Alexander Rosendahl

Deletion of Exon I of SMAD7 in Mice Results in Altered B Cell Responses

The members of the TGF-β superfamily, i.e., TGF-β isoforms, activins, and bone morphogenetic proteins, regulate growth, differentiation, and apoptosis, both during embryonic development and during postnatal life. Smad7 is induced by the TGF-β superfamily members and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. In addition, Smad7 is induced by other stimuli. Thus, it can fine-tune and integrate TGF-β signaling with other signaling pathways. To investigate the functional role(s) of Smad7 in vivo, we generated mice deficient in exon I of Smad7, leading to a partial loss of Smad7 function. Mutant animals are viable, but significantly smaller on the outbred CD-1 mouse strain background. Mutant B cells showed an overactive TGF-β signaling measured as increase of phosphorylated Smad2-positive B cells compared with B cells from wild-type mice. In agreement with this expected increase in TGF-β signaling, several changes in B cell responses were observed. Mutant B cells exhibited increased Ig class switch recombination to IgA, significantly enhanced spontaneous apoptosis in B cells, and a markedly reduced proliferative response to LPS stimulation. Interestingly, LPS treatment reverted the apoptotic phenotype in the mutant cells. Taken together, the observed phenotype highlights a prominent role for Smad7 in development and in regulating the immune system’s response to TGF-β.

Immune response during tumor therapy with antibody-superantigen fusion proteins.

To engineer superantigens (SAg) to express tumor reactivity, we genetically fused the Fab‐part of the tumor‐reactive MAb C215 and the bacterial SAg staphylococcal enterotoxin A (SEA). Treatment of mice carrying established lung micrometastases of the C215‐transfected syngeneic B16 melanoma with 3‐4 daily injections of C215Fab‐SEA resulted in strong anti‐tumor effects, while only moderate effects were seen when treatment was given every 4th day (intermittent treatment). High serum levels of IL‐2, TNF‐α, IFN‐γ and strong induction of CTLs (cytotoxic T lymphocytes) were noted after priming with the fusion protein. T cells responded well to 3 daily injections of C215Fab‐SEA and then gradually entered a hyporesponsive state, characterized by a reduced ability to produce IL‐2, TNF‐α and IFN‐γ and failure to mediate cytotoxicity in vitro. Intermittent treatment was characterized by increased levels of IL‐10, concomitant with accentuated loss of IL‐2, TNF‐α and IFN‐γ production. A 10‐fold increase in SEA‐reactive TCR VB3+ CD4+ cells was observed in the spleen, while a loss of TCR VB3+ CD8+ and VB11+ CD8+ cells was noted. This is in striking contrast to injections of native SEA which induced a marked deletion of TCR VB3+ CD4+ T cells, but not of CD8+ cells. Recovery of the THI cytokine profile occurred within 1–2 weeks, while restoration of cytotoxicity required several months and correlated with recovery of TCR VB3+ CD8+ and TCR VB11+ CD8+ T cells. These results show that the temporal relationship of SAg stimulations dictates the cytokine profile. Moreover, different mechanisms appear to regulate hyporesponsiveness in CD4+ and CD8+ T cells.

Long-term survival and complete cures of B16 melanoma–carrying animals after therapy with tumor-targeted IL-2 and SEA

The bacterial superantigen (SAg) staphylococcal enterotoxin A (SEA) is a potent inducer of CTL activity and cytokine production in vivo. To engineer SAg for cancer immunotherapy, we genetically fused SEA to a Fab fragment of the C215 tumor‐reactive antibody. Strong reduction of lung metastasis was seen in mice carrying established lung metastases of the poorly immunogenic B16‐C215 melanoma after Fab–SEA therapy. However, important anti‐tumor effector functions, such as IFN‐γ secretion and CTL activity, gradually declined during therapy. In this study, we show that Fab–SEA immunotherapy is strongly potentiated by Fab–IL‐2 co‐administration. Combined Fab–IL‐2 and Fab–SEA therapy prolongs the immune response in vivo, limits the development of immunological unresponsiveness and promotes maximal anti‐tumor effects. Significantly prolonged survival was noted in tumor‐carrying animals treated with Fab–SEA/ Fab–IL‐2 as compared with Fab–SEA or Fab–IL‐2 alone. Combination therapy resulted in complete cure in 90% of tumor‐bearing animals, whereas only 10% long‐term survival was seen in Fab–SEA or Fab–IL‐2‐treated animals. Single Fab–SEA therapy induced a hyporesponsive state after 2 cycles of treatment. In contrast, the immune response after combination therapy was characterized by substantially augmented IFN‐γ and TNF‐α production and strong CTL activity. Our data demonstrate that combined Fab–SEA and Fab–IL‐2 therapy prolongs the immune response in vivo and induced long‐term survival of more than 90% of the animals carrying the highly aggressive B16 melanoma. Int. J. Cancer 81:156–163, 1999.

Repeated treatment with antibody-targeted superantigens strongly inhibits tumor growth.

Superantigens (SAg) are microbial proteins with the capacity to activate a large proportion of T cells. We have developed a novel approach for cancer immunotherapy by genetically fusing the SAg staphylococcal enterotoxin A (SEA) to a Fab‐fragment of a tumor‐specific antibody. Repeated exposure to SEA induces a state of unresponsiveness including cell deletion and functional hyporesponsiveness, i.e., anergy. In this study we have developed improved therapeutic schedules to allow repeated injections of Fab‐SEA, limit development of immunological unresponsiveness and promote maximal anti‐tumor response. Four daily injections of Fab‐SEA to mice carrying B16‐C215 lung metastases resulted in 90–95% reduction in the number of metastases. However, the animals did retain a minimal residual tumor disease. The immune system was in a hyporesponsive state after 4 daily Fab‐SEA injections, and further injections did not improve therapy. Two repeated cycles, each comprising 4 daily injections of Fab‐SEA, significantly prolonged the survival and resulted in complete cure of a fraction of the animals. A rest period of 10 days between the cycles was required to mount an efficient secondary anti‐tumor response. This secondary immune response was characterized by partial recovery of cytokine production i.e., interleukin‐2, interferon‐γ and tumor necrosis factor‐α. Strong CTL activity was detected in animals that had rested for 8 weeks between the 2 cycles. Interestingly, irrespective of the resting period, the CD4+ SEA‐reactive T cells expanded in response to all 4 additional Fab‐SEA injections both locally and in spleen. In contrast, only marginal expansion of CD8+ T cells was seen if restimulation was given within 1 month. Our data show that potent anti‐tumor effector functions can be induced after repeated stimulation cycles with a SAg‐monoclonal antibody fusion protein resulting in a CD4+ T cell‐dependent cytokine release, prolonged survival and induction of complete cures. Int. J. Cancer 76:274‐283, 1998.© 1998 Wiley‐Liss, Inc.

Increasing Selectivity of CC Chemokine Receptor 8 Antagonists by Engineering Nondesolvation Related Interactions with the Intended and Off-Target Binding Sites

The metabolic stability and selectivity of a series of CCR8 antagonists against binding to the hERG ion channel and cytochrome Cyp2D6 are studied by principal component analysis. It is demonstrated that an efficient way of increasing metabolic stability and selectivity of this series is to decrease compound lipophilicity by engineering nondesolvation related attractive interactions with CCR8, as rationalized by three-dimensional receptor models. Although such polar interactions led to increased compound selectivity, such a strategy could also jeopardize the DMPK profile of compounds. However, once increased potency is found, the lipophilicity can be readjusted by engineering hydrophobic substituents that fit to CCR8 but do not fit to hERG. Several such lipophilic fragments are identified by two-dimensional fragment-based QSAR analysis. Electrophysiological measurements and site-directed mutagenesis studies indicated that the repulsive interactions of these fragments with hERG are caused by steric hindrances with residue F656.

RhoB deficiency in thymic medullary epithelium leads to early thymic atrophy.

RhoB, a member of the Rho subfamily of small GTPases, mediates diverse cellular functions, including cytoskeletal organization, cell transformation and vesicle trafficking. The thymus undergoes progressive decline in its structure and function after puberty. We found that RhoB was expressed in thymic medullary epithelium. To investigate a role of RhoB in the regulation of thymic epithelial organization or thymocyte development, we analyzed the thymi of RhoB-deficient mice. RhoB-deficient mice were found to display earlier thymic atrophy. RhoB deficiency showed significant reductions in thymus weight and cellularity, beginning as early as 5 weeks of age. The enhanced expression of TGF-β receptor type II (TGFβRII) in thymic medullary epithelium was observed in RhoB-null mice. In addition, the expression of fibronectin, which is shown to be regulated by TGF-β signaling, was accordingly increased in the mutant thymic medulla. Since there is no age-related change of RhoB expression in the thymus, it is unlikely that RhoB in thymic epithelium directly contributes to age-related thymic involution. Nevertheless, our findings strongly support a physiological role of RhoB in regulation of thymus development and maintenance through the inhibition of TGF-β signaling in thymic medullary epithelium.

CCR8 signaling influences Toll-like receptor 4 responses in human macrophages in inflammatory diseases.

ABSTRACT CCR8 immunity is generally associated with Th2 responses in allergic diseases. In this study, we demonstrate for the first time a pronounced attenuated influx of macrophages in ovalbumin (OVA)-challenged CCR8 knockout mice. To explore whether macrophages in human inflamed lung tissue also were CCR8 positive, human lung tissue from patients with chronic obstructive pulmonary disease (COPD) was evaluated. Indeed, CCR8 expression was pronounced in invading monocytes/macrophages from lungs of patients with Global Initiative for Obstructive Lung Disease (GOLD) stage IV COPD. Given this expression pattern, the functional role of CCR8 on human macrophages was evaluated in vitro. Human peripheral blood monocytes expressed low levels of CCR8, while macrophage colony-stimulating factor (M-CSF)-derived human macrophages expressed significantly elevated surface levels of CCR8. Importantly, CCL1 directly regulated the expression of CD18 and CD49b and hence influenced the adhesion capacity of human macrophages. CCL1 drives chemotaxis in M-CSF-derived macrophages, and this could be completely inhibited by lipopolysaccharide (LPS). Whereas both CCL1 and LPS monotreatment inhibited spontaneous superoxide release in macrophages, CCL1 significantly induced superoxide release in the presence of LPS in a dose-dependent manner. Finally, CCL1 induced production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and could inhibit LPS-induced cytokine production in a dose-dependent manner. Our data demonstrate, for the first time, the presence of CCR8 on inflammatory macrophages in human COPD lung tissue. Importantly, the functional data from human macrophages suggest a potential cross talk between the CCR8 and the Toll-like receptor 4 (TLR4) pathways, both of which are present in COPD patients.

Small molecule antagonists of CCR8 inhibit eosinophil and T cell migration

In this study, we demonstrate that in addition to T lymphocytes, human naïve eosinophils and the differentiated eosinophil-like cell line, AML14.3D10 express CCR8 and respond to CCL1 through CCR8 engagement. The responsiveness of cells was dependent on maturation stage, since CCL1 induced pronounced chemotaxis only in differentiated CCR8 positive AML14.3D10 cells. Despite the low CCR8 surface expression, human naïve eosinophils respond with a chemotaxis to high concentration CCL1. We further describe that Th2 clones in a maturation dependent fashion produce autocrine CCL1, which renders them unresponsive to further stimulation. An innovative method to enrich primary CCR8 reactive T cells was developed which demonstrates that primary peripheral CCR8 expressing T cells respond significantly to CCL1. We have developed novel small molecule CCR8 antagonists that are effective in inhibiting calcium mobilization and chemotaxis in differentiated AML cells as well as in human primary CCR8 positive T cells. Importantly, we demonstrate that the compounds can be divided into two subgroups: (i) compounds that are functional agonists for calcium mobilization and chemotaxis (ii) compounds that are pure antagonists. We demonstrate that agonism of these compounds does not correlate with their antagonistic potency. Taken together, we have identified a novel set of CCR8 compounds with antagonistic properties that inhibit CCL1 driven chemotaxis in both CCR8 expressing eosinophils as well as primary human T cells.

Orally bioavailable allosteric CCR8 antagonists inhibit dendritic cell, T cell and eosinophil migration

The chemokine receptor CCR8 is associated with asthma. Herein, we describe that both mature and immature dendritic cells (DC) express CCR8, whereas only mature DC migrate towards CCL1. Moreover, transient LPS challenge significantly down-regulates CCR8 expression hence attenuating CCL1 chemotaxis. To inhibit CCR8 pathophysiology, we recently developed a novel series of small molecule CCR8 antagonists containing a diazaspiroundecane scaffold, which had micromolar potency. However, these first generation antagonists had high lipophilicity that endowed the compounds with poor physicochemical properties, and were thus not suitable for further development. By introducing polar bicyclic groups on the N-benzyl substituent and building in further polar interactions on the amide group we now show second generation diazospiroundecane antagonists with significantly improved overall properties. Potency is substantially improved from micromolar to nanomolar potency in CCR8 binding and inhibition of chemotaxis in human primary T cells, DC and in an eosinophil cell line. In addition to high potency, the most attractive antagonist, AZ084 showed excellent selectivity, high metabolic stability in vitro and an attractive in vivo PK profile with a long half-life in rat. Interestingly, in ligand saturation experiments and in wash-off experiments, CCL1 was shown to have two binding sites to CCR8 with K(d) at 1.2/68pM respectively, and on-off rates of 0.004 and 0.0035/0.02pMmin, respectively. The lead antagonist, AZ084, appears to act as an allosteric inhibitor with a K(i) at 0.9nM. Taken together, we herein report a novel oral allosteric CCR8 antagonist with predicted low once-daily dosing capable of potent inhibition of both human T cell and DC functions.