Randomized, phase II, placebo-controlled trial of onartuzumab and/or bevacizumab in combination with weekly paclitaxel in patients with metastatic triple-negative breast cancer.

BACKGROUND: Increased hepatocyte growth factor/MET signaling is associated with an aggressive phenotype and poor prognosis in triple-negative breast cancer (TNBC). We evaluated the benefit of adding onartuzumab, a monoclonal anti-MET antibody, to paclitaxel with/without bevacizumab in patients with TNBC. PATIENTS AND METHODS: Women with metastatic TNBC were randomized to receive onartuzumab plus placebo plus weekly paclitaxel (OP; n = 60) or onartuzumab plus bevacizumab plus paclitaxel (OBP; n = 63) or placebo plus bevacizumab plus paclitaxel (BP; n = 62). The primary end point was progression-free survival (PFS); additional end points included overall survival (OS), objective response rate (ORR), and safety. This trial was hypothesis generating and did not have power to detect minimum clinically meaningful differences between treatment arms. RESULTS: There was no improvement in PFS with the addition of onartuzumab to BP [hazard ratio (HR), 1.08; 95% confidence interval (CI) 0.69-1.70]; the risk of a PFS event was higher with OP than with BP (HR, 1.74; 95% CI 1.13-2.68). Most patients had MET-negative tumors (88%); PAM50 subtype analysis showed basal-like tumors in 68% of samples. ORR was higher in the bevacizumab arms (OBP: 42.2%; 95% CI 28.6-57.1; BP: 54.7%; 95% CI 41.0-68.4) compared with OP (27.5%; 95% CI 15.9-40.6). Median OS was shorter with OBP (HR, 1.36; 95% CI 0.75-2.46) and OP (HR, 1.92; 95% CI 1.03-3.59), than with BP. Peripheral edema was more frequent in the onartuzumab arms (OBP, 51.8%; OP, 58.6%) versus BP (17.7%). CONCLUSION: This study did not show a clinical benefit of the addition of onartuzumab to paclitaxel with/without bevacizumab in patients with predominantly MET-negative TNBC. CLINICALTRIALSGOV: NCT01186991.

The role of autoantigens in autoimmune disease.

Many autoantigens have been identified in human patients and in rodent models. In numerous experimental settings, these autoantigens or related autoreactive lymphocytes can transfer autoimmunity. Although autoreactivity spreads to new epitopes during the course of disease, single-epitope-specific therapies show considerable efficacy in multi-epitope-induced models of autoimmunity. These observations may indicate that epitope-specific therapies operate at the level of regulating mechanisms of immune tolerance rather than exerting a direct effect on autoreactive T lymphocytes.

A phase I trial of solubilized DR2:MBP84-102 (AG284) in multiple sclerosis.

OBJECTIVE: To assess the safety, tolerability, and biologic and clinical activity of a solubilized complex comprised of human leukocyte antigen-DR2 with myelin basic protein84-102 (AG284)in patients with secondary progressive MS. BACKGROUND: Soluble species-specific major histocompatibility complex myelin basic protein91-103 complexes ameliorate disease in a dose-dependent manner when administered to SJL/J mice with chronic relapsing experimental allergic encephalomyelitis. Preincubation with AG284 reduces the proliferative response of a DR2-restricted, myelin basic protein84-102-reactive T cell clone, derived from a MS patient, to myelin basic protein84-102 in the presence of autologous antigen-presenting cells. METHODS: Thirty-three patients with secondary progressive MS were randomly assigned to receive three alternate day IV doses of AG284 or placebo in a double-masked dose escalation study. The primary outcome was safety and tolerability. Secondary outcomes included a comparison of pre- and post-treatment gadolinium-enhanced brain MRI activity, Kurtzke Expanded Disability Status Scale, and Nine Hole Peg Test scores. RESULTS: The frequency of adverse events was similar in the AG284 and placebo recipients. No significant treatment effect was detected by Expanded Disability Status Scale, Nine Hole Peg Test, or number of new gadolinium-enhancing MRI lesions. CONCLUSIONS: AG284 as administered during this study was safe and well tolerated. Further studies are warranted to determine the biologic activity and clinical efficacy of this potential treatment for MS.

Chemokines in autoimmune diseases.

Autoimmune diseases like multiple sclerosis (MS) and insulin-dependent diabetes (IDD) are believed to be mediated by pathogenic CD4+ autoreactive T cells which mediate selective destruction of specific host cells. Interrupting the trafficking of such T cells from host circulation to the sites of pathology, such as the central nervous system in the case of MS and the pancreas in the case of IDD, potentially offers a novel opportunity for therapeutic intervention in these diseases. The following summarizes our evolving thoughts on the role of the chemokine network in MS and IDD, and focuses on the chemokine receptor CXCR3 as a potential target for impeding T-cell-mediated destruction in these disease settings.

The cytokine stew and innate resistance to L. monocytogenes.

The Listeria monocytogenes (L. monocytogenes) infection model has been a useful system to evaluate the cellular interactions leading to host immunity. The initiation of the innate immune response in naive animals and subsequent progression to acquired immunity represent an integrated system with numerous layers of complexity. Coincident with experimental infection is the induction of cytokines. Cytokines, which are soluble mediators of cell growth, maintenance and function, from a network of pleiotropic stimuli that serve as one of the main driving forces for the progressive development of cellular responses. A variety of in vivo approaches, such as injection of the recombinant cytokines themselves or antibodies to neutralize their activity, have been used to define these stimuli. Perhaps one of the most useful tools is that of germline-manipulated animals. One of the many cytokines implicated in resistance to L. monocytogenes infection is interleukin (IL)-6, a molecule associated with diverse infectious and pathophysiological disease states. This review concentrates on various cytokines (IL-1, TNF alpha, IFN-gamma, IL-12, IL-10 and the colony-stimulating factors (CSF)) thought to play a role during the innate host response to L. monocytogenes infection, with a special emphasis on studies using IL-6-deficient mice. Additionally, we show unpublished data obtained when the concepts learned from L. monocytogenes infection in IL-6-deficient mice were applied to other infection models.

Effect of the colony-stimulating factor-1 null mutation, osteopetrotic (csfm(op)), on the distribution of macrophages in the male mouse reproductive tract.

Macrophages are found throughout the male reproductive tract and its accessory glands. Mice homozygous for a null mutation (csfm(op)) in the gene for the mononuclear phagocytic growth factor colony-stimulating factor-1 (CSF-1) have a significantly lower density of macrophages, defined by the mononuclear phagocytic antigen F4/80, in the testis, cauda and caput epididymis, prostate, seminal vesicles, and vas deferens. These data indicate that CSF-1 is the major growth factor regulating the occurrence of macrophages in male reproductive tissues. The residual macrophages were correctly located in the tissue except in the caput epididymis, where they failed to take up positions adjacent to the tubular epithelium. Restoration of circulating CSF-1 concentrations in csfm(op)/csfm(op) males totally restored F4/80+ cell density in the testis and caput and cauda epididymis and partially restored their density in the vas deferens and seminal vesicles but failed to affect density in the prostate. This failure to correct all populations with circulating CSF-1 suggests the requirement for local synthesis of CSF-1 at appropriate developmental stages and/or its expression in a cell surface-associated form. The absence of macrophages in the testis and epididymis of csfm(op)/csfm(op) mice correlates with dysfunction in these tissues, suggesting that macrophages play important nonimmunological roles in these tissues.

The mechanism of in vitro T helper cell type 1 to T helper cell type 2 switching in highly polarized Leishmania major-specific T cell populations.

We have previously demonstrated that highly polarized CD4+ Th1 cells isolated from Leishmania major-infected mice could be switched to a Th2-like phenotype when cultured for 1 wk in the presence of APC, L. major Ag, and IL-4, suggesting that the reversion of a differentiated Th response could occur at the population level. To investigate the cellular basis for this population switch, CD4+ lymph node cells from Th1-polarized L. major-infected mice were separated into two subsets based on the level of expression of L-selectin (Mel-14), and each subset was stimulated with APC and IL-2 for 1 wk in the presence or the absence of IL-4. Mel-14low T cells contained all of the initial Th1 activity and retained their Th1 phenotype when cultured with IL-4. In contrast, Mel-14high T cells did not produce cytokines upon challenge with L. major Ag, but gave rise to a Th2-like population after culture with IL-4. Thus, the newly induced Th2 population was derived from undifferentiated cells distinct from the Th1 cells present in the starting population. This undifferentiated Th precursors could be induced to develop into either Th1 or Th2 cells and were not recent thymic emigrants as they were present in mice thymectomized before infection. These experiments show that a chronically stimulated and highly polarized Th1 population consisted of both precursor T cells able to differentiate into Th2 cells and cells fully differentiated into Th1 cells that could not be induced to switch their pattern of cytokine production.

Mouse gamma delta TCR+NK1.1+ thymocytes specifically produce interleukin-4, are major histocompatibility complex class I independent, and are developmentally related to alpha beta TCR+NK1.1+ thymocytes.

Mouse T cells co-expressing an alpha beta T cell receptor (TCR) and the NK1.1 antigen have been shown to be major interleukin (IL)-4-producing cells and could therefore regulate cell-mediated immune responses. We have identified a related sub-set of thymocytes co-expressing a gamma delta TCR and NK1.1 which also produce IL-4. Unlike alpha beta +NK1.1+ thymocytes, the selection of gamma delta +NK1.1+ thymocytes is not dependent upon beta 2-microglobulin (beta 2m)-associated class I molecule expression because these cells are present in beta 2m-deficient mice. This suggests that gamma delta +NK1.1+ T cells may regulate immune responses to a different variety of antigens. However, the development of alpha beta +NK1.1+ and gamma delta +NK1.1+ thymocytes appears to be related. Analysis of different mutant mice lacking alpha beta +NK1.1+ thymocytes revealed a specific increase in gamma delta +NK1.1+ thymocyte production when the block in alpha beta +NK1.1+ thymocyte differentiation occurs after beta TCR rearrangement.

Induction of a Th2 population from a polarized Leishmania-specific Th1 population by in vitro culture with IL-4.

The infection of mice with Leishmania major parasite induces polarized Th1 and Th2 responses that cannot be significantly changed in vivo after 2 to 3 wk of infection by using either cytokines or anti-cytokine Abs. It is not clear, however, whether the T cell populations are irreversibly differentiated or whether the inability to modify the cytokine production reflects inefficiencies in the experimental treatments or complications of the infection itself. To study this further, we have cultured CD4+ T cells from L. major-infected mice with specific Ag, APC, and IL-2, in the presence or absence of different cytokines and/or anti-cytokine Abs. Th1 cells cultured for 1 wk in the presence of IL-4 produced very low levels of IFN-gamma but, instead, produced high levels of IL-4 and IL-10, suggesting that IL-4 was able to cause the conversion of a Th1 into a Th2 population. The Th2-like population generated in vitro was stable and retained its phenotype in vivo when transferred into L. major-infected C.B-17 scid mice. In contrast, the presence of IFN-gamma and IL-12 during the Th2 cell stimulation enhanced IFN-gamma production but was not sufficient to induce a complete conversion of a Th2 into a Th1-like population. Taken together, these data show that highly polarized murine Th populations can be modified and even converted to the opposite cytokine phenotype in vitro, suggesting possible therapeutic applications for cytokines.

Internalization of Candida albicans and cytoskeletal organization in macrophages and fibroblasts treated with concanavalin A.

This paper investigates the ability of macrophages and of non-typically phagocitic cells such as fibroblasts to internalize 51Cr-labelled C. albicans in presence or in absence of lectin concanavalin A (Con A). The results obtained demonstrate that fibroblasts are also able to internalize C. albicans and that this property is potentiated by the presence of Con A. Lectin modifies only the phenotype of the fibroblast, which, poorly attached to the substrate, is globular in shape. Despite reduced cellular spreading, phagocytosis is stimulated by the lectin. In both cell populations, changes in the organization of some cytoskeletal proteins such as tubulin, actin and alpha-actinin are evident during the C. albicans infection; such rearrangements are more evident and longlasting in the fibroblasts treated with Con A.

Reversal of polarized T helper 1 and T helper 2 cell populations in murine leishmaniasis.

T helper 1 (Th1) and Th2 cells are the major subsets of fully differentiated CD4+ T cells in the mouse. The spectrum of cytokines characteristic of each subset determines the distinctive regulatory and effector functions mediated by each subset. We have used the murine model of Leishmania major infection to study the question of whether highly polarized populations of normal T cells are as stable in their cytokine phenotype as Th clones or whether the phenotype can be altered with regulatory cytokines. Interleukin 4 (IL-4) appears to be a key cytokine for Th2 responses as it is necessary for both the initial differentiation of Th responses to L. major and the stability of ongoing responses. Furthermore, IL-4 is capable of converting highly polarized Th1 responses to Th2 responses either in vitro or when adoptively transferred to severe combined immunodeficiency mice.

Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse.

Colony stimulating factor-1 (CSF-1) regulates the survival, proliferation and differentiation of mononuclear phagocytes. The osteopetrotic (op/op) mutant mouse is devoid of CSF-1 due to an inactivating mutation in the CSF-1 gene and is deficient in several mononuclear phagocyte subpopulations. To analyze more fully the requirement for CSF-1 in the establishment and maintenance of mononuclear phagocytes, the postnatal development of cells bearing the macrophage marker antigens F4/80 and MOMA-1, in op/op mice and their normal (+/op or +/+) littermates, were studied during the first three months of life. In normal mice, maximum expression of tissue F4/80+ cells was generally correlated with the period of maximum organogenesis and/or cell turnover. Depending on the tissue, the F4/80+ cell density either decreased, transiently increased or gradually increased with age. In op/op mice, tissues that normally contain F4/80+ cells could be classified into those in which F4/80+ cells were absent and those in which the F4/80+ cell densities were either reduced, normal or initially normal then subsequently reduced. To assess which F4/80+ populations were regulated by circulating CSF-1 in normal mice, op/op mice in which the circulating CSF-1 concentration was restored to above normal levels by daily subcutaneous injection of human recombinant CSF-1 from day 3 were analyzed. These studies suggest that circulating CSF-1 exclusively regulates both the F4/80+ cells in the liver, spleen and kidney and the MOMA-1+ metallophilic macrophages in the spleen. Macrophages of the dermis, bladder, bone marrow and salivary gland, together with a subpopulation in the gut, were partially restored by circulating CSF-1, whereas macrophages of the muscle, tendon, periosteum, synovial membrane, adrenals and the macrophages intimately associated with the epithelia of the digestive tract, were not corrected by restoration of circulating CSF-1, suggesting that they are exclusively locally regulated by this growth factor. Langerhans cells, bone marrow monocytes and macrophages of the thymus and lymph nodes were not significantly affected by circulating CSF-1 nor decreased in op/op mice, consistent with their regulation by other growth factors. These results indicate that important differences exist among mononuclear phagocytes in their dependency on CSF-1 and the way in which CSF-1 is presented to them. They also suggest that the prevalent role of CSF-1 is to influence organogenesis and tissue turnover by stimulating the production of tissue macrophages with local trophic and/or scavenger (physiological) functions. Macrophages involved in inflammatory and immune (pathological) responses appear to be dependent on other factors for their ontogenesis and function.(ABSTRACT TRUNCATED AT 400 WORDS)

Neutralizing antibody to interleukin 4 induces systemic protection and T helper type 1-associated immunity in murine candidiasis.

An interleukin 4 (IL-4)-specific monoclonal antibody (mAb) was administered to mice infected systemically with the yeast Candida albicans, and the animals were monitored for mortality, development of delayed-type hypersensitivity, production of antibodies of different isotypes, release of IL-2, IL-4, IL-6, and interferon gamma (IFN-gamma) in vitro by splenic CD4+ lymphocytes, and levels of IL-4 and IFN-gamma mRNA in these cells. Neutralization of IL-4 by three weekly injections of mAb in several independent experiments resulted in an overall cure rate of 81% versus 0% of controls. Cure was associated with efficient clearance of the yeast from infected organs and histologic evidence of disease resolution, detection of strong T helper type 1 (Th1) responses, and establishment of long-lasting protective immunity. Soon after infection, and as a result of the first or second injection of mAb, there was a decrease in IL-4 mRNA in CD4+ cells, which was accompanied by an increase in the levels of IFN-gamma-specific transcripts. Our data thus indicate that the production of IL-4 by Th2 cells may limit Th1-associated protective immunity in murine candidiasis.

Antigen-specific cytolysis of infected cells in murine candidiasis.

Immune L3T4+ and Lyt-2+ lymphocytes play an important role in the acquired resistance of mice to challenge with virulent Candida albicans, and release macrophage-activating cytokines in response to yeast cells in vitro. To determine whether antigen (Ag)-specific cytotoxic T lymphocytes are generated during fungal infection, purified L3T4+ and Lyt-2+ lymphocytes from immunized mice were cultured in the presence of syngeneic accessory cells, Candida Ag, and IL-2. Yeast-infected bone marrow macrophages and peritoneal exudate neutrophils were used as target cells in a standard 51Cr release assay. Ag-specific, MHC-unrestricted lysis of infected macrophages was evident with immune Lyt-2+ cells after 5-10 days in culture. Under the same experimental conditions, the cytotoxic activity of L3T4+ cells was negligible, but its expression could be induced by the addition of anti-CD3 antibody. Culturing immune Lyt-2+ cells for shorter periods of time (1-2 days) resulted in preferential lysis of infected neutrophils. In addition, at limiting effector cell numbers, Ag-specific MHC-restricted lymphocytes with cytotoxic activity to infected macrophages could be identified. We suggest that C. albicans infection stimulates multiple cytotoxic T-cell precursors with varying recognition stringency, which may have an important role in antifungal resistance in vivo.

Th1 and Th2 cytokine secretion patterns in murine candidiasis: association of Th1 responses with acquired resistance.

Two chemically mutagenized agerminative variants of Candida albicans were used to immunize mice against challenge with highly virulent cells of the parent strain. Although both mutants (Vir- 3 and Vir- 13) resulted in nonlethal infection and could be recovered from mouse organs for many days after the intravenous inoculation of 10(7) to 10(6) cells, significant protection to systemic challenge with virulent C. albicans was induced by only one (Vir- 3) of the two variants. Anticandidal resistance in Vir- 3-infected mice was associated with the occurrence in vivo of strong delayed-type hypersensitivity to Candida antigen, detection in vitro of highly fungicidal effector macrophages, and presence in the serum of a large proportion of Candida-reactive antibodies of the immunoglobulin G2a isotype. Bulk cultures of purified CD4+ lymphocytes from mice infected with either mutant were compared for their ability to produce gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-6 in vitro. After stimulation with specific antigen, CD4+ cells from Vir- 3-immunized mice released large amounts of the Th1-specific cytokines, IFN-gamma and IL-2, at a time when CD4+ cells from Vir- 13-infected mice predominantly secreted the characteristic Th2 cytokines, IL-4 and IL-6. These results were confirmed by quantitative analysis of cytokine-producing Th1 and Th2 cells. In addition, only mice infected with Vir- 3 displayed a high frequency of CD8+ cells with the potential for in vitro lysis of yeast-primed bone marrow macrophages. Purified CD4+ cells from Vir- 3-infected mice, but not a mixture of these cells with CD4+ lymphocytes from mice infected with Vir- 13, could adoptively transfer delayed-type hypersensitivity reactivity onto naive mice. Taken together, these data suggest that both Th1 and Th2 CD4+ lymphocytes may be activated during experimental C. albicans infection in mice.

Candida albicans-specific Ly-2+ lymphocytes with cytolytic activity.

To determine whether antigen (Ag)-specific cytotoxic T lymphocytes are generated during experimental Candida albicans infection, purified L3T4+ and Ly-2+ lymphocytes from immunized mice were cultured in the presence of syngeneic accessory cells, C. albicans Ag, and interleukin 2. Yeast-infected bone marrow macrophages were used as target cells in a standard 51Cr-release assay. Freshly isolated L3T4+ and Ly-2+ lymphocytes failed to lyse either target cell type. However, Ag-specific, major histocompatibility complex (MHC)-unrestricted lysis of infected macrophages was evident with immune Ly-2+ cells after 7-10 days in culture. The cultured cells were greater than 98% Thy-1+, CD3+, L3T4-, Ly-2+, T cell receptor alpha/beta + T cells, and their lytic activity was potentiated by the addition of anti-CD3 monoclonal antibodies. At limiting effector cell numbers, Ag-specific MHC-restricted lymphocytes with cytotoxic activity against infected macrophages could be identified. We suggest that C. albicans infection stimulates multiple cytotoxic cell precursors with varying recognition stringency, which include MHC class I-restricted, Ag-specific cytotoxic T lymphocytes.

Macrophage colony-stimulating factor in murine candidiasis: serum and tissue levels during infection and protective effect of exogenous administration.

Serum and tissue concentrations of the macrophage-specific colony-stimulating factor (CSF-1) and the number of CSF-1-responsive cells in bone marrow were investigated in mice chronically infected with a low-virulence strain of the opportunistic zoopathogenic yeast Candida albicans. CSF-1 levels in serum, brain, kidney, liver, and lung were significantly increased shortly after infection and remained elevated during the 2 weeks preceding the onset of specific T cell-dependent immunity. The number of monocytic precursor cells was also increased in the bone marrow of infected mice. When macrophages from naive donors were exposed in vitro to purified murine CSF-1, their anticandidal activity in vitro appeared to be enhanced. CSF-1 was also administered in vivo to prospective recipients of a lethal C. albicans challenge. The results showed that the factor could effectively potentiate the animals' resistance to the yeast, as shown by increased survival times and reduced recovery of viable C. albicans from the organs of the CSF-1-treated mice. Therefore, the present data suggest that CSF-1 is likely to contribute to early resistance to fungal infection and could be successfully exploited in experimental models of antifungal immunotherapy.