/c mice,Hu Rt-TEXO vaccine more efficiently stimulated HER2-specific CD8+ CTL responses than HER2-TEXO did.The percentage of HER2-specific CD8+ CTL in total CD8+ T cell population(2.28%)was occurred in mouse peripheral blood after Hu Rt-TEXO vaccination,leading to killing 90% of HER2-specific target cells in vivo.However,there was only 1.31% HER2-specific CD8+ CTL which killed 53% of HER2-specific target cells in HER2-TEXO vaccination group(P<0.05).Moreover,Hu Rt-TEXO vaccine stimulated enhanced HER2-specific CTL memory.HER2-specific recall responses(3.51%)in Hu Rt-TEXO-immunized mice was much higher than that(1.54%)in HER2-TEXO-immunized ones(P<0.05).4)Hu Rt-TEXO vaccine treatment was capable of significantly inhibiting growth of pre-established early stage HER2+ 4T1HER2 mouse breast cancer tumors(lung metastatic tumor and s.c.tumor)in wild-type BALB/c mice.Compared with HER2-TEXO control group,Hu Rt-TEXO induced an enhanced therapeutic immunity against HER2+ 4T1HER2 breast cancer tumors(P<0.05).5)Hu Rt-TEXO vaccination completely prevented 100%(8/8)of double Tg HLA-A2/HER2 mice from growth of HLA-A2+HER2+ BL6-10A2/HER2 tumor cells,whereas HER2-TEXO vaccination only prevented 3/8 of mice from tumor challenge(P<0.05).Hu Rt-TEXO more effectively overcame HER2 immune tolerance and stimulated more potent protective immunity against HLA-A2+HER2+ BL6-10A2/HER2 tumor than HER2-TEXO in double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance.6)Ex vivo expanded HLA-A2-restricted and HER2-specific CTLs were obviously cytolytic to trastuzumab-resistant HLA-A2+HER2+ BT474A2 human breast cancer cells in vitro and in athymic nude mice in vivo.These CTLs almost completely eradicated pre-established HLA-A2+HER2+ BT474A2 breast cancer transplanted tumors(3-4 mm in diameter).Conclusions: The heterologous human/rat HER2-specific exosome-targeted T cell vaccine Hu Rt-TEXO is capable of stimulating stronger HER2-specific humoral and T cell immune responses and more effectively breaking immune tolerance to HER2,thus leading to an enhanced protective antitumor immunity.Hu Rt-TEXO,a potential therapeutic vaccine for HER2+ breast cancer,may provide a new therapeutic alternative for patients with trastuzumab-resistant HER2+ breast tumor.