Bilha Schechter

Persistent elimination of ErbB-2/HER2-overexpressing tumors using combinations of monoclonal antibodies: Relevance of receptor endocytosis

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.

Reduction of cytotoxicity to normal tissues by new regimens of cell-cycle phase-specific drugs

Abstract A new policy for minimizing cytotoxicity to normal tissues of cell-cycle phase-specific drugs is suggested. This policy is based on short drug pulses and on an integer relation between the period of drug application and the cell-cycle length of the host susceptible tissue. The new protocol is expected to be effective even if the cell-cycle parameters of the malignant population have the same mean as those of the host cells but their variance is larger, and as long as the variance in the duration of the drug pulse is not too large. Under these conditions protocols can be determined by the host temporal parameters alone. The method has been verified in vitro , treating a leukemic cell line with cytosine arabinoside (Ara-C).

Combining sites of antibodies with l-alanine and d-alanine peptide specificity and the effect of serum proteolytic activity on their estimation

Abstract 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.

Aptamer to ErbB-2/HER2 enhances degradation of the target and inhibits tumorigenic growth

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.

Functional polymers in drug delivery: carrier-supported CDDP (cis-platin) complexes of polycarboxylates — effect on human ovarian carcinoma

Abstract 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).

Cancer therapeutic antibodies come of age: Targeting minimal residual disease

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.

Immunoreactivities of polyclonal and monoclonal anti‐T and anti‐Tn antibodies with human carcinoma cells, grown in vitro and in a xenograft model

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.

Polymers in drug delivery: immunotargeting of carrier-supported cis-platinum complexes

Abstract 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.

Effect of the dosing interval on myelotoxicity and survival in mice treated by cytarabine

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.

Immunological studies on specific antibodies against trypsin

Abstract Immunization of rabbits with trypsin in Freunds 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.