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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.

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.

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.

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...

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.

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.

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.

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 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.

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.