Christopher J. Dean

Synergistic interaction between ligands binding to the CD4 binding site and V3 domain of human immunodeficiency virus type I gp120

Abstract We demonstrate that soluble CD4 (sCD4) or a monoclonal antibody (mAb), 39.138, binding to a conformational epitope of gpl20 involved in CD4 binding, and mAbs binding to the V3 domain of gpl20, can synergistically neutralize human immunodeficiency virus type I (HIV-1). In contrast, a neutralizing mAb binding to a linear epitope within the CD4 binding domain was unable to exert a synergistic effect in combination with V3 mAbs, suggesting that synergism is dependent on ligands binding to the critical, discontinuous, gp120 residues constituting the CD4 binding site. A number of V3 mAbs showed increased binding to virion gpl20 in the presence of sCD4, suggesting a mechanism for the synergistic neutralization. This effect was not observed with recombinant or detergent solubilized viral gp120, suggesting that the oligomeric structure of gpl20 on viral particles affects V3 epitope exposure. This hypothesis is supported by the ability of two new V3 mAbs, 8/38c and 8/64b, to only neutralize HIV-1 in the presence of sCD4 or mAb 39.138; binding studies demonstrate that these mAbs only bind to virion gpl20 in the presence of sCD4. Thus, V3 epitope exposure is modulated by the interaction of virion gpl20 with ligands specific for the CD4 binding domain and results in enhanced antibody-mediated neutralization.

Targeting of cells expressing wild‐type EGFR and type‐III mutant EGFR (EGFRvIII) by anti‐EGFR MAb ICR62: A two‐pronged attack for tumour therapy

With a view to their use in cancer therapy, we have produced rat monoclonal antibodies (MAbs) directed against 5 distinct epitopes (A–E) on the external domain of the wild‐type human EGF receptor (EGFR). Here, we have investigated the relative binding and anti‐tumour activity of our anti‐EGFR MAbs against HC2 20d2/c cells, which have been engineered to overexpress the type‐III mutated form of the human EGFR (EGFRvIII). We found that anti‐EGFR MAbs that are the most effective antagonists of EGFR ligands (e.g., ICR16, ICR62 and ICR80) also bind to cells that overexpress the EGFRvIII. Although these antibodies are potent inhibitors of the growth of cells which express wild‐type EGFR, they did not directly inhibit the growth in vitro of EGFRvIII expressing HC2 20d2/c cells, or the constitutive tyrosine kinase activity of this receptor. However, in the presence of human peripheral blood mononuclear cells (PBMC), the rat IgG2b MAb ICR62 induced strong antibody‐dependent cell‐mediated cytotoxicity (ADCC) against HC2 20d2/c cells in culture. Interestingly, MAb ICR62 also inhibited very effectively experimental lung metastases of HC2 20d2/c cells in athymic nude mice. Our results suggest that anti‐EGFR MAb ICR62, which binds to the EGFRvIII, may have potential in the treatment of tumors which overexpress the EGFRvIII via immunological mechanisms such as ADCC. Since tumours that are EGFRvIII positive may also overexpress the wild‐type EGFR, the use of anti‐EGFR MAbs that target both wild‐type and mutant receptors may have advantages over those that target only1form.

The human EGF receptor as a target for cancer therapy: six new rat mAbs against the receptor on the breast carcinoma MDA-MB 468

Using the breast carcinoma cell line MDA-MB 468 as immunogen, we have produced six new rat monoclonal antibodies (mAbs) against the human EGF receptor (EGFR) and are investigating their use for diagnostic and therapeutic applications in cancer patients whose tumours overexpress these receptors. The mAbs (three IgG2b and one each of IgG2a, IgG1 and IgA) were selected on the basis that they bound to the extracellular domain of the EGFR and blocked growth factor-receptor interaction. Competitive assays showed that, with the exception of antibody ICR65, the mAbs bound to one of two distinct epitopes on the external domain of the EGFR. ICR65, however, cross-reacted with mAbs binding to both epitopes. All of the mAbs immunoprecipitated the 170 kDa glycoprotein from cells expressing the EGFR but not the 185 kDa product of the related c-erbB-2 proto-oncogene. Unlike EGF and TGF alpha none of the mAbs stimulated the growth of quiescent human foreskin fibroblasts but they inhibited the EGF and TGF alpha induced growth stimulation of these cells in vitro. When tested for their effect on tumour cells the mAbs were found to inhibit the growth in vitro of a number of human tumours that overexpressed the EGFR (e.g. HN5, HN6, HN15, A431, MDA-MB 468) but they were without effect on tumour cell lines expressing low or undetectable amounts of the receptor. Our initial results indicate that this new generation of antibodies which bind with high affinity to the EGFR, block growth factor-receptor interaction and inhibit the growth of human squamous carcinoma cell lines overexpressing the receptor have potential for clinical application.

Immunotherapy of human tumour xenografts overexpressing the EGF receptor with rat antibodies that block growth factor-receptor interaction

Athymic mice bearing xenografts of human tumours that overexpress the receptor (EGFR) for EGF and TGF alpha have been used to evaluate the therapeutic potential of three new rat monoclonal antibodies (mAbs) directed against two distinct epitopes on the extracellular domain of the human EGFR. The antibodies, ICR16 (IgG2a), ICR62 (IgG2b) and ICR64 (IgG1), have been shown (Modjtahedi et al., 1993) to be potent inhibitors of the growth in vitro of a number of human squamous cell carcinomas because they block receptor-ligand interaction. When given i.p. at 200 micrograms dose, the three antibodies were found to induce complete regression of xenografts of the HN5 tumour if treatment with antibody commenced at the time of tumour implantation (total doses: ICR16, 3.0 mg; ICR62, 1.2 mg; ICR64, 2.2 mg). More importantly when treatment was delayed until the tumours were established (mean diam. 0.5 cm) both ICR16 and ICR62 induced complete or almost complete regression of the tumours. Furthermore, treatment with a total dose of only 0.44 mg of ICR62 was found to induce complete remission of xenografts of the breast carcinoma MDA-MB 468, but ICR16 was less effective at this dose of antibody and only 4/8 tumours regressed completely. ICR16 and ICR62 were poor inhibitors of the growth in vitro of the vulval carcinoma A431, but both induced a substantial delay in the growth of xenografts of this tumour and 4/8 tumours regressed completely in the mice treated with ICR62 (total dose 2.2 mg). Although ICR16 and ICR64 were more effective than ICR62 as growth inhibitors in vitro, ICR62 was found to be substantially better at inducing regression of the tumour xenografts due perhaps to additional activation of host immune effector functions by the IgG2b antibody. We conclude that these antibodies may be useful therapeutic agents that can be used alone without conjugation to other cytotoxic moieties.

Antitumor activity of combinations of antibodies directed against different epitopes on the extracellular domain of the human EGF receptor.

The receptor (EGFR) for epidermal growth factor (EGF) and transforming growth alpha (TGFα) is often overexpressed in certain types of human malignancy and high levels of expression of the receptor and/ or coexpression of growth factors. EGF and TGFα have also been correlated with poor prognosis in many patients. We have produced a number of rat monoclonal antibodies (MAbs) against four distinct epitopes on the external domain of the EGF receptor and are currently evaluating their potential for therapeutic use. Nine of these of MAbs were found to inhibit the binding of TGF and EGF to the receptor on tumor cells and these MAbs were able to inhibit the growth in vitro and in vivo of tumor cells that overexpress the EGF receptor. Here, we describe the results of experiments to determine the antitumor activity of combination treatment with two antibodies directed against separate epitopes on the external domain of human EGFR. Our results showed that treatment of human tumor xenografts with a combination of two anti-EGFR MAbs that bind to two distinct epitopes on the external domain of EGF receptor was not as effective as treatment with ICR62 alone, which binds to epitope C on the EGFR and is of IgG2b isotype. A phase I clinical trial with antibody ICR62 is currently underway in Royal Marseden Hospital (UK) in patients with head and neck and lung carcinomas.

Rat monoclonal antibodies to nonoverlapping epitopes of human immunodeficiency virus type 1 gp120 block CD4 binding in vitro.

Monoclonal antibodies (MAbs) to a recombinant form of the envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1 IIIB) were raised in rats and screened for their ability to block recombinant gp120 binding to recombinant, soluble CD4 (sCD4) in vitro. Four such MAbs were identified and characterised. Each MAb bound strongly to gp120 from eight widely divergent HIV-1 strains from the United States and Africa. Two MAbs were mapped to the fourth conserved (C4) region of gp120, whereas the other two recognised an as yet undefined, conformationally sensitive epitope. MAbs to the latter epitope were the more potent in blocking the gp120-sCD4 interaction. None of the MAbs, however, had potent neutralising activity.

Anti‐EGFR monoclonal antibodies which act as EGF, TGFα, HB‐EGF and BTC antagonists block the binding of epiregulin to EGFR‐expressing tumours

Epiregulin is the newest member of the epidermal growth factor (EGF) family of ligands that was isolated from conditioned medium of the murine fibroblast‐derived tumour cell line NIH3T3/T7. Here, using a panel of anti‐EGFR receptor (EGFR) monoclonal antibodies (MAbs) directed against 4 distinct epitopes on the external domain of the receptor, we have investigated the importance of the EGFR in transmitting the biological action of epiregulin. We found that MAb ICR9, which enhances the binding of EGF, TGFα, HB‐EGF and betacellulin to the EGFR, also increases the binding of 125I‐epiregulin to a number of EGFR‐expressing tumour cell lines, including EJ, SKBR3, SKOV3, MDA‐MB468 and HN5. In addition, anti‐EGFR MAbs ICR15, ICR16, ICR61, ICR62 and ICR80, which block the binding of 125I‐EGF to the EGFR, inhibit the binding of 125I‐epiregulin to these tumour cell lines. Like EGF, we found that both the epiregulin‐induced growth inhibition of HN5 and MDA‐MB468 cells and tyrosine phosphorylation of the 170 kDa EGFR on HN5 cells are reversed in the presence of anti‐EGFR MAbs ICR62 and ICR80. Surprisingly and unlike 125I‐EGF, radiolabelled epiregulin bound very poorly to human bladder carcinoma EJ cells and its binding to SKOV3 cells was not inhibited efficiently in the presence of blocking antibodies. We conclude that the EGFR plays an important role in transmitting the biological action of epiregulin and that these effects could be blocked in the presence of anti‐EGFR MAbs. The low level of binding of epiregulin compared with EGF to EJ cells suggests that the EGFR may not be the primary receptor for epiregulin. Int. J. Cancer 75:310–316, 1998.

Preclinical models for the evaluation of targeted therapies of metastatic disease.

It has been estimated that approx 60–70% of cancer patients harbor overt or subclinical metastases at diagnosis, and it is the eradication of such systemic disease that largely determines survival. Preclinical tumor model systems employed to evaluate potential new treatment strategies should aim to represent the process and patterns of metastasis of their clinical counterparts as closely as possible. Severe combined immune-deficient (SCID) andnu/nu mice have been extensively used as hosts for the growth of human tumor cell lines and in some cases fresh tumor material. However, in most instances the resulting neoplasms fail to metastasize, and the aberrant immune systems of such animals has limited their use mainly to passive therapies of localized disease. Recently, the development of specially selected tumor variants and the use of appropriate orthotopic sites for implantation has provided several models in which dissemination can be demonstrated. Where the gene coding for a potential target antigen has been cloned, and where its overexpression or mutation is associated with malignancy (e.g., c-erbB-2, H-ras), transgenic mice may yield tumors that will develop in these immunocompetent hosts. In some cases such tumors exhibit metastasis. A third approach is to transfect human genes of interest into appropriate rodent tumors expressing the desired metastatic phenotype. These various approaches are compared with particular reference to mammary carcinoma biology.

Radioimmunolocalisation in breast cancer using the gene product of c-erbB2 as the target antigen.

Lymph node status is still the single most important prognostic factor in breast cancer. Axillary surgery remains the only reliable means of providing this information. This pilot study evaluates using a highly specific radiolabelled monoclonal antibody to provide equivalent information by a non-invasive technique. After optimisation of labelling conditions, our first antibody, ICR12 (against the gene product of c-erbB-2) was evaluated in a mouse model system. Twenty-four hours post i.v. injection the mice were killed and their organs, blood and tumours harvested for counting. Tumour localisation was four times greater than that into normal tissues, reaching 20% injected dose per gram of tumour. Eight patients have had this Tc99m-ICR12. Patient selection was by immunocytochemical staining of fine needle aspirates from the patients own breast cancer. After intravenous administration of the immunoconjugate, tomographic images were obtained at 24 h. These results were compared to the subsequent histopathological examinations. Three patients acted as normal controls, one patient was negative due to inappropriate sampling, and two patients had strong membrane staining and provided excellent tumour localisation to both breast primary and regional node metastases. A further two patients only had moderate antigen expression on staining and did not localise well. The good performance of this radiolabelled antibody with patients that strongly stain for the antigen encourages the development of this system as both a method of staging breast cancer and a potential means of immunotherapy in this subgroup of patients.

Metastasis in the nude rat associated with lack of immune response.

EVI1DENCE is accumulating that in experimental animals there is an inverse relationship between the host response to a tumour and its metastatic capacity. Earlier work (Eccles & Alexander, 1974) has shown that T-cell deprivation (e.g. by thymectomy and sublethal irradiation) leads to a defective recruitment of mononuclear phagocytes to the primary tumour site and a concomitant increase in the incidence of distant metastases. Although this effect can be reversed by reconstitution of the T-deprived animals with lymphoid cells before tumour implantation (Eccles, 1978), the possibility remains that surgical stress and irradiation may interfere with other aspects of the animals physiology and contribute to the increased development of metastases. The reappearance of the mutant nude Rowett rat which is congenitally athymic (Festing et al., 1978) has provided an ideal system in which to evaluate the role of T cells without recourse to stressful immunosuppressive procedures. In this communication we describe our initial observations on the growth, metastatic behaviour, hostcell infiltration of, and antibody responses to a Hooded rat fibrosarcoma in normal and athymic animals. The athymic rat popuilation used in this study was provided by the MRC Laboratory Animal Centre, Carshalton, and was of mixed genetic background (Table I). Because heterozygous litter-mates of the homozygous athymic animals were not available at the time, we used for controls Lister Hooded/Cbi rats which are geneticTABLE I.-Host-cell infiltration: estimation of number of Fc-positive phagocytic cells as %0 of total cells recovered from tumour by enzymatic digestion