A novel form of 4-1BBL has better immunomodulatory activity than an agonistic anti-4-1BB Ab without Ab-associated severe toxicity

Abstract

Agonistic Abs to select costimulatory members of CD28 and TNFR family have shown efficacy in various preclinical cancer immunotherapeutic settings. However, the use of agonistic Abs is often associated with severe toxicity due to non-specific activation of lymphocytes. We hypothesized that natural costimulatory ligands may serve as more potent and safer alternative to agonistic Abs for immunotherapy. In this communication, we focused on 4-1BBL as the molecule of choice because of the pleiotropic effects of 4-1BB signaling in the immune system and the demonstrated therapeutic efficacy of 4-1BB agonistic Abs in preclinical cancer and infection models. We report that a novel form of soluble ligand, SA-4-1BBL, delivered more potent and qualitatively different signals to T cells than an agonistic Ab. Importantly, while treatment of naïve mice with the agonistic Ab resulted in severe toxicity, as assessed by enlarged spleen and peripheral LNs, non-specific T cell proliferation, hepatitis, and systemic inflammatory cytokine production, treatment with SA-4-1BBL lacked these immune anomalies. Agonistic Ab treatment produced full toxicity in FcgammaR(-/-) or complement C1q(-/-) or C3(-/-) knockout mice, suggesting lack of involvement of stimulatory FcgammaRs or complement system in the observed toxicity. Naïve and memory T cells served as direct targets of anti-4-1BB Ab-mediated toxicity. Potent immunostimulatory activity combined with lack of toxicity rationalizes further development of soluble SA-4-1BBL as an immunomodulatory component of therapeutic vaccines against cancer and chronic infections.

Fig 1

SA-4-1BBL demonstrates better immune activity than an agonistic anti-4-1BB Ab without Ab-associated toxicity. (A) In vitro CD8⁺ T cell (left) and CD4⁺ T cell (right) proliferation. Flow sorted CD8⁺ and CD4⁺ T cells were stimulated for 3 days with a suboptimal dose of an agonistic anti-CD3 Ab in conjugation with various doses of SA-4-1BBL or 3H3 Ab in the presence of irradiated syngeneic splenocytes. Cells were pulsed with [³H]-thymidine for the last 16 hrs of culture. (B) In vivo OT-I and OT-II T cell proliferation. One million flow sorted OT-I T cells and OT-II (CD45.2⁺) were labeled with CFSE and injected i.v. into naïve congenic C57BL/6.SJL (CD45.1⁺) mice. Mice were vaccinated i.v. with OVA (10 µg) in combination with various doses (µg) of SA-4-1BBL or 3H3 Ab as indicated. Proliferation was assessed using multiparameter flow cytometry 3 days after vaccination. (C) In vivo killing response. C57BL/6 mice were immunized i.v. with OVA (50 µg) in combination with 3H3 Ab (25 µg) or SA-41BBL (25 µg). Seven days post vaccination, mice received SIINFEKL-pulsed syngeneic splenocytes and peptide-specific killing was assessed 2 days later and expressed as percent lysis for each histogram. (D) In vivo toxicity as assessed by the weight of spleen and lymph nodes harvested from mice vaccinated in panel C. p values determined by Student’s t test. Data are representative of a minimum of three independent experiments for Panels A–C.

Fig 2

SA-4-1BBL treatment lacks Ab associated toxicity. Naïve C57BL/6 mice were injected s.c. once (○) or once weekly over a 3-wk period (●) with 100 µg of SA-4-1BBL, equivalent doses of anti-4-1BB Ab, or other appropriate controls. Animals were euthanized one week after the final treatment and assessed for various indicators of toxicity, including peripheral LN and spleen weights (A) and total peripheral LN and spleen cell numbers (B). (C) Anti-4-1BB Ab demonstrates super-agonistic activity in vivo. In vivo proliferation of spleen CD8⁺ and CD4⁺ T cells was determined by adoptively transferring 2 × 10⁶ CFSE-labeled CD45.1⁺ congenic splenocytes into naïve C56BL/6 (CD45.2⁺) mice one day prior to the indicated treatments. Percentage of proliferating cells was determined by CFSE dilution using FACS analysis. p values determined by ANOVA, anti-4-1BB vs. all other groups within same treatment schedule. Data were obtained from a minimum of 3 mice per group.

Fig 3

Treatment with anti-4-1BB Ab, but not SA-4-1BBL, results in a drastic increase in the production of inflammatory cytokines. Lymphocytes and sera harvested from mice that underwent treatment as described in Figure 2 were analyzed for percentage (A) and absolute numbers (B) of splenic CD8⁺ and CD4⁺ T cells expressing IFN-γ. Spleen cells were stimulated with PMA and ionomycin for 6 hours and then surface stained with anti-CD8 and anti-CD4 Abs followed by intracellular staining for IFN-γ. (C) Cytokine levels in the serum of various treatment group, as determined using a CBA kit and flow cytometry. p values determined by ANOVA, anti-4-1BB vs. all other groups within same treatment schedule. Data were obtained from a minimum of 3 mice per group.

Fig 4

Anti-4-1BB Ab associated toxicity is FcR and complement independent. Naïve C57BL/6 Fcer1g^−/−, C57BL/6 C3^−/−, and C57BL/6 C1q^−/− male mice were injected once s.c. with 100 µg of anti-4-1BB Ab or control rat IgG, sacrificed one week after treatment, and assessed for various indicators of toxicity. Peripheral LN and spleen weights of treated mice (A), total peripheral LN and spleen cell numbers (B), as well as total T cell and B cell numbers as determined by FACS analysis (C). p values for anti-4-1BB vs. rat IgG for each type of KO mice was determined by Student’s t test. Data were obtained from a minimum of 3 mice per group.

Fig 5

T cells are the direct target of anti-4-1BB Ab mediated toxicity. C57BL/6 4-1BB^−/− mice were adoptively transferred with the indicated numbers and types of lymphocytes and treated one day later using 100 µg of anti-4-1BB Ab. Mice were sacrificed one week after treatment, and assessed for various indicators of toxicity. All the cells, except splenocytes, were sorted by flow cytometry to ensure purity before adoptive transfer. Peripheral LN and spleen weights (A) and total peripheral LN and spleen cell numbers (B) of the treated mice. p values determined by ANOVA, 4-1BB^−/− without adoptive transfer vs. all other groups. (C–F) Anti-4-1BB Ab directly activates monoclonal T cells resulting in toxicity. A single treatment of OT-I Rag^−/− and OT-II Rag^−/− mice with 100 µg of anti-4-1BB Ab results in enlarged peripheral lymph nodes (C), total cell numbers (D), and CD8⁺ T cell numbers (E) as determined by flow cytometry. (F) As compared to controls, more CD8⁺ T cells from anti-4-1BB Ab treated OT-I Rag^−/− mice produce IFN-γ and TNF-α as determined by intracellular cytokine staining. p values determined by Student’s t test. Data were obtained from a minimum of 3 mice per group.