4-1BB ligand as an effective multifunctional immunomodulator and antigen delivery vehicle for the development of therapeutic cancer vaccines

Abstract

Therapeutic subunit vaccines based on tumor-associated antigens (TAA) represent an attractive approach for the treatment of cancer. However, poor immunogenicity of TAAs requires potent adjuvants for therapeutic efficacy. We recently proposed the tumor necrosis factor family costimulatory ligands as potential adjuvants for therapeutic vaccines and, hence, generated a soluble form of 4-1BBL chimeric with streptavidin (SA-4-1BBL) that has pleiotropic effects on cells of innate, adaptive, and regulatory immunity. We herein tested whether these effects can translate into effective cancer immunotherapy when SA-4-1BBL was also used as a vehicle to deliver TAAs in vivo to dendritic cells (DCs) constitutively expressing the 4-1BB receptor. SA-4-1BBL was internalized by DCs upon receptor binding and immunization with biotinylated antigens conjugated to SA-4-1BBL resulted in increased antigen uptake and cross-presentation by DCs, leading to the generation of effective T-cell immune responses. Conjugate vaccines containing human papillomavirus 16 E7 oncoprotein or survivin as a self-TAA had potent therapeutic efficacy against TC-1 cervical and 3LL lung carcinoma tumors, respectively. Therapeutic efficacy of the vaccines was associated with increased CD4(+) T and CD8(+) T-cell effector and memory responses and higher intratumoral CD8(+) T effector/CD4(+)CD25(+)Foxp3(+) T regulatory cell ratio. Thus, potent pleiotropic immune functions of SA-4-1BBL combined with its ability to serve as a vehicle to increase the delivery of antigens to DCs in vivo endow this molecule with the potential to serve as an effective immunomodulatory component of therapeutic vaccines against cancer and chronic infections.

Figure 1

SA-4-1BBL targets conjugated antigen in vivo to DCs for enhanced uptake and cross-presentation. A, Bone marrow-derived DCs internalize SA-4-1BBL following 4-1BB binding. DCs from wild type and 4-1BB^-/- C57BL/6 mice were incubated with SA-4-1BBL-FITC at 4°C for receptor binding, washed, and then incubated for 1 h at 4°C or 37°C for internalization. CD11c (red) DCs from wild type, but not 4-1BB^-/-, mice internalize SA-4-1BBL (green) at 37°C, but not 4°C. A minimum of 3 fields per slide were analyzed. B, SA-4-1BBL-Ova conjugates enhance antigen uptake by DCs in vivo. C57BL/6 mice were injected s.c. with conjugated SA-4-1BBL-Ova-FITC (25-10 μg), nonconjugated SA-4-1BBL+Ova-FITC (25+10 μg), Ova-FITC alone (10 μg), or left untreated. Draining lymph node cells were harvested 24 h later, stained with CD11c-PE, CD11b-PErcp-Cy5.5, B220-PEcy7, and CD8α-APC-cy7 Abs, and analyzed in flow cytometry. Data for Panels A and B are representative of two independent experiments. C, SA-4-1BBL-Ova conjugates increase antigen cross-presentation by DCs in vivo. C57BL/6 mice were vaccinated as in B, except Ova without FITC was used as antigen. Draining lymph node cells were harvested 24 h later and subjected to Ab staining as in B. APC-conjugated 25D1.16 Ab was used to detect K^b/SIINFEKL on the surface of DCs. D, SA-4-1BBL-Ova conjugates result in increased antigen presentation to OT-I CD8⁺ T cells in vivo. C57BL/6.SJL (CD45.1⁺) mice were immunized s.c. with Ova (10 μg) conjugated or nonconjugated with various doses (μg) of SA-4-1BBL as indicated. 2×10⁶ sorted OT-I T cells (CD45.2⁺) were labeled with CFSE and injected i.v. 24 or 48 h later. Proliferation was assessed using flow cytometry 3 days later. Data are representative of a minimum of three independent experiments for Panels C and D.

Figure 2

SA-4-1BBL conjugate vaccine has improved immunostimulatory activity than nonconjugate vaccine. A, in vivo OT-I and OT-II proliferation. 2×10⁶ sorted OT-I T and OT-II T cells (CD45.2⁺) were labeled with CFSE and injected i.v. into naïve congenic C57BL/6.SJL (CD45.1⁺) mice. Twenty-four h later mice were vaccinated s.c. with Ova (10 μg) conjugated or nonconjugated to various doses of SA-4-1BBL as indicated. Proliferation was assessed 3 days after vaccination by gating on CD4⁺CD45.2⁺ and CD8⁺CD45.2⁺ cells using flow cytometry. Data is representative of four independent experiments. B, as in A, but flow sorted OT-I T cells were injected into 4-1BB^-/- mice and OT-I T cell proliferation was assessed by gating on Vβ_5.1/5.2⁺CD8⁺ T cells. Data is representative of two independent experiments. C, in vivo endogenous CTL killing response. C57BL/6 (CD45.2⁺) mice were immunized s.c. with Ova (50 μg) conjugated or nonconjugated with SA-4-1BBL (25 μg). Seven days post vaccination, mice received SIINFEKL-pulsed syngeneic splenocytes from C57BL/6.SJL (CD45.1⁺) and peptide-specific killing was assessed 2 days later and expressed as percent lysis for each histogram (n = 5; * P < 0.05 compared with each other and Ova control). D, as in C, but mice were immunized with a recombinant HPV-16 E7 protein (10 μg) conjugated or nonconjugated to SA-4-1BBL (25 μg). n = 5, * P < 0.05 compared with each other and E7 control.

Figure 3

SA-4-1BBL conjugate vaccine has robust efficacy in TC-1 therapeutic tumor model. A, immunization with SA-4-1BBL conjugate vaccine demonstrates potent efficacy in eradicating established TC-1 tumors. C57BL/6 mice were challenged with live TC-1 cells and vaccinated once s.c. on day 6 post-tumor challenge with E7 (10 μg) conjugated or nonconjugated with SA-4-1BBL (25 μg) or an equimolar quantity of SA (10 μg). ** P < 0.001 for conjugated SA-4-1BBL-E7 vs. all other groups. Long-term surviving animals develop E7-specific CD8⁺ T-cell effector and memory pool (B) as determined by in vivo killing response (n = 3, * P < 0.05 vs. naïve control), (C) increased CD4⁺ and CD8⁺ T cell intracellular IFN-γ and IL-2 expression (n = 3, * P < 0.05 vs. naïve control), and (D) increased percentages of total memory CD44^hiCD43^-CD8⁺ T cells (n = 3, * P < 0.05 vs. each other and naïve control), and effector memory CD44^hiCD62L^lowCD4⁺ T cells (n = 3, * P < 0.05 vs. naïve control). Data for (B-D) are representative of two independent experiments.

Figure 4

SA-4-1BBL conjugate vaccine increases the intratumoral CD8⁺ Teff/Treg ratio. A, vaccination with SA-4-1BBL-E7 conjugates increased intratumoral CD8⁺ T cells and decreased CD4⁺Foxp3⁺ Treg cells, as determined by confocal microscopy, resulting in increased ratio of intratumoral CD8⁺ Teff/Treg cells (B; n = 4, * P < 0.05 vs. each other and naïve and SA-E7 controls). A minimum of 3 fields/tumor section were analyzed and pictures were taken under 20× objective.

Figure 5

SA-4-1BBL conjugate vaccine has robust efficacy in eradicating established 3LL tumors. C57BL/6 mice were challenged with 3LL tumor cells and vaccinated s.c. on day 6 post-tumor challenge with survivin (10 μg) conjugated or nonconjugated with SA-4-1BBL (25 μg) or an equimolar quantity of SA (10 μg). ** P < 0.001 for conjugated SA-4-1BBL-survivin vs. all other groups.

Figure 6

SA-4-1BBL conjugates serve as an effective therapeutic vaccine in TC-1 lung metastasis model. C57BL/6 mice (n = 3 per group) were challenged with live TC-1 cells by i.v. tail injection and vaccinated i.v. on day 6 post-tumor challenge with E7 (10 μg) conjugated with SA-4-1BBL (25 μg) or an equimolar quantity of SA (10 μg). A, mice were euthanized 21 d after tumor challenge and lungs were weighed, and (B) photographed after injection with Indian ink to assess tumor nodules. ** P < 0.001 and * P < 0.05. Data are representative of a minimum of three independent experiments for each panel.