An autoimmune-mediated strategy for prophylactic breast cancer vaccination

Abstract

Although vaccination is most effective when used to prevent disease, cancer vaccine development has focused predominantly on providing therapy against established growing tumors. The difficulty in developing prophylactic cancer vaccines is primarily due to the fact that tumor antigens are variations of self proteins and would probably mediate profound autoimmune complications if used in a preventive vaccine setting. Here we use several mouse breast cancer models to define a prototypic strategy for prophylactic cancer vaccination. We selected alpha-lactalbumin as our target vaccine autoantigen because it is a breast-specific differentiation protein expressed in high amounts in the majority of human breast carcinomas and in mammary epithelial cells only during lactation. We found that immunoreactivity against alpha-lactalbumin provides substantial protection and therapy against growth of autochthonous tumors in transgenic mouse models of breast cancer and against 4T1 transplantable breast tumors in BALB/c mice. Because alpha-lactalbumin is conditionally expressed only during lactation, vaccination-induced prophylaxis occurs without any detectable inflammation in normal nonlactating breast tissue. Thus, alpha-lactalbumin vaccination may provide safe and effective protection against the development of breast cancer for women in their post-child-bearing, premenopausal years, when lactation is readily avoidable and risk for developing breast cancer is high.

Figure 1. Immunogenicity of recombinant mouse a-lactalbumin

(a) Western blot showing purified recombinant mouse α-lactalbumin detected with His antibody. LNC taken 10 days after immunization of SWXJ female mice with α-lactalbumin showed recall responses that were: (b) antigen-specific to recombinant mouse α-lactalbumin but not to recombinant human cochlin over a broad dose range; (c) elicited from both purified CD4+ and CD8+ T cells in response to 25 μg/ml α-lactalbumin; and (d) consistent with a proinflammatory type 1 cytokine profile with high production of IFN-γ and IL-2 and low production of the type 2 cytokines, IL-4, IL-5, and IL-10 in response to 25 μg/ml α-lactalbumin. (e) Six weeks after α-lactalbumin immunization CD3+ T cells were detected only as isolated individual surveillance cells in mammary parenchyma of non-lactating α-lactalbumin immunized mice (arrows) and never appeared as part of any inflammatory infiltrate in non-lactating breast tissues. (f) Extensive inflammatory infiltrates of CD3+ T cells were consistently found in mammary parenchyma of lactating α-lactalbumin immunized mice (arrows). (g) CD3+ infiltrates were never observed in mammary tissue from lactating control mice immunized with CFA alone. (h) Flow cytometry analysis of breast infiltrates showed high frequencies of CD3+CD4+ (left panel) and CD3+CD8+ T cells (right panel) expressing the CD44^high activated phenotype. (i) Real-time RT-PCR analysis of lactating mammary tissue showed significantly elevated expression levels of IFN-γ (P = 0.001) but not IL-10 (P > 0.10). All error bars show ±SE. Each * indicates significance.

Figure 2. α-lactalbumin vaccination delays and treats breast tumor growth

(a) Growth of autochthonous breast tumors was significantly inhibited (P = 0.0004) in ten month old MMTV-neu mice immunized with α-lactalbumin at eight weeks of age. (b) Growth of transplanted 4T1 tumors was significantly inhibited (P = 0.0006) following prophylactic immunization with α-lactalbumin 13 days prior to tumor inoculation. (c) H&E staining of tissue extracted from the flank of a representative mouse 5 days after s.c. inoculation of 2×10⁴ 4T1 tumor cells confirms the presence of an established growing tumor (arrows) infiltrating the underlying connective tissue. Size bar = 100 μm. Significant inhibition of 4T1 tumor growth occurred when α-lactalbumin immunization occurred (d) 5 days after tumor inoculation (P < 0.01) and (e) 13 days after tumor inoculation (P < 0.01), (f) but not 21 days after tumor inoculation (P > 0.10). (g) Significant inhibition (P < 0.0006) in the growth of very aggressive autochthonous tumors occurred following α-lactalbumin immunization of MMTV-PyVT transgenic mice at 6 weeks of age. Tumors in MMTV-PyVT mice were amenable to measurement in only one direction. All error bars show ±SE. Each * indicates significance.

Figure 3. α-lactalbumin specific T cells induce tumor inflammation and cytotoxicity

(a) Thirty-two days after BALB/c mice were vaccinated with α-lactalbumin and inoculated with 4T1 cells, tumors were extensively infiltrated with CD3+ T cells. (b) T cell infiltrates were never observed in control inoculated mice immunized with CFA alone. (c) Flow cytometry analysis of TILs showed a predominance of CD4+ (64.3%) over CD8+ (14.4%) T cells. (d) TILs showed a type-1 proinflammatory phenotype involving high production of IFN-γ in response to 50 μg/ml α-lactalbumin. (e) ELISPOT analysis of TILs showed that CD4+ rather than CD8+ T cells produced IFN-γ in response to 50 μg/ml α-lactalbumin since its secretion by cultured T cells was inhibited by treatment with class II but not class I antibodies. (f) CD8+ T cells mediated 4T1 specific cytotoxicity since death of cultured 4T1 tumor cells was inhibited by treatment of cultured α-lactalbumin primed LNC with antibodies specific for mouse CD8 but not CD4. All data shown are representative of three experiments providing similar results. All error bars show ±SE.

Figure 4. Inhibition of tumor growth by α-lactalbumin vaccination is mediated by T cells

Transfer of α-lactalbumin primed LNC into naive recipient BALB/c mice on the same day as inoculation with 4T1 tumors resulted in (a) highly significant (P < 0.0001) inhibition of tumor growth, (b) significantly decreased (P < 0.03) incidence of tumor bearing mice, and (c) significantly decreased (P < 0.0008) final tumor weight. (d) Significant tumor growth inhibition occurred in naive mice receiving either CD4+ (left panel; P = 0.002) or CD8+ (right panel; P = 0.003) T cells enriched by magnetic bead separation from α-lactalbumin primed LNC. All error bars show ±SE. Each * indicates significance.