31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

GDF-5 can act as a context-dependent BMP-2 antagonist

Background Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-β family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5—as evident from its alternative name, cartilage derived morphogenetic protein 1—plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a “BMP-2 mimic” with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor. Results Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites. Conclusions Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5′s signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs

Identification of a Novel TGF-beta-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β\beta-Receptor-3 (TGFR-3)

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor beta (TGF-$\beta$) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-beta-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 angstrom crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3

Identification of a Novel TGF-β-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β-Receptor-3 (TGFR-3)

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3

Data and refinement statistics of mouse TGFR-3-ZP-C.

<p>+ Numbers in parenthesis are for the highest-resolution shell. # 5% of reflections have been chosen as <i>R</i>_free set. <i>R</i>_sym is calculated as where <i>I_i</i> is the <i>i^th</i> observation of the n^th reflection and <<i>I</i>> the mean of all observations of the n^th reflection. ## Calculated with program COOT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067214#pone.0067214-Emsley1" target="_blank">[35]</a>.</p

Crystal structure of murine TGFR-3-ZP-C.

<p>(<b>A</b>) Stereographic ribbon representation of the murine TGFR-3-ZP-C structure (in blue and red). The ZP-C domain in the crystals extends from Thr591 to Asp757. No density is visible for residues 730 to 744. The FG loop connecting β-strands F to G (residues 711 to 746) and β-strand G are shown in red. The EHP segment that is part of β-strand G is marked. For comparison reason the structure of rat ZP-C (PDB entry 3QW9, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067214#pone.0067214-Lin1" target="_blank">[13]</a>) that was used to solve the structure of murine ZP-C is shown in transparent grey and light red. In contrast to the murine ZP-C structure, the entire FG loop is visible in the rat ZP-C structure. Chain breaks in the murine ZP-C structure are marked with black dots. (<b>B</b>) Stereographic representation of the rat and murine ZP-C structure identical to that in panel (A) but after application of an approximately 110° rotation around a vertical axis.</p

Binding affinities determined by SPR from the interaction of TGFR-3 and variants thereof with immobilized TGF-β ligands TGF-β2 and BMP-2.

*<p>NB no detectable binding.</p

Analysis of the TGF-β2-binding activity of TGFR-3-ZP variants and peptides.

<p>(<b>A</b>) Analytical size-exclusion chromatography of TGFR-3-ZP in the presence (blue) and absence of TGF-β2 (deep blue). The migration behaviour of TGFR-3-ZP is also compared to that of the truncated protein TGFR-3-ZP(ΔC) either with (green) or without TGF-β2 (dark green). In absence of TGF-β2, TGFR-3-ZP and TGFR-3-ZP(ΔC) show similar retention times. The absorbance spectra of the samples are shown together with the elution profile of molecular weight standards (grey dotted curve, major peaks from left to right: ferritin (440 kDa), aldolase (158 kDa), BSA (67 kDa), ovalbumin (43 kDa) and myoglobin (17 kDa)). <i>K_av</i>-values derived from peak maxima are listed in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067214#pone.0067214.s005" target="_blank">Fig. S5</a>. (<b>B</b><i>) In vitro</i> interaction analysis of TGFR-3-ZP binding with TGF-β2 immobilized on a biosensor surface using SPR. The analyte TGFR-3-ZP was injected at time point 300 s, six different analyte (TGFR-3ZP) concentrations (indicated by different colors) were used. At time point 600 s buffer was perfused to record dissociation of TGFR-3-ZP from the TGF-β2 biosensor surface. (<b>C</b>) Same as (B) but using TGFR-3-ZP(ΔC) as analyte. No TGF-β2 binding activity is observed for the truncated mutant TGFR-3-ZP(ΔC). (<b>D</b>) to (<b>H</b>) Sensorgrams obtained with peptides mimicking the C-terminus of human and mouse TGFR-3-ZP immobilized onto a neutravidin-coated biosensor via an N- or C-terminal chemically attached biotin moiety and using TGF-β2 as analyte. (<b>D</b>) Interaction TGF-β2 with immobilized P1-mZP comprising residues 735–782. Six different analyte concentrations (indicated by color and numbers) were used for analysis. (<b>E</b>) Same as in (D) but for the peptide P2-hZP comprising residues 737–788 of human TGFR-3. (<b>F</b>) Same as in (D) but for peptide P3-mZP (residues 749–782). P3-mZP does not show any TGF-β2 binding. (<b>G</b>) Sensorgram for the interaction of TGF-β2 (as analyte, five different concentrations were used) and P4-mZP comprising residues 732–748. (<b>H</b>) Same as in (G) but for the peptide P5-hZP comprising residues 734–750. The fit of the SPR data is indicated by a solid line.</p

Secondary structure and oligomerization of TGFR-3-ZP proteins.

<p>(<b>A</b>) The CD spectra of all protein variants resemble spectra of properly folded proteins with predominantly antiparallel β-sheet structures. TGFR-3-ZP black, TGFR-3-ZP(ΔC) grey, TGFR-3-ZP(234) blue and TGFR-3-ZP(234,ΔC) green. (<b>B</b>) Monomodal size-distribution histogram of TGFR-3-ZP (2.0 mg⋅ml⁻¹, at 20°C) monitored by dynamic light scattering. The intensity (in %) is plotted versus discrete particle sizes (<i>R</i>). In the histogram one peak with a hydrodynamic radius of 4.0 nm and a polydispersity of 20.9% is obtained. The apparent molecular weight of 88 kDa indicates a dimeric assembly of TGFR-3-ZP in solution. (<b>C</b>) Analytical size-exclusion chromatography of TGFR-3-ZP (black line) and TGFR-3-ZP(234) (grey dotted line) on a Superdex 200 10/300 column. The apparent molecular weight of the elution peak was calculated to be 75 kDa for TGFR-3-ZP and 70 kDa for TGFR-3-ZP(234), suggesting a dimeric assembly of TGFR-3-ZP.</p