M. J. Embleton

RADIOIMMUNODETECTION OF HUMAN COLORECTAL CANCERS BY AN ANTI-TUMOUR MONOCLONAL ANTIBODY

In 10 out of 11 patients with colorectal cancer radiolabelled antitumour monoclonal antibody (791T/36) was localised to the tumour. The mean tumour to non-tumour uptake ratio of antibody demonstrated by imaging with a gamma camera was 4.4/1 after subtraction of background radioactivity. The antibody did not localise in one patient who had received radiotherapy to his tumour two weeks previously. In 5 patients with primary neoplasms localisation of the antibody was confirmed by further imaging of the resected specimens and in-vitro radioactivity counting of the tumour and comparison with the activity in adjacent normal colon.

Antitumor properties of vindesine-monoclonal antibody conjugates

SummaryThe anticancer alkaloid vindesine (VDS) was conjugated to four mouse monoclonal antibodies recognizing human tumor-associated antigens. The antibodies were 96.5 (antimelanoma, IgG2a); 791T/36 (antiosteogenic sarcoma, IgG2b); 11.285.14, and 14.95.55 (anticarcinoembryonic antigen, IgG1 and IgG2a respectively). Conjugates VDS-96.5 and VDS-791T/36 were tested in vitro and shown to be specifically cytotoxic for target cells expressing the appropriate antigen. The in vivo effects of the antibodies and conjugates were tested against human tumor xenografts in athymic or immunodeprived mice using multiple treatments. Conjugate VDS-96.5 retarded the initial growth of a melanoma xenograft, whereas free antibody was without effect. Similarly, VDS-791T/36 but not free antibody retarded the growth of osteogenic sarcoma 791T. The most marked antitumor effects observed were those obtained with VDS conjugates of the anti-CEA antibodies against a colorectal tumor xenograft. Antibody 14.95.55 suppressed tumor growth both alone and as a VDS conjugate, whereas 11.285.14 produced only a slight effect alone but an almost complete and lasting suppression of tumor growth as a VDS conjugate. Free VDS had little effect at nontoxic levels. Acute studies showed that VDS-11.285.14 conjugate was considerably less toxic than free VDS in Balb/c mice.

Selective cytotoxicity against human tumour cells by a vindesine-monoclonal antibody conjugate.

The anti-mitotic drug vindesine was coupled chemically to a monoclonal antibody raised originally against the human osteogenic sarcoma cell line, 791T. The cytotoxicity of the conjugate in vitro was tested, in comparison with free vindesine, against sarcoma 791T and other antigenically cross-reactive osteogenic sarcoma-cell lines, and also against tumour cell lines which have no detectable reaction with the monoclonal antibody. Continuous exposure of cultured 791T cells indicated that the vindesine was partially inactivated following conjugation since the conjugate was less toxic than the free drug. However, antibody-binding activity was essentially preserved following conjugation. Despite diminished drug activity in the conjugate, assays designed to mimic antibody binding to tumour in which target cells were treated with conjugate and washed before culture, showed selective cytotoxicity for osteogenic sarcoma lines with little or no effect on non-cross reactive control cells. In comparison, free vindesine was toxic equally for all cell lines and free antibody was non-toxic. These studies indicate that conjugation of a cytotoxic agent to a monoclonal antibody can confer on that agent selectivity for a particular target cell type which is recognised by the antibody.

Tumour localization of monoclonal antibody against a rat mammary carcinoma and suppression of tumour growth with adriamycin-antibody conjugates

SummaryMonoclonal antibody to the rat mammary carcinoma Sp4 isolated from hybridoma supernatants and labelled with 125I showed preferential in vivo localization into subcutaneous growths of Sp4 compared with normal tissues and a range of other transplanted tumours. No specific localization was seen with 125I-labelled normal rat immunoglobulin. Adriamycin conjugated to monoclonal antibody significantly retarded Sp4 tumour growth at 1/25th of the effective dose of the free drug, and in some cases brought about total tumour regression. Normal rat immunoglobulin-adriamycin conjugates were relatively ineffective. These studies indicate that monoclonal antibody directed against tumour cell surface antigens may be highly effective for tumour targeting of therapeutic agents.

Immunity in the tumor‐bearing host and its modification by serum factors

Tumor‐bearing individuals initiate immune responses to tumor‐associated neoantigens. These responses are demonstrable by in vitro analyses of cell‐mediated and humoral immune reactions, although it is emphasized that in most human studies, their correlation with positive tumor rejection reactions has not as yet been fully substantiated. Detection of cell‐mediated immunity in tumor‐bearing hosts has led to the concept that its effectiveness in controlling tumor growth may be modified or impaired by antagonistic humoral factors. This proposal is discussed with regard to the role of blocking reactions operative at the level of the target tumor cell, and with respect to direct inhibition of lymphocyte cytotoxicity by interaction with humoral factors. Available evidence indicates that blocking reactions are probably mediated by tumor‐specific immune complexes, and although antibody‐mediated blocking has been detected, its relevance in abrogating cell‐mediated immunity in the tumor‐bearing host is questionable. Alternatively, inhibition of the reactivity of sensitized lymphocytes, by tumor antigen or immune complexes, may prove to be of more importance in the immunologic control of tumor growth.

A bispecific monoclonal antibody against methotrexate and a human tumour associated antigen augments cytotoxicity of methotrexate-carrier conjugate.

A bispecific monoclonal antibody, reactive with methotrexate (MTX) and a tumour associated antigen (gp72) has been produced by fusing spleen cells from MTX immunised mice with 791T/36/3 (anti-gp72) hybridoma. The hybrid antibody was purified from anti-MTX and anti-gp72 antibodies present in the hybridoma culture supernatant by combinations of affinity chromatography on a MTX-agarose immunoabsorbent and stepwise acid elution from Sepharose-Protein A. A particular feature of the present antibody is that it reacts with conjugated MTX; this would allow in vivo targeting of conjugates, increasing many fold the number of molecules of drug carried or localising to pre-targeted antibody. Dual binding between tumour cell surface antigen and MTX was demonstrated by the ability of the hybrid antibody to bridge between tumour cells and MTX as MTX-HSA conjugate, reaction here being detected by flow cytofluorimetry. Purified hybrid antibody specifically enhanced the in vitro cytotoxicity of MTX-HSA for gp72 positive tumour cells.

Monoclonal Antibodies for Targeted Therapy with Vindesine

Abstract Four different vindesine monoclonal antibody conjugates recognising human tumours were prepared. The molar conjugation ratio of drug to antibody varied according to the monoclonal, in the range 3:1 to 10:1. Full antigen binding was retained by two conjugates and markedly reduced in one. Pre-treatment of target cells with the conjugates produced dose-related inhibition of cell growth in subsequent culture. Specificity could be demonstrated and was associated with the presence of target antigens on the tumour cells.

A CD66a-specific, activation-dependent epitope detected by recombinant human single chain fragments (scFvs) on CHO transfectants and activated granulocytes.

Antibodies to CD66 recognize at least five members (CD66a–e) of the carcinoembryonic antigen (CEA) family. Recombinant human single‐chain Fv fragments (scFvs) that bind specifically to CD66a (biliary glycoprotein) were obtained from a naive human scFv library. The scFvs bound to the N‐domain of CD66a on Chinese hamster ovary (CHO) transfectants but did not bind to freshly isolated peripheral granulocytes or to dimethylsulfoxide‐treated HL‐60 cells. In contrast, scFvs bound well to granulocytes that were short‐term activated with N‐formyl‐Met‐Leu‐Phe or phorbol 12‐myristate 13‐acetate and to human HL‐60 cells that were treated with all‐trans‐retinoic acid to induce granulocytic differentiation. Quantification of antigenic sites showed that the activation‐dependent CD66a epitopes were expressed on nearly all of the CD66a molecules on CHO‐biliary glycoprotein transfectants, but they were detected only on a portion of the molecules on activated polymorphonuclear neutrophils and differentiated HL‐60 cells. Binding of CD66a scFvs to their neoepitopes on prestimulated PMNs induced respiratory burst, suggesting that CD66a is capable of delivering transmembrane signals in these cells.

Antigenicity and drug susceptibility of human osteogenic sarcoma cells “escaping” a cytotoxic methotrexate-albumin-monoclonal antibody conjugate

Cells of osteogenic sarcoma line 791T were treated in vitro with a selectively cytotoxic methotrexate-human serum albumin-monoclonal antibody conjugate at concentrations which were toxic but allowed the “escape” of a small number of tumour cell colonies (less than 0.3% compared with controls). These colonies were propagated as clones in order to test their expression of the monoclonal antibody ( 791T /36)-defined antigen and their resistance to methotrexate (MTX) by comparison with parental cells. Most of the conjugate-treated clones were incapable of prolonged growth and died out, in contrast to untreated 791T clones which virtually always grow progressively. Only four treated clones grew at rates comparable with the parental line. Flow cytofluorometric analysis indicated that the surviving clones expressed normal or enhanced amounts of 791T /36-defined antigen and clonogenic assays demonstrated that they were sensitive to cytotoxicity by MTX. As could be predicted from these results, further exposure to the conjugate inhibited growth of the clones at doses comparable with those active against parental 791T cells. It is concluded that tumour cell clones emerging after exposure to a toxic concentration of a drug-antibody conjugate are not necessarily modified resistant clones, but may have severely impaired long-term growth potential or be susceptible to further contact with the same conjugate.

Recombinant ricin toxin A chain cytotoxicity against carcinoembryonic antigen expressing tumour cells mediated by a bispecific monoclonal antibody and its potentiation by ricin toxin B chain.

A bispecific monoclonal antibody recognising both carcinoembryonic antigen (CEA) and ricin toxin A chain (RTA) was tested for its ability to target recombinant RTA (r-RTA) to CEA-expressing tumour cells, alone and in combination with ricin B chain (RTB). The antibody, 636 (Robins et al., 1990), induced significant RTA cytotoxicity against MKN45 gastric carcinoma cells which express high levels of CEA, using the r-RTA at a concentration below that known to be intrinsically cytotoxic. The addition of ricin toxin B chain (RTB) also potentiated cytotoxicity of r-RTA, and there was an additive increase in potentiation against CEA-positive cells when both RTB and 636 were included. The bispecific antibody restored potentiation by RTB after blocking of its binding site with excess galactose, and also the cytotoxic activity of whole ricin which had been blocked with galactose. It was concluded that the 636 bispecific antibody was highly effective in targeting the toxic moiety of the molecule to CEA-expressing cells, and allowed exploitation of the additional ability of the B chain to facilitate cellular incorporation. The facilitating function of the B chain was equally effective whether or not its lectin site was active.