Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome.

We have developed a method for the introduction of yeast artificial chromosomes (YACs) into transgenic mice. An 85 kilobase (kb) fragment of the human heavy chain immunoglobulin gene was cloned as a YAC, and embryonic stem cell lines carrying intact, integrated YACs were derived by co-lipofection of the YAC with an unlinked selectable marker. Chimaeric founder animals were produced by blastocyst injection, and offspring transgenic for the YAC were obtained. Analysis of serum from these offspring for human heavy chain antibody subunits demonstrated expression of the YAC-borne immunoglobulin gene fragment. Co-lipofection may prove to be a highly-successful means of producing transgenic mice containing large gene fragments in YACs.

Immunohistochemical detection of the Ca antigen in normal and tumor tissues of humans by use of Ca1 monoclonal antibody.

The Ca1 monoclonal antibody was used to detect Ca antigen in paraffin sections with the use of a two-stage indirect immunoperoxidase technique. A range of normal and malignant human tissues was examined. The antibody reacted with squamous cell carcinomas, small cell carcinomas, adenocarcinomas, ductal carcinomas, and anaplastic carcinomas from gastrointestinal, lung, and breast tissues. Melanomas were negative. Reactivity was also observed against normal tissues: urinary transitional epithelium, fallopian tube epithelium, breast ducts, sweat glands, sebaceous glands, alveolar epithelium, bile duct, gallbladder, kidney tubules, and some digestive tract epithelial cells. Although the study confirms earlier reports that Ca1 is a marker for some normal, nonciliated epithelial cells and for epithelial and some other cancers, a more widespread distribution for normal tissues was found.

Intratumor localization of monoclonal antibody in patients with melanoma treated with antibody to a 250,000-dalton melanoma-associated antigen.

Antibody localization at the tumor site was assessed in melanoma patients who received the murine monoclonal antibody 9.2.27. Antibody was administered twice weekly in escalating doses from 1 to 500 mg. Localization was assessed by biopsies of cutaneous and lymph node lesions obtained 24-96 hours following therapy. The percentage of tumor cells that bound the antibody in vivo was dose dependent, with similar findings obtained by either flow cytometry or immunoperoxidase staining techniques. Little or no in vivo binding of the 9.2.27 antibody to tumor cells was found following 1- and 10-mg doses, whereas all specimens demonstrated in vivo binding of the antibody following 200- and 500-mg doses. Fluorescence staining intensity, as quantitated by flow cytometry, was employed to determine the degree of in vivo saturation of antibody binding sites following therapy. The degree of saturation was found to vary substantially among patients: Some patients demonstrated nearly 100% saturation after 200-mg doses of 9.2.27 antibody, whereas others demonstrated only half maximal saturation after doses of 500 mg. Although immunoperoxidase staining provided important qualitative information regarding the distribution of antigen and antibody within the tumor, these studies demonstrated the usefulness of immunofluorescent flow cytometry for quantitative assessment of antibody localization in solid tumors and provided information necessary for the design of further trials of monoclonal antibodies and immunoconjugates.

Murine monoclonal IgG3 antibodies to human colorectal tumor-associated antigens: production and characterization of antibodies active in both antibody-dependent cellular cytotoxicity and complement-mediated cytolysis.

Murine monoclonal antibodies have been developed against human colorectal tumors using immunogens consisting of extracts from xenografted human colon carcinoma bound to lectin-conjugated agarose beads. Antibodies of the IgG3 isotype were selected from the resulting fusions, since we and other investigators have reported that this class of antibody can mediate antibody-dependent cellular cytotoxicity (ADCC). They were characterized with respect to specificity, ADCC activity, and complement-mediated cytolytic activity. One antibody, NR-Co-01, was produced using wheat germ agglutinin-agarose, whereas NR-Co-03, NR-Co-04, and NR-Co-05 were independently derived after immunization with a Dolichos biflorus lectin-agarose carrier. All four antibodies reacted intensely with the cytoplasm and cell surface of colonic adenocarcinomas and showed a restricted normal tissue reactivity. Recognition of epithelial cells in some normal adult tissues was revealed by immunoperoxidase staining of frozen tissues. No binding was found to normal lymphocytes, monocytes, granulocytes, and erythrocytes by immunofluorescence flow cytometry. All four of the antibodies were reactive with neutral glycolipids partitioned by Folch extraction of human colon carcinoma cell lines and also with a glycoprotein extracted from ovarian cyst mucin, suggesting that they may recognize one or more carbohydrate structures. The NR-Co-01, NR-Co-03, NR-Co-04, and NR-Co-05 are potent inducers of ADCC with human peripheral blood mononuclear effector cells and of complement-mediated cytolysis with human complement. However, they exhibited no ADCC activity with nude or normal mouse peripheral blood mononuclear cells at target:effector ratios ranging from 40:1 to 160:1. Their tumor reactivity, restricted normal tissue distribution, and ability to direct ADCC and complement-mediated cytolysis make these antibodies promising candidates for cancer therapy.

Murine monoclonal IgG3 to human colorectal tumor-associated antigens: enhancement of antibody-dependent cell-mediated cytotoxicity by interleukin 2.

Murine monoclonal antibodies (MAB) of the IgG3 subclass generated to colorectal tumor-associated glycolipids were assessed for enhancement of antibody-dependent cell-mediated cytotoxicity (ADCC) by interleukin 2 (IL-2). Mononuclear cell preparations containing large granular lymphocyte effectors required only low doses of IL-2 (0.1-10 units/ml) and short exposure (3 h) for maximal enhancement of ADCC. Exposure of mononuclear cells for longer periods (1-6 days) to higher levels of IL-2 (100-1000 units/ml) resulted in the generation of lymphokine-activated killer N cells which were lytically active without antibody. A non-ADCC MAB of the IgG1 subclass showed no ADCC even after stimulation of effector cells with IL-2. Effector cells pretreated with IL-2 showed an enhanced rate of cytolysis of target cells in the presence of antibody. Pretreatment of effector cells with IgG3 MAB resulted in lower ADCC, but pretreatment of target cells or simultaneous addition of MAB and effectors to target cells gave higher levels. These results indicated that ADCC with murine IgG3 is enhanced by levels of IL-2 achievable in current patient trials. The combination of antibody and IL-2-boosted effector cells gives comparable levels of killing to lymphokine-activated killer cells but should not suffer from similar toxicity. The NR-Co-04 antibody in combination with lymphokine may prove effective in treating colon cancer, a disease for which both chemotherapeutic and current biological therapies suggest a need for improved forms of therapies.

Cobalamin analogues modulate the growth of leukemia cells in vitro.

Analogues of cyanocobalamin (CN-Cbl), with functional groups attached to either the various propionamide groups of the corrin ring or to the ribose-nucleotide linker arm, have been evaluated in a cobalamin (Cbl)-dependent in vitro cell growth assay. In this bioassay, CN-Cbl supported, in a dose-dependent manner, the growth of the murine lymphoma BW5147 and the Cbl carrier protein, human apo-transcobalamin II, reduced the required concentration of Cbl by 100-1000-fold. Any chemical modification of Cbl decreased its ability to support cellular viability and proliferation, with several of the modifications abrogating activity completely. All of the Cbl analogues that promoted growth required the presence of apo-transcobalamin II for the optimal support of cell growth. Generally, Cbl analogues modified at the d-position of the corrin ring and, to a lesser degree, analogues modified at the b- position supported cell growth, whereas analogues with modifications at the e-position did not support cell growth. Mixing experiments demonstrated an inverse order of potency of Cbl analogues to inhibit cell growth. Thus, Cbl analogues with modifications at the e-position were potent inhibitors, whereas b-analogues exhibited only partial inhibitory activity at high molar excess, and d-analogues had no inhibitory activity at all. These results indicate that modifications at the e-position of Cbl abolish the ability of Cbl to support cell growth and generate potent inhibitors of Cbl-dependent cell growth.

Induction of apoptosis in neoplastic cells by depletion of vitamin B12.

Methionine synthase, a critical enzyme in deoxyribonucleotide biosynthesis for DNA replication, requires vitamin B12 as a cofactor. We have tested the hypothesis that depletion of cells of vitamin B12 would block growth of neoplastic cells and divert them into apoptosis and could form the basis of a new therapeutic strategy for cancer treatment. Using nitrous oxide to inactivate vitamin B12 we show that, in a variety of cell lines in vitro, methionine synthase is rapidly inhibited, the cells cease proliferation and undergo apoptosis. The kinetics of cell death, once started, are similar to those observed following methotrexate treatment or serum withdrawal. This is the first observation of apoptosis being induced following depletion of an essential metabolite as opposed to the more conventional strategy of adding a toxic drug to damage cells thereby triggering apoptosis. Moreover, vitamin B12 depletion has no effect on the nonproliferating cell population.

Monoclonal antibody therapy of malignant melanoma: in vivo localization in cutaneous metastasis after intravenous administration.

The murine antimelanoma monoclonal antibody, 9.2.27, was administered intravenously to eight patients with metastatic malignant melanoma. Biopsies of metastatic nodules clearly demonstrate the selective localization of this antibody on the melanoma cell surface with a dose-response relationship to the quantity of administered antibody. The antibody infusions were clinically well tolerated and the pharmacokinetics of the antibody and the antiglobulin responses are described. This study indicates that murine monoclonal antibodies have potential as selective targeting agents in the design of future therapeutic trials using monoclonal antibodies or conjugates thereof in the treatment of cancer.

Effect of antibody dose on the imaging and biodistribution of indium-111 9.2.27 anti-melanoma monoclonal antibody.

Eleven patients with metastatic melanoma underwent serial gamma camera imaging and biodistribution measurements after i.v. injection of escalating doses of [111In]9.2.27, an antimelanoma murine monoclonal antibody. Patients received a fixed dose of 1 mg of [111In]9.2.27, with no additional 9.2.27 (five patients), or co-infused with 49 mg (five patients) or 99 mg (one patient) of unlabeled, unconjugated 9.2.27. Higher doses resulted in prolonged blood-pool retention, less uptake in spleen and bone marrow, and appeared to have a positive effect in improving tumor imaging. A dose of 1 mg of 9.2.27 permitted detection of tumors in two of five patients and two of ten lesions, while with greater than or equal to 50 mg, tumors were detected in all patients and in 24 of 32 lesions. Human gamma globulin injected prior to administration of [111In]9.2.27 failed to block the prominent liver, spleen, and bone marrow uptake. No toxicity was observed. These results indicate the feasibility of imaging metastatic melanoma with [111In]9.2.27 and suggest that antibody dose may be a critical determinant of biodistribution and tumor uptake.

Successful imaging of malignant melanoma with technetium-99m-labeled monoclonal antibodies.

F(ab')2 and Fab fragments of murine monoclonal antibody 9.2.27, that recognizes the 250 kD melanoma-associated antigen, were labeled with 99mTc using the bifunctional chelate method of Fritzberg et al. Twenty-seven (27) patients received, intravenously, 10 mg of either F(ab')2 (8), or the Fab (27), labeled with up to 30 mCi of 99mTc. These doses were preceded by an infusion of cold irrelevant antibody. The average serum T1/2 of the F(ab')2 and the Fab were 11 hr and 2 hr, respectively. Twenty-two percent (22%) of the total injected F(ab')2 dose was excreted in the urine in 20 hr, compared to 55% for the Fab group. Imaging was optimal 6-9 hr postinjection for the Fab patients. No nonspecific uptake in liver, spleen, bone marrow, or lung was observed for either antibody form. Overall, (43/53) 81% of known metastases were seen with visualization of tumors as small as 250 mg and tumor localization as high as 0.03% injected dose/g. Immunoperoxidase staining of freshly-frozen tumor nodules removed 24 hr postinjection confirmed antibody deposition in the tumor. Thirty-six previously unknown ("occult") metastatic sites were detected. To date, 12/36 of these sites have been confirmed. We conclude that 99mTc-labeled antibody to melanoma produces high resolution images with a high sensitivity of detecting metastatic melanoma. The detection of previously unknown sites of disease has proven helpful in directing additional diagnostic studies (i.e., CT) as well as planning of therapeutic options.

Transcobalamin II and in vitro proliferation of leukemic cells.

We have recently shown that antibodies to transcobalamin II (TCII) inhibit the in vitro growth of human and murine leukemic cells. This antiproliferative strategy targets the uptake of cobalamin (Cbl), an essential cofactor for two biochemical reactions in humans. To date there has been no appropriate cell culture model available to study antagonism of Cbl as a potential antiproliferative strategy. We have established cell culture conditions which allow reproducible measurements of cell proliferation that is dependent on Cbl and its carrier protein, TCII. This bioassay has allowed us to demonstrate that several monoclonal antibodies, raised against TCII, are potent inhibitors of cell proliferation and that excess Cbl abrogates this inhibitory effect. Thus, supporting our hypothesis that interference with Cbl uptake or metabolism will result in inhibition of cell proliferation. Furthermore, Cbl metabolism appears to provide a useful target for antiproliferative strategies which now involve the use of inactive Cbl analogs. In this review, we update our work on the role of targeting TCII and Cbl as an antiproliferative strategy for leukemic cells. We suggest that this strategy may provide a novel direction for anti cancer reagents.

Monoclonal antibodies to human colorectal tumor-associated antigens: improved elicitation and subclass restriction.

Monoclonal antibodies to tumor-associated antigens (TAA) of human colorectal cancer were elicited using immunosorbents of lectins combined with peripheral protein extracts of xenografted colon adenocarcinoma. This method of immunization was compared with whole cells from surgical specimens and to crude membranes from xenografted tumors. The immunosorbent immunogens were superior to the other immunogens in three ways: (1) the number of hybrids reactive with colon tumor cells or extracts, but not with lymphoid cells or extracts, (2) the number of stable hybrids after cloning, and (3) the number of hybridoma clones reactive with tissue sections of colon tumors, but not normal colonic mucosa. In addition, lectin immunosorbents elicited primarily IgG antibodies, especially IgG3, with almost 50% of the clones of interest reacting to seemingly less immunogenic glycoproteins. The improved elicitation of monoclonal antibodies to TAA by the use of lectin immunosorbents and peripheral protein extracts has considerable potential for generating reagents useful in diagnosis and therapy of human tumors.

Community critical care course: blueprint for success.

For 10 years, a group of 19 California hospitals has supported a community-based critical care educational program for entry-level critical care nurses. Program management is rotated every two years. Two courses are offered quarterly: a 32-hour ECG course and a 96-hour Critical Care Nursing course. The community-based approach is very well received because it provides broad-based critical care educational programs in a very cost-effective manner.

Monoclonal antibodies in the treatment of cancer: preliminary observations and future prospects.

The need for improved specificity in cancer therapy is apparent. With the advent of monoclonal antibodies, the possibility of specifically targeted therapy is here. Early trials with monoclonal antibody in experimental animals and in man have demonstrated antibody can travel to specific tumor sites and localize on or around the tumor cells displaying antigens to which the antibody is directed. This evidence of specific targeting, along with early evidence of therapeutic efficacy for monoclonal antibodies and antibody immunoconjugates with drugs, toxins and isotopes, is encouraging. Some preliminary clinical observations from two monoclonal antibody trials are presented and the current status of clinical trials with monoclonal antibodies is reviewed.

Monoclonal antibody therapy in malignant melanoma: factors effecting in vivo localization.

Thirteen patients with metastatic malignant melanoma received intravenous therapy with the murine antimelanoma monoclonal antibody 9.2.27. Five patients were entered on a dose escalation protocol with twice weekly escalating doses of 10-500 mg, in an extension of a previously reported trial. These patients demonstrated near saturation of available antibody binding sites in vivo following the 500 mg dose, with minimal toxicity. The remaining patients were entered onto a dose schedule comparison study, with a 500 mg dose administered either in a single 2 h infusion or as five daily 2 h infusions of 100 mg to examine the effects of different dose schedules and of an interrupted schedule on subsequent therapy with the same antibody. Intratumor localization of the monoclonal antibody did not appear to vary with respect to the dose schedule; however, interruption in therapy for 4 weeks was accompanied by somewhat poorer localization of antibody. This effect appeared to be primarily attributable to development of human antimurine antiglobulin in 25-30% of patients with resultant decrease in intratumor localization of antibody and more rapid clearance of the 9.2.27 antibody from the circulation. Earlier reports with other antibodies notwithstanding, initial infusions of 500 mg of 9.2.27 did not induce tolerance to the murine immunoglobulin. This study confirms and extends the findings of our initial trial of the 9.2.27 antibody by demonstrating that, although clinical responses were not observed, the antibody can be safely administered at doses up to 500 mg, with good intratumor localization of antibody. The diminished localization of antibody associated with antiglobulin responses indicates the importance of monitoring antiglobulin levels during therapy, and the necessity of controlling or preventing this phenomenon when monoclonal antibodies are administered in multiple doses as drug, toxin, or radionuclide immunoconjugates.

Characterization of a human-mouse chimeric antibody reactive with a human melanoma associated antigen.

The data presented demonstrate that a human-murine chimeric antibody has been generated that retains the immunoreactivity and has similar pharmacokinetic properties of its parent murine monoclonal antibody NRML-05. A competitive ELISA assay demonstrated that antigen reactivity of both parent and chimera were nearly identical. In a direct cell binding assay, NRML-05 and chimeric antibodies were immunoreactive, 81% and 83%, respectively. Scatchard Analysis of the antibodies indicate very similar affinities for NRML-05 (4.4 x 10(9) M-1) and chimera (2.7 x 10(9) M-1). Localization of the two antibodies to tumor xenografts in nude mice were very similar in biodistribution studies. The chimeric antibody is not as well recognized by antiglobulin from patients who have responded to non-idiotypic murine antibody determinants. Although this data does not predict how immunogenic a chimera would be in the clinical setting, it does suggest that patients without significant anti-idiotypic antiglobulin response could benefit from second and subsequent administrations with chimeric antibody. Even though anti-idiotypic responses may occur with the chimera, these may be reduced as a result of the presentation of these epitopes on the less immunogenic human constant domains.

Localisation and toxicity study of a vindesine-anti-CEA conjugate in patients with advanced cancer.

Safety of administration of a vindesine (VDS)-anti-CEA conjugate and its ability to localise after radiolabelling were investigated in patients with advanced metastatic carcinoma (4 colorectal and 4 ovarian). For imaging, patients received between 230 and 520 micrograms of 131I labelled antibody. In 5, localisation of conjugate was demonstrated, in another it was equivocal and in 2 patients, undetectable. For assessment of safety each patient also received a single dose of conjugate increasing from 1.2 to 42 mg antibody linked to 24 to 1800 micrograms VDS. The in vitro activity of the anti-CEA antibody and its ability to localise in vivo were preserved after conjugation. There was no obvious toxicity or hypersensitivity attributable to either the radiolocalisation or escalated doses of conjugate in any of the patients. The feasibility of the preparation and administration to patients of a vindesine-antibody conjugate has been demonstrated.

Carcinoembryonic antigen (CEA) expression and heterogeneity in primary and autologous metastatic gastric tumours demonstrated by a monoclonal antibody.

The expression of carcinoembryonic antigen (CEA) in gastric malignancies has been assessed using a monoclonal antibody in an immunoperoxidase technique. Of 119 primary tumours examined, 92% reacted with the antibody. Metastases were available for 81 of the patients and 83% were CEA positive. A noteworthy observation was the detection of malignant cells in the lymph nodes of two patients, as a result of the presence of CEA, who were originally reported to be free of metastases. Of those patients whose primary tumours expressed CEA, 86% had at least one CEA positive metastasis. Two or more metastases were available from 60 of the patients and in 20% the secondaries were a mixture of positive and negative for CEA. Consequently, the CEA status of a single lesion does not enable confident prediction of expression in other metastases. In addition to variation between multiple lesions removed from the same patient phenotypic diversity of expression was observed between tumour cells of a given mass. Such distribution of the CEA detected by this monoclonal antibody may impose certain restrictions on its application. However, the high frequency of expression by gastric cancers indicate that it is a potentially useful antigen as a target for radiolocalisation or therapeutic agents.

The generation of monoclonal anti-idiotype antibodies to human B cell-derived leukemias and lymphomas.

We developed murine anti-idiotype monoclonal antibodies for each of four patients with B cell-derived leukemias and lymphomas. Idiotypic immunoglobulin was isolated from mouse X human tumor-cell hybridomas or from patients' serum and was used to immunize mice for the development of murine anti-idiotype monoclonal antibodies. Each patient's anti-idiotype antibodies demonstrated reactivity restricted to the immunizing immunoglobulin, thereby limiting their therapeutic utility to a single individual. In addition, we isolated isotype switch variants of hybridomas producing monoclonal anti-idiotypic antibody. The restricted specificity of these antibodies was found to be of value for the analysis of the extent of malignant B cell infiltration in a variety of tissues from several patients. Large populations of idiotype-bearing cells were detectable in biopsy specimens from patients K.T. and L.H. In contrast, although bone marrow specimens from patient G.D. were apparently devoid of morphologically abnormal cells, a small, highly fluorescent population of cells was demonstrable underscoring the potential utility of these antibodies for posttreatment evaluation as well as for therapy. In a fourth patient, H.M., anti-idiotype antibodies developed against the circulating macroglobulin isolated from his plasma failed to react with either his circulating or bone marrow hairy cell leukemia cells. However, examination of an enlarged inguinal lymph node revealed the presence of a large number of idiotype-bearing cells. Thus, the presence of two distinct malignant B cell clones were discovered in this individual through the use of anti-idiotype monoclonal antibodies. Anti-idiotype antibodies, therefore, represent a highly specific tool for the evaluation and potential therapy of B cell malignancies in individual patients.

Detection of two distinct malignant B cell clones in a single patient using anti-idiotype monoclonal antibodies and immunoglobulin gene rearrangement.

Immunoglobulin gene rearrangement analysis and somatic cell hybridization techniques were used to examine the malignant cell population in an unusual patient with hairy cell leukemia and macroglobulinemia (N Engl J Med 296:92, 1977). Although previous investigations suggested that the IgM macroglobulin was secreted by the circulating leukemia cells, anti-idiotype monoclonal antibodies raised to the IgM macroglobulin failed to react with the malignant cells in the circulation and bone marrow. In contrast, approximately 50% of the mononuclear cells from an enlarged inguinal lymph node reacted strongly with the anti-idiotype antibodies. Subsequent reanalysis of all cell populations demonstrated that whereas the circulating and bone marrow cells were IgM kappa-bearing, the macroglobulin was IgM gamma-bearing and the lymph node cells were evenly divided among IgM kappa-bearing and IgM gamma-bearing. Immunofluorescence flow cytometry indicated that those lymph node cells that reacted strictly with the anti-idiotype antibody were IgM gamma-bearing, demonstrating that they were the source of macroglobulin. An analysis of immunoglobulin gene DNA confirmed the coexistence of two distinct malignant B cell populations in the lymph node and indicated that the IgM kappa-bearing lymph node cells were identical to the circulating and bone marrow leukemic cells.