Search

By year 2025 pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer related death. However, other than improved chemotherapy and a small molecule inhibitor of the epidermal growth factor receptor (EGFR), no targeted drugs are currently available. Repurposing of approved drugs might offer a rapid solution. We employed an animal PDAC model, expressing a mutant and a wild type form of p53 and KRAS, respectively. Cetuximab, a clinically approved anti-EGFR monoclonal antibody (mAb) weakly inhibited PDAC xenografts, similar to trastuzumab, a mAb against HER2, a co-receptor of EGFR. Because the combination of cetuximab and trastuzumab only moderately enhanced the anti-tumor effects, we combined each with a home-made mAb to the same receptor and identified two cooperative pairs. The pair of trastuzumab and a murine anti-HER2 mAb better than the anti-EGFR pair inhibited PDAC xenografts, although HER2's abundance in our model is 15-fold lower than the level of EGFR. In vitro studies attribute cooperation to forced receptor endocytosis/degradation and inhibition of both DNA synthesis and cell migration. Taken together, our results identify cooperative pairs of anti-PDAC antibodies and highlight potential mechanisms of anti-tumor effects.

Due to intrinsic aggressiveness and lack of effective therapies, prognosis of pancreatic cancer remains dismal. Because the only molecular targeted drug approved for pancreatic ductal adenocarcinoma is a kinase inhibitor specific to the epidermal growth factor receptor (EGFR), and this receptor collaborates with another kinase, called HER2 (human EGF-receptor 2), we assumed that agents targeting EGFR and/or HER2 would effectively retard pancreatic ductal adenocarcinoma. Accordingly, two immunological strategies were tested in animal models: (i) two antibodies able to engage distinct epitopes of either EGFR or HER2 were separately combined, and (ii) pairs of one antibody to EGFR and another to HER2. Unlike the respective single monoclonal antibodies, which induced weak effects, both types of antibody combinations synergized in animals in terms of tumor inhibition. Immunological cooperation may not depend on receptor density, antigenic sites, or the presence of a mutant RAS protein. Nevertheless, both types of antibody combinations enhanced receptor degradation. Future efforts will examine the feasibility of each strategy and the potential of combining them to achieve sustained tumor inhibition.

Aptamers, oligonucleotides able to avidly bind cellular targets, are emerging as promising therapeutic agents, analogous to monoclonal antibodies. We selected from a DNA library an aptamer specifically recognizing human epidermal growth factor receptor 2 (ErbB-2/HER2), a receptor tyrosine kinase, which is overexpressed in a variety of human cancers, including breast and gastric tumors. Treatment of human gastric cancer cells with a trimeric version (42 nucleotides) of the selected aptamer (14 nucleotides) resulted in reduced cell growth in vitro, but a monomeric version was ineffective. Likewise, when treated with the trimeric aptamer, animals bearing tumor xenografts of human gastric origin reflected reduced rates of tumor growth. The antitumor effect of the aptamer was nearly twofold stronger than that of a monoclonal anti–ErbB-2/HER2 antibody. Consistent with aptamer-induced intracellular degradation of ErbB-2/HER2, incubation of gastric cancer cells with the trimeric aptamer promoted translocation of ErbB-2/HER2 from the cell surface to cytoplasmic puncta. This translocation was associated with a lysosomal hydrolase-dependent clearance of the ErbB-2/HER2 protein from cell extracts. We conclude that targeting ErbB-2/HER2 with DNA aptamers might retard the tumorigenic growth of gastric cancer by means of accelerating lysosomal degradation of the oncoprotein. This work exemplifies the potential pharmacological utility of aptamers directed at cell surface proteins, and it highlights an endocytosis-mediated mechanism of tumor inhibition.

Monoclonal antibodies (mAbs) to ErbB-2/HER2 or to its sibling, the epidermal growth factor receptor (EGFR), prolong survival of cancer patients, especially when combined with cytotoxic therapies. However, low effectiveness of therapeutic mAbs and the evolution of patient resistance call for improvements. Here we test in animals pairs of anti-ErbB-2 mAbs and report that pairs comprising an antibody reactive with the dimerization site of ErbB-2 and an antibody recognizing another distinct epitope better inhibit ErbB-2-overexpressing tumors than other pairs or the respective individual mAbs. Because the superiority of antibody combinations extends to tumor cell cultures, we assume that nonimmunological mechanisms contribute to mAb synergy. One potential mechanism, namely the ability of mAb combinations to instigate ErbB-2 endocytosis, is demonstrated. Translation of these lessons to clinical applications may enhance patient response and delay acquisition of resistance.

Aptamers represent a promising new treatment modality for cancer. Specificity and high affinity are two parameters that characterize aptamers. In this work, we elucidated physicochemical parameters of an ERBB2/HER2 specific aptamer and determined an optimal multimerization state, leading to higher binding and improved avidity. We applied biochemical, immunochemical and biophysical methodologies to characterize binding behaviors of multimerized versions of an ERBB2/HER2 specific aptamer and demonstrate structural integrity. Finally, we show that the trimeric ERBB2/HER2 specific aptamer instigates no immunogenic response in vivo. In summary, the set of methodologies we employed establishes a way to enhance activity of a model HER2-aptamer.

Vaccines are by definition prophylactic, but in recent years an interest has developed in therapeutic vaccines for infectious diseases such as AIDS and tuberculosis, as well as gastric ulcers, cancer (with different approaches to combat various types of malignancy) and autoimmune diseases (a definite success was the development of a vaccine against multiple sclerosis) and there are potential vaccines in development for myasthenia gravis, lupus and diabetes. Therapeutic vaccines are also being developed against cognitive diseases such as Alzheimer's disease, prion diseases and Huntington's disease. All of these efforts are based on the therapeutic vaccine being closely related chemically to the etiological agent that causes the disease.

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. © 2000 Wiley-Liss, Inc.

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.

Complexing cis -dichlorodiammineplatinum (II) (CDDP) to the polycarboxylic carriers carboxymethyl dextran (CMdex, M r = 40 kDa) and poly- l -glutamic acid (p-Glu, M r = 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.

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.

Renal accumulation of streptavidin: Potential use for targeted therapy to the kidney. 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.

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, Tyr 1 Tyr 2 Glu 3 Glu 4 -poly- dl -Ala- -poly-Lys and Tyr 1 Glu 2 Tyr 3 Glu 4 -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, Tyr 1 and Tyr 2 rings are free, non-interacting and undergo fast internal rotation. Computed minimum energy conformations confirm these conclusions and indicate that Tyr 1 and Tyr 2 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 Tyr 1 -Tyr 3 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.

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.

Cis-diamminedichloroplatinum (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-MAb108 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.

Ten years after the first clinical application of Rituximab, an anti-CD20 recombinant monoclonal antibody, immunotherapy has become common practice in oncology wards. Thanks to the great diversity of the immune system and the powerful methodology of genetic engineering, the pharmacologic potential of antibody-based therapy is far from exhaustion. The recent application of Trastuzumab, an antibody to a receptor tyrosine kinase, in adjuvant breast cancer therapy marks the beginning of a new phase in cancer treatment. Here we discuss molecular mechanisms of antibody-based therapy, the emerging ability to target minimal disease and the therapeutic potential of combining antibodies with other modalities.

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.

Vascular endothelial growth factor (VEGF) is one of the key growth factors regulating tumor angiogenesis and thus it is one of the primary targets for antiangiogenic therapy. The long-term effects of VEGF include induction of proliferation and migration of endothelial cells, tube formation and maintenance of the immature capillaries. The early effects of VEGF include vasodilation and increased permeability. We hypothesize that the early responses to VEGF can serve to develop a quantitative measure of the activity of VEGF, and therefore may be applicable for monitoring the efficacy of systemic suppression of VEGF signaling during antiangiogenic therapy. For that end we tested the ability of MRI and fluorescence microscopy to detect the early response to intradermal VEGF165 in nude mice. VEGF-induced local vasodilation and increased permeability was detected by intravenous administration of macromolecular biotin-BSA-GdDTPA(23) 30 min after intradermal administration of VEGF. Contrast leak showed saturation kinetics. Delayed contrast administration (90 min after intradermal administration of VEGF) resulted in low contrast leak and demonstrated that the saturation kinetics is not due to contrast equilibration between plasma and the interstitial space, but rather is due to suppression of vascular permeability. Permeability was restored by a second bolus of VEGF, showing that the saturation kinetics is primarily due to inactivation of the growth factor. Confocal microscopy of fluorescent BSA-FITC confirmed the permeability changes monitored by MRI. Moreover, confocal microscopy showed efficient lymphatic uptake of the extravasated contrast material specifically in regions of VEGF induced hyper-permeability.

ErbB-2/HER2 is a member of the epidermal growth factor receptor (EGFR) family and when trans-activated, it stimulates several downstream signaling cascades, including the mitogen-activated protein kinase cascade. This ligand-less receptor is moderately expressed in normal adult tissues, where it regulates cell growth and differentiation, but gene amplification and consequent overexpression of the HER2/ErbB-2 protein have been associated with tumors of the breast and ovary, enhanced metastatic potential and poor prognosis. Monoclonal antibodies (mAbs) to ErbB-2/HER2 prolong survival of cancer patients, especially when combined with cytotoxic therapies. However, low effectiveness of therapeutic mAbs and the evolution of patient resistance call for further exploitation of the potential of mAbs. We found that combinations of anti-ErbB-2 mAbs comprising an antibody reactive with the dimerization site of ErbB-2 and an antibody recognizing another distinct epitope form an efficient and synergistic inhibitory sys...

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.

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.

A major problem in cancer chemotherapy is how to enhance the effectiveness of currently available anti cancer drugs. Due to the lack of selectivity of cytotoxic agents, the administration of single drug doses is restricted to sub-toxic levels. Drug clearance and excretion and/or metabolic conversion of the drug result in only transient increased levels at the tumor site which are generally insufficient to effect complete cure. The problem is therefore, how to attain sufficient amounts of drug at the tumor site for the required period of time, without using drug doses which are above the threshold of toxicity. One possible way is to use molecules with inherent or acquired ability to interact selectively with the target organ, e.g. anti-tumor antibodies, as carriers for the drug, thus leading to specific targeting to the tumor site. An alternative approach is to attach anti-cancer agents to polymeric carriers that will act as control release units by affecting the biodistribution and maintenance of the drug. These delivery devices may cause retardation of drug clearance or inactivation as well as prolonged or sustained release of the drug at non-toxic levels, thus overcoming the problem of harmful peak concentrations. Such polymeric drug systems, in addition to being advantageous to the free drug as such, may be also used for conjugation to antitumor antibodies for the purpose of immunotargeting.

Radioionated avidin and streptavidin 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.

Prompted by early observations of the cytotoxic and antineoplastic properties of certain ferrocene and ferricenium derivatives, efforts in this laboratory were focused on the synthesis of carrier-bound ferrocene compounds. Subsequent cell culture tests carried out with selected conjugates obtained in that program showed these polymers to be highly active antiproliferative agents. In the present project toxicological work has been performed in vivo on several ferrocene conjugates in an effort to assess their toxic effects in experimental animals (CD-1 mice). The conjugates, all based on an α,β-DL-polyaspartamide backbone structure, comprise the ferrocene drug model as a terminal on short side chains containing biofissionable amide or ester links for intracellular drug release. The polymers, dissolved in phosphate-buffered saline, have been injected in predetermined concentrations into the vein of the mice, and the maximum tolerated dose (MTD) levels have been determined, the latter referring to the highest dose levels administered that would allow long-term survival of the test animals. For the five conjugates tested, MTD levels range from about 3 to 30 mg Fe/kg or 0.05–0.66 mmol Fe/kg. Compared on a molar metal-to-metal basis with similarly structured platinum conjugates tested previously (MTD, ∼0.14–2.66 mmol Pt/kg), these values are indicative of comparatively high toxicity of the ferrocene polymers. Some implications of these findings are discussed.

Cis-diamminedichloroplatinum(II) (cis-DDP) and cis diamminediaquoplatinum(II) nitrate (cis-aq) were complexed to poly-L-glutamic acid (PG) of Mr 13,000 or 58,000. Soluble complexes were formed by mixing the platinum(II) compounds with the PG preparations in water at a molar ratio of one platinum(II) to 5 glutamyl residues of the PG. Compared to free cis-DDP, the complexes were about 2 to 6 times less cytotoxic to F9 embryonal carcinoma cells in vitro. In vivo studies with this tumor indicated that when administered i.p. one day after the tumor cells, the complexes were somewhat less active than cis-DDP. However, in comparison to the free drug which, due to its toxicity, was effective only within a very narrow dose range, the complexes were much less toxic, manifesting a wide therapeutic range in which no mortality occurred either from the tumor or as a result of the toxic effects of the drug. Cis-DDP and cis-aq complexes of PG Mr-58,000 were somewhat more toxic than the complexes of PG Mr, 13,000. The in vivo anti-tumor activity of most complexes was superior to that of free cis-DDP when tested in mice carrying high tumor loads. Due to their wide therapeutic range, the complexes could be given at high drug doses resulting in increased life span of the tumor-bearing mice. Moreover, 3 repeated injections of these high doses did not cause death from toxicity and resulted in partial or total eradication of advanced tumors.

We investigated the interaction between T lymphocytes and macrophages in the in vitro sensitization of lymphocytes against tumor cells. Spleen cells were sensitized in vitro by syngeneic peritoneal macrophages that had been fed with cell-free antigen preparation of syngeneic tumor cells. The sensitized T lymphocytes acquired specific cytotoxic activity in vitro and the capacity to inhibit tumor development in vivo. Allogeneic macrophages, syngeneic fibroblasts, or the antigen preparation by itself were not able to sensitize the lymphocytes against the tumor.

The present study was undertaken in order to evaluate the possibility of using anti-tumor anti-bodies as specific carriers of chemotherapeutic drugs to tumor metastases. Xenogeneic and syngeneic antisera were prepared against a metastasizing C57BL tumor, the Lewis lung carcinoma (3LL). The binding of absorbed xenogeneic and syngeneic anti-3LL sera or their Ig fractions to 3LL tumor cells was assayed by direct and indirect methods. Antisera prepared against tumor cells from a local growth reacted to a similar extent with cells obtained from the local tumor and with those obtained from the lung metastases. Radioiodinated immunoglobulins from syngeneic antisera injected into mice bearing metastases localized preferentially in metastasis-bearing lungs as compared to several other organs tested. No such preferential uptake was observed either in mice bearing metastases injected with iodinated normal Ig or in normal mice injected with iodinated anti-3LL Ig. This relative accumulation in the metastasis-bearing lungs was observed 3–4 days after the inoculation, when the whole Ig fraction was used, whereas a specific antibody-enriched preparation localized as early as 24 h after injection. Daunomycin—anti-3LL-Ig conjugates were effective inhibitors of tumor cells from both local and the metastatic growths. They were more active than either the free drug or drug conjugates of normal Ig, in in vitro assays.

A method is described for measuring cell-mediated cytotoxicity, based on the incorporation of labeled leucine into actively synthesized proteins in viable target cells which have survived interaction with effector lymphocytes. The method was studied with in vitro or in vivo sensitized lymphocytes in xenogeneic or allogeneic systems. This method was found to be applicable to quantitative determination of cell-mediated cytotoxicity by in vitro sensitized lymphocytes against a syngeneic tumor.

Experiments are reported on immunotherapy of lethal lung metastasis by lymphocytes sensitized in culture against a syngenic tumor. We used the Lewis lung carcinoma (3LL) which had originated in a C57Bl mouse. C57Bl mice were footpad inoculated with tumor cells. Seven days liter, the tumor-bearing leg was amputated, and the animals were inoculated with syngenic lymphocytes which had been sensitized on monolayers of 3LL-tumor cells. The sensitized lymphocytes significantly reduced the development of lethal lung metastasis of the 3LL-tumor cells.

Plasma levels and serum protein binding of cis-diamminedichloro-platinum(II) (cis-DDP) or cis-diamminediaquoplatinum(II) (cis-aq) complexed to carboxymethyl-dextran (CM-dex) with a molecular weight of 10,000 (T-10), 40,000 (T-40), and 250,000 (T-250) were investigated in BALB/c mice. Levels of active drug in the circulation after the i.v. or i.p. administration of the free or complexed drugs, as well as the loss of drug activity due to serum protein binding following incubation with mouse serum, were monitored by an antitumor in vitro assay using a drug-sensitive tumor cell line. Following i.v. injection of the complexes, active platinum(II) was maintained in the circulation at higher levels and for a longer period, whereas the free drug disappeared rapidly. The rate of disappearance of the complexed drug from the circulation was markedly influenced by the molecular size of the carrier CM-dex, since the retained amount of drug was considerably higher with the T-40 and T-250 complexes than with the T-10 complex. An i.p. injection resulted in a rapid and transient appearance of low levels of the free drugs in the blood, whereas in the case of the complexes, transport to the circulation was slower and their maintenance in the blood system was markedly higher. Serum protein binding was much slower with CM-dex-complexed drugs (regardless of the molecular size of the CM-dex carrier) than with the free drugs.

Immunization of rabbits with trypsin in Freund's adjuvant results in the formation of specific antisera, which give positive precipitin reactions with trypsin as well as with several derivaties of the enzyme. The antibodies which are precipitated by the enzyme are intact molecules, as practically no enzymatic degradation occurs prior to formation of antige-antibody complexes. Isolation of the γG globulin fraction from the antiserum results in effective elimination of any nonspecific anti-trypsin activity and allows the study of the inhibitory effect of the antibodies. The antibodies against trypsin inhibit the proteolytic activity of the enzyme, as well as, to a more limited extent, that of chymotrypsin. The inhibition is directly related to the size of the substrate: the higher its molecular weight the higher the extent of inhibition. It is concluded from the data that although a very small portion of the antibodies might be directed towards a region including the catalytic site of the enzyme, the bulk of the antibodies interacts with other regions of the molecule and, therefore inhibitory effects is a result of steric hindrance.

The production of tumor-specific cell-mediated cytotoxicity following in vitro sensitization of C57BL spleen cells against a syngeneic 3LL Lewis lung carcinoma was studied. Lymphocytes were sensitized on monolayers of the tumor cells for 4-5 days. The cytotoxicity was assayed by measuring the reduction in 3H-leucine and 3H-thymidine incorporation by target cells after interaction with the sensitized lymphocytes. Spleen lymphocytes sensitized on monolayers of 3LL tumor cells caused a high extent of lysis; such cells tested on C57BL or C3H fibroblast targets evoked only a low level of cytotoxicity. C57BL spleen cells sensitized on C57BL fibroblasts caused a low level of cytotoxicity when tested on a 3LL target. Thus cytotoxicity appeared to be tumor specific. The reduced incorporation into protein and DNA of target tumor cells caused by the sensitized lymphocytes was a measure of cell injury, which was more sensitive than direct cell count or uptake of 51CR. Lymphocytes from syngeneic tumor-bearing mice, tested 13-25 days after tumor inoculation, did not manifest in vitro cytotoxicity. On the contrary, such lymphocytes sometimes appeared to have a promoting effect on the tumor cells.

Cis-diamminedichloroplatinum (II) (cis-DDP) and its structural analogue cis-diamminediaquoplatinum(II) nitrate (cis-aq) were complexed via an intermediate dextran carrier to antibodies specifically reactive with B lymphoma cells (38C-13). The potential use of these drugs in site-directed immunotargeting was evaluated. The two platinum(II) compounds were previously shown to form pharmacologically active complexes with carboxymethyl dextran (CM-dex). For the purpose of preparing drug-antibody complexes, CM-dex was first conjugated to idiotypic antibodies that recognize a specific membrane IgM on the B lymphoma cells. The conjugates were prepared by a modified water-soluble carbodiimide method in which N-hydroxysuccinimide was used to enhance the coupling reaction. The conjugation was followed by separation of the CM-dex-IgG conjugates from unconjugated CM-dex or IgG. The platinum(II) compounds were then complexed to the CM-dex-IgG resulting in complexes carrying up to 50 mole drug/mole IgG. Both cis-DDP and cis-aq complexes of CM-dex-antibody conjugates maintained most of the original cell-binding activity of the antibodies. An in vitro assay was used to demonstrate selective binding to tumor cells in which the target cells were treated with specific immune complexes and washed before culture. In this assay the specific complexes showed preferential cytotoxicity for the B lymphoma cells in comparison to the free drugs, drug CM-dex, or nonspecific immune complexes.

The role of immunosuppressor cells in preventing host immune rejection of tumor cells is described. The growth of Lewis lung carcinoma (3LL) in syngeneic C57BL mice is accompanied by a weak and transient anti-tumor cytotoxic response that is later on replaced by tumor-enhancing activity. This enhancing activity is correlated with the generation of suppressor cells in 3LL-bearing mice. Such suppressors were demon strated in two ways: (a) Elimination of the hydrocortisone(HC)-sensitive lymphoid population from tumorbearing mice (TBM) resulted in a significant increase in the anti-tumor cytotoxic response and in a marked delay in tumor development. It is assumed that HC inactivates precursors of suppressor lymphocytes whereas the mature suppressor cells themselves are HC-resistant. (b) The increased resistance against the tumor could be partially re-suppressed by restoring the HC-treated TBM with spleen or thymus cells from normal C57BL. Suppression, however, was more pronounced if the resistant mice were restored with spleen or thymus cells from TBM. HC-resistant spleen cells from TBM that appear to be enriched for mature suppressor cells were capable of suppressing in vitro the secondary sensitization of spleen cells from TBM against tumor cells.

Suppressor cells in spleens of mice bearing the Lewis lung carcinoma (3LL) suppressed the anti-tumor immune response in vivo but not in vitro. The hydrocortisone (HC)-resistant fraction of spleen cells from tumor-bearing mice (TBM) was, on the other hand, suppressive in vitro, due to enrichment for HC-resistant suppressor cells. This cellular fraction suppressed the tumor-induced differentiation of memory cells as well as the tumor-independent in vitro activation of TBM spleen cells. The in vitro lymphoproliferative response of TBM spleen cells to mitogens was also subjected to suppression: these cells showed a lower response to a mitogen such as Concanavalin A (Con A) and were capable of suppressing the response of normal spleen cells to this mitogen. HC treatment of TBM 2 days prior to spleen harvest enriched the splenic population for suppressor cells that suppressed Con A-induced activation.

Spleen cells from C57BL mice carrying the metastatic Lewis lung carcinoma (3LL) developed a transient cytotoxic response towards the tumor shortly after tumor transplantation. Later on, the cytotoxic activity was lost and enhancing lymphocytes could be demonstrated in the spleens of the tumor-bearing mice (TBM). Spleen cells that were transferred together with tumor cells into syngeneic recipients enhanced tumor growth. The enhancing activity could be eliminated by the removal of a cell population that bound to histamine/rabbit serum albumin/Sepharose (HRS). The adherent population was enriched for enhancing lymphocytes, since it enhanced tumor growth more than the unfractionated population. The non-adherent cells, on the other hand, lost their enhancing activity in vivo and were sometimes protective against tumor growth. In addition, these cells manifested in vitro cytotoxi-city against tumor cells. Hence the suppresssion of the cytotoxic expression in TBM is, at least in part, due to suppressor lymphocytes that bind to unsolubilized histamine. These cells seem to enhance tumor growth by suppressing host reactivity. Thus the enhancing lymphocyte populations can be separated into two subpopulations, of which one is enriched while the other is depleted of suppressor cells.

Suppressor lymphocytes responsible for the suppression of anti-tumor immune response were previously demonstrated in spleens of C57BL mice carrying the syngeneic Lewis lung carcinoma (3LL). Based upon earlier observations on the inactivating effect of hydrocortisone (HC) on precursors of suppressor lymphocytes, we have now studied the effect of the steroid on the anti-tumor cellular immune response and on tumor development in 3LL-bearing mice. Mice were given a single injection of HC before, together with, or after the inoculation of 3LL tumor cells. Tumor development and mouse mortality were delayed when HC was administered after tumor inoculation. The highest delay was observed when the steroid was injected to the mice 3 to 5 days after the tumor cells. HC-resistant spleen cells (15 to 30% of the total number of cells) obtained from such tumor-bearing mice (TBM) 2 or 3 days after HC treatment enhanced tumor development significantly more than cells from untreated TBM, when transferred together with tumor cells into syngeneic recipients. However, this enhancing activity rapidly disappeared upon spleen regeneration. Spleen cells obtained 7 to 14 days after HC injection manifested a protective activity against tumor growth. These results suggest that HC affects the balance between lymphocyte subpopulations which participate in either the rejection or the enhancement of the tumor. HC-resistant spleen cells recovered from the spleens of TBM shortly after HC treatment seem to be enriched for preformed mature suppressor cells, but are deficient for their precursors. This deficiency, which is transient due to thymus and spleen regeneration, is later on expressed in an increased cytotoxic response against the tumor.

The immune competency of peripheral blood (cord) lymphocytes of newborn infants has been investigated by a battery of assays in vitro. Number of T cells was enumerated by E-rosette formation and a cytotoxicity assay by using an anti-human-thymocyte antiserum. Lymphokine production as an indicator of T cell function was evaluated by leukocyte migration inhibition factor production following stimulation with various mitogens, and "viral" and "immune" interferon production in response to stimulation with polyriboinosinic -cytidilic acid and phytohemagglutinin, respectively. Ability to respond to mitogens was also tested by means of 3H-thymidine uptake into DNA, and by measuring the early synthesis of protein by lymphocytes by means of 3H-leucine uptake. Lymphocyte cyclic adenosine monophosphate levels in resting cells and after trypsin treatment was also used as a test of cellular competency. Total B cells was evaluated by EAC-rosette formation, and class specific surface membrane immunoglobulin-bearing cells were assayed by the peroxidase immunoenzymatic method. Normal children and adults served as controls. The results indicated that no significant differences in immune competency could be shown between newborns and older people except for lymphocyte cyclic adenosine monophosphate levels which were lower in the newborns, both in resting and in stimulated cells. The relationship between this latter finding and the immune status is as yet not clear. It is concluded that neonatal lymphocytes are essentially immunocompetent, with the expression of their immune capabilities in vivo becoming apparent only after encounter with environmental antigenic stimuli.

The mitogenic activity of soybean agglutinin was found to depend on the presence of lectin aggregates formed in lectin preparations stored in the lyophilized state. Such soybean agglutinin preparations gave maximal stimulation of untreated pig lymph node cells and neuraminidase-treated mouse spleen cells at relatively high concentrations, ranging from 100 to 2000 mug/ml. After separation into unaggregated (divalent) and polymeric (tetra-and multivalent) fractions, it was found that the unaggregated lectin did not stimulate the cells, while the tetravalent and multivalent fractions were active and gave maximal stimulation at a concentration of 10 mug/ml. These results suggest that soybean agglutinin must have at least four sugar binding sites in order to be able to stimulate lymphocytes.

The high molecular weight copolymer (mol. wt. = 75 000) containing the repeating sequence–(l-Tyr-l-Ala-l-Glu)–and denoted as polypeptide (Tyr-Ala-Glu)n has previously been shown to possess an α-helical structure under physiological conditions. Studies were performed to check whether monovalent fragments of antibodies derived from anti-(Tyr-Ala-Glu)n antibodies could convert the partially helical oligopeptide (Tyr-Ala-Glu)13 into a more helical conformation. Circular dichroic spectra of mixtures of the monovalent antibody fragments with (Tyr-Ala-Glu)13 were measured at two different concentrations of both components. Analogous experiments were done with the same fragments and the polypeptide (Tyr-Ala-Glu)n. When anti-(Tyr-Ala-Glu)n antibody fragments were mixed with (Tyr-Ala-Glu)13. When the same fragments were mixed with the polypeptide (Tyr-Ala-Glu)n, a difference spectrum was observed at the ellipticity band of the polypeptide centered at 275 nm. It was concluded that anti-(Tyr-Ala-Glu)n monovalent antibody fragments could not only convert the (Tyr-Ala-Glu)13 into a structure more similar to the polypeptide (Tyr-Ala-Glu)n, but could also stabilize the helical content of this polypeptide.

Polyalanyl determinants, when attached to acidic or basic carriers, provoke a specific anti-polyalanyl antibody population, the majority of which possesses an electrical charge which is inversely related to that of the carrier. The alanine chains in the immunogens form complete determinants and the contribution of the carrier to the specificity of the antibodies is minimal. The anti-polyalanyl antibodies, when fractionated into a more acidic and a more basic fraction, possess similar properties of their combining sites, as judged from hapten inhibition and equilibrium dialysis studies. Thus, the specificity of the antibodies and the combining site features are determined by the hapten determinants, whereas the net charge of the antibodies is determined by the net charge of the immunogen. The difference in the net electrical charge of the antibodies may reflect differences in areas of the molecule other than the combining site. A comparison of anti-polyalanyl antibodies obtained with different carriers has shown that the specificity and size of the combining sites are similar regardless of the nature of the carrier molecule, whereas different affinities of the antibodies are obtained with different immunogens.

A series of peptides possessing the structure (l-Tyr-l-Ala-l-Glu)n, with n = 1,2,3,4,7,9, and 13 was synthesized. The oligomers up to n = 4 were prepared by stepwise synthesis using the N-hydroxysuccinimide ester of the tripeptide derivative for the elongation of the chain. The oligomers with n = 7,9, 13 were prepared by a polycondensation technique, followed by gel filtration. The circular dichroism spectra of the above oligopeptides were measured in the wavelength range 200–330 nm in a solution of 0.15 M sodium chloride –0.02 M sodium phosphate, pH 7.4, conditions at which the high molecular weight polytripeptide (Tyr-Ala-Glu)n exists in a helical conformation. The circular dichroic spectrum of the high molecular weight polymer exhibits two negative ellipticity bands: an intense band at 220 nm ([Θ]max= 8700) and a weak band at 273 nm ([Θ]max= 360). The tripeptide (n = 1) exhibits a positive ellipticity band at 227 nm ([Θ]max= 4000) and a broad, positive, weak band at 270 nm ([Θ]max= 115). The other oligopeptides (n = 2 to 13) exhibit positive bands in the 227 nm region, with ellipticity values that decrease as the degree of polymerization increases. Except for (Tyr-Ala-Glu)13, which has a negative peak centered at 216 nm, circular dichroic spectra of the other oligomers (n = 1 to 9) in the 200–210 nm region resemble that of random coils. In the 260–290 nm region, the circular dichroic curves of the oligopeptides gradually approach the 272 nm band of the polypeptide (Tyr-Ala-Glu)n, as the degree of polymerization increases. Under physiological conditions, only the (Tyr-Ala-Glu)13 shows an indication of helical content.

Antibodies with specificity towards poly- l -alanine and poly- d -alanine were prepared in rabbits by immunization with the respective polyalanyl proteins. From a study of the inhibition of preciption reactions with various alanine peptides it appeared that peptides composed exclusively of the d -isomer were much more efficient inhibitor in the poly- d -alanine immune system than were those composed exclusively of the l -isomer in the poly- l -alanine system. This apparent difference was due to proteolytic activity in rabbi sera. When immunoglobulin G preparations devoid of proteolytic activity were used, no significant differences between the antipodal systems were found. The size of the specific combining region of the antibodies was found to be such as to accommodate a maximum of 3–4 alanine residues, as concluded from the inhibition results. From the extent of inhibition of the stereo-specific antigen-antibody reactions with alanine peptides composed of l and d residues at determined positions, it was concluded that the region of the antigenic determinant furthest removed from the protein carrier is of paramount importance in determining the specificaties of the antibodies formed.

Antibodies were provoked against a random copolymer of l-tyrosine, l-alanine, and l-glutamic acid, denoted as(Tyr,Ala,Glu)n, and against the α-helical high molecular weight polymer containing the repeating sequence l-tyrosyl-l-alanyl-l-glutamyl, denoted as (Tyr-Ala-Glu)n. Significant crossreactions were found between anti-(Tyr-Ala-Glu)n antibodies and (Tyr,Ala,Glu)n, as well as between anti-(Tyr,Ala,Glu)n antibodies and (Tyr-Ala-Glu)n. Small peptides, obtained from a 45-min pronase digestion of (Try,Ala,Glu)n efficiently inhibited precipitin reactions between anti-(Tyr,Ala,Glu)n and (Tyr,Ala,Glu)n, anti-(Tyr,Ala,Glu)n and (Tyr-Ala-Glu)n, and anti-(Tyr-Ala-Glu)n and (Tyr,Ala,Glu)n. The peptides did not affect at all the reaction between anti-(Tyr-Ala-Glu)n and (Try-Ala-Glu)n. It was concluded that the cross-reaction between the (Tyr-Ala-Glu)n and (Tyr,Ala,Glu)n systems were due to sequential determinants in (Tyr-Ala-Glu)n rather than helix-dependent determinants in (Tyr,Ala,Glu)n. The cross-reactive fraction from anti-(Tyr-Ala-Glu)n sera was isolated on a (Tyr,Ala,Glu)n immunoadsorbent and the purified antibodies were found to precipitate to a much larger extent with (Tyr,Ala,Glu)n than with (Tyr-Ala-Glu)n. These antibodies were probably produced against some degradation products of (Tyr-Ala-Glu)n produced in vivo.