Engineered soluble, trimerized 4-1BBL variants as potent immunomodulatory agents

Figures

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  Fig. 1. Multimerized s4-1BBL proteins exert potent co-stimulatory effects in 4-1BB expressing reporter cells. a Left: 4-1BB expression on control reporter cells (open histogram) and 4-1BB-reporter cells (grey histogram). Right: TCS control and TCS-4-1BBL were analyzed for expression of membrane-bound anti-CD3-scFv; 4-1BBL expression on control TCS (open histogram) and TCS-4-1BBL (grey histogram). b, e, h Schematic representation of synthetic genes encoding s4-1BBL, s4-1BBL-TNC, s4-1BBL-TriXVIIILL (long 23-mer linker; L*), Triple-s4-1BBL, Triple-s4-1BBL-TriXVIII, s4-1BBL-TriXVIII (short GS linker L) and s4-1BBL-TriXV. c, f, i 4-1BB reporter cells were probed with supernatants derived from HEK293T cells transfected with expression constructs encoding multimerized s4-1BBL proteins. Bound s4-1BBL was detected using a biotinylated Strep-tag II antibody in conjunction with streptavidin-PE via flow cytometry. Data are presented as fold induction relative to the values obtained upon binding of biotinylated Strep-tag II antibody on 4-1BB expressing reporter cells without soluble 4-1BBL protein (binding of s4-1BBL- Strep-tag II antibody/binding of Strep-tag II antibody (without s41BBL); MFI values…

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  Fig. 2. Shortened versions of 4-1BBL ectodomain retain strong agonistic effects a Schematic representation of a synthetic gene encoding a short version of trimerized s4-1BBL (s4-1BBLsh-TriXVIII; aa 90–242). b 4-1BB reporter cells were incubated with supernatants containing s4-1BBL-TriXVIII and s4-1BBLsh-TriXVIII (90–242). Bound s4-1BBL was detected using a biotinylated Strep-tag II antibody in conjunction with streptavidin-PE. c 4-1BB reporter cells were stimulated with TCS control alone or in presence of urelumab (1 μg/ml) or cell culture supernatants containing s4-1BBL proteins used at indicated dilutions. TCS expressing 4-1BBL served as an additional positive control for 4-1BB activation. Data are presented as fold change relative to the values obtained upon stimulation with TCS control alone. d 4-1BB reporter cells were stimulated with TCS control alone or in presence of urelumab (1 μg/ml) or cell culture supernatants containing different short versions of s4-1BBLsh-TriXVIII (90–242, 90–241, 90–240, 90–239, 91–242, 91–241, 91–240, 92–242, 92–241, 92–240, 93–242, 93–241 and 93–240, respectively) proteins used at indicated dilutions. TCS expressing 4-1BBL served as an additional…

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  Fig. 3. Soluble 4-1BBL-TriXVIII efficiently co-stimulates proliferation and activation of primary human CD4+ and CD8+ T cell (a, b, c) 1 × 105 CFSE-labeled PBMCs were co-cultured with 3 ng/ml anti-human CD3 (UCHT1) in the absence or presence of different dilutions of culture supernatants containing s4-1BBL-TriXVIII or 1 µg/ml urelumab. After 5 days, PBMCs were harvested and stained for CD4, CD8, and CD25 and analyzed by flow cytometry. a Schematic representation of the gating strategy of one representative experiment is depicted. b T cell proliferation of 5 healthy volunteers is depicted. Data show percentages of CFSElow gated on live CD4+ or CD8+ T cells normalized to 3 ng/ml of UCHT1 antibody alone. c T cell activation by analysis of geometric mean of fluorescence intensity (gMFI) of CD25 expression is shown for 5 healthy volunteers for the CD4+ and CD8+ T cell subsets. Data are normalized to values obtained to UCHT1 antibody alone. d Cell culture supernatants of PBMCs co-cultured with 300 ng/ml anti-human CD3 (OKT3) in the absence or presence of different dilutions of culture supernatants containing s4-1BBL-TriXVIII or 1 µg/ml urelumab were collected and analyzed by Luminex™-based…

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  Fig. 4. A murine 4-1BBL-TriXVIII protein strongly co-stimulates reporter cells expressing m4-1BB a Schematic representation of a synthetic gene encoding murine s4-1BBL-TriXVIII (ms4-1BBL-TriXVIII) that was cloned into the mammalian expression vector pCEP4. b Analysis of cell surface expression of murine 4-1BB on reporter cells and murine 4-1BBL on T cell stimulator cells (TCS). c Control (Ctrl) reporter cells and m4-1BB reporter cells were probed with diluted supernatants derived from HEK293T cells transfected with expression constructs encoding ms4-1BBL-TriXVIII. Bound ms4-1BBL-TriXVIII was detected using a biotinylated Strep-tag II antibody in conjunction with streptavidin-PE. d, e Control reporter cells and m4-1BB reporter cells were stimulated with TCS alone or with TCS in the presence of cell culture supernatants containing ms4-1BBL-TriXVIII used at indicated dilutions. TCS expressing m4-1BBL served as positive control for m4-1BB activation. Data of one representative experiment is shown

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  Fig. 5. Assessment of co-stimulatory capacity of supernatants derived from cells infected with measles vectors encoding soluble 4-1BBL a Schematic representation of oncolytic measles viral vector encoding human s4-1BBL-TriXVIII. b, c Control (Ctrl) reporter cells and 4-1BB reporter cells were probed with diluted supernatants derived from cells infected with control measles virus (TMV-018), or virus encoding human s4-1BBL-TriXVIII (TMV-110). TCS expressing 4-1BBL as well as urelumab (1 μg/ml) in combination with TCS ctrl served as positive control for 4-1BB activation. Activation of NFκB-eCFP was assessed via flow cytometry. Data of one representative experiment is shown. Data are presented as fold induction relative to the values obtained upon stimulation with TCS control alone (reporter cells stimulated with TCS-Ctrl in the presence of Urelumab or TMV-110 (s4-1BBLsh-TriXVIII) supernatant or TMV-018 supernatant divided by reporter cells stimulated with TCS alone (control)). N, nucleocapsid gene; P, phosphoprotein gene; C/V, non-structural proteins, M, matrix protein gene; F, fusion protein gene; H, hemagglutinin gene; L, large protein gene

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  Fig. 6. Efficacy of an oncolytic measles virus expressing s4-1BBL-TriXVIII in a CD34 + humanized mouse tumor model a Top: Experimental study design depicting the treatment of TMV-018 (MV-SCD; Control)/TMV-110 (MV-s4-1BBL-TriXVIII) as monotherapy and in combination with pembrolizumab in a CD34+ humanized mouse tumor model. b Average tumor volume form D0 to D20 is shown (tumor volume expressed in mm.3). N = 5 mice per group. Means ± SEM is shown. A Two-way ANOVA followed by a Tukey post-test was used to compare the treatment groups. Mock control versus TMV-110 (***), mock control versus TMV-110 + pembrolizumab (***), pembrolizumab versus TMV-018 + pembrolizumab (***), pembrolizumab versus TMV-110 + pembrolizumab (*), # TMV-018 versus TMV-110 (***), TMV-018 versus TMV-110 + pembrolizumab (***), TMV-018 + pembrolizumab versus TMV-110 (***), TMV-018 + pembrolizumab versus TMV-110 + pembrolizumab (***). ns: p > 0.12, * p < 0.033, ** p < 0.002, *** p < 0.001. Bottom: Percentage of survival of TMV-110 alone or in combination with pembrolizumab. Log-rank Mantel Cox test was used to compare survival curves. *TMV-110 + pembrolizumab versus mock control. #TMV-110 + pembrolizumab versus…