We are entering an era in which biological macromolecules can be designed to order and genetically or synthetically produced. If the experience with classical low molecular weight drugs is any guide, it will be possible in fact, easy to produce many more such agents than can be tested for effect in animals, let alone in humans. Predictive criteria are required for the process of rational design, and these must be based on an understanding of fundamental physiological and pharmacological principles. Monoclonal antibodies illustrate the point. Hundreds of new monoclonal reagents with possible clinical use are being developed every year. Each of them could be administered as whole antibody, F(ab')*, or Fab. Each could be conjugated to a toxin, a drug, a radionuclide, or a liposome containing one of those agents. Each could be class-switched to change effector functions or could be mutated in its binding site. As noted previously,' the combinatorial aspects of trying every possibility are out of the question even before one has considered such current and likely future developments as ( I ) chimeric molecules combining mouse variable and human constant domains; (2) Fv (variable domain) fragments; (3) recombinants consisting of murine hypervariable segments and human variable domain framework; (4) conjugates formed by genetically grafting a linker peptide onto the molecule; and (5) enzyme-antibody or toxin-antibody chimeras. At the end of this developmental process will be ligand molecules consisting of antibody-derived binding sites grafted onto molecules designed de novo. A predictive pharmacology of biologicals must be based on theoretical and experimental information from several hierarchical levels: global (whole body), regional, local, cellular, and molecular. Here we will focus on one study of whole body pharmacokinetics, one development in regional delivery, and one issue at the local, or tissue, level.

The pharmacology of monoclonal antibodies.

During the past decade, proof of the principle that peptide receptors can be used successfully for in vivo targeting of human cancers has been provided. The molecular basis for targeting rests on the in vitro observation that peptide receptors can be expressed in large quantities in certain tumors. The clinical impact is at the diagnostic level: in vivo receptor scintigraphy uses radiolabeled peptides for the localization of tumors and their metastases. It is also at the therapeutic level: peptide receptor radiotherapy of tumors emerges as a serious treatment option. Peptides linked to cytotoxic agents are also considered for therapeutic applications. The use of nonradiolabeled, noncytotoxic peptide analogs for long-term antiproliferative treatment of tumors appears promising for only a few tumor types, whereas the symptomatic treatment of neuroendocrine tumors by somatostatin analogs is clearly successful. The present review summarizes and critically evaluates the in vitro data on peptide and peptide receptor expression in human cancers. These data are considered to be the molecular basis for peptide receptor targeting of tumors. The paradigmatic peptide somatostatin and its receptors are extensively reviewed in the light of in vivo targeting of neuroendocrine tumors. The role of the more recently described targeting peptides vasoactive intestinal peptide, gastrin-releasing peptide, and cholecystokinin/gastrin is discussed. Other emerging and promising peptides and their respective receptors, including neurotensin, substance P, and neuropeptide Y, are introduced. This information relates to established and potential clinical applications in oncology.

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Peptide receptors as molecular targets for cancer diagnosis and therapy.

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the "abbreviated" CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients' sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.

Grafting of “Abbreviated” Complementarity-Determining Regions Containing Specificity-Determining Residues Essential for Ligand Contact to Engineer a Less Immunogenic Humanized Monoclonal Antibody

A current trend in cell biology is to study the mechanisms of regulatory cellular function through specific structural and func tional molecules, i.e., growth factors, their receptors, membrane transporters, cytoskeletal systems, surface membrane and pericellular components, etc., rather than classical enzymes. The key molecules involved in such functional regulation are modified by 2 major chemical processes, phosphorylation and glycosylation. A greal deal of interest has been focused on phosphorylation as a ubiquitous and essential modulatory mechanism of cellular function. This idea has been greatly reinforced by the fact that many transforming genes (cellular as well as viral oncogenes) encode phosphoproteins, which by themselves are protein kinases, although the functions of many of their substrates have not been identified (see, for a review, Ref. 9). Less attention has been paid to the functional significance of glycosylation than to that of phosphorylation; nevertheless, the changes in glycosylation occur as quickly and dramatically as the process of phosphorylation at various stages of develop ment, differentiation, and oncogenesis (see, for a review, Ref. 38). While phosphorylation patterns are few, the patterns of glycosylation are numerous; over 100 types of glycosylated structures have been characterized among those bound to lipids (ceramides), i.e., glycolipids (see, for a review, Ref. 39). Similar variation of glycosylation can also be found in the peripheral region of carbohydrates in glycoproteins, in addition to the structural variation in the core region of carbohydrate chains bound to proteins through Nor O-glycosides (see, for reviews, Refs. 42 and 65). In contrast to phosphorylation, which directly modulates pro tein function, glycosylation affects the conformation, localization, and organization of functional and structural proteins; thus, glycosylation determines the turnover and organizational frame work of proteins within cytoplasm, membranes, and the pericellular matrix (see, for reviews, Refs. 42 and 87). LJpidglycosylation in membranes, particularly cell surface membranes, offers cell recognition sites for cell-social events and may regulate the function of membrane proteins such as receptors and adhesive proteins (38, 115) (Table 1). With the functional significance of

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Aberrant glycosylation in cancer cell membranes as focused on glycolipids: overview and perspectives.

Targeting of iodine-123-labelled tumour-associated monoclonal antibodies to ovarian, breast, and gastrointestinal tumours

To determine whether tumors containing carcinoembryonic antigen could be detected by administration of a Radio-labeled, affinity-purified, goat IgG having 70 per cent immunoreactivity against carcinoembryonic antigen, 18 patients with a history of cancer of diverse histopathology received an average total dose of 1.0 mCi of 131l-labeled IgG. Total-body photoscans were performed with a gamma scintillation camera at various intervals after administration of the radioactive antibody. Ordinary photoscans proved difficult to interpret because of blood-pool background radioactivity, thus necessitating the computer subtraction of radioactive blood-pool agents from the antibody's 131I activity. Tumor location could be demonstrated at 48 hours after injection in almost all cases studied. The scans were negative in patients without demonstrable tumors or with tumors apparently devoid of carcinoembryonic antigen. Circulating antigen levels of up to 350 ng per milliliter did not prevent successful tumor imag...

Use of Radiolabeled Antibodies to Carcinoembryonic Antigen for the Detection and Localization of Diverse Cancers by External Photoscanning

Our recent clinical experience in the radioimmunodetection of cancer in 142 patients with a proven history of cancer is summarized. 131 I-labeled affinity-purified goat immunoglobulin G having 70% immunoreactivity with carcinoembryonic antigen (CEA), representing a radiation dose ranging from 0.7 to 2.1 mCi/70 kg patient (2 to 3 µg/kg immunoglobulin G protein), was administered i.v. Anterior, posterior, and lateral radioscans of the chest and abdomen were made routinely at 24 and 48 hr with a γ-scintillation camera. Computer-assisted processing of the images in order to subtract 99m Tc background radioactivity was used to enhance the tumor-related activity, this method resulting in an average 2.5-fold enhancement of the tumor images. A very high percentage of tumor detection was achieved in most of the epithelial cancers studied. The overall sensitivity (true-positive rate) in the four major cancer types studied was as follows: colorectal cancer, 85%; ovarian cancer, 88%; cervical cancer, 90%; and lung cancer, 71%. The specificity (true-negative rate) of the method was likewise very high, ranging from 83 to 100%. In some cases, tumor radioimmunodetection was positive when other detection methods failed. The smallest tumors detectable by this method of radioimmunodetection appeared to be about 2 cm. Metastatic tumors could be localized in a number of patients with normal plasma CEA titers, although in colorectal, cervical, and lung cancer patients there appeared to be a correlation between positive radioimmunodetection and plasma CEA elevation. In general, however, radioimmunodetection was more reliable in detecting cancer among the patient population studied than were the plasma CEA assay results. High amounts of circulating CEA did not appear to prevent successful cancer radioimmunodetection. No untoward or hypersensitivity reactions were found among the patients studied, even when radioimmunodetection was repeated. Evaluation of the clinical findings in the 142 cancer patients studied revealed 116 nonneoplastic benign disease conditions, of which only 2 showed some evidence of radioantibody localization (less than 2%). Administration of radiolabeled normal goat immunoglobulin G to 22 cancer patients, some of whom were already shown to be positive for radioimmunodetection with radioactive CEA antibody, resulted in only 4 of 32 tumor sites with some evidence of transient radiolocalization; 3 of these 4 sites were massive lesions at least 10 cm in diameter.

https://cancerres.aacrjournals.org/content/canres/40/8_Part_2/2984.full.pdf

Radioimmunodetection of cancer with radioactive antibodies to carcinoembryonic antigen

https://www.gastrojournal.org/article/0016-5085(83)90076-8/pdf

Carcinoembryonic antigen radioimmunodetection in the evaluation of colorectal cancer and in the detection of occult neoplasms

External in vivo imaging of tumors with radioactively labeled antibodies to tumor-associated antigens requires processing to remove interfering nontarget radioactivity. Processing entails the use of simulated nontarget compounds labeled with a radionuclide physically different from that on the antibody and substraction of the simulated nontarget activity from the nontargeted antibody. A 2-fold increase of the target to nontarget radioactivity is achieved that has made possible a greater than 70% tumor detection rate.

Imaging Approach in Radioimmunodetection

Sixteen patients with histologically proven malignant neoplasia were investigated by radioimmunodetection, using goat anti-alpha-fetoprotein (AFP) antibody radiolabeled with 131I. Images of the chest and abdomen were made with a scintillation camera, usually at 24 and 48 hr following injection of 1 to 2.5 mCi of radioiodinated antibody. Computer-assisted processing for the subtraction of 99mTc background radioactivity was used to enhance the detection and localization of tumors visualized by immune scintigraphy. All 12 sites involved by five AFP-producing tumors could be demonstrated by radioimmunodetection, while normal goat immunoglobulin G labeled with 131I failed to show similar results in one of the patients in whom the radioactive AFP antibody achieved tumor radiolocalization. Five patients in whom the tumors were not expected to produce AFP also had their large tumors demonstrated by immune scintigraphy in 6 of 16 tumor sites. The average tumor to non-tumor 131I image count density ratios were 3.20 and 1.96 for the AFP-containing and putatively AFP-deficient tumors, respectively. The image contrast was significantly greater for the AFP-containing tumors, and the subtraction technique enhanced the image contrast more than 2-fold. Based upon these initial results, the sensitivity of the method (true-positive rate) was 100%, its specificity (true-negative rate) was 80%, and the accuracy of the technique was 85%. This study thus indicates that radioimmunodetection of cancer with radioactive AFP antibodies can be useful in the evaluation of patients with AFP-containing neoplasms.

https://cancerres.aacrjournals.org/content/canres/40/8_Part_2/3008.full.pdf

E. Edmund Kim

Papers

Use of Radiolabeled Antibodies to Carcinoembryonic Antigen for the Detection and Localization of Diverse Cancers by External Photoscanning

Radioimmunodetection of cancer with radioactive antibodies to carcinoembryonic antigen

Carcinoembryonic antigen radioimmunodetection in the evaluation of colorectal cancer and in the detection of occult neoplasms

Imaging Approach in Radioimmunodetection

Radioimmunodetection of cancer with radiolabeled antibodies to alpha-fetoprotein.