Mark X. Sliwkowski

HER2 is a validated target in breast cancer therapy. Two drugs are currently approved for HER2-positive breast cancer: trastuzumab (Herceptin), introduced in 1998, and lapatinib (Tykerb), in 2007. Despite these advances, some patients progress through therapy and succumb to their disease. A variation on antibody-targeted therapy is utilization of antibodies to deliver cytotoxic agents specifically to antigen-expressing tumors. We determined in vitro and in vivo efficacy, pharmacokinetics, and toxicity of trastuzumab-maytansinoid (microtubule-depolymerizing agents) conjugates using disulfide and thioether linkers. Antiproliferative effects of trastuzumab-maytansinoid conjugates were evaluated on cultured normal and tumor cells. In vivo activity was determined in mouse breast cancer models, and toxicity was assessed in rats as measured by body weight loss. Surprisingly, trastuzumab linked to DM1 through a nonreducible thioether linkage (SMCC), displayed superior activity compared with unconjugated trastuzumab or trastuzumab linked to other maytansinoids through disulfide linkers. Serum concentrations of trastuzumab-MCC-DM1 remained elevated compared with other conjugates, and toxicity in rats was negligible compared with free DM1 or trastuzumab linked to DM1 through a reducible linker. Potent activity was observed on all HER2-overexpressing tumor cells, whereas nontransformed cells and tumor cell lines with normal HER2 expression were unaffected. In addition, trastuzumab-DM1 was active on HER2-overexpressing, trastuzumab-refractory tumors. In summary, trastuzumab-DM1 shows greater activity compared with nonconjugated trastuzumab while maintaining selectivity for HER2-overexpressing tumor cells. Because trastuzumab linked to DM1 through a nonreducible linker offers improved efficacy and pharmacokinetics and reduced toxicity over the reducible disulfide linkers evaluated, trastuzumab-MCC-DM1 was selected for clinical development.

The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from the carboxyl-terminal tail has been determined to 2.6-Å resolution, both with and without an EGFRK-specific inhibitor currently in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, TarcevaTM). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the carboxyl-terminal tail are discussed with respect to receptor oligomerization. The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from the carboxyl-terminal tail has been determined to 2.6-Å resolution, both with and without an EGFRK-specific inhibitor currently in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, TarcevaTM). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the carboxyl-terminal tail are discussed with respect to receptor oligomerization. Growth factor interactions with cell surface receptors influence proliferation, survival, differentiation, and metabolism (1Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1292) Google Scholar). The loss of control over these vital cellular processes is a hallmark of oncogenesis (2Hunter T. Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2269) Google Scholar). For instance, aberrant signaling from overexpressed growth factor receptor ErbB2 is causal in approximately 30% of invasive breast cancers (3Ross J.S. Fletcher J.A. Stem Cells. 1998; 16: 413-428Crossref PubMed Scopus (614) Google Scholar). Growth factors bind to a cognate membrane-bound receptor system and mediate changes in the intracellular portion of the receptor, often through the formation of dimers or oligomers of receptors that initiate signal transduction cascades. The epidermal growth factor receptor (EGFR, 1The abbreviations used for: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; RTK, receptor tyrosine kinase; SH2, Src homology 2; p-Tyr, phosphotyrosine; A-loop, activation loop; DTT, dithiothreitol; P38, mitogen-activated protein kinase p38; MES, 4-morpholineethanesulfonic acid; FGFRK, fibroblast growth factor receptor kinase; EGFRK, epidermal growth factor receptor kinase; N-lobe, NH2-terminal lobe; C-lobe, COOH-terminal lobe; r.m.s., root mean square; LCK, lymphocyte tyrosine kinase; p-IRK, insulin receptor kinase-phosphorylated form; FGF, fibroblast growth factor; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; CDK2, cyclin-dependent kinase 2. also ErbB1 or HER1) and its ligands, epidermal growth factor (EGF) and transforming growth factor-α, are among the earliest characterized members of the growth factor/receptor tyrosine kinase (RTK) family. In contrast to the widely applicable ligand-induced receptor dimerization paradigm, there is evidence that EGFR family members exist as preformed dimers (4Moriki T. Maruyama H. Maruyama I.N. J. Mol. Biol. 2001; 311: 1011-1026Crossref PubMed Scopus (277) Google Scholar) and form higher oligomer signaling complexes (5Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3538) Google Scholar). Normal signaling in the EGFR system involves ligand-induced homo-oligomerization or hetero-oligomerization with the closely related RTKs ErbB2 (HER2), ErbB3 (HER3) and/or ErbB4 (HER4) (6Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell. Biol. 2001; 2: 127-137Crossref PubMed Scopus (5633) Google Scholar). Autophosphorylation of key tyrosine residues within the carboxyl-terminal portion of the receptor provides sites for direct interaction with SH2-containing proteins, leading to subsequent signal transduction events. The EGFR system, including receptor homologues and relevant ligands, is complex. There are at least 12 different ligands that bind to the EGF receptor family with partially redundant specificity for certain receptors. Several of the ligands including EGF, transforming growth factor-α, heparin-binding EGF, and betacellulin are reported to bind to EGFR with nanomolar dissociation constants (7Jones J.T. Akita R.W. Sliwkowski M.X. FEBS Lett. 1999; 447: 227-231Crossref PubMed Scopus (320) Google Scholar). Betacellulin also binds ErbB4 with high affinity. Similarly, heregulin binds to ErbB3 or ErbB4 with dissociation constants in the nanomolar range. So far, a ligand that binds ErbB2 alone has not been identified, although the affinity of an ErbB2/ErbB3 heterodimer for heregulin is high, ∼1011m (8Karunagaran D. Tzahar E. Beerli R.R. Chen X. Graus-Porta D. Ratzkin B.J. Seger R. Hynes N.E. Yarden Y. EMBO J. 1996; 15: 254-264Crossref PubMed Scopus (588) Google Scholar, 9Sliwkowski M.X. Schaefer G. Akita R.W. Lofgren J.A. Fitzpatrick V.D. Nuijens A. Fendly B.M. Cerione R.A. Vandlen R.L. Carraway III., K.L. J. Biol. Chem. 1994; 269: 14661-14665Abstract Full Text PDF PubMed Google Scholar). In addition, the kinase domain of ErbB3 has non-canonical amino acids at some key positions, which render it catalytically inactive (10Guy P.M. Platko J.V. Cantley L.C. Cerione R.A. Carraway III., K.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8132-8136Crossref PubMed Scopus (596) Google Scholar). Taken together, these factors point to a complicated interplay between cross-reacting ligands, functional diversity among receptors, and differential expression in the EGFR signaling system. In the non-signaling state, most RTKs possess low basal kinase activity that increases substantially upon growth factor binding. This results from receptor oligomerization and subsequent transphosphorylation of tyrosine residues within a partner kinase domain. Specifically, initial phosphotyrosine (p-Tyr) modification of the “activation loop” (A-loop) generates optimal catalytic activity and subsequent rapid phosphorylation at substrate docking sites elsewhere on the receptor intracellular domain. The EGFR, ErbB2, and ErbB4 receptors are the only known RTKs that do not require this initial phosphorylation of kinase domain residues for full catalytic competency. This unique feature may partially explain why EGFR family members are frequently involved in cellular transformation. In the RTKs for which crystal structures of both unphosphorylated and phosphorylated versions of the kinase domain are available, phosphorylation in the A-loop causes it to undergo a large structural reorganization that relieves steric and/or chemical restraints on the catalytic active site (11Hubbard S.R. Till J.H. Annu. Rev. Biochem. 2000; 69: 373-398Crossref PubMed Scopus (892) Google Scholar). Distinguishing the EGFR family further is an intracellular dimerization motif that has been roughly assigned to reside between the kinase domain and the carboxyl-terminal phosphorylation sites. The greatest effects on receptor function seem to be concentrated in the Leu955-Val956-Ile957 segment of EGFR (“LVI”) and other ErbB receptors. This motif is necessary for ligand-independent dimerization of EGFR intracellular domains (12Chantry A. J. Biol. Chem. 1995; 270: 3068-3073Abstract Full Text Full Text PDF PubMed Google Scholar) and for transphosphorylation in ErbB2/ErbB3 heterodimers (13Schaefer G. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 1999; 274: 859-866Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Moreover, alanine substitutions in this region override mutations in the transmembrane segment of ErbB2 that would otherwise lead to constitutive signaling via non-ligand induced dimerization (14Penuel E. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 2002; 277: 28468-28473Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The molecular mechanism by which these residues influence receptor activity is not well understood. Members of the EGFR family are frequently overactive in solid tumors (15Khazaie K. Schirrmacher V. Lichtner R.B. Cancer Metastasis Rev. 1993; 12: 255-274Crossref PubMed Scopus (267) Google Scholar). A number of therapeutic approaches that interfere with aberrant EGFR family signaling are being investigated (16Shawver L.K. Slamon D. Ullrich A. Cancer Cells. 2002; 1: 117-123Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). A relatively new therapeutic approach to kinase inhibition is the use of ATP-competitive small molecules (17Druker B.J. Sawyers C.L. Kantarjian H. Resta D.J. Reese S.F. Ford J.M. Capdeville R. Talpaz M. N. Eng. J. Med. 2001; 344: 1038-1042Crossref PubMed Scopus (2442) Google Scholar, 18Schindler T. Bornmann W. Pellicena P. Miller W.T. Clarkson B. Kuriyan J. Science. 2000; 289: 1938-1942Crossref PubMed Scopus (1621) Google Scholar, 19Arteaga C.L. J. Clin. Oncol. 2001; 19: S32-S40PubMed Google Scholar, 20Kelloff G.J. Fay J.R. Steele V.E. Lubet R.A. Boone C.W. Crowell J.A. Sigman C.C. Cancer Epidemiol. Biomark. Prev. 1996; 5: 657-666PubMed Google Scholar). Several groups have shown that certain 4-anilinoquinazoline derivatives are both selective and effective inhibitors of the EGFR kinase (21Woodburn J.R. Pharmacol. Ther. 1999; 82: 241-250Crossref PubMed Scopus (780) Google Scholar). Structural data exist for compounds of this general class bound to the distantly related intracellular kinases CDK2 and mitogen-activated protein kinase p38 (P38) (22Shewchuk L. Hassell A. Wisely B. Rocque W. Holmes W. Veal J. Kuyper L.F. J. Med. Chem. 2000; 43: 133-138Crossref PubMed Scopus (207) Google Scholar, 23.Deleted in proof.Google Scholar, 24.Deleted in proof.Google Scholar). 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Chem. 2000; 43: PubMed Scopus Google direct structural evidence has been we the of the EGFR kinase alone and in with the inhibitor residues from EGFR A. L. J.S. A. J. Yarden Y. J. PubMed Scopus Google Scholar) by an NH2-terminal site and a COOH-terminal and The with and and the on the to the and site an NH2-terminal with a site and a COOH-terminal with The with and with the the system to the of and in at with of EGFRK and at for by at for on and of DTT, to the The The at for to The over a with a The with for EGFRK protein from the with of EGFRK as by with to the and EGFRK concentrated to a of and a with EGFRK as by and DTT, that the protein any The EGFRK concentrated to for of of EGFRK over in protein with the MES, in a used as in a of a as to in of EGFRK with erlotinib by of in a MES, for with and without the erlotinib treatment in a with with data at of the Structural and at of the for Structural to for both and with and W. 1996; Scopus Google Scholar). The high of the very high in a the high of data used in on a on for and and is the number of is the number of unique at least is the of at least is the of a and the for The for the of is not reported by and are and structure factor for or from of in is number of assigned is the number of is the number of unique at least is the of at least is the of a and the for The for the of is not reported by and are and structure factor for or from in is number of assigned in a new and cell and The structure D. 1994; PubMed Scopus Google Scholar) a of in data to from an data with of the data for the of and the used to higher for the data with A. Scopus Google Scholar) and a with of and and with and The structure used as a point for the and the of The of structural to any of the The is to be assigned with which a for the amino would of or The of the is well it intermolecular contacts with molecules within the The activation loop is for its factors and a The factors are high and the by of the lack of and are from the J. G. H. I.N. 2000; PubMed Scopus Google Scholar) and The EGFR kinase domain (EGFRK) adopts the of all reported protein kinase domains The NH2-terminal is from and the COOH-terminal is The are by a similar to in which and ATP-competitive inhibitors have been found to of the catalytic the on the the loop the the motif the catalytic the catalytic loop and the A-loop The NH2-terminal of EGFRK adopts a structure similar to structures of RTKs for with the kinase domain from the fibroblast growth factor receptor is although a the of EGFRK from other kinase domains. The of amino acids the of the loop and amino acids to that from the expression The we in the is with the residues an The NH2-terminal amino acids are by intermolecular contacts including involving of residues and although between and both and the a similar to of the lymphocyte tyrosine kinase H. 1996; PubMed Scopus Google the insulin receptor kinase-phosphorylated form S.R. EMBO J. 16: PubMed Scopus Google Scholar) and the unphosphorylated form of the receptor kinase M. J. S.R. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) EGFRK the motif found in these related and has In the interactions of the and an between the and a or and the the NH2-terminal region to the In EGFRK, contacts and on the and a similar the the of active of kinases is a between that with the and or a close is In both the and of EGFRK, we find a between of this in that EGFR not require large within the for catalytic The COOH-terminal domain of EGFRK the of in other kinase domain of the of kinase domains from both and a of as with the N-lobe, a key from In most protein the activation loop its catalytically conformation only it phosphorylated on a or For these the unphosphorylated activation loop is from the active conformation and may a direct For instance, the unphosphorylated A-loop in is with substrate and the unphosphorylated insulin receptor kinase A-loop as well as the substrate tyrosine The A-loop in from other unphosphorylated A-loop has shown that of the EGFRK A-loop, at a that is phosphorylated in other be by without loss of function N. A. M. Y. M. Biochem. 1992; PubMed Scopus Google Scholar). with this we that the A-loop of EGFRK adopts an conformation similar to the phosphorylated A-loop of Many interactions this most of which are also found in other active kinase well with of and contact with the of a that the of in the between of and that between and the of in p-IRK, the this conformation is This interaction may be for the loop there is of The between and the catalytic is the as between the residues in and other tyrosine This is to the catalytic and substrate for In p-IRK, is and in EGFRK, the a closely to that of the of in from the at in of the A-loop with residues the catalytic and to of is The of the is not by is among low at the that it with a or with from phosphorylation on the in the shown the of of the insulin receptor kinase to between and are key for the conformation by the of the EGFRK A-loop, and interactions in in an interaction not in the the of to the of This interaction further to the unphosphorylated EGFRK A-loop active and it is that the of at among kinases is the of the A-loop, an from kinases have a the catalytic its interactions with a in are and the or of the with in EGFRK with the very similar to found in There are other protein kinases with known molecular structures that do not require phosphorylation in their A-loop for optimal catalytic among kinase D.J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) kinase G. J. X. EMBO J. 1995; PubMed Scopus Google Scholar) carboxyl-terminal Src kinase A. Y. H. S. A. S. M. T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) and kinase B. J. EMBO J. 1996; 15: PubMed Scopus Google Scholar) The most relevant of these is the kinase in which a at an A-loop to sites of phosphorylation with the the catalytic and the or The EGFRK A-loop is in and the of of these residues and to EGFRK function amino substitutions that the in of M. Biochem. J. 1995; PubMed Scopus Google Scholar). In EGFRK, the structural of and are a between the contacts of in and the effects of in the conformation by the EGFRK A-loop to from an for the active conformation an for an For instance, there is to the EGFRK A-loop the conformation found for from which it by to intermolecular crystal contacts do not seem to a interactions of this do not exist in structures and would not we to an A-loop in crystal The of EGFRK also the Leu955-Val956-Ile957 found to in a the transphosphorylation of substrate in EGFR family the amino substitutions for are the most for A direct contact between this segment and receptor or an has been for the ErbB2/ErbB3 system (13Schaefer G. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 1999; 274: 859-866Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In EGFRK is in close contact with the The region is to the by the This that the not contact is that the of with alanine it and residues from the is that the segment effects in the intracellular region that the of the COOH-terminal tail as a substrate for the the and residues have The residues are of an signal that receptors to or via Cell. Full Text PDF PubMed Scopus Google Scholar, A. Science. PubMed Scopus Google Scholar). The residues at well and are in close contact with molecules with The amino acids in are not There is structure in the COOH-terminal with the for a The crystal contacts by the well COOH-terminal residues molecules The contact surface by approximately the other is characterized by an The in to being of is also with respect to The COOH-terminal region is and the contact region of the molecules is The region of the is at the of the The contact for the COOH-terminal region is in the of a different The well of the EGFRK also in and FGFRK, structural with these RTKs as well with a of most of only The for adopts the The site of would require only to the in the The between the and EGFRK structures in this region is in the in both and the loop is is in the erlotinib as it is to the as a general of inhibitor binding. The between the of kinase structures has been to on the or of or a close with the catalytic kinase the between EGFRK is most similar to of LCK, p-IRK, and for which of of on the the of both and with of and of FGFRK, and the of the is between of and and erlotinib the for is and the for the are and between the and erlotinib structures with and be is at are The of this of the by an from the erlotinib complex. This the at and a of the that the in This is and between are on the by have that as erlotinib inhibition through to the site by There is a ATP-competitive inhibitors the EGFRK S. J.M. J. Med. Chem. PubMed Scopus Google Scholar, A. N. R. R. E. N. J. Med. Chem. 2000; 43: PubMed Scopus Google Scholar, Chem. Rev. 2001; PubMed Scopus Google Scholar, P. Med. Rev. 2000; PubMed Scopus Google Scholar). have been to to EGFRK S. J.M. J. Med. Chem. PubMed Scopus Google Scholar, A. N. R. R. E. N. J. Med. 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PubMed Scopus Google contact for both are In the and direct their protein to of and with of In the only the of these contacts is In we both and contacts and the of erlotinib with respect to the and contacts is a of the CDK2 and The catalytic activity of the EGFR family kinases is unique among The of the vital cellular processes by EGFR signaling be by control of the of COOH-terminal substrate to the active The structure we have determined this as we find all of the catalytic and for without or substrate This from other RTKs the that the between the or kinase domains in an EGFR family may from of closely related RTKs for The in the upon the of erlotinib to that would have a similar the of effects require with the of the catalytic from the we in this of the erlotinib by EGFRK in the of inhibitor and in the any from the crystal is to have the to have of protein kinase domains are found both A-loop phosphorylation and are The constitutive catalytic of EGFRK and of the A-loop in an active conformation are the crystal and the between erlotinib and are to have the of the we upon inhibitor and that erlotinib may not the form an of the that is in the COOH-terminal a direct with the receptors or A for the on transphosphorylation by for is an by the loss of the that effects would in the COOH-terminal tail The intermolecular contact in crystal are is in protein are to form these The of these contacts involving the COOH-terminal region is to The lack of for the residues the that of and is The of the contacts of the COOH-terminal region is at a site on the protein that from both and and the segment between and large changes in the may this a is with results in upon M. A. A. U. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The data do not to that these contacts also in a cellular the high of membrane-bound EGFR and evidence for non-ligand induced dimers (4Moriki T. Maruyama H. Maruyama I.N. J. Mol. Biol. 2001; 311: 1011-1026Crossref PubMed Scopus (277) Google Scholar) that close among some of the intracellular domains is M. S. and M. for data for with as well as and the at Structural and T. K. and at of the Structural at the by the U. S. of of and The is by the of of the U. S. of at