In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.

Bcl-2 family proteins are important regulators of apoptosis that function during development and other physiological processes but also contribute to pathological conditions associated with inappropriate cell survival such as cancer (1, 2). This still expanding family contains both pro-and anti-apoptotic members characterized by the presence of one or more Bcl-2 homology (BH) 1 domains. Of these domains, BH3 has proven to be a key element in pro-apoptotic Bcl-2 homologs that mediate both protein binding and cell death functions (1, 3). In Caenorhabditis elegans, the BH3-containing protein EGL-1 functions at the most proximal point in a pathway required for all programmed cell death, and in mammalian cells BH3 proteins transduce signals from cell surface receptors to a central cell death pathway regulated by Bcl-2 (4 -6). BAD is a "BH3-only" pro-apoptotic Bcl-2 family member whose function is regulated by phosphorylation in response to survival factors such as nerve growth factor, insulin-like growth factor-1, and interleukin-3 (7-9). In its unphosphorylated state, BAD forms heterodimers with anti-apoptotic Bcl-2 homologs and promotes cell death. These activities are inhibited by phosphorylation of BAD on either of two serine residues, Ser 112 or Ser 136 (8). The serine/threonine kinase Akt that is activated by growth factors through a PI 3-kinase-dependent mechanism phosphorylates BAD on Ser 136 (9, 10). The ribosomal S6 kinases and a mitochondria-localized cAMP-dependent protein kinase (PKA) have been reported to phosphorylate BAD on Ser 112 (11, 12) following stimulation by growth factors and interleukin-3, respectively. When BAD is phosphorylated on these sites, it is sequestered in the cytosol in a complex with 14-3-3 and fails to interact with Bcl-x L at mitochondrial membranes (8). The present study provides genetic, biochemical, and biological evidence that growth factor-induced phosphorylation on the novel site Ser 155 , which is within the functionally critical BH3 domain, directly blocks BAD binding to Bcl-x L and may represent another major regulatory mechanism of BAD inactivation. EXPERIMENTAL PROCEDURESConstructs-A cDNA for murine BAD was obtained by reverse transcriptase-PCR using mRNA isolated from FL5.12 cells and the following PCR primers: 5Ј-GCCTCCAGGATCCAAGATGGGAACC-3Ј and 5Ј-GG-AGCGGGTAGAATTCCGGGATG-3Ј. When this cDNA, which encodes the 204-amino acid BAD protein (7), was cloned into pcDNA3 (Invitrogen) and was expressed in cells, the protein product was significantly larger than the endogenous BAD protein (approximately 30 kDa versus 23 kDa, respectively) detected with an anti-BAD antibody (C-20, Santa Cruz Biotechnology, Inc.). A methionine residue at position 43 of the mouse sequence corresponds to the first methionine residue of the human BAD. We generated a cDNA for the shorter form of murine BAD by using an upstream PCR primer surrounding the second methionine residue (5Ј-TGGAGACCAGGATCCCAGAGTAGCT-3Ј) and the same downstream primer as above. The expressed construct co-migrated with the ...

Denaturation of DNA photolyase (deoxyribodipyrimidine photolyase, EC 4.1.99.3) from Escherichia coli with guanidine hydrochloride or acidification to pH 2 released, in addition to FAD, a chromophore with the spectral and chromatographic properties of a reduced pterin. Treatment of the enzyme with iodine prior to acidification converted the chromophore to a stable, oxidized derivative, which was resolved by HPLC into four species with identical spectral properties. The same species, in the same distribution, were obtained from the yeast enzyme. The material isolated from the iodine-oxidized enzyme was shown to be a pterin by conversion to pterin-6-carboxylic acid with alkaline permanganate and was found to release glutamate upon acid hydrolysis. The presence of 10-formylfolate in the isolated, oxidized chromophore was demonstrated by absorption and fluorescence spectroscopy and by deformylation and conversion to folic acid. Analysis of the distribution of polyglutamates revealed that the four species identified by HPLC corresponded to the tri-, tetra-, penta-, and hexaglutamate derivatives of 10-formylfolate. The results were consistent with Y linkages in the triglutamate derivative with additional glutamates linked via the a-carboxyl group of the preceding residue. Treatment with rat plasma hydrolase produced the monoglutamate derivative of 10-formylfolate. The native, enzyme-bound form of the folate cofactor was identified as 5,10-methenyltetrahydrofolylpolyglutamate by effecting release and isolation at low pH to protect the 5,10-methenyl bridge and preserve the reduced pyrazine ring structure. DNA photolyase (deoxyribodipyrimidine photolyase, EC 4.1.99.3) catalyzes the repair of pyrimidine dimers that are formed between adjacent pyrimidine bases in the DNA as a result of damage by UV light. The repair reaction is driven by visible or long wavelength UV light and appears to depend on electron transfer from a photoexcited reduced flavin in the photolyase to the dimer (1). DNA photolyase has been purified from a number of different organisms. The action spectrum of the enzyme varies somewhat with the species as does the state of the flavin in the isolated enzyme. The enzyme purified from Escherichia coli contains a blue neutral flavin semiquinone radical, but the flavin can be reduced with a concomitant increase in activity (1, 2). The flavin in the isolated yeast enzyme is in the fully reduced state (3). The mechanism by which visible or long wavelength UV light energy is gathered, transferred to the flavin, and subsequently utilized in the DNA repair reaction is not fully understood. It is becoming clear, however, that a second enzyme-bound cofactor participates in catalysis. In the experiments described below, the second chromophore of E. coli and yeast photolyase is characterized as a conjugated pterin. A stable, oxidized species was isolated and identified as 10-formylfolic acid; however, evidence is presented that indicates that in the enzyme the folate cofactor exists in a reduced, fluorescent sta...

A majority of ovarian and non-small cell lung adenocarcinoma cancers overexpress folate receptor a (FRa). Here, we report the development of an anti-FRa antibody-drug conjugate (ADC), consisting of a FRa-binding antibody attached to a highly potent maytansinoid that induces cell-cycle arrest and cell death by targeting microtubules. From screening a large panel of anti-FRa monoclonal antibodies, we selected the humanized antibody M9346A as the best antibody for targeted delivery of a maytansinoid payload into FRa-positive cells. We compared M9346A conjugates with various linker/maytansinoid combinations, and found that a conjugate, now denoted as IMGN853, with the N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB) linker and N