Combined treatment of the experimental human papilloma virus-16-positive cervical and head and neck cancers with cisplatin and radioimmunotherapy targeting viral E6 oncoprotein.

BACKGROUND: Human papilloma virus (HPV) is implicated in >99% of cervical cancers and ∼40% of head and neck squamous cell carcinoma (HNSCC). We previously targeted E6 oncogene with (188)Rhenium-labelled monoclonal antibody (mAb) C1P5 to HPV16 E6 in cervical cancer and HNSCC. Intranuclear E6 can be accessed by mAbs in non-viable cells with leaky membranes. As radioimmunotherapy (RIT) efficacy depends on the availability of target protein-we hypothesised that pretreatment with cisplatin will kill some tumour cells and increase E6 availability for RIT. METHODS: Mice with subcutaneous HPV16+ cervical (CasKi) and HNSCC (2A3) tumours were pretreated with 0-7.5 mg kg(-1) per day cisplatin for 3 days followed by (188)Re-C1P5 and biodistribution was performed 24 h later. For RIT, the animals were treated with: 5 mg kg(-1) per day cisplatin for 3 days; or 5 mg kg(-1) per day cisplatin for 3 days followed 200 or 400µCi (188)Re-C1P5 mAb; or 200 or 400µCi (188)Re-C1P5 mAb; or left untreated, and observed for tumour growth for 24 days. RESULTS: Pretreatment with cisplatin increased the uptake of (188)Re-C1P5 in the tumours 2.5 to 3.5-fold and caused significant retardation in tumour growth for CasKi and 2A3 tumours in both RIT alone and cisplatin, and RIT groups in comparison with the untreated control and cisplatin alone groups (P<0.05). The combined treatment was more effective than either modality alone (P<0.05). CONCLUSION: Our study demonstrates that preceding RIT targeting E6 oncogene with chemotherapy is effective in suppressing tumour growth in mouse models of HPV16+ cancers.

Increased chitinase expression and fungal-specific antibodies in the bronchoalveolar lavage fluid of asthmatic children.

BACKGROUND: Increasing evidence highlights the contribution of chitinases and fungal infection to the development of asthma. OBJECTIVE: The purpose of this study was to characterize chitinase expression and serological markers of fungal infection in children with severe asthma. METHODS: Bronchoalveolar lavage fluid (BALF) was collected from children undergoing clinically indicated flexible bronchoscopy. A diagnosis of asthma was confirmed by pulmonary function testing. BALF was tested for chitinase activity and YKL-40 (an enzymatically inactive chitinase) concentrations. Specimens were cultured for fungal organisms and tested for cryptococcal antigen by ELISA. IgG and IgA reactivity to whole extract fungal (Aspergillus fumigatus, Alternaria alternata, Cryptococcus neoformans and Candida albicans) proteins were determined by immunoblot assay. RESULTS: Among the 37 patients studied, 30 were asthmatic and 7 were non-asthmatic. Asthmatics exhibited elevated serum IgE levels (median: 748 IU/mL, IQR: 219-1765 IU/mL). Chitinase activity was greater in the BALF of asthmatics (mean, 0.85 ± 1.2 U/mL) compared with non-asthmatics (mean: 0.23 ± 0.21 U/mL, P = 0.012). Likewise YKL-40 concentrations were higher in the BALF of asthmatics and correlated with chitinase activity. There was a trend towards increased fungal-specific IgG in the BALF of asthmatics compared with non-asthmatics and for C. albicans this difference reached statistical significance. IgA reactivity to C. neoformans and A. fumigatus was greater in the BALF of asthmatics compared with non-asthmatics. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with non-asthmatics, asthmatic children exhibited increased chitinase activity and increased YKL-40 levels in BALF. Increased IgG and IgA reactivity to fungal proteins in the BALF of asthmatics may reflect a local response to fungal infection. Our findings are consistent with and suggest a role for chitinases in asthma pathogenesis among Bronx children and provide serological evidence of an association between fungal infection and severe asthma.

Toward developing a universal treatment for fungal disease using radioimmunotherapy targeting common fungal antigens.

BACKGROUND: Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall. METHODS: MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls. RESULTS: (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing. CONCLUSION: Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

The mouse antibody response to infection with Cryptococcus neoformans: VH and VL usage in polysaccharide binding antibodies.

Cryptococcus neoformans is a ubiquitous fungus that can cause serious infections in humans. The fungus has a polysaccharide (C. neoformans capsular polysaccharide; CNPS) capsule that contributes to its pathogenicity and can elicit an antibody response. Nevertheless, only 4 of 60 BALB/c mice chronically infected with C. neoformans had a detectable increase in serum anti-CNPS. The sera of three responder mice contained both IgM and IgG anti-CNPS antibody, and the titers of lambda and kappa anti-CNPS antibody were approximately equal. Eight IgM and one IgG3 monoclonal antibodies (mAbs) were generated from the spleen of one responder mouse, and one IgA was generated from the spleen of another mouse. Seven of the IgMs, the IgG3, and the IgA mAb had lambda light chains and were specific for serotype D CNPS. Molecular analysis confirmed that this was a highly restricted antibody response. All of the D-specific antibodies used VH441, JH3, and either V lambda 2/J lambda 2 or V lambda 1/J lambda 1, and all had the same heavy chain CDR3 amino acid sequence, even though there were differences in the nucleotide sequence of the N/D segment. One IgM mAb reacted with both serotype A and D CNPS, and this mAb used different VH and JH genetic elements and had kappa light chains. All the anti-CNPS mAbs used J proximal VH gene elements that have previously been shown to bind dextran and other polysaccharides. Sequence and Southern blot analysis indicate that the serotype-D CNPS-specific mAbs arose from only a few precursor B cells.

Molecular characterization of the humoral responses to Cryptococcus neoformans infection and glucuronoxylomannan-tetanus toxoid conjugate immunization.

The molecular characteristics of the humoral immune response to a serotype A Cryptococcus neoformans infection were compared with the response elicited by a cryptococcal glucuronoxylomannan-tetanus toxoid (GXM-TT) conjugate. Anticryptococcal monoclonal antibodies (mAbs) isolated from both responses have previously been shown to recognize the same antigenic determinant of cryptococcal GXM. Southern blot and sequence analyses indicate that the hybridomas isolated from each response arose from only a few precursor B cells. All the mAbs generated from the infected and GXM-TT conjugate-immunized mice utilize the same VH7183 family member: JH2/JH4, v kappa 5.1, and J kappa 1; mAbs generated by different B cells had complementarity-determining region 3's (CDR3s) composed of seven amino acids with a common sequence motif. Thus, the molecular analysis of these anticryptococcal mAb-producing hybridomas indicated that the response to both cryptococcal infection and conjugate immunization was oligoclonal and highly restricted with regard to immunoglobulin gene utilization. The GXM-TT conjugate primarily stimulated isotype switching and clonal proliferation, and did not result in hybridomas expressing additional immunoglobulin repertoires. The mAbs from both responses had a number of replacement mutations at the 5' end of CDR2 that appear to be the result of antigen-driven selection. Somatic mutation also resulted in altered epitope specificity for one mAb, 13F1. Passive administration of representative mAbs from different clones generated in response to the GXM-TT conjugate prolonged survival of lethally infected mice.

Human astrocytes inhibit Cryptococcus neoformans growth by a nitric oxide-mediated mechanism.

Cryptococcus neoformans is an opportunistic fungus that causes life-threatening meningoencephalitis in 5-10% of patients with acquired immune deficiency syndrome. Cryptococcal meningoencephalitis is characterized by a lymphohistiocytic infiltrate, accumulation of encapsulated forms of C. neoformans, and varying degrees of glial reaction. Little is known about the contribution of endogenous central nervous system cells to the pathogenesis of cryptococcal infections. In this study, we investigated the role of astrocytes as potential effector cells against C. neoformans. Primary cultures of human fetal astrocytes, activated with interleukin 1 beta plus interferon gamma inhibited the growth of C. neoformans. The inhibition of C. neoformans growth was paralleled by production of nitrite, and reversed by the inhibitors of nitric oxide (NO.) synthase, NG-methyl-mono-arginine and NG-nitro-arginine methyl ester. The results suggest a novel function for human astrocytes in host defence and provide a precedent for the use of NO. as an antimicrobial effector molecule by human cells.

Epitope location in the Cryptococcus neoformans capsule is a determinant of antibody efficacy.

Monoclonal antibodies (mAbs) to the polysaccharide capsule of Cryptococcus neoformans can prolong survival in mice. However, the properties of antibodies that mediate protection are not fully understood. The IgM mAbs 12A1 and 13F1 originated from the same B cell and differ only by somatic mutations in their variable regions; yet mAb 12A1 protects against serotype D infection, while mAb 13F1 does not. Phage peptide display libraries were used to analyze the fine specificity of these two mAbs. The selection of distinct peptide motifs from identical libraries confirmed that mAbs 12A1 and 13F1 bound to two distinct epitopes. Immunofluorescence and immunoelectron microscopy studies revealed differences in antibody localization within the capsule of serotype D strain; mAb 12A1 bound to the outer rim of the capsule resulting in an annular pattern, whereas mAb 13F1 bound throughout the capsule and had a punctate appearance. The difference in the binding pattern of mAb 12A1 and 13F1 was not observed on serotype A organisms, where both mAbs bound to the capsule with an annular fluorescence pattern. The fluorescence pattern of binding correlated with protective efficacy; mAb 13F1 prolonged survival of mice infected with the J11 serotype A strain (annular fluorescence), but not serotype D strains (punctate pattern). Annular binding, but not punctate binding, was associated with increased opsonic efficacy for phagocytosis of C. neoformans by J774.16 macrophage-like cells. The correlation between capsular binding pattern, opsonic activity, and ability to prolong survival suggests that the efficacy of anticryptococcal antibodies is dependent upon where they bind in the polysaccharide capsule.

Immunoglobulin G3 blocking antibodies to the fungal pathogen Cryptococcus neoformans.

Vaccination and infection can elicit protective and nonprotective antibodies to the fungus Cryptococcus neoformans in mice. The effect of nonprotective antibodies on host defense is unknown. In this study we used mixtures of protective and nonprotective monoclonal antibodies (mAbs) to determine if nonprotective mAbs blocked the activity of the protective mAbs. Antibody isotype and epitope specificity are important in determining the ability to prolong survival in mice given a lethal C. neoformans infection. Three different nonprotective immunoglobulin (Ig) G23 mAbs to cryptococcal capsular polysaccharide were used to study the interaction between the IgG3 isotype and protective IgG1 and IgG2a mAbs in murine cryptococcal infection. One IgG3 mAb reduced the protective efficacy of an IgG1 with identical epitope specificity. A second IgG3 mAb with different epitope specificity also reduced the protection provided by the IgG1 mAb. The protective efficacy of an IgG2a mAb was also dramatically decreased by still another IgG3 mAb. To our knowledge this is the first report of blocking antibodies to a fungal pathogen. The results have important implications for the development of vaccines and passive antibody therapy against C. neoformans.

Protective and nonprotective monoclonal antibodies to Cryptococcus neoformans originating from one B cell.

Two immunoglobulin M monoclonal antibodies (mAbs) derived from the same B cell recognize different epitopes on the capsular polysaccharide of the pathogenic yeast, Cryptococcus neoformans. Their respective epitopes are located in spatially distinct regions of the capsule. Passive administration of one mAb prolonged survival whereas the other mAb did not. The results indicate that specificity is an important determinant of antibody efficacy against C. neoformans and that somatic mutations occurring during the antibody response can affect the protective efficacy of antibodies to C. neoformans.

Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy.

Metastatic melanoma remains difficult to treat despite recent approvals of several new drugs. Recently we reported encouraging results of Phase I clinical trial of radiolabeled with 188Re murine monoclonal IgM 6D2 to melanin in patients with Stage III/IV melanoma. Subsequently we generated a novel murine IgG 8C3 to melanin. IgGs are more amenable to humanization and cGMP (current Good Manufacturing Practice) manufacturing than IgMs. We performed comparative structural analysis of melanin-binding IgM 6D2 and IgG 8C3. The therapeutic efficacy of 213Bi- and 188Re-labeled 8C3 and its comparison with anti-CTLA4 immunotherapy was performed in B16-F10 murine melanoma model. The primary structures of these antibodies revealed significant homology, with the CDRs containing a high percentage of positively charged amino acids. The 8C3 model has a negatively charged binding surface and significant number of aromatic residues in its H3 domain, suggesting that hydrophobic interactions contribute to the antibody-melanin interaction. Radiolabeled IgG 8C3 showed significant therapeutic efficacy in murine melanoma, safety towards healthy melanin-containing tissues and favorable comparison with the anti-CTLA4 antibody. We have demonstrated that antibody binding to melanin relies on both charge and hydrophobic interactions while the in vivo data supports further development of 8C3 IgG as radioimmunotherapy reagent for metastatic melanoma.

Phenotypic switching of Cryptococcus neoformans occurs in vivo and influences the outcome of infection.

Phenotypic switching has been linked to the virulence of many pathogens, including fungi. However, it has not been conclusively shown to occur in vivo or to influence the outcome of infection. Cryptococcus neoformans undergoes phenotypic switching in vitro to colony types that differ in their virulence in mice. In this study, we asked whether C. neoformans undergoes phenotypic switching in vivo and whether this phenomenon contributes to virulence. By using a small inoculum to preclude the introduction of variants that had already switched during in vitro propagation, we demonstrated that in vivo switching to a mucoid phenotype occurred in two mice strains and was associated with a lethal outcome. Phenotypic switching resulted in changes of the capsular polysaccharide that inhibited phagocytosis by alveolar macrophages. This promoted a more vigorous inflammatory response and rapid demise. These data document in vivo switching in a fungus and associate this phenomenon with enhanced virulence and a lethal outcome. The importance of this finding is underscored by the increased likelihood of phenotypic switching in chronic cryptococcosis; thus this mechanism may account for the inability to eradicate the organism in immunocompromised hosts.

Melanization of Cryptococcus neoformans in murine infection.

Cryptococcus neoformans is a fungus that is pathogenic in humans and that can produce melanin in vitro. Melanization is associated with virulence, but there is no evidence that melanin is made during infection. Melanins are difficult to study because they are amorphous and insoluble. Melanin-binding peptides from a phage display library were used to demonstrate that C. neoformans makes melanin-like compounds in tissue. Melanin-binding peptides were characterized by a high proportion of positively charged and aromatic residues. Two other methods, demonstration of an antibody response to melanin in mice infected with C. neoformans and analysis of yeast cell walls in infected tissue by light microscopy, were used to support these findings. The demonstration that C. neoformans melanizes in tissue has important implications for pathogenesis and drug discovery.

Melanin and virulence in Cryptococcus neoformans.

Melanin synthesis has been associated with virulence for the human pathogenic fungus Cryptococcus neoformans. Recent evidence indicates that C. neoformans cells synthesize melanin during infection and that this pigment protects the fungus against immune defense mechanisms.

Insights into mechanisms of antibody-mediated immunity from studies with Cryptococcus neoformans.

At first glance Cryptococcus neoformans appears an unlikely microbe to provide a new understanding of mechanisms of antibody-mediated immunity (AMI), because it is a facultative intracellular fungal pathogen for which the role of naturally acquired AMI in host defense is uncertain. However, numerous studies have now established that certain antibodies (Abs) against C. neoformans are protective in certain hosts. Studies with Abs to C. neoformans have provided new insights into AMI and generated new precedents with implications for other pathogens. The following concepts have emerged: 1) susceptibility to C. neoformans may be related to qualitative and quantitative aspects of the Ab response; 2) protective monoclonal Abs can be generated against pathogens even when the role of humoral immunity is uncertain; 3) Abs to C. neoformans mediate protection by immunomodulatory effects, thereby linking Ab efficacy to the overall host immune response; 4) Ab efficacy is critically dependent on fine specificity, which in turn is affected by immunoglobulin variable region usage, somatic mutation and constant region usage; 5) the efficacy of passive Ab therapy is a function of Ab dose and infecting innoculum, with lack of efficacy at the extremes of Ab concentration; 6) Ab-mediated toxicity resulting from antigen-Ab complex-induced release of platelet activating factor is isotype dependent. Observations with C. neoformans have stimulated a reappraisal of the role of humoral immunity for other pathogens and highlighted the limitations in current methods of assessing the role of Ab in host defense.

Antibody-mediated protection against intracellular pathogens.

The view that antibody-mediated protection is unimportant against intracellular pathogens is not supported by the literature. In fact, there is convincing evidence that antibody can protect against many important intracellular pathogens. The challenge now is to identify antigens that elicit protective antibodies, use them in vaccine design and understand how humoral and cellular immune mechanisms cooperate.

Intracellular parasitism of macrophages by Cryptococcus neoformans.

Cryptococcus neoformans, an encapsulated fungal pathogen, causes meningoencephalitis in immunocompromised patients. Recent in vivo studies have demonstrated that C. neoformans is a facultative intracellular pathogen, as was previously suggested by in vitro studies. For survival in macrophages, C. neoformans utilizes a novel strategy for intracellular parasitism that includes the accumulation of intracellular polysaccharide in cytoplasmic vesicles. Confirmation of the fact that C. neoformans is a facultative intracellular pathogen could provide new insights into several poorly understood areas of cryptococcal pathogenesis, including mechanisms for latency and persistence and the lack of efficacy of humoral immunity. The finding that C. neoformans replicates inside macrophages in vitro in a manner similar to that observed in vivo provides an excellent system to dissect the molecular mechanisms responsible for this unique pathogenic strategy.

The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neoformans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes.

The three-dimensional structure of 2H1, a protective monoclonal antibody to Cryptococcus neoformans, has been solved at 2.4 A resolution, in both its unbound form and in complex with the 12 amino acid residue peptide PA1 (GLQYTPSWMLVG). PA1 was previously identified as a potential mimotope of the cryptococcal capsular polysaccharide by screening of a phage display peptide library. Peptide binding is associated with only minor rearrangements of some side-chains and a small shift in the H2 loop of the antibody. The peptide assumes a tightly coiled conformation consisting of one inverse gamma-turn and one type II beta-turn that serves to place the entire peptide motif, consisting of ThrP5, ProP6, TrpP8, MetP9 and LeuP10, into a depression in the antibody combining site. A small number of H-bonds between peptide and antibody contribute to the affinity and specificity. Poor steric complementarity between PA1 and the antibody heavy chain along with the fact that the majority of the interactions between 2H1 and PA1 involve van der Waals interactions with the light chain may explain why this peptide acts as only a partial mimotope of the capsular polysaccharide epitope.

Immune complexes increase nitric oxide production by interferon-gamma- stimulated murine macrophage-like J774.16 cells.

Murine macrophage-like J774.16 cells were tested for changes in nitric oxide production upon incubation with immune complexes. Cryptococcus neoformans capsular polysaccharide and polysaccharide-specific monoclonal antibodies were added to J774.16 cells in the presence and absence of recombinant murine interferon-gamma (IFN-gamma). The effect of immune complexes on nitrite synthesis was both concentration dependent and isotype dependent. In the presence of IFN-gamma, immune complexes of IgG1, IgG2, IgG2b, or IgG3 isotype increased nitrite levels, whereas complexes of IgM isotype did not. Immune complexes did not alter nitrite production by unstimulated macrophages. Antibody alone, antigen alone, and antigen with irrelevant IgG1 antibody did not augment nitrite formation, either in the presence or absence of IFN-gamma, indicating a requirement for Fc gamma R cross-linking. These results suggest that IgG isotypes may offer additional protection against pathogens by enhancing macrophage nitric oxide production.

Mu switch region deletion is associated with both T cell independent and T cell dependent responses.

Isotype switching is a process by which immunoglobulin variable gene regions initially proximal to and expressed with the mu constant region gene (C mu) can rearrange downstream to other constant region genes. Thus the same antigen binding site can be expressed with each of the other constant region isotypes and perform the full panoply of effector functions. Although isotype switching is thought to involve highly reiterated 'switch site' sequences located 5' to constant region genes, the exact role of these switch sites is unknown. It has been reported that prior to switching, the 'donor' switch site 5' of C mu occasionally undergoes deletions, but it is not known whether this is an adventitious event or one which predisposes to or prevents isotype switching. Since T cell independent (TI) immune responses are dominated by IgM while T cell dependent (TD) responses are associated with switching to IgG, we have examined the state of the mu switch site in 51 IgM-producing hybridomas isolated from a variety of TI and TD responses. Although more hybridomas from the TI responses studied exhibited S mu deletions, deletion of S mu also occurred in hybridomas isolated from TD responses. Analysis of a well-characterized clonally related subset of IgM hybridomas also revealed that mu switch region deletion can be associated with the productive allele.

Human and murine immunoglobulin expression vector cassettes.

We describe the construction of new immunoglobulin (Ig) expression vectors and their use in the production of recombinant chimeric Ig molecules in transfected mammalian cells. The vectors contain the cDNA encoding the constant regions of human (mu, alpha1, gammal, gamma2, gamma3, gamma4, kappa) and murine (mu, gamma2a, kappa) Ig heavy and light chains. Unique restriction sites flanking the Ig variable region allow for replacement of variable regions generated by PCR. The CMV promoter allows for the transfection and expression of Ig in non-lymphoid cells. Distinct drug selection markers for heavy chain and light chain expression vectors allows for sequential or co-transfection of the vectors. We show that secretion of recombinant Ig can reach 1.2 microg/ml per million cells per day for transfected B cells. Replacement of the variable region results in the production of functional Ig retaining antigen specificity.