mAbs to receptor tyrosine kinases such as EGF receptor/ErbB-1 and HER2/ErbB-2 inhibit the tumorigenic growth of certain cancer cells, but although recombinant versions of such Abs are already used in oncology wards, the mechanism underlying immunotherapy remains unknown. We report that anti-EGF receptor Abs promote a slow endocytic process distinct from the rapid EGF-induced receptor internalization. Combining mAbs that engage distinct epitopes significantly accelerates receptor degradation. In addition, mAb combinations are more effective than single Abs in inhibiting HER2 signaling in vitro and tumorigenesis in animals. We present a model attributing efficacy of immunotherapy to the size of Ab-receptor lattices formed at the cell surface, which dictates the rate of endocytic clearance and extent of signaling blockade.

The objective of this preliminary investigation of a number of water-soluble carrier-bound platinum(II) complexes for potential use in cancer chemotherapy was to assess the toxicological behavior of representative platinum coordination compounds anchored to, or incorporated into, polymeric carriers via polymer-attached amine ligands, The conjugates included linear polyaspartamides (1-4, 6, 7), each composed of a major fraction of subunits featuring side-chain-attached tertiary amino groups as water-solubilizing entities, and a minor fraction of subunits comprising the anchored platinum complexes, again as side-chain components. Whereas in 1-4 the platinum atom was polymer-bound through a single amino group, both 6 and 7 contained polymer-attached cis-diamine-chelating ligands coordinating to the metal center, Also included in this study was a linear polyamidoamine (5), which contained a poly(ethylene oxide) segment in the backbone in addition to intrachain ethylenediamine segments acting as cis-diamine chelating ligands for coordination to the platinum center. The compounds were injected as aqueous (phosphate-buffered saline) solutions into the tail veins of CD-1 mice (four to eight mice per conjugate), and the maximally tolerated dose was determined for each compound. For polyaspartamides 1-4 the dose levels ranged from about 25 mg Pt (kg body weight(-1)) (in conjugate 4) to 500 mg Pt kg(-1) (in compound 1), the latter conjugate proving some 100-fold less toxic than cisplatin (3-4 mg Pt kg(-1)), which was included in this study for comparison. Low toxicity (tolerated dose 160 mg Pt kg(-1)) was also observed for the intrachain cis-diamineplatinum complex polymer (5), The polyaspartamide conjugates 6 and 7, on the other hand, both characterized by a cis-diamineplatinum complex system in the side chain, were toxic even below the dose level of 20-25 mg Pt kg(-1). The preliminary findings of this study, while providing a basis for more extensive and broad-based toxicological studies, will serve to direct and optimize structural conjugate designs in forthcoming synthetic programs. Copyright (C) 2000 John Wiley & Sons, Ltd.

Selective delivery of biologically active substances is designed to overcome nonspecific biodistribution of drugs and increase their local concentration at the target tissue. Certain strategies are based on carrier-mediated delivery to selective tissues and organs through ligands recognized by receptors or other address molecules on target cells. Avidins offer an attractive approach to organ- or tissue-selective targeting. Avidin and streptavidin, two biotin-binding proteins, can be targeted to specific tissues when modified with appropriate tissue markers. Their resistance to proteolytic enzymes supports long-term accumulation at the target tissue or organ, and their biotin binding sites permit the delivery of biotinylated molecules or carriers loaded with cytotoxic drugs or other bioactive substances. Modification of the two proteins with tissue-specific markers (lactose for parenchymal and trinitrophenyl for nonparenchymal liver cells) resulted in high and prolonged accumulation in the target tissue. The modified proteins could target high doses of chemotherapeutic drugs (CDDP and 5-fluorouridine) to the liver through biotinyl dextran-derived carriers. Drug Dev. Res. 50:258-271, 2000. (C) 2000 Wiley-Liss, Inc.

Trinitrophenyl (TNP) modification of streptavidin (St) resulted in high and prolonged accumulation in mouse liver following intravenous administration of radioiodinated TNP streptavidin (TNP-St). Uptake, which is correlated with increased TNP substitution, was first observed at 2-3 h, increased to 40-50% of injected dose/gram tissue (%/g) at 24 h and slowly declined later on. A low degree of accumulation (10%/g) was observed in the spleen. TNP substitution of other proteins such as bovine serum albumin (BSA) or ovalbumin (Ova) led to a transient short-term liver uptake. The enzyme-resistance property of streptavidin and its biotin binding sites render TNP-modified streptavidin a potential targeting vehicle to the liver. 5-Fluorouridine (FUR) was attached to high molecular weight carrier carboxymethyldextran (CMdex, derived from 40 kDa dextran) and the dextran FUR conjugate was charged with 2-4 biotinyl groups (in the form of biotinyl-diaminopropionyl-tyrosine, BDT) for complexing to TNP-St. Biodistribution monitoring of the BDT-CMdex-FUR ligand, radiolabeled at the tyrosyl residue of BDT and targeted via non-radiolabeled TNP-St, showed that ligand accumulation in the liver was similar to TNP-St itself. Liver targeting of FUR was demonstrated by trace-labeling FUR with its structural analog 5,6-[3H]uridine prior to conjugation to dextran hydrazide. Specific liver accumulation of [3H] radioactivity occurred following administration of the conjugate only when complexed to TNP-St. Hepatic levels of [3H] radioactivity were in the range of 25%/g or 35% per whole liver during a period of at least 8 h, as compared to the rapid elimination of free FUR+[3H]uridine (4%/g at 20 min). [3H]-drug radioactivity disappeared at a faster rate as compared to 125I-dextran radioactivity, suggesting that metabolic processes required to generate the 5,6-[3H]uracil-containing active metabolites took place.

Complexing cis-dichlorodiammineplatinum (II) (CDDP) to the polycarboxylic carriers carboxymethyl dextran (CMdex, Mr = 40 kDa) and poly-l-glutamic acid (p-Glu, Mr = 40 kDa) yielded pharmacologically active platinum (II) multi-complexes of decreased drug toxicity. Displacement of CDDP chlorine atoms by hydrogens of carboxyl groups on polymer side-chains could give rise to mono-functional linkages capable of releasing the drug in favor of ligandsl exhibiting higher affinity toward Pt (II) (e.g. DNA, the target for drug activity in tumor cells). The CDDP-CM-dex complex, carrying up to 40 mol releasable CDDP per mol carrier, was cytotoxic against ovarian carcinoma cells in vitro with activity comparable to that of free CDDP (1.5 times lower). Its in vivo toxicity was influenced by drug: carrier molar ratio with a direct correlation between drug load and toxicity (a complex of 15: 1 was 4-fold less toxic than CDDP). The CDDP-p-Glu complex (60 mol drug/mol p-Glu) was characterized by higher thermodynamic stability, its reduced in vivo toxicity was not affected by CDDP load and its in vitro activity was lower than that of free CDDP (2.6-fold). Both CDDP complexes were effective in suppressing the growth of human ovarian carcinoma (OVCAR-3) in athymic mice. Due to them, decreased toxicity the complexes exerted a wider therapeutic dose range of activity (80% survival at 3–12 mg/kg) as compared to the narrow and inconsistent effective dose range of free CDDP (80% survival at 1–2.5 mg/kg).

Human polyclonal, monospecific anti‐T and ‐Tn antibodies were found to be reactive in ELISA tests with human ovarian (IGROV‐1, OVCAR‐3 and SKOV‐3), breast (SKBr‐3 and T47D)‐ and oral (KB)‐carcinoma cell lines, but less so or non‐reactive with normal epithelia and fibroblasts. The direct binding radioimmunoassay, using 125I‐labeled human antibodies, to the IGROV‐1 cancer cells was inhibited by homologous unlabeled antibodies of the same concentration, but not by the respective immunodominant haptenic monosaccharides (Gal for T and GalNAc for Tn). Rodent ascitic monoclonal anti‐T (Ca3114 and Ca3741) and anti‐Tn (Ca3250, Ca3268 and Ca3638) antibodies were also reactive with the ovarian‐ and breast‐cancer cells, as measured by FACS and ELISA tests, but to a lower extent than the polyclonal human antibodies. Both the monoclonal anti‐T (Ca3741) and anti‐Tn (Ca3250 and Ca3638) antibody‐binding reactivities were significantly inhibited by the haptenic free monosaccharides. Addition of the above MAbs to IGROV‐1 ovarian‐cancer or T47D breast‐cancer cells cultured in vitro resulted in significant cytological change and inhibition of the viability of the tumor cells, but not of normal epithelial breast cells. This effect on viability was shown to be complement‐independent, yet it was profoundly influenced by the concentration of the serum added to the assay medium. In vivo biodistribution of the anti‐T (Ca3114) and anti‐Tn (Ca3638) MAbs administered i.p. to athymic IGROV‐1 tumor‐bearing CD1 female nude mice revealed higher 125I‐labeled antibody accumulation in the tumor xenografts and in their lung tissues, as compared with other organs of the same mice tested. The above results thus suggest the feasibility of utilizing these antibodies in immunotherapy and drug targeting. Int. J. Cancer 72:119–127, 1997. © 1997 Wiley‐Liss Inc.

Two laser spectroscopists from IBM have put forward a controversial theory to explain one of the greatest enigmas of astronomical spectroscopy: unexplained dark absorption bands in the spectra of all bright stars. Astronomers have in general been skeptical, but this radical theory is winning converts.

How does the brain generate the rhythms that may link separate sets of neurons responding to the same object? Particular neurons seem to act as pacemakers for the rhythms, while networks of other cells spread and synchronize them. Results from one group were reported in the 4 October issue of Science .

Hepatic metastases of malignant tumors is a major problem in the treatment of cancers for which the liver is the most common site for recurrences. In the present study we describe a selective delivery system to the liver which may facilitate specific hepatic targeting of anti-cancer agents. Avidin and streptavidin are two biotin-binding proteins with extreme resistance to proteolytic activity. Trinitrophenyl (TNP) modification of these two proteins resulted in specific accumulation in mouse liver with levels of 40-50 percent per gram tissue (%/g) during a period of several days. The two modified proteins could target to the liver high doses of covalently bound radionuclide iodine-125, a biotinylated ligand such as biotinyl-tyrosine (BT) or large biotinylated carriers such as carboxymethyl dextran (CMdex, 40kDa). Appropriately derivatized dextrans serve as carriers for various chemotherapeutic drugs, as demonstrated here for cis-dichlorodiammineplatinum (CDDP). Specific liver targeting of CDDP complexed to CMdex-TNP-streptavidin could be monitored by flame atomic absorption spectrometry of the Pt metal: High levels of the Pt drug were concentrated in the liver for at least 15hr following its targeted delivery as compared to essentially undetectable levels after administration of the free drug.

The Concorde trial shows an increase in CD4 + lymphocytes, but not a higher survival, in AZT treated asymptomatic patients. Our murine bone-marrow experiments show that in chronic AZT treatments a large increase in the lymphoids/erythroids compartment is due to host cytotoxicity. We put forward a mathematical formula for predicting cytotoxicity of given drug protocols, which can be used for modulating the schedule so as to increase its efficacy while maintaining its toxicity low. Our study suggests that in chronic treatments a large, single, daily dose will be less toxic than the same dose divided into several daily dosings.

Cisplatin (CDDP), a most powerful anticancer agent, was complexed to a polycarboxylic carrier carboxymethyldextran to form a platinum(II) multicomplex. Complexing occurs by displacement of the chlorine atoms of the platinum coordination complex by hydrogen of polymer side-chains to form mono- or bifunctional anchoring to adjacent carboxyls on the carrier. The carrier-complexed drug interacted with DNA and was pharmacologically active against tumor cells. The drug-carrier complex was immunotargeted to human epidermoid carcinoma (KB) tumors, using the monoclonal antibody (mAb) 108 directed against the epidermal growth factor receptor that is overexpressed on KB cells. Biotinyl-monoclonal antibody was bound to a platinum(II)-carboxymethyldextran-avidin conjugate and the immune complex was administered into established subcutaneous KB tumors to evaluate its effects upon intratumor treatment. The results showed that the immune complex was specifically effective in inhibiting tumor growth. The antibody in the complex must be tumor-specific to anchor the drug-carrier multicomplex to the tumor site since an unbiotinylated antibody, or replacing the anti-KB antibody by a biotinylated antibody of a different specificity, resulted in reduced or abolished inhibitory effects.

Streptavidin exhibits a remarkable accumulation in the kidney. Biodistribution studies with radio-iodinated streptavidin showed that 70 to 80% of the injected dose per gram tissue (%/g) were retained in kidneys of Balb/C mice for three to four days compared to less than 5%/g levels in other tissues. This observation means that 15 to 20% of the injected dose is accumulated in the kidney, an organ that constitutes less than 1% of total body weight. Similar results of percent radioactivity per total kidney were obtained in other mouse strains as well as in rats and rabbits. Avidin, or the post-secretory form of streptavidin which is of a higher molecular weight, do not show any preferential affinity to the kidney. The kidney-accumulated streptavidin was mostly confined to the cortex, concentrated in the proximal tubular cells. Accumulation of streptavidin in the kidney was independent of biotin, since addition of biotin to radio-iodinated streptavidin prior to injection did not affect its kidney uptake. Therefore, streptavidin, which aquires its kidney accumulation property following truncation of the native form, may be utilized for renal specific delivery of chemotherapeutic agents, radioactive isotopes and other effector molecules. Such ligands can be linked to streptavidin via conventional coupling methods or following their biotinylation. Preliminary experiments showed that streptavidin can target to the kidney biotinylated ligands or high doses of chemically linked radionuclides.

Spectroscopic methods have been applied to elucidate conformational differences responsible for the immunological diversity of two synthetic multichain copolymers, Tyr1Tyr2Glu3Glu4-poly-DL-Ala--poly-Lys and Tyr1Glu2Tyr3Glu4-poly-DL-Ala--poly-Lys. Despite their far-reaching structural similarity in the epitope peptide and complete identity in the poly-Ala--poly-Lys carrier, these two copolymers manifest a wide range of opposed immunological attributes. Different genetic control mechanisms govern their immunogenic properties, and their interactions with antigen presenting cells or T cells and B cells are mediated via different immunological routes. Following previous photoCIDNP (photoChemically Induced Dynamic Nuclear Polarization) investigations, we applied NMR and fluorescence measurements to these two copolymers in order to search for structural differences that could account for their opposed immunological behaviour. The differences between the two antigens are traced to the spatial orientation of the tyrosine residues. Hydrophobic Tyr1--Tyr3 intramolecular inter-side-chain interactions characterize the Tyr1Glu2Tyr3Glu4 polymer, whereas Tyr1 and Tyr2 in the Tyr1Tyr2Glu3Glu4 polymer are non-interacting and freely rotating. It is thus inferred that Tyr1 and Tyr2 are distant and point to different directions in space, whereas Tyr1 and Tyr3 are in close proximity, as was suggested by a previous CIDNP study and by molecular structure computations. We infer that these structural differences may relate to the different immunological behaviour of the TyrTyrGluGlu and TyrGluTyrGlu polymers.

Many antineoplastic drugs are cell-cycle-phase-specific. These drugs are often highly toxic to the host, as they have the potential to impair replication, not only in the cancer cells, but also in the normal tissues. Using mathematical models it has been shown how selectivity of these drugs can be increased by exploiting the relatively large variability in cell-cycle parameters of the neoplasia. These models predict that toxicity to the host of cell-cycle-phase-specific drugs can be minimised if the dosing interval is an integer multiple of the average intermitotic interval of the susceptible host cells. Experimental evidence supporting this prediction is presented in this work. Our results show that a constant duration of the dosing interval yields higher survival rates in mice treated by cytarabine, as compared with random dosing intervals. Minimal myelotoxicity is exerted when the dosing interval is an exact multiple of the inter-mitotic time of bone marrow stem cells and erythroid progenitors (i.e. 7 h). Survival is significantly lower in mice treated every 8 h, or its multiple, as compared with that of mice treated at a 7 h or 10 h dosing interval.

The streptavidin-biotin system has been used to immunotarget whole ricin to tumor cells in a system that overcomes ricin-nonspecific cytotoxicity. Biotin was linked to ricin via a disulfide-containing reagent, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3'-dithiopropionate. The product, biotinyl-S,S-ricin (b-ricin), retained most of its in vitro cytotoxic activity against human epidermoid carcinoma (KB) cells. Complexing b-ricin to streptavidin resulted in greater than 99% loss of its cellular toxicity which is associated with loss of cell-binding activity. The streptavidin-b-ricin complex could, however, be targeted to KB cells via the biotinylated monoclonal antibody 108 which is specific to the epidermal growth factor receptor overexpressed on KB cells. The complex did not regain its activity if the specific antibody was not biotinylated or if the biotinylated antibody was of a different specificity. Streptavidin is thus used to block b-ricin, presumably due to a steric restraint of the streptavidin on the ricin B-chain, and to bridge it to biotinyl antibody recognizing the target cell. Avidin could not replace streptavidin in this system since a complex between b-ricin and avidin retained a major part (60%) of ricin cytotoxic activity. This is attributed to the nonspecific binding of avidin to cells in vitro, including the KB cells. It is suggested that b-ricin is blocked by both streptavidin and avidin, but once the complex gains access to the cell surface, its cytotoxic activity is specifically retrieved.

Photochemically induced dynamic nuclear polarization (photoCIDNP) measurements, specific for exposed tyrosine residues, have been applied to elucidate conformational differences responsible for the immunological diversity of the synthetic multichain copolymers, Tyr1Tyr2Glu3Glu4-poly-dl-Ala- -poly-Lys and Tyr1 Glu2 Tyr3 Glu4-poly-dl-Ala-poly-Lys. These two copolymers are essentially identical in their molecular weight, size, shape and composition, and differ only in the order of the two internal amino acid residues within the sequence of the tetrapeptide epitopes. Nonetheless, previous studies have shown that the two macromolecules behave differently, as evidenced by their immunological and immunogenic properties. As immunogens they act under different genetic control mechanisms, and differ in their interactions with antigen presenting cells, T cells and B cells. Antibodies elicited against these two antigens do not cross react. The photoCIDNP measurements of these two polymers, intended to elucidate discrete structural differences controlling immune recognition, showed that in the TyrTyrGluGlu polymer, Tyr1 and Tyr2 rings are free, non-interacting and undergo fast internal rotation. Computed minimum energy conformations confirm these conclusions and indicate that Tyr1 and Tyr2 point to different regions in space. In TyrGluTyrGlu, however, CIDNP measurements give rise to one broad tyrosine 3,5 proton signal, the result of a strong Tyr1-Tyr3 hydrophobic interaction. These two tyrosine residues are thus close in space, and undergo slow internal rotation. These results are in agreement with the computed minimum energy conformations.

Cis-diamminedichoroplatinum (II) (cis-Pt) complexed to a carboxymethyl dextran-avidin conjugate was targeted to biotin-monoclonal antibody 108 (b-MAb 108). This MAb recognizes the extracellular domain of the epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma (kB) cells over-expressing EGF-R. Cis-Pt-carboxymethyl-dextran-avidin (Pt-dex-Av) containing 60-90 M cis-Pt/M avidin was administered 24 hr following b-MAb 108 containing 3-5 M biotin/M MAb. This treatment was potentially more effective in suppressing the growth of established KB tumor xenografts, or in inhibiting the development of lung metastases in nude mice, than free MAb 108, free drug or MAb 108 followed by drug. Replacing b-MAb 108 by unbiotinylated antibody or by b-MAb of a different specificity also yielded lower suppressive effects. The sequential administration of Pt-dex-Av following b-MAb was more effective than introduction of the Pt-dex-Av when already complexed to b-MAb 108. The results presented in this preliminary investigation suggest that Pt-dex-Av is specifically removed from the circulation by b-MAb 108 concentrated at the tumor site.

Zidovudine (azidothymidine, AZT) toxicity to the bone marrow (BM) is a major hindrance to its widespread clinical application in the treatment of the acquired immunodeficiency syndrome (AIDS). In this work we verify the prediction of a mathematical model that cytotoxicity to the host can be reduced when the frequency of drug administration is an integer multiple of the target cell average cycle time (ca. 7 h in murine BM cells). We report in vivo experiments in mice showing that a 7-h frequency of AZT administration is significantly less toxic than other frequencies when peripheral blood parameters and the proportion of BM cells arrested at the S-phase gate of the DNA content distribution are considered.

Radioionated avidin and steptavidin were characterized for their biodistribution and tissue association in Balb/c mice, in comparison to their interaction with cells in vitro. Binding of avidin to spleen and bone‐marrow cells in vitro was up to 20‐fold higher than that of streptavidin, but when tested in vivo avidin clearance from blood and tissues was considerably faster than that of streptavidin. Levels of avidin at 24 h after an intravenous injection were below 1% (of the injected dose/mass tissue) in most organs. Non‐glycosylated avidin was similar in its biodistribution to native avidin. Native streptavidin exhibited higher and prolonged tissue association with 5–10% levels in lung, liver, spleen, kidney and blood, whereas its truncated form showed low tissue levels (1–3%) but a remarkably high affinity to the kidney (80%). Exogenous biotin did not affect streptavidin distribution in vivo but caused a 2–7‐fold increase in the retention of avidin (but not non‐glycodylated avidin) in some of the organs.