Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries.

Fusion phage libraries expressing single-chain Fv antibodies were constructed from the peripheral blood lymphocytes of two melanoma patients who had been immunized with autologous melanoma cells transduced the gamma-interferon gene to enhance immunogenicity, in a trial conducted at another institution. Anti-melanoma antibodies were selected from each library by panning the phage against live cultures of the autologous tumor. After two or three rounds of panning, clones of the phage were tested by ELISA for binding to the autologous tumor cells; > 90% of the clones tested showed a strong ELISA reaction, demonstrating the effectiveness of the panning procedure for selecting antimelanoma antibodies. The panned phage population was extensively absorbed against normal melanocytes to enrich for antibodies that react with melanoma cells but not with melanocytes. The unabsorbed phage were cloned, and the specificities of the expressed antibodies were individually tested by ELISA with a panel of cultured human cells. The first tests were done with normal endothelial and fibroblast cells to identify antibodies that do not react, or react weakly, with two normal cell types, indicating some degree of specificity for melanoma cells. The proportion of phage clones expressing such antibodies was approximately 1%. Those phage were further tested by ELISA with melanocytes, several melanoma lines, and eight other tumor lines, including a glioma line derived from glial cells that share a common lineage with melanocytes. The ELISA tests identified three classes of anti-melanoma antibodies, as follows: (i) a melanoma-specific class that reacts almost exclusively with the melanoma lines; (ii) a tumor-specific class that reacts with melanoma and other tumor lines but does not react with the normal melanocyte, endothelial and fibroblast cells; and (iii) a lineage-specific class that reacts with the melanoma lines, melanocytes, and the glioma line but does not react with the other lines. These are rare classes from the immunized patients' repertoires of anti-melanoma antibodies, most of which are relatively nonspecific anti-self antibodies. The melanoma-specific class was isolated from one patient, and the lineage-specific class was isolated from the other patient, indicating that different patients can have markedly different responses to the same immunization protocol. The procedures described here can be used to screen the antibody repertoire of any person with cancer, providing access to an enormous untapped pool of human monoclonal anti-tumor antibodies with clinical and research potential.

Human anti-idiotypic antibodies induced a humoral and cellular immune response against a colorectal carcinoma-associated antigen in patients.

Induction of immunity against antigens expressed on tumor cells might prevent or delay recurrence of the disease. Six patients operated on for colorectal carcinoma were immunized with human monoclonal anti-idiotypic antibodies (h-Ab2) against the mouse 17-1A anti-colon carcinoma antibody, mimicking a nominal antigen (GA733-2). All patients developed a long-lasting T-cell immunity against the extracellular domain of GA733-2 (GA733-2E) (produced in a baculovirus system) and h-Ab2. This was shown in vitro by specific cell proliferation (DNA-synthesis) assay as well as by interleukin 2 and interferon gamma production and in vivo by the delayed-type hypersensitivity reaction. Five patients mounted a specific humoral response (IgG) against the tumor antigen GA733-2E (ELISA) and tumor cells expressing GA733-2. Epitope mapping using 23 overlapping peptides of GA733-2E revealed that the B-cell epitope was localized close to the N terminus of GA733-2. Binding of the antibodies to the tumor antigen and to one 18-aa peptide was inhibited by h-Ab2, indicating that the antibodies were able to bind to the antigen as well as to h-Ab2. The results suggest that our h-Ab2 might be able to induce an anti-tumor immunity which may control the growth of tumor cells in vivo.

Invariance and restriction toward a limited set of self-antigens characterize neonatal IgM antibody repertoires and prevail in autoreactive repertoires of healthy adults.

Analysis of the reactivity of IgM with self-antigens in tissues by a quantitative immunoblotting technique showed striking invariance among newborns in the human and in the mouse. The self-reactive repertoire of IgM of adults was also markedly conserved; it comprised most anti-self reactivities that prevailed among neonates. Multivariate analysis confirmed the homogeneity of IgM repertoires of neonates toward self- and non-self-antigens. Multivariate analysis discriminated between newborn and adult repertoires for reactivity with two of five sources of self-proteins and with non-self-antigens. Our observations support the concept that naturally activated B lymphocytes are selected early in development and throughout life for reactivity with a restricted set of self-antigens.

Modulation of the antibody response by Porphyromonas gingivalis and Fusobacterium nucleatum in a mouse model

Successive immunization of mice with Fusobacterium nucleatum and Porphyromonas gingivalis has been shown to modulate the specific serum IgG responses to these organisms. The aim of this study was to investigate these antibody responses further by examining the IgG subclasses induced as well as the opsonizing properties of the specific antibodies. Serum samples from BALB/c mice immunized with F. nucleatum (gp1-F), P. gingivalis (gp2-P), P. gingivalis followed by F. nucleatum (gp3-PF) F. nucleatum followed by P. gingivalis (gp4-FP) or saline alone (gp5-S) were examined for specific IgG1 (Th2) and IgG2a (Th1) antibody levels using an ELISA and the opsonizing properties measured using a neutrophil chemiluminescence assay. While IgG1 and IgG2a subclasses were induced in all immunized groups, there was a tendency towards an IgG1 response in mice immunized with P. gingivalis alone, while immunization with F. nucleatum followed by P. gingivalis induced significantly higher anti-P. gingivalis IgG2a levels than IgG1. The maximum light output due to neutrophil phagocytosis of P. gingivalis occurred at 10 min using nonopsonized bacteria. Chemiluminescence was reduced using serum-opsonized P. gingivalis and, in particular, sera from P. gingivalis-immunized mice (gp2-P), with maximum responses occurring at 40 min. In contrast, phagocytosis of immune serum-opsonized F. nucleatum demonstrated peak light output at 10 min, while that of F. nucleatum opsonized with sera from saline injected mice (gp5-S) and control nonopsonized bacteria showed peak responses at 40 min. The lowest phagocytic response occurred using gp4-FP serum-opsonized F. nucleatum. In conclusion, the results of the present study have demonstrated a systemic Th1/Th2 response in mice immunized with P. gingivalis and/or F. nucleatum with a trend towards a Th2 response in P. gingivalis-immunized mice and a significantly increased anti-P. gingivalis IgG2a (Th1) response in mice immunized with F. nucleatum prior to P. gingivalis. Further, the inhibition of neutrophil phagocytosis of immune serum-opsonized P. gingivalis was modulated by the presence of anti-F. nucleatum antibodies, while anti-P. gingivalis antibodies induced an inhibitory effect on the phagocytic response to F. nucleatum.

Effect of periodontal treatment on the serum antibody levels to heat shock proteins

We have shown previously that both humoral and cellular immune responses to heat shock protein 60 (HSP60) are elevated in chronic periodontitis patients compared with non-diseased subjects. The aim of the present study was to determine whether periodontal treatment could influence the level of serum antibodies to human HSP60 and Porphyromonas gingivalis GroEL, a bacterial homologue of human HSP60. Sera were obtained from 21 patients with moderate to advanced chronic periodontitis at the baseline examination and again after completion of treatment. Antibody levels were determined using an enzyme-linked immunosorbent assay. The mean anti-P. gingivalis GroEL antibody levels were down-regulated significantly by periodontal treatment when recombinant P. gingivalis GroEL was used as an antigen, whereas antibody levels to P. gingivalis GroEL-specific peptide were significantly elevated following successful periodontal therapy. The mean level of anti-human HSP60 antibody remained unchanged although individual levels of antibody either increased or decreased after periodontal treatment, suggesting that synthesis of these antibodies might be regulated independently during the course of periodontal infection. Although their regulatory mechanisms in chronic infection are not understood, further study would provide insight not only into the role of these antibodies in the pathogenesis of periodontitis but also into the possible link between periodontitis and systemic diseases such as coronary heart disease.

Targeted delivery of heparin and LMWH using a fibrin antibody prevents restenosis

This study investigates a stent-less local delivery system for anti-restenotic agents utilizing antibodies to cross-linked fibrin (XLF). Heparin and low molecular weight heparin (LMWH) were conjugated to an antibody to cross-linked fibrin D-dinner (1D2). Rabbit right carotid arteries were injured with a balloon catheter, then the animals were given a bolus injection of 40 mug/k,g 1D2-heparin (26-70 mug/kg heparin) or 1D2-LMWH (29-80 mug/kg LMWH) conjugates or controls of saline (0.5 ml/kg), heparin (150 U/kg), LMWH (2 mg), or 1D2 (40 mug/kg), with or without a heparin bolus and sacrificed after 2 weeks (8 groups, n = 6/group). The injured artery of rabbits given 1D2-heparin or 1D2-LMWH conjugates had reduced neointimal development, with decreased luminal narrowing and positive remodelling compared with animals given control drugs. Animals given 1D2-heparin conjugate (with a heparin bolus) had three to five times more endothelial cells than the rabbits given saline or unconjugated heparin, while rabbits given 1D2-LMWH conjugate had up to 59% fewer neointimal cells than those given unconjugated drugs. There was little difference in extracellular matrix organization or composition. Thus cross-linked fibrin-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall where they influence wall remodelling and endothelial and neointimal cell number, reducing neointimal formation without systemic complications. Local delivery of anti-restenotic agents should minimise systemic effects, bleeding complications and potentially the cost of treatment due to a single, lower dose. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Transient production of recombinant proteins by chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG(4) encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 mug mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn(297) was not significantly affected by nocodazole during transient production by this method. (C) 2004 Wiley Periodicals, Inc.

The role of CD4(+) and CD8(+) T cells on antibody production by murine Peyer's patch cells following mucosal presentation of Actinomyces viscosus

The aim of this study was to determine the role of CD4 and CD8 cells on specific antibody production by murine Peyer's patch (PP) cells after oral immunization with Actinomyces viscosus in mice. Female DBA/2 mice were orally immunized with three low doses of heat-killed A. viscosus. Sham-immunized mice served as a control group. Mice were depleted of CD4 or CD8 cells by intraperitoneal injection of anti-CD4 or anti-CD8 antibodies daily for 3 days before oral immunization. One week after the last oral immunization, PPs were removed and cell suspensions were cultured with A. viscosus. Specific antibody production in the culture supernatants was assessed by enzyme-linked immunosorbent assay. The results showed that oral immunization with A. viscosus induced a predominant specific immunoglobulin A (IgA) response by PP cells and, to a lesser extent, IgM antibodies. Depletion of CD4 but not CD8 cells suppressed the production of specific antibodies. These results suggest that oral immunization with low doses of A. viscosus may induce the production of specific antibodies by murine PP cells in a CD4-cell-dependent fashion.

Innate recognition of apoptotic cells:novel apoptotic cell-associated molecular patterns revealed by crossreactivity of anti-LPS antibodies

Cells dying by apoptosis are normally cleared by phagocytes through mechanisms that can suppress inflammation and immunity. Molecules of the innate immune system, the pattern recognition receptors (PRRs), are able to interact not only with conserved structures on microbes (pathogen-associated molecular patterns, PAMPs) but also with ligands displayed by apoptotic cells. We reasoned that PRRs might therefore interact with structures on apoptotic cells-apoptotic cell-associated molecular patterns (ACAMPs)-that are analogous to PAMPs. Here we show that certain monoclonal antibodies raised against the prototypic PAMP, lipopolysaccharide (LPS), can crossreact with apoptotic cells. We demonstrate that one such antibody interacts with a constitutively expressed intracellular protein, laminin-binding protein, which translocates to the cell surface during apoptosis and can interact with cells expressing the prototypic PRR, mCD14 as well as with CD14-negative cells. Anti-LPS cross reactive epitopes on apoptotic cells colocalised with annexin V-and C1q-binding sites on vesicular regions of apoptotic cell surfaces and were released associated with apoptotic cell-derived microvesicles (MVs). These results confirm that apoptotic cells and microbes can interact with the immune system through common elements and suggest that anti-PAMP antibodies could be used strategically to characterise novel ACAMPs associated not only with apoptotic cells but also with derived MVs. © 2013 Macmillan Publishers Limited All rights reserved.

A Therapeutic Antibody for Cancer, Derived from Single Human B Cells.

Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

Synthesis and evaluation of novel ellipticines as potential anti-cancer agents

Ellipticine is a natural product which possesses multimodal anti-cancer activity. This thesis encompasses the synthesis and biological evaluation of novel ellipticine and isoellipticine derivatives as anti-cancer agents. Expanding on previous work within the group utilising vinylmagnesium bromide, derivatisation of the C5 position of ellipticine was accomplished by reaction of a key ketolactam intermediate with Grignard reagents. Corresponding attempts to introduce diverse substitution at the C11 position were unsuccessful, although one novel C11 derivative was produced using an alkyllithium reagent. A panel of novel ellipticinium salts encompassing a range of substitutions at the N2, C9 and N6 positions were prepared. Extensive derivatisation of the N10 position of isoellipticine was undertaken for the first time. Novel substitution in the form of acid and methyl ester functionalities were introduced at the C7 position of isoellipticine while novel C7 aldehyde and alcohol derivatives were synthesised. A large panel of isoellipticinium salts were prepared with conditions adjusted for the reactivity of the alkyl halide. Novel coupling reactions to increase the yield of isoellipticine were attempted but proved unsuccessful. A panel of 54 novel derivatives was prepared and a multimodal analysis of their anti-cancer activity was conducted. The NCI 60-human tumour cell lines screen was a primary source of information on the in vitro activity of compounds with derivatives found to exert potent anticancer effects, with mean GI50 values as low as 1.01 μM across the full range of cancer types and as low as 16 nM in individual cell lines. A second in vitro screen in collaboration with researchers in the University of Nantes identified derivatives which could potently inhibit growth in a p53 mutant NSCLC cell line. The cell cycle effects of a selected panel of isoellipticines were studied in leukaemia cell lines by researchers in the Department of Biochemistry and Cell Biology, UCC. Emerging from this, the therapeutic potential of one of the derivatives in AML was then assessed in vivo in an AML xenograft mouse model, with tumour weight reduced by a factor of 7 in treated mice relative to control.

Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library.

B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of potential therapeutic interest. To generate anti-B7-H4 targeting reagents, we isolated antibodies by differential cell screening of a yeast-display library of recombinant antibodies (scFvs) derived from ovarian cancer patients and we screened for functional scFvs capable to interfere with B7-H4-mediated inhibition of antitumor responses. We found one antibody binding to B7-H4 that could restore antitumor T cell responses. This chapter gives an overview of the methods we developed to isolate a functional anti-B7-H4 antibody fragment.

The Burkholderia Cenocepacia OmpA-Like Protein BCAL2958: Identification, Characterization, and Detection of Anti-BCAL2958 Antibodies in Serum from B. Cepacia Complex-Infected Cystic Fibrosis Patients

Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain an important cause of morbidity and mortality among cystic fibrosis patients, highlighting the need for novel therapeutic strategies. In the present work we have studied the B. cenocepacia protein BCAL2958, a member of the OmpA-like family of proteins, demonstrated as highly immunogenic in other pathogens and capable of eliciting strong host immune responses. The encoding gene was cloned and the protein, produced as a 6× His-tagged derivative, was used to produce polyclonal antibodies. Bioinformatics analyses led to the identification of sequences encoding proteins with a similarity higher than 96 % to BCAL2958 in all the publicly available Bcc genomes. Furthermore, using the antibody it was experimentally demonstrated that this protein is produced by all the 12 analyzed strains from 7 Bcc species. In addition, results are also presented showing the presence of anti-BCAL2958 antibodies in sera from cystic fibrosis patients with a clinical record of respiratory infection by Bcc, and the ability of the purified protein to in vitro stimulate neutrophils. The widespread production of the protein by Bcc members, together with its ability to stimulate the immune system and the detection of circulating antibodies in patients with a documented record of Bcc infection strongly suggest that the protein is a potential candidate for usage in preventive therapies of infections by Bcc.

Clinically-relevant threshold of preformed donor-specific anti-HLA antibodies in kidney transplantation.

BACKGROUND: Pretransplant anti-HLA donor-specific antibodies (DSA) are recognized as a risk factor for acute antibody-mediated rejection (AMR) in kidney transplantation. The predictive value of C4d-fixing capability by DSA or of IgG DSA subclasses for acute AMR in the pretransplant setting has been recently studied. In addition DSA strength assessed by mean fluorescence intensity (MFI) may improve risk stratification. We aimed to analyze the relevance of preformed DSA and of DSA MFI values. METHODS: 280 consecutive patients with negative complement-dependent cytotoxicity crossmatches received a kidney transplant between 01/2008 and 03/2014. Sera were screened for the presence of DSA with a solid-phase assays on a Luminex flow analyzer, and the results were correlated with biopsy-proven acute AMR in the first year and survival. RESULTS: Pretransplant anti-HLA antibodies were present in 72 patients (25.7%) and 24 (8.6%) had DSA. There were 46 (16.4%) acute rejection episodes, 32 (11.4%) being cellular and 14 (5.0%) AMR. The incidence of acute AMR was higher in patients with pretransplant DSA (41.7%) than in those without (1.6%) (p<0.001). The median cumulative MFI (cMFI) of the group DSA+/AMR+ was 5680 vs 2208 in DSA+/AMR- (p=0.058). With univariate logistic regression a threshold value of 5280 cMFI was predictive for acute AMR. DSA cMFI's ability to predict AMR was also explored by ROC analysis. AUC was 0.728 and the best threshold was a cMFI of 4340. Importantly pretransplant DSA>5280 cMFI had a detrimental effect on 5-year graft survival. CONCLUSIONS: Preformed DSA cMFI values were clinically-relevant for the prediction of acute AMR and graft survival in kidney transplantation. A threshold of 4300-5300 cMFI was a significant outcome predictor.