Inhibition of integrin αVß6 changes fibril thickness of stromal collagen in experimental carcinomas.

BACKGROUND: Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF-ß1 and -ß3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma. METHODS: The potential role of αVß6 integrin-mediated activation of latent TGF-ß was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal αVß6 integrin-specific monoclonal antibody 3G9 was used to inhibit the αVß6 integrin activity. RESULTS: Both KAT-4 and Capan-2 cells expressed the αVß6 integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-ß in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-ß1 and -ß3 reduced collagen fibril thickness in both tumor models. CONCLUSION: Our data demonstrate that the αVß6-directed activation of latent TGF-ß plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to αVß6 inhibition depends on the simultaneous presence of alternative paths for latent TGF-ß activation and the extent of desmoplasia.

Evaluation of Tyro3 expression, Gas6-mediated Akt phosphorylation, and the impact of anti-Tyro3 antibodies in melanoma cell lines.

Tyro3, a member of the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases, has emerged as a potential oncogene in melanoma. Here, we confirm that Tyro3 is specifically overexpressed in primary melanoma samples and show that Tyro3 is expressed at varying levels in numerous melanoma cell lines. Short hairpin RNA-mediated knockdown of Tyro3 led to significant cell death via apoptotic mechanisms in nearly all melanoma cell lines tested, regardless of the BRAF or NRAS mutation status or co-expression of Axl and/or Mer. We generated soluble and monomeric versions of the human Tyro3 extracellular domain and human Gas6 for affinity measurements and correlated these values with the level of Gas6 required to induce Tyro3 signaling in cellular assays. Calcium was critical for the correct folding of Gas6 and its binding to Tyro3. In melanoma cell lines, Gas6 induced Tyro3 phosphorylation and downstream Akt phosphorylation without apparent effects on Erk. We generated monoclonal antibodies (mAbs) against Tyro3 to examine their effect on survival signaling in melanoma cell lines. The mAbs generated against Tyro3 included nonligand blockers, partial blockers, and competitive ligand blockers. A number of weak and partial ligand blockers (all recognizing the Tyro3 Ig domains) were the most effective at blocking ligand-mediated downstream signaling of Tyro3. Overall, these data indicate that Tyro3 may confer increased survival signals in melanoma cells and can be stymied using inhibitory mAbs. These mAbs may be useful for further investigations of the role of Tyro3 in melanoma.

Mechanistic and pharmacodynamic studies of DuoBody-CD3x5T4 in preclinical tumor models.

CD3 bispecific antibodies (bsAbs) show great promise as anticancer therapeutics. Here, we show in-depth mechanistic studies of a CD3 bsAb in solid cancer, using DuoBody-CD3x5T4. Cross-linking T cells with tumor cells expressing the oncofetal antigen 5T4 was required to induce cytotoxicity. Naive and memory CD4+ and CD8+ T cells were equally effective at mediating cytotoxicity, and DuoBody-CD3x5T4 induced partial differentiation of naive T-cell subsets into memory-like cells. Tumor cell kill was associated with T-cell activation, proliferation, and production of cytokines, granzyme B, and perforin. Genetic knockout of FAS or IFNGR1 in 5T4+ tumor cells abrogated tumor cell kill. In the presence of 5T4+ tumor cells, bystander kill of 5T4- but not of 5T4-IFNGR1- tumor cells was observed. In humanized xenograft models, DuoBody-CD3x5T4 antitumor activity was associated with intratumoral and peripheral blood T-cell activation. Lastly, in dissociated patient-derived tumor samples, DuoBody-CD3x5T4 activated tumor-infiltrating lymphocytes and induced tumor-cell cytotoxicity, even when most tumor-infiltrating lymphocytes expressed PD-1. These data provide an in-depth view on the mechanism of action of a CD3 bsAb in preclinical models of solid cancer.

Discovery of CC-99677, a selective targeted covalent MAPKAPK2 (MK2) inhibitor for autoimmune disorders.

As an anti-inflammatory strategy, MAPK-activated protein kinase-2 (MK2) inhibition can potentially avoid the clinical failures seen for direct p38 inhibitors, especially tachyphylaxis. CC-99677, a selective targeted covalent MK2 inhibitor, employs a rare chloropyrimidine that bonds to the sulfur of cysteine 140 in the ATP binding site via a nucleophilic aromatic substitutions (SNAr) mechanism. This irreversible mechanism translates biochemical potency to cells shown by potent inhibition of heat shock protein 27 (HSP27) phosphorylation in LPS-activated monocytic THP-1 cells. The cytokine inhibitory profile of CC-99677 differentiates it from known p38 inhibitors, potentially suppressing a p38 pathway inflammatory response while avoiding tachyphylaxis. Dosed orally, CC-99677 is efficacious in a rat model of ankylosing spondylitis. Single doses, 3 to 400 mg, in healthy human volunteers show linear pharmacokinetics and apparent sustained tumor necrosis factor-α inhibition, with a favorable safety profile. These results support further development of CC-99677 for autoimmune diseases like ankylosing spondylitis.

Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential.

Natural killer cells constitute 50-90% of lymphocytes in human uterine decidua in early pregnancy. Here, CD56(bright) uterine decidual NK (dNK) cells were compared with the CD56(bright) and CD56(dim) peripheral NK cell subsets by microarray analysis, with verification of results by flow cytometry and RT-PCR. Among the approximately 10,000 genes studied, 278 genes showed at least a threefold change with P < or = 0.001 when comparing the dNK and peripheral NK cell subsets, most displaying increased expression in dNK cells. The largest number of these encoded surface proteins, including the unusual lectinlike receptors NKG2E and Ly-49L, several killer cell Ig-like receptors, the integrin subunits alpha(D), alpha(X), beta1, and beta5, and multiple tetraspanins (CD9, CD151, CD53, CD63, and TSPAN-5). Additionally, two secreted proteins, galectin-1 and progestagen-associated protein 14, known to have immunomodulatory functions, were selectively expressed in dNK cells.

Enapotamab vedotin, an AXL-specific antibody-drug conjugate, shows preclinical antitumor activity in non-small cell lung cancer.

Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of levels of AXL, a member of the TAM receptor tyrosine kinase family, in NSCLC and demonstrate potent antitumor activity of the AXL-targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the patient-derived xenograft (PDX) models and was associated with AXL mRNA expression levels. Significant single-agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin-mediated cytotoxicity. Enapotamab vedotin also showed antitumor activity in vivo in 3 EGFR-mutant, EGFR inhibitor-resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.

Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors.

Intratumor heterogeneity is a key factor contributing to therapeutic failure and, hence, cancer lethality. Heterogeneous tumors show partial therapy responses, allowing for the emergence of drug-resistant clones that often express high levels of the receptor tyrosine kinase AXL. In melanoma, AXL-high cells are resistant to MAPK pathway inhibitors, whereas AXL-low cells are sensitive to these inhibitors, rationalizing a differential therapeutic approach. We developed an antibody-drug conjugate, AXL-107-MMAE, comprising a human AXL antibody linked to the microtubule-disrupting agent monomethyl auristatin E. We found that AXL-107-MMAE, as a single agent, displayed potent in vivo anti-tumor activity in patient-derived xenografts, including melanoma, lung, pancreas and cervical cancer. By eliminating distinct populations in heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumor growth. Furthermore, by inducing AXL transcription, BRAF/MEK inhibitors potentiated the efficacy of AXL-107-MMAE. These findings provide proof of concept for the premise that rationalized combinatorial targeting of distinct populations in heterogeneous tumors may improve therapeutic effect, and merit clinical validation of AXL-107-MMAE in both treatment-naive and drug-resistant cancers in mono- or combination therapy.

Inflammatory cytokine and thrombin regulation of interleukin-8 and intercellular adhesion molecule-1 expression in first trimester human decidua.

CONTEXT: Decidual neutrophil infiltration complicates spontaneous abortions associated with inflammation and hemorrhage. Transendothelial neutrophil migration into inflamed tissues involves IL-8-mediated chemoattraction, then neutrophil attachment to endothelial cell-expressed intercellular adhesion molecule-1 (ICAM-1). OBJECTIVE: The aim of this study was to assess IL-8 and ICAM-1 regulation in decidual inflammation and hemorrhage; the effects of the proinflammatory cytokines, TNFalpha, IL-1beta and the hemostatic, proinflammatory cytokine thrombin were measured on IL-8 expression in first trimester decidual cells (DCs), and ICAM-1 immunostaining was compared in normal, inflamed, and hemorrhagic first trimester decidua. DESIGN: Immunohistochemistry of human decidua and in vitro treatment of human decidual cells were performed. SETTING: The study was conducted at the Academic Medical Center. INTERVENTION: . DCs were passaged until they were leukocyte-free (CD45 free by FACS), then were incubated with estradiol or medroxyprogesterone acetate alone or with TNFalpha or IL-1beta or thrombin. Normal, inflamed, and hemorrhagic decidua were immunostained for ICAM-1 and the neutrophil marker CD14. MAIN OUTCOME MEASURE: ICAM-1 immunostaining was performed in decidua. IL-8 levels in DC-conditioned media were assessed by ELISA and immunoblotting; IL-8 mRNA levels were measured by quantitative RT-PCR. RESULTS: Endothelial cell ICAM-1 immunostaining was similar in normal and inflamed or hemorrhagic decidua. In cultured DCs, optimal concentrations of IL-1beta, TNFalpha, and thrombin elevated secreted IL-8 levels by 1083 +/- 261-, 370 +/- 77-, and 45 +/- 15-fold, respectively (mean +/- SEM; P < 0.05; n = 8). The effects were dose dependent and were unaffected by medroxyprogesterone acetate. Western blotting confirmed the ELISA results, and corresponding effects on IL-8 mRNA levels were observed. CONCLUSIONS: Cytokine/thrombin-enhanced DC IL-8 expression interacts with constitutively expressed ICAM-1 in decidual endothelium to modulate neutrophil trafficking into hemorrhagic and inflamed first trimester decidua.

TGFbeta promotes conversion of CD16+ peripheral blood NK cells into CD16- NK cells with similarities to decidual NK cells.

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56(superbright)CD16(-)CD9(+)KIR(+) distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16(+)CD9(-) peripheral blood NK cells in medium containing TGFbeta1 resulted in a transition to CD16(-)CD9(+) NK cells resembling decidual NK cells. Decidual stromal cells, when isolated and cultured in vitro, were found to produce TGFbeta1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFbeta1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFbeta. In addition, Lin(-)CD34(+)CD45(+) hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor.

Regulation of monocyte chemoattractant protein-1 expression by tumor necrosis factor-alpha and interleukin-1beta in first trimester human decidual cells: implications for preeclampsia.

The current study describes a statistically significant increase in macrophages (CD68-positive cells) in the decidua of preeclamptic patients. To elucidate the regulation of this monocyte infiltration, expression of monocyte chemoattractant protein-1 (MCP-1) was assessed in leukocyte-free first trimester decidual cells. Confluent decidual cells were primed for 7 days in either estradiol or estradiol plus medroxyprogesterone acetate to mimic the decidualizing steroidal milieu of the luteal phase and early pregnancy. The medium was exchanged for a serum-free defined medium containing corresponding steroids +/- tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta. After 24 hours, enzyme-linked immunosorbent assay measurements indicated that the addition of medroxyprogesterone acetate did not affect MCP-1 output, whereas 10 ng/ml of TNF-alpha or IL-1beta increased output by 83.5-fold +/- 20.6 and 103.1-fold +/- 14.7, respectively (mean +/- SEM, n = 8, P < 0.05). Concentration-response comparisons revealed that even 0.01 ng/ml of TNF-alpha or IL-1beta elevated MCP-1 output by more than 15-fold. Western blotting confirmed the enzyme-linked immunosorbent assay results, and quantitative reverse transcriptase-polymerase chain reaction confirmed corresponding effects on MCP-1 mRNA levels. The current study demonstrates that TNF-alpha and IL-1beta enhance MCP-1 in first trimester decidua. This finding suggests a mechanism by which recruitment of excess macrophages to the decidua impairs endovascular trophoblast invasion, the primary placental defect of preeclampsia.

Viral evasion of natural killer cells.

Viruses have evolved mechanisms to avoid the host immune system, including means of escaping detection by both the innate and adaptive immune responses. Natural killer (NK) cells are a central component of the innate immune system and are crucial in defense against certain viruses. To attain a state of chronic infection, some successful viruses have developed specific mechanisms to evade detection by and activation of NK cells. These NK cell-specific evasion mechanisms fall into distinct mechanistic categories used in numerous virus families.

CD1d and invariant NKT cells at the human maternal-fetal interface.

Invariant CD1d-restricted natural killer T (iNKT) cells comprise a small, but significant, immunoregulatory T cell subset. Here, the presence of these cells and their CD1d ligand at the human maternal-fetal interface was investigated. Immunohistochemical staining of human decidua revealed the expression of CD1d on both villous and extravillous trophoblasts, the fetal cells that invade the maternal decidua. Decidual iNKT cells comprised 0.48% of the decidual CD3+ T cell population, a frequency 10 times greater than that seen in peripheral blood. Interestingly, decidual CD4+ iNKT cells exhibited a striking Th1-like bias (IFN-gamma production), whereas peripheral blood CD4+ iNKT clones exhibited a Th2-like bias (IL-4 production). Moreover, compared to their peripheral blood counterparts, decidual iNKT clones were strongly polarized toward granulocyte/macrophage colony-stimulating factor production. The demonstration of CD1d expression on fetal trophoblasts together with the differential pattern of cytokine expression by decidual iNKT cells suggests that maternal iNKT cell interactions with CD1d expressed on invading fetal cells may play an immunoregulatory role at the maternal-fetal interface.

Disulfide bond-mediated dimerization of HLA-G on the cell surface.

HLA-G is a nonclassical class I MHC molecule with an unknown function and with unusual characteristics that distinguish it from other class I MHC molecules. Here, we demonstrate that HLA-G forms disulfide-linked dimers that are present on the cell surface. Immunoprecipitation of HLA-G from surface biotinylated transfectants using the anti-beta2-microglobulin mAb BBM.1 revealed the presence of an approximately equal 78-kDa form of HLA-G heavy chain that was reduced by using DTT to a 39-kDa form. Mutation of Cys-42 to a serine completely abrogated dimerization of HLA-G, suggesting that the disulfide linkage formed exclusively through this residue. A possible interaction between the HLA-G monomer or dimer and the KIR2DL4 receptor was also investigated, but no interaction between these molecules could be detected through several approaches. The cell-surface expression of dimerized HLA-G molecules may have implications for HLA-Greceptor interactions and for the search for specific receptors that bind HLA-G.