Folate receptor is over-expressed in a variety of carcinomas. To design a cytotoxic drug that would selectively target these carcinomas, we synthesized folate-maytansinoids. These drugs showed high affinity toward folate receptor, appeared to enter cells exclusively via the folate receptor-mediated caveolar pathway and displayed high cytotoxic potency (in the range of 10 ؊11 to 10 ؊10 M) and remarkable selectivity for folate receptor-expressing carcinoma cell lines. Folatemaytansinoids represent a new class of tumor-specific agents in which the targeting and the cytotoxic function can be altered independently. Int. J. Cancer 73:859-864, 1997. Wiley-Liss, Inc.The therapeutic effectiveness of anti-cancer drugs in current clinical use is limited by their low selectivity for tumors and associated high systemic toxicity. Enormous research efforts, therefore, were undertaken to develop tumor-selective drugs by conjugating a toxin or a cytotoxic drug to an antibody or a hormone that binds selectively to cancer cells (Pastan et al., 1992;Pietersz and Krauer, 1994). One cell surface-expressed molecule that is very attractive for targeting cytotoxic agents is folate receptor because it is greatly over-expressed in a variety of carcinomas and has a highly restricted distribution of expression in normal tissues (Stein et al., 1991;Franklin et al., 1994;Garin-Chesa et al., 1993; Weitman et al., 1992a,b;Ross et al., 1994). The first vehicles used to deliver cytotoxic moieties to folate receptor-expressing cells were anti-folate receptor monoclonal antibodies (MAbs) (Cogliati et al., 1991;Coney et al., 1994). Antibodies are large proteins whose targeting capacity and therapeutic usefulness may be hampered by their poor tumor penetration (Jain, 1989;Fujimori et al., 1989;Cassidy et al., 1993;Debinski and Pastan, 1995) and immunogenicity (Ghetie and Vitetta, 1994). These problems could be avoided by using small molecules for targeting drugs. Folate receptor binds its natural low m.w. ligand, folic acid, with high affinity (K d in the range of 1 nM or less [Kamen, 1987;Sirotnak et al., 1987]), and only low concentrations of folic acid derivatives (collectively called folates) that can bind to the receptor are found in blood (Kamen, 1987;Sirotnak et al., 1987). Therefore, it was proposed that extraneously administered folic acid could compete easily with the endogenous folates and serve as a targeting vehicle. Folic acid would preferentially target folate receptor and not folate transporter since the latter binds folic acid with an affinity that is at least 10 4 -fold lower (Kamen, 1987;Sirotnak et al., 1987) than the affinity toward folate receptor. Folic acid was conjugated to protein toxins (Leamon and Low, 1992;Leamon et al., 1993). These agents, however, also may be prone to the limitations typical for large protein-based therapeutic agents. We wished to design a low m.w. cytotoxic drug targeted to folate receptor. To achieve this goal, we synthesized the maytansinoid DM1, a highly cytotoxic drug that inhibits the polymerizat...

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A protein carboxyl methyltransferase (PCMT), which specifically modifies atypical protein L-isoaspartyl and D-aspartyl residues, is widely distributed in eucaryotic cells, but the factors that regulate its activity in vivo have not been identified. It has been proposed that the PCMT initiates the repair of structurally damaged proteins. To test the possibility that the concentration of structurally abnormal cellular proteins affects PCMT activity, protein carboxyl methylation reactions were studied in HeLa cells exposed to various stresses that increase the extent of protein unfolding in cells. Protein carboxyl methylation rates increased 70-80% during incubations at 42 degrees C and remained elevated for periods of up to 8 hr. This sustained increase was greater than that predicted from thermal effects on the enzyme alone and may reflect the exposure of atypical aspartyl sites as proteins unfold as well as increased rates of protein deamidation and isomerization at elevated temperatures. Methylation rates showed no increases following 12 hr incubations with the amino acid analogs L-azetidine-2-carboxylic acid or L-canavanine. Northern blot analysis of RNA preparations from control and stressed cells revealed three major transcripts for the PCMT in HeLa cells, which are 1.6, 2.6, and 4.5 kb in length. The concentrations of all three transcripts decreased by approximately 20% from control levels during heat shock. No changes in PCMT transcript concentrations were observed during incubation with the amino acid analogs. By contrast, large increases in the concentrations of hsp70 and ubiquitin transcripts were observed following either heat or chemical stresses. The results demonstrate that the PCMT is a constitutive component of cells whose function is required under normal conditions as well as during stress conditions, which accelerate structural damage to cellular proteins.

Folate receptor is over‐expressed in a variety of carcinomas. To design a cytotoxic drug that would selectively target these carcinomas, we synthesized folate‐maytansinoids. These drugs showed high affinity toward folate receptor, appeared to enter cells exclusively via the folate receptor–mediated caveolar pathway and displayed high cytotoxic potency (in the range of 10−11 to 10−10 M) and remarkable selectivity for folate receptor–expressing carcinoma cell lines. Folate‐maytansinoids represent a new class of tumor‐specific agents in which the targeting and the cytotoxic function can be altered independently. Int. J. Cancer 73:859–864, 1997. © 1997 Wiley‐Liss, Inc.

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