Glenn M. Smith

Identification of Epitope Regions Recognized by Tumor Inhibitory and Stimulatory Anti-ErbB-2 Monoclonal Antibodies: Implications for Vaccine Design

The self-oncoprotein ErbB-2 is overexpressed in a number of malignancies. The presence of endogenous anti-ErbB-2 Ab and T cell immune responses to this protein in cancer patients has made ErbB-2 an attractive target for active immunization. However, the finding that murine anti-ErbB-2 Abs can have stimulatory, inhibitory, or no effects on cancer cell growth suggests that an inappropriately induced immune response may have an adverse effect. To ensure the induction of a beneficial Ab response, it is important to identify the epitopes recognized by these Abs. In this study we have used phage-displayed ErbB-2 gene fragment libraries and synthetic peptides to epitope-map a panel of anti-ErbB-2 mAbs. The epitopes of three mAbs, N12, N28, and L87, were successfully located to C531-A586, T216-C235, and C220-C235 of ErbB-2, respectively. It was found that while N12 inhibited tumor cell proliferation, N28 stimulated the proliferation of a subset of breast cancer cell lines overexpressing ErbB-2. The peptide region recognized by N12, (C531-A586; EP531), was used as an immunogen to selectively induce an inhibitory immune response in mice. Mice immunized with the GST fusion peptide (GST-EP531) recognized the peptide region EP531 as well as native ErbB-2. More importantly, Igs purified from mouse sera were able to inhibit up to 85% of tumor cell proliferation. In conclusion, our study provides direct evidence of the function-epitope relationship of anti-ErbB-2 Abs and also emphasizes the value of inducing a potent tumor inhibitory polyclonal Ab response by rationally selecting regions of ErbB-2 used for immunization.

Detection of a soluble form of the leukocyte surface antigen CD48 in plasma and its elevation in patients with lymphoid leukemias and arthritis

Proteins with glycosylphosphatidylinositol (GPI) anchors exhibit a range of activities and some of these proteins exist in both a membrane-associated and a soluble form. CD48 is a 47-kd GPI-linked glycoprotein which is expressed on T and B lymphocytes, monocytes, and many lymphoid malignancies. The biological function of CD48 is unknown. We describe the detection of a soluble form of CD48 in plasma and serum. Its level was quantified by an immunoenzymometric assay (IEMA) specific for soluble CD48. While soluble CD48 was detected in the plasma of healthy individuals (median = 29 ng/ml; range, 15–48 ng/ml), elevated levels were detected in some patients with lymphoproliferative disease (median = 41 ng/ml; range, 9–213 ng/ml), arthritis (median = 42 ng/ml; range, 13–67 ng/ml), and acute EBV infection (174 ng/ml). Soluble CD48 was also detectable in tissue culture supernatants from the Raji lymphoid cell line. The mechanism of CD48 release from cells is unclear. The finding of significant levels of soluble CD48 in plasma and the development of a sensitive IEMA for its measurement will facilitate further studies on its normal function and its role in disease.

Biodistribution of filamentous phage-Fab in nude mice.

In vivo panning of peptide libraries in mice has allowed the isolation of peptides which target the vasculature of specific organs. The application of this approach to phage displaying Fab fragments (phage-Fab) could lead to the isolation of antibodies which recognize novel tumor antigens. In this study, we have evaluated the biodistribution of phage-Fab in nude mice. Balb/c nude mice were injected intravenously with 10(9) TU of phage displaying the anti-colon cancer Fab c30.6. Blood samples were collected at nine time points over a period of 72 h and three groups of four mice were sacrificed at 4 min, 24 h and 72 h. Normal tissues (liver, colon, spleen, kidneys, lungs, skeletal muscle) and faeces were collected at these time points and the number of viable phage in each sample was determined. The distribution of phage in tissues was also examined by immunohistochemical analysis of paraffin-embedded tissues. Regression analysis of plasma kinetic data showed that the half-life and the volume of distribution of phage was 3.6 h and 1 ml, respectively. Phage uptake occurred predominantly in lungs, kidneys, spleen and liver. Relatively few phage were distributed to colon and muscle, and phage were eliminated from the circulation by 72 h. Immunohistochemical analysis showed phage to be mainly within the vasculature at 4 min, whereas notable phage extravasation was observed at 24 h and 72 h. In conclusion, this study provides information on the in vivo behavior of phage-Fab which will be useful in the design of in vivo panning strategies. By choosing appropriate time points for tissue collection, it may be possible to isolate novel Fabs against both intra- and extravascular targets.

Comparison of phage pIII, pVIII and GST as carrier proteins for peptide immunisation in Balb/c mice

Carrier proteins are important for improving the efficiency of synthetic peptide vaccines. Recently, genetically-based systems such as filamentous phage display or glutathione S-transferase (GST)-fusion proteins have been employed for immunisation. Whilst these carrier systems can facilitate the evaluation of a potential vaccine by reducing the time and cost of production, their relative efficacy and the kinetics of the immune response to each carrier has not been directly compared. In this study, we have displayed the epitopes of the anti-ErbB-2 Mabs N12 (C531-A586, EP531) and N28 (T216-C235, EP216) on phage minor coat protein pIII, major coat protein pVIII and GST. Balb/c H-2(d) mice were immunised with the constructs and the sera were tested after the initial, the 3rd, 5th and 6th immunisations for an anti-peptide, an anti-ErbB-2 and an anti-carrier response. The specificity of the antibody response was also mapped using synthetic peptides. It was found that GST was the best of the three carriers, both in terms of the magnitude and the kinetics of the induced anti-peptide and anti-ErbB-2 response. Multiple (five) administrations of the immunogens were necessary to obtain a high titre of antibodies specific for ErbB-2. It was further noted that whilst an anti-EP531 response was induced using all three carriers, EP216 was not immunogenic irrespective of the carrier used. The lack of immunogenicity of EP216 implies it does not contain a H-2(d) T cell recognition site. All three carriers provide a useful system for vaccination and consequently facilitate the identification of T-cell epitopes in Balb/c inbred mice.

Lipid composition of an ethanol-tolerant strain of Zymomonas mobilis

Abstract The lipid composition of exponentially growing Zymomonas mobilis, strain ZM4, has been examined. 31P-and 13C-NMR was used for identification and quantitation of the major lipid components. The major neutral lipid was identified as hopan-22-ol, with lesser amounts of squalene and hop-22(29)-ene. The remaining minor neutral lipids consisted of a series of hopanoids and oxygenated hydrocarbons. The major phospholipids were identified as: phosphatidylethanolamine (46%), phosphatidylglycerol (24%), phosphatidylcholine (9%), cardiolipin (6%) and phosphatidylserine (6%). Two unidentified phospholipids accounted for the remaining 9%. The fatty acid composition was determined by GLC and GC-MS to be: 14:0 (12%), 14:1 (0.5%), 16:0 (13%), 16:1 (2%), 18:0 (trace), 18:1 (70%) and C-19 (cyclopropane (3%)).

Antitumour activity of a chimeric antibody against the leucocyte antigen CD48.

Abstract Preclinical studies with the murine anti-CD48 antibody, mHuLym3 (IgG2a) have shown it to be a potentially useful therapeutic reagent in the treatment of human leukaemia and lymphoma. For clinical use, humanised antibodies can have a number of advantages over their original murine version, including mediation of higher effector cell function with human cells, longer serum half-life and lower immunogenicity. In this study, we have produced a mouse/human chimeric HuLym3 antibody (cHuLym3) where the murine antibody constant regions have been replaced with human constant regions. We report the production and preclinical characterisation of the antibody, cHuLym3, with potent in vitro and in vivo antitumour activity. The genes encoding the variable heavy and light chains were amplified by the polymerase chain reaction, sequenced and cloned into eukaryotic expression vectors containing the human light- and heavy-chain constant regions (κ and IgG1). The chimeric and murine HuLym3 antibodies had similar cell-binding specificity and affinity. In the human Raji cell severe combined immunodeficient mouse model the i.v. injection of cHuLym3 and mHuLym3 produced similar antitumour responses. Doses of cHuLym3 and mHuLym3 (100 μg) on days 1, 2 and 4 after i.v. Raji cell injection produced a 40% longer time to hind-leg paralysis than when a control antibody was used. cHuLym3 had more potent activity than mHuLym3 in antibody-dependent cellular cytotoxicity (ADCC) assays in vitro, with human peripheral blood mononuclear cells as effectors. Up to 60% specific cell lysis was observed with cHuLym3 in ADCC assays. These properties suggest that anti-CD48 antibodies may be useful in the treatment of a number of diseases including lymphoid leukaemias and lymphoma.

A recombinant GM-CSF-PE40 ligand toxin is functionally active but not cytotoxic to cells

A granulocyte/macrophage colony‐stimulating factor (GM‐CSF)‐Pseudomonas exotoxin (PE) 40 fusion protein was constructed for potential use in the treatment of myeloid leukaemias, as a conditioning agent prior to allogeneic bone marrow transplantation or for ex vivo purging of malignant cells prior to autologous bone marrow transplantation. The GM‐CSF‐PE40 fusion protein successfully binds to the GM‐CSF receptor and is capable of initiating a mitogenic signal similar to native GM‐CSF in the GM‐CSF‐dependent TF1 cell line. The toxin component also appears to be fully functional as determined by an in vitro adenosine diphosphate‐ribosylation assay. The GM‐CSF‐PE4n fusion protein, however, was not cytotoxic to a number of myeloid leukaemia cell lines. It is suggested that the mechanism of internalization of the GM‐CSF receptor is not appropriate for the translocation of PE to the cytosol where it can fulfill its cytotoxic potential.

Characterization of a novel leucocyte surface membrane antigen recognized by the monoclonal antibody WM65

WM65 is a murine mAb whieh recognizes a novel surface membrane antigen present on leukaemic and normal leucocytes. The present study further investigates the nature of this antigen, especially those features which relate to the possible therapeutic applications of the WM65 antibody. There are 1–3 × 104 molecules of this antigen present on normal leucocytes, and the same or greater numbers of antigen molecules are present on a variety of leukaemic cells. In vitro data showed that the WM65 antibody is internalized following interaction with its antigen on normal leucocytes. The affinity of this antibody was calculated using an FLISA method which required neither labelling of the antibody nor purification of the antigen and the affinity constant was found to be 3 × 107±2 × 107 (mol/L)−1. Further data are presented which suggest that this antigen is a differentiation antigen and an integral membrane protein. Despite the relatively low affinity of the WM65 antibody, a number of characteristics of the antigen suggest the antibody may possibly have therapeutic applications. These characteristics include its cellular distribution, the number of antigen molecules expressed on the cell surface and its ability to internalize in vitro.