Immunization with human monoclonal antiidiotypic antibody in colorectal cancer.
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Antibodies, Anti-Idiotypic↗
Biomedical subjects
L G Durrant
Publications and source records attributed to L G Durrant.
Measurement of antibody binding to antigenic peptides conjugated in situ to albumin-coated microtitre plates.
Monoclonal antibodies have been prepared against a synthetic peptide with a sequence corresponding to a repeated hydrophilic region of the protein core of the human MUC-2 gastrointestinal mucin. Peptide conjugates, prepared by glutaraldehyde cross-linking with keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), were employed as the immunogen and target antigen (for screening by ELISA), respectively. However, for the measurement of antibody binding to peptide by an ELISA procedure, an alternative strategy was developed and is described in this report: peptides were conjugated directly to BSA immobilized by physical adsorption to the surface of microtitre plate wells. This procedure permits peptides to be tested as target antigens by ELISA without prior preparation of peptide-carrier conjugates.
Amino Acid Sequence↗
The influence of epitope presentation on the immunorecognition of human colon carcinoma related mucin peptides.
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Amino Acid Sequence↗
Development of second generation monoclonal antibodies recognising Lewisy/b antigen by anti-idiotypic immunisation.
Five new monoclonal antibodies (Mabs) recognising the Lewisy/b hapten of the IgG isotype have been produced following immunisation with rat anti-idiotypic antiserum to C14, an IgM Lewisy/b hapten Mab and boosting with C14gp200 antigen. They have the same fine specificity as the original Mab binding to a cell surface and secreted antigen preferentially expressed by colorectal tumour cells.
Animals↗
Cell kinetics of the in vitro metaphase arrest technique and the clinical applications.
The in vitro metaphase arrest technique (crypt cell production rate-CPPR) has been used to measure human rectal mucosal proliferation. Study of preincubation times, dose response curves and lag phases suggest that a concentration of vincristine of 5 micrograms/ml and 16 hour preincubation with time point increments between 25 and 125 minutes give optimal conditions for measuring rectal mucosal proliferation. Twenty individuals had rectal CCPR repeated without intervention of any kind. Close correlation was found between the two values (r = 0.89 and P = 0.0001). The effect of polyethylene glycol bowel preparation was also studied in 35 subjects. There was good correlation (r = 0.66, P = 0.007). There was close correlation between rectal and caecal CCPR as measured in 20 patients who had colonoscopy (r = 0.72, P = 0.0003). The in vitro metaphase arrest technique is a useful parameter of rectal mucosal proliferation and may be used with confidence in a number of different clinical situations.
Biopsy↗
An idiotypic replica of carcinoembryonic antigen inducing cellular and humoral responses directed against human colorectal tumours.
A monoclonal anti-idiotypic antibody (anti-Id MAb) 708 (IgG2b), which inhibited the binding of NCRC23 (IgG1) MAb to CEA and prevented radiolabelled CEA from binding to MAb NCRC23, was produced. No recognition of 3 other anti-CEA antibodies, 3 other IgG1 or 2 IgM MAbs was observed with this anti-idiotypic antibody. When an immunoblotting technique was used, 708 anti-Id MAb failed to bind to isolated heavy or light chains of MAb NCRC23, whereas binding was observed with intact antibody. Mouse, rat and human lymphocytes (in vitro) were immunized with 708 anti-Id MAb and the resultant Ab3 antibodies all inhibited binding of labelled 708 anti-Id MAb to MAb NCRC23 and also reacted with CEA, showing that 708 anti-Id MAb induced anti-CEA antibody responses. Similarly, mice immunized with 708 anti-Id MAb could be restimulated in vitro with either CEA or tumour cells expressing CEA which induced specific T-cell proliferative responses. Human tumour-infiltrating lymphocytes isolated from colorectal tumours or peripheral blood T cells from cancer patients were stimulated in vitro with 708 anti-Id MAb or an irrelevant IgG2b antibody. Six days later both sets of lymphocytes were restimulated with CEA, and lymphocytes primed to 708 anti-Id MAb proliferated in response to CEA. These results suggest that 708 anti-Id MAb can act as an idiotypic replica of CEA and stimulate cellular and humoral anti-CEA immune responses. It is therefore of great interest as an idiotypic vaccine against colorectal cancer.
Animals↗
A rapid method for separating tumour infiltrating cells and tumour cells from colorectal tumours.
As solid tumours are composed of a heterogeneous mixture of cells the role of any particular cell type is difficult to analyse. A rapid method of sorting freshly disaggregated highly viable single cell suspensions of colorectal tumours has therefore been developed. Infiltrating cells were initially sorted from tumour cell suspensions using magnetic beads coated with a cocktail of monoclonal antibodies recognising leucocyte common antigen, CD45 (lymphocytes), CDw14 (macrophages and granulocytes) and Thy-1 antigen (stromal cells). Between 4-10 x 10(6) cells, 70-87% pure, were rapidly sorted. The tumour cells were then sorted from the remaining cell suspension using a cocktail of monoclonal antibodies which recognise 100% of colorectal tumours. Between 2-7 x 10(6) tumour cells, 70-87% pure, were rapidly sorted. Both populations of cells were 95% viable and able to grow in vitro.
Antibodies, Monoclonal↗
Inhibition of gastrin-stimulated growth of gastrointestinal tumour cells by octreotide and the gastrin/cholecystokinin receptor antagonists, proglumide and lorglumide.
The rat pancreatic cell line, AR42J possessed high-affinity gastrin and somatostatin receptors and its growth was stimulated by physiological gastrin-17 concentrations between 5 x 10(-11) mol/l and 10(-9) mol/l as measured by [75Se]selenomethionine uptake. The somatostatin analogue, octreotide (2 x 10(-7) to 2 x 10(-11) mol/l), reduced this stimulated growth. Gastrin-stimulated AR42J growth was also inhibited by proglumide (3 x 10(-4) mol/l) and lorglumide (3 x 10(-5) mol/l) at maximal G17 concentrations of 5 x 10(-11) and 10(-10) mol/l, respectively, and the analogues competed with [125I] gastrin-17 (5 x 10(-10) mol/l) for binding to gastrin receptors on AR42J (50% inhibitory concentrations, less than or equal to 10(-3) mol/l and 4 x 10(-6) mol/l, respectively. Octreotide reduced the basal growth of the human gastric cell line, MKN45G, (which is associated with intracellular gastrin immunoreactivity) in serum-free medium to 73% of control at a concentration of 2 x 10(-8) mol/l, which was reversed by gastrin-17 (10(-10) mol/l). Lorglumide (3 x 10(-5) mol/l) also reduced the basal growth to 30% of control, which was reversed to 78% by 10(-5) mol/l gastrin. Proglumide had no effect on the basal growth of MKN45G.
Animals↗
Antitumor immune response and interleukin 2 production induced in colorectal cancer patients by immunization with human monoclonal anti-idiotypic antibody.
The immunogenicity of human anti-idiotypic antibody has been investigated using a human monoclonal anti-idiotypic antibody (105AD7) which interacts with the binding site of 791T/36, a mouse monoclonal antibody against gp72 antigen. This antigen is frequently expressed in gastrointestinal cancer, therefore, six patients with advanced colorectal cancer have been immunized with 105AD7 as an aluminum hydroxide gel precipitate in a phase I clinical study. Cryopreserved blood mononuclear cells were tested for in vitro proliferative responses by [3H]thymidine incorporation; plasma samples were tested by enzyme-linked immunosorbent assay for anti-anti-idiotypic and antitumor antibodies, and for interleukin 2. Proliferative responses to gp72 positive tumor cells were seen in four of five patients tested; parallel in vitro responses to 105AD7 anti-idiotypic antibody were seen in most of these patients. Interleukin 2 was detected in the plasma of four of six patients after 105AD7 immunization, with peak levels up to 7 units/ml. No toxicity related to anti-idiotype immunization and no antitumor or anti-anti-idiotype antibodies were seen. This study shows that human monoclonal anti-idiotype 105AD7 is immunogenic in cancer patients, inducing cellular antitumor responses and interleukin 2 production. This suggests that human monoclonal anti-idiotype antibodies may have considerable potential for immunotherapy of human cancer.
Adult↗
Induction of delayed hypersensitivity to human tumor cells with a human monoclonal anti-idiotypic antibody.
There is considerable interest in the development of anti-idiotypic antibodies as vaccines in a number of diseases, including cancer. We have developed a human anti-idiotypic monoclonal antibody (105AD7) which binds at or very near to the binding site of mouse antitumor monoclonal antibody 791T/36. The 791T/36 antibody binds to a tumor-associated antigen (gp72) expressed on a number of human tumors, including colorectal and ovarian carcinomas and osteogenic sarcoma. This study shows that, in rats and mice, 105AD7 induces delayed-type hypersensitivity to human tumor cells bearing the gp72 antigen. Local transfer of delayed hypersensitivity was also demonstrated using lymphocytes from mice primed with 105AD7. These findings show that the human monoclonal anti-idiotypic antibody 105AD7 is likely to induce cellular immune responses to tumors in cancer patients.
Animals↗
Intracellular gastrin in human gastrointestinal tumor cells.
Flow cytometry and immunohistochemical analyses of the human gastric adenocarcinoma cell line MKN45G identified an intracellular peptide recognized by an anti-gastrin-17 (G17) antiserum but not by an anti-cholecystokinin-specific antiserum. Staining was not associated with the parental line MKN45, of which MKN45G is a clonal variant. The MKN45G cell line had elevated in vitro growth in serum-free medium in which the proliferation of MKN45G cells but not MKN45 cells was reduced to 58% of the control value by treatment with a rabbit anti-G17 antiserum. This inhibition of proliferation was reversed by preabsorbing the antiserum with excess G17. Disaggregated primary human gastric and colorectal tumors were screened for gastrin immunoreactivity by flow cytometry, and 6 of 28 colorectal and 8 of 22 gastric tumors had greater than 20% positively staining cells.
Adenocarcinoma↗
Reactivity of an anti-(human gastric carcinoma) monoclonal antibody with core-related peptides of gastrointestinal mucin.
A murine anti-(human gastric carcinoma) monoclonal antibody, GL-013 (IgG1), which reacts with a high-molecular-mass glycoprotein from colorectal tumour tissue [Yang and Price (1989) Anticancer Res 9: 1707], was examined for reactivity against a panel of purified and partially purified antigens associated with tumours of the gastrointestinal tract. These included carcinoembryonic antigen (CEA), normal cross-reacting antigen, Y-hapten glycoproteins, and perchloric acid extracts and glycolipid preparations from colorectal tumours. While the GL-013 antibody failed to bind to these antigens, it was found to react strongly with synthetic peptides with sequences based upon that reported for the protein core of a human gastrointestinal mucin [Barnd et al. (1989) Proc Natl Acad Sci USA 86: 7159; Gum et al. (1989) J Biol Chem 264: 6480]. In control tests, a series of other anti-(colorectal tumour) antibodies (IgG1 and IgG3), with broad reactivity towards gastrointestinal carcinomas, as well as an anti-CEA antibody, (IgG1) failed to react with the synthetic peptides. It is concluded that the anti-(gastric carcinoma) monoclonal antibody GL-013 binds to a threonine-rich peptide epitope expressed within the protein core of gastrointestinal mucins.
Adenocarcinoma↗
Co-stimulation of gastrointestinal tumour cell growth by gastrin, transforming growth factor alpha and insulin like growth factor-I.
Epidermal growth factor receptors and insulin like growth factor-I receptors were co-expressed on two gastric and three colorectal tumour cell lines. Previous studies have shown that gastrin receptors were also expressed at a low level or two of these cell lines. Both TGF alpha and IGF-I promoted cell growth in all of the cell lines tested. The cell doubling time of a colorectal cell line was reduced from 48 to 30-34 h. Furthermore the effects of the growth factors were additive. Each growth factor also increased the response of the cells to gastrin, but a combination of both growth factors and gastrin did not further increase growth.
Cell Division↗
The effect of the E2 prostaglandin enprostil, and the somatostatin analogue SMS 201 995, on the growth of a human gastric cell line, MKN45G.
The effect of enprostil and the somatostatin analogue SMS 201 995 on the growth of a clonal variant of the human gastric adenocarcinoma cell line, MKN45, was studied. The derived cell line grew twice as fast as MKN45 when grown as a xenograft line in nude mice. However, it did not respond trophically to gastrin either in vitro or in vivo (unlike MKN45) although it possessed the same number of gastrin receptors as the parental line. Gastrin production by the cell line during in vitro culture was twice that of MKN45; thus, the cell line was denoted MKN45G. When MKN45G was grown as xenografts in nude mice (n = 10/group), enprostil (20 micrograms/kg/day) significantly inhibited tumour growth when administered continuously by an osmotic mini-pump from day 1 to day 7 of a 20-day experiment, and induced tumour regression when administered from day 7 to day 14. Enprostil reduced postprandial serum gastrin levels when administered from day 7 to day 14 and prevented gastrin release by MKN45 in vitro. SMS 201 995 at doses of 25 and 240 micrograms/kg/day induced tumour regression when administered from day 1 to day 7 and the former dose reduced post-prandial serum gastrin levels at day 5. Gastrin release by MKN45G was not affected by SMS 201 995 in vitro, thus its effect may not be mediated directly via gastrin, requiring interaction between other hormones or growth factors in the in vivo situation.
Adenocarcinoma↗
Production of a human monoclonal antibody recognising a determinant on mouse IgG2b from a patient receiving mouse monoclonal antibody for diagnostic imaging.
The development of human antibodies recognising mouse immunoglobulins represents an obstacle to effective antibody therapy. This study shows that patients produce modest titres of antibodies (predominantly anti-mouse rather than anti-idiotypic) after a single low-dose injection for immunoscintigraphy, suggesting that repeated imaging with the same or a different antibody could be a problem. Fusion of the lymphocytes from a patient who had been imaged twice previously resulted in a monoclonal antibody that specifically binds to an IgG2b isotypic determinant. Anti-IgG2b antibodies predominated in this patient's serum. Production of human monoclonal antibodies from patients given mouse monoclonal antibodies not only allows a finer dissection of the immune repertoire but also provides possible reagents for controlling the human anti-(mouse Ig) response, for selection of class-switch variants of mouse monoclonal antibodies and enhancing tumour imaging.
Animals↗
Phase I study of monoclonal antibody-ricin A chain immunotoxin XomaZyme-791 in patients with metastatic colon cancer.
Monoclonal antibody 791T/36, recognizing a Mr 72,000 antigen on the surface of colon carcinoma cells, has been used to construct an immunotoxin by conjugating to it the ribosomal inhibitor protein, ricin toxin A chain. The antibody 791T/36 has been shown to bind to membranes of freshly disaggregated tumor cells from human colon tumors, and to localize in tumors in vivo. Subacute toxicology testing in rats receiving immunotoxin i.v. showed, at highest doses, weight loss, decreased serum albumin, and hepatocyte vacuolization without elevation in liver function tests. A Phase I dose escalation study was carried out in which 17 patients with metastatic colorectal cancer were treated with doses of immunotoxin ranging from 0.02 to 0.2 mg/kg/day in 1-h i.v. infusions for a 5-day course. Side-effects included a composite of signs and symptoms thought to be generic to ricin A chain immunotoxins, including decreased serum albumin, mild fever, and flu-like symptoms, all being reversible. Two additional findings, reversible proteinuria and mental status changes, were also noted which may be characteristic of this immunotoxin. By 10-20 days after therapy, most patients developed IgM and IgG antibodies against both the ricin toxin A chain and the immunoglobulin portion of the immunotoxin, which were asymptomatic. A strong anticombining site antibody response was seen. Biological activity manifest as mixed tumor regression was seen in five patients.
Adult↗
Flow cytometric screening of monoclonal antibodies for drug or toxin targeting to human cancer.
Monoclonal antibodies (MAbs) that are candidates for antibody-directed therapy were evaluated by a flow cytometric method. This method accurately quantitates the intensity of staining and the percentage of cells from freshly derived primary tumors expressing the relevant cell surface antigens. This method was applied to human colorectal, gastric, and ovarian carcinomas. It allowed calculations of the number of drug molecules that potentially could be delivered by each MAb as well as selection of the optimal combinations of antibodies for treatment of each type of cancer. The binding of all the MAbs varied among the tumors, although combinations of antibodies reduced this problem. A combination of MAbs C14 and NCRC-23 recognized 97% of colorectal tumors. A combination of C14, NCRC-23, and 791T/36 recognized 95% of gastric tumors. Combinations of either 791T/36 and C14 or 791T/36 and NCRC-11 recognized 80% of ovarian tumors. The number of cells binding with a single MAb varied within the tumor. The optimal anti-colorectal tumor antibody was NCRC-23 (anti-carcinoembryonic antigen), which recognized a mean of 65% of the large cells within a tumor at a mean antigen density of 4.9 X 10(5) sites/cell. The optimal anti-gastric tumor antibody was C14 (anti-Y hapten), which recognized a mean of 66% of the large cells within a tumor at a mean antigen density of 4.4 X 10(5) sites/cell. The optimal anti-ovarian antibody was 115/D8, which recognized 54% of the large cells at a mean antigen density of 4.2 X 10(5) sites/cell. These antigen densities were similar to those calculated for HLA/ABC antigens in colorectal and ovarian cancers. However, the gastric tumors expressed elevated levels of major histocompatibility complex class I antigens, with a mean density of 7.3 X 10(5) sites/cell. Combinations of antibodies that recognize a high proportion of tumor cells are likely to be necessary for MAb-drug targeting to prevent tumor recurrence and/or metastases.
Antibodies, Monoclonal↗
The in vitro growth response of primary human colorectal and gastric cancer cells to gastrin.
A series of 31 colorectal and 13 gastric primary human tumours were screened for their growth response to human gastrin-17 in vitro, as assessed by 75Se-seleno-methionine incorporation. Fifty-five percent of colorectal and 69% of gastric tumours showed a significant trophic response to the hormone. The responses were achieved at physiological gastrin concentrations (post-prandial circulating gastrin levels) in 35% of colorectal and 55% of gastric tumours. Lymphocytes from tumour-associated lymph nodes showed no response to the hormone and "normal" mucosal cells (obtained from the resection margin of the surgical specimen) showed lower mean levels of 75Se-seleno-methionine uptake (colorectal: 110%; gastric: 119%, expressed as a percentage of the control) when compared to tumours (colorectal: 151%; gastric: 147%). The small number of well differentiated and/or Dukes' stage A colorectal tumours examined were gastrin-responsive, but all the responsive gastric tumours were poorly differentiated. With respect to ploidy, 89% of diploid and 67% of aneuploid colorectal tumours responded trophically to gastrin. Patients with colorectal or gastric tumours may benefit from treatment with gastrin antagonists.
Animals↗