Enhanced recognition of human colorectal tumour cells using combinations of monoclonal antibodies.

Murine monoclonal antibodies directed against tumour associated antigens are potentially useful in tumour diagnosis and therapy. However, all the antigens they recognise may be heterogeneously expressed on tumours and this may allow escape of cells from therapy if a single monoclonal antibody is used. One approach is to use combinations of monoclonal antibodies recognising complementary cell surface antigens. A flow cytometric method which allows accurate quantitation of the intensity of staining and the percentage of fresh primary tumour cells binding a series of monoclonal antibodies has therefore been developed. This allows calculations as the number of drug molecules which could be potentially delivered by each monoclonal antibody and the optimal combination of antibodies which should be used. Monoclonal antibodies recognising Y hapten (C14), CEA (228, 161) and 791T-p72 antigen (791T/36) have been screened as a possible combination for colorectal cancer. There was inter-tumour variation in the binding of all the monoclonal antibodies although combinations could reduce or abrogate this problem. A combination of the monoclonal antibodies C14, 228, 791T/36 and 161 would recognise 100% of tumours. Sixty per cent of tumours bound all four antibodies, 78% any three, 90% any two and 100% any one antibody. There was also intra-tumour variation in the number of tumour cells per lesion that were recognised, the best monoclonal antibody, 161, stained a mean of 59% of cells per tumour whereas the anti-cytokeratin monoclonal antibody stained a mean of 74% of cells per tumour. An increased intensity of staining of tumour membranes was observed when a combination of C14 and 228 was used compared to binding of individual antibodies. Furthermore there was still no significant binding to normal colon membranes. Combinations of monoclonal antibodies which recognise a high percentage of tumours are likely to be necessary for monoclonal antibody drug targeting to prevent tumour recurrence and/or metastases.

Intraperitoneal 131I- and 111In-791T/36 monoclonal antibody in recurrent ovarian cancer: imaging and biodistribution.

An examination of the biokinetics and biodistribution of i.p. administered 131I- or 111In-labelled 791T/36 antibody (1 mg) has been carried out in five patients with stage III/IV ovarian cancer. Blood kinetics and urinary excretion of the radiolabels were assayed. Scintigraphy was performed immediately following administration and before and after peritoneal lavage at 48 h. Blood levels of both preparations rose over the first 20-40 h reaching 8-14% of the administered dose in the circulation and then declined (T1/2 of 40 h). Circulating radiolabel was still attached partially to antibody as shown by precipitation with anti-mouse IgG antiserum. The rapid appearance of radiolabel in the bloodstream meant that any tumour localization could be from circulating antibody rather than local infiltration. Interpretation of the images was difficult and the distribution of the tracer was different from that previously observed using i.v. administration of antibody. In some cases the images were confusing and the uptake of activity did not fit in with the clinical knowledge of the disease or the findings from laparoscopy. Tumour specimens resected at 4-5 days showed up to 0.02% of the dose g-1.

Humoral immune responses to XMMCO-791-RTA immunotoxin in colorectal cancer patients.

Monoclonal antibody 791 (XMMCO-791) recognizes a colorectal tumour-associated antigen. Antibody 791-ricin A chain immunotoxin (XMMCO-791-RTA) inhibits growth of human tumour xenografts and it is therefore being evaluated for the treatment of colorectal cancer. One of the problems with therapy with mouse monoclonal antibodies is they stimulate humoral responses in patients. However antigens linked to ricin are cytotoxic for B cells and therefore XMMCO-791-RTA may not be immunogenic. The humoral antibody response to murine monoclonal antibody XMMCO-791 (IgG2b) conjugated to the plant toxin, ricin A chain (RTA), was measured in colorectal cancer patients in a phase I clinical trial. All patients produced strong responses to the XMMCO-791 immunoglobulin and to RTA. The predominant response to the antibody was against the idiotypic determinant although anti-subclass and anti-mouse antibodies were also detected. A component of the anti-idiotypic immunoglobulin response in the colorectal cancer patients was directed against the combining site of XMMCO-791. These antibodies inhibited in-vitro binding of XMMCO-791 to target 791 cells and so may be inhibitors of repeated immunotoxin therapy. Immunotoxins do not abrogate the immune response to mouse immunoglobulin in vivo but instead are highly immunogenic.

Abrogation of antibody responses in rats to murine monoclonal antibody 791T/36 by treatment with daunomycin-cis-aconityl-791T/36 conjugates.

The majority of monoclonal antibodies in clinical use are of murine origin. It is now well-established that patients generate an antibody response to the mouse immunoglobulin which restricts repeated administration. Pre-sensitization of patients to mouse antibody is screened by hypersensitivity to i.d. administered antibody. This study shows that low doses of mouse antibody administered either i.d. or s.c. are highly immunogenic and suggests that a serological assay would be a safer method of screening for anti-mouse antibodies. Rats treated with monoclonal antibody linked via an acid labile cis-aconityl bond to daunomycin failed to produce a primary response to this conjugate. They were also rendered immunologically unresponsive to subsequent challenges with the unconjugated monoclonal antibody. The induced state of immunological unresponsiveness to free antibody persisted in the rats for 18 weeks and although antibody-cis-aconityl-daunomycin pre-treated animals eventually responded to the fourth challenge with free antibody, at week 25, the response was still significantly less than in the free antibody-pre-treated and challenged animals. These studies show that the use of antibody-cis-aconityl-daunomycin conjugates may provide an approach for the control of human responses to mouse immunoglobulin.

Sensitivity of newly established colorectal cell lines to cytotoxic drugs and monoclonal antibody drug conjugates.

A major problem in the chemotherapy of colorectal cancers is their resistance to most cytotoxic drugs which may be due to insufficient cellular transport. Drugs conjugated to monoclonal antibodies recognising tumour antigens may overcome these difficulties by providing access of active agents to the tumour cells. The anti-tumour monoclonal antibody shown to localise in patients with colorectal cancer, 791T/36, has been investigated as a potential targeting antibody. Eight cell lines were established from surgically resected material and were shown to bind 791T/36 antibody. They were screened for their sensitivity to methotrexate, 5-fluorouracil and daunomycin. Although 5-fluorouracil is the drug of choice for chemotherapy of colorectal cancer it was the most cytotoxic drug in only 2 of the 8 cell lines. Only the 4 cell lines which were resistant to methotrexate showed less cytotoxicity with methotrexate than 5-fluorouracil. The cell lines which were resistant to methotrexate were more sensitive to 791T/36-methotrexate conjugates. Daunomycin was the most cytotoxic drug in 4 of the 8 cell lines. However, a similar cytotoxicity was observed for free drug and 791T/36 daunomycin in the two lines tested. Selective monoclonal antibody drug conjugates may offer a solution to treatment of tumours which are resistant to classical chemotherapeutic agents. This is the first report to show that newly established cell lines that are resistant to classical chemotherapeutic agents are rendered sensitive when the drug enters the cell as a drug monoclonal antibody carrier.