WO2008154439A1 - Procédés et compositions pour induire une apoptose dans des cellules cancéreuses - Google Patents

Procédés et compositions pour induire une apoptose dans des cellules cancéreuses Download PDF

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WO2008154439A1
WO2008154439A1 PCT/US2008/066202 US2008066202W WO2008154439A1 WO 2008154439 A1 WO2008154439 A1 WO 2008154439A1 US 2008066202 W US2008066202 W US 2008066202W WO 2008154439 A1 WO2008154439 A1 WO 2008154439A1
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seq
antibody
amino acid
light chain
heavy chain
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Peter Flynn
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Irm Llc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to antibodies that specifically bind to Death Receptor 5 ("DR5").
  • Apoptosis is a highly conserved cell suicide program essential for development and tissue homeostasis of all metazoan organisms. Changes to the apoptotic pathway that prevent or delay normal cell turnover can be just as important in the pathogenesis of diseases as are abnormalities in the regulation of the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either prevent or induce cell death.
  • TNF Tumor necrosis factor
  • TRAIL -related apoptosis-inducing ligand
  • TRAIL Upon binding to DR5, TRAIL induces cell death by apoptosis. See, e.g., Pan et al, Science 277:815-8 (1997); Sheridan, et al , Science 277:818-21 3 (1997); Walczak ef ⁇ /, EMBO J. 16:5386-97 4 (1997). In vitro, TRAIL has been shown to kill a broad spectrum of tumor cells that express DR5, but is relatively non-toxic to normal cells.
  • the invention provides antibodies that bind Death Receptor 5 (DR5).
  • the antibodies comprise (a) a heavy chain variable region comprising a human heavy chain V- segment, a heavy chain complementary determining region 3 (CDR3), and a heavy chain framework region 4 (FR4), and
  • a light chain variable region comprising a human light chain V segment, a light chain CDR3, and a light chain FR4, wherein i) the heavy chain CDR3 comprises the amino acid sequence HEEGI (SEQ ID NO:
  • the light chain CDR3 variable region comprises the amino acid sequence QXHXXTP (SEQ ID NO:50), wherein X denotes any amino acid.
  • the invention provides antibodies that bind Death Receptor 5 (DR5) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise the following three complementary determining regions (CDRs): CDRl, CDR2 and CDR3; wherein: i) the CDRl of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2 and SEQ ID
  • the CDR2 of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:
  • the CDR3 of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:
  • the CDRl of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; v) the CDR2 of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO:17; vi) the CDR3 of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO:20 and SEQ ID NO:21, with the proviso that the antibody does not comprise all of SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NO: 19.
  • the antibodies comprise a heavy chain V-segment sharing at least 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:44. In some embodiments, the antibodies comprise a light chain V-segment sharing at least 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:46 or SEQ ID NO:48.
  • the heavy chain CDR3 comprises the amino acid sequence motif HEEGIYFX 1 X 2 (SEQ ID NO:51), wherein X 1 is D, T or K and X 2 is Y, K or V; and ii) the light chain CDR3 comprises the amino acid sequence motif
  • QX 3 HX 4 X 5 TP (SEQ ID NO:52) or QX 3 HX 4 X 5 TPFT (SEQ ID NO:53), wherein X 3 is Q or H, X 4 is Y, L or K, and X 5 is T, Q, I, E, H or G.
  • the heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of HEEGIYFDY (SEQ ID NO:7), HEEGIYFDK (SEQ ID NO:8),
  • HEEGIYFDV (SEQ ID NO:54), HEEGIYFTY (SEQ ID NO:55) and HEEGIYFKY (SEQ ID NO:54).
  • the light chain CDR3 comprises an amino acid sequence selected from the group consisting of QQHYTTP (SEQ ID NO:57), QQHYQTP (SEQ ID NO:58), QQHYITP (SEQ ID NO:59), QQHYETP (SEQ ID NO:60), QQHYHTP (SEQ ID NO:61), QQHYGTP
  • QHHYTTP (SEQ ID NO: 65); or ii) the light chain CDR3 comprises an amino acid sequence selected from the group consisting of QQHYTTPFT (SEQ ID NO: 19), QQHYQTPFT (SEQ ID NO:66), QQHYITPFT (SEQ ID NO:20), QQHYETPFT (SEQ ID NO:67), QQHYHTPFT (SEQ ID NO:
  • the heavy chain FR4 is a human germline FR4. In some embodiments, the FR4 is human germline JH4 (SEQ ID NO:28).
  • the heavy chain comprises a human germline J-segment.
  • the heavy chain human germline J-segment is JH4.
  • the heavy chain human germline J-segment is the amino acid sequence YFD(YZK)WGQGT(LZT)(VZL)TVSS (SEQ ID NO:73).
  • the light chain FR4 is a human germline FR4. In some embodiments, the FR4 is human germline JK2 (SEQ ID NO: 38).
  • the light chain comprises a human germline J-segment.
  • the light chain human germline J-segment is JK2.
  • the light chain human germline J-segment is the amino acid sequence (YZF)TFG(QZS)GTKLEIK (SEQ ID NO:74).
  • the CDRl of the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3; ii) the CDR2 of the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO:6; iii) the CDRl of the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; and iv) the CDR2 of the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
  • the heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO:9; and ii) the light chain CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22.
  • the CDRl of the heavy chain V-segment comprises SEQ ID NO:2; ii) the CDR2 of the heavy chain V-segment comprises SEQ ID NO: 5; iii) the heavy chain CDR3 comprises SEQ ID NO:8; iv) the CDRl of the light chain V-segment comprises SEQ ID NO: 11; v) the CDR2 of the light chain V-segment comprises SEQ ID NO: 16; and vi) the light chain CDR3 comprises SEQ ID NO:20.
  • the CDRl of the heavy chain V-segment comprises SEQ ID NO:2; ii) the CDR2 of the heavy chain V-segment comprises SEQ ID NO: 5; iii) the heavy chain CDR3 comprises SEQ ID NO:8; iv) the CDRl of the light chain V-segment comprises SEQ ID NO: 12; v) the CDR2 of the light chain V-segment comprises SEQ ID NO: 17; and vi) the light chain CDR3 comprises SEQ ID NO:20.
  • the CDRl of the heavy chain V-segment comprises SEQ ID NO:2; ii) the CDR2 of the heavy chain V-segment comprises SEQ ID NO: 5; iii) the heavy chain CDR3 comprises SEQ ID NO:8; iv) the CDR 1 of the light chain V-segment comprises SEQ ID NO : 11 ; v) the CDR2 of the light chain V-segment comprises SEQ ID NO: 17; and vi) the light chain CDR3 comprises SEQ ID NO:20.
  • the heavy chain variable region shares at least 90%, 93%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the variable region of SEQ ID NO:43. In some embodiments, the light chain variable region shares at least 90%, 93%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the variable region of SEQ ID NO:45 or SEQ ID NO:47.
  • the antibody binds to DR5 with an equilibrium dissociation constant (KD) of less than 1 x 10 "8 M.
  • KD equilibrium dissociation constant
  • the antibody is a FAb' fragment.
  • the antibody is an IgG.
  • the antibody is a single chain antibody (scFv).
  • the antibody comprises human constant regions.
  • the antibody is a DR5 agonist. In some embodiments, the antibody is a DR5 antagonist. [0024] In some embodiments, the antibodies comprise a heavy chain having at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO:39 and a light chain having at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42.
  • the antibodies comprise a heavy chain comprising SEQ ID NO:39 and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42.
  • the light chain comprises SEQ ID NO:40.
  • the light chain comprises SEQ ID NO:41.
  • the light chain comprises SEQ ID NO:42.
  • the invention provides pharmaceutically acceptable compositions comprising an anti-DR5 antibody of the invention and a physiologically compatible excipient.
  • the embodiments of the antibodies in the compositions are as described above and throughout the application.
  • the invention provides methods of inducing apoptosis in a cancer cell comprising contacting the cell with an anti-DR5 antibody of the invention, wherein the antibody is an agonist of DR5.
  • an anti-DR5 antibody of the invention wherein the antibody is an agonist of DR5.
  • the invention provides methods of inducing apoptosis in a cancer cell in a subject comprising administering to the subject a therapeutically effective amount of an anti-DR5 antibody of the invention, wherein the antibody is an agonist of DR5.
  • the cell is further contacted with an apoptosis-inducing agent.
  • an "antibody” refers to a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen.
  • An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy" chain (about 50-70 kD), connected through a disulfide bond.
  • the recognized immunoglobulin genes include the K, ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either K or ⁇ .
  • Heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these regions of light and heavy chains respectively.
  • an "antibody” encompasses all variations of antibody and fragments thereof that possess a particular binding specifically, e.g., for DR5.
  • CDRs complementary-determining regions
  • the CDRs are the target protein-binding site of the antibody chains that harbors specificity for such target protein. There are three CDRs (CDRl-3, numbered sequentially from the N-terminus) in each human V L or V H , constituting about 15-20% of the variable domains.
  • the CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for the binding specificity.
  • the remaining stretches of the V L or V H the so-called framework regions, exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).
  • the positions of the CDRs and framework regions are determined using various well known definitions in the art, e.g. , Kabat, Chothia, international ImMunoGeneTics database (IMGT) (on the worldwide web at imgt.cines.fr/), and AbM (see, e.g., Johnson et «/. , Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987); Chothia et al, Nature, 342:877-883 (1989); Chothia et al, J. MoI.
  • IMGT international ImMunoGeneTics database
  • binding specificity determinant or “BSD” interchangeably refer to the minimum contiguous amino acid sequence within a complementary determining region necessary for determining the binding specificity of an antibody.
  • a minimum binding specificity determinant can be within one or more CDR sequences.
  • the minimum binding specificity determinants reside within (i.e., are determined solely by) a portion or the full-length of the CDR3 sequences of the heavy and light chains of the antibody.
  • an "antibody light chain” or an “antibody heavy chain” as used herein refers to a polypeptide comprising the V L or V H , respectively.
  • the endogenous V L is encoded by the gene segments V (variable) and J (junctional), and the endogenous V H by V, D (diversity), and J.
  • Each of V L or V H includes the CDRs as well as the framework regions.
  • antibody light chains and/or antibody heavy chains may, from time to time, be collectively referred to as "antibody chains.” These terms encompass antibody chains containing mutations that do not disrupt the basic structure of V L or V H , as one skilled in the art will readily recognize.
  • Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab' which itself is a light chain joined to V H -C H I by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region. Paul, Fundamental Immunology 3d ed. (1993).
  • antibody While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, supra; Marks et al, Biotechnology, 10:779-783, (1992)).
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol.
  • humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some complementary determining region ("CDR") residues and possibly some framework (“FR”) residues are substituted by residues from analogous sites in rodent antibodies.
  • a "chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, and drug; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • variable region or "V-region” interchangeably refer to a heavy or light chain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. See, Figure 2.
  • An endogenous variable region is encoded by immunoglobulin heavy chain V-D-J genes or light chain V-J genes.
  • a V-region can be naturally occurring, recombinant or synthetic.
  • variable segment or “V-segment” interchangeably refer to a subsequence of the variable region including FR1-CDR1-FR2-CDR2-FR3. See, Figure 2.
  • An endogenous V-segment is encoded by an immunoglobulin V-gene.
  • a V-segment can be naturally occurring, recombinant or synthetic.
  • J-segment refers to a subsequence of the variable region encoded comprising a C-terminal portion of a CDR3 and the FR4.
  • An endogenous J-segment is encoded by an immunoglobulin J-gene. see, Figure 2.
  • a J-segment can be naturally occurring, recombinant or synthetic.
  • a "humanized” antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al, Proc. Natl. Acad. ScL USA, 81:6851- 6855 (1984); Morrison and Oi, Adv. Immunol, 44:65-92 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994).
  • corresponding human germline sequence refers to the nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences.
  • the corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences.
  • the corresponding human germline sequence can be framework regions only, complementary determining regions only, framework and complementary determining regions, a variable segment (as defined above), or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods described herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art.
  • the corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 92%, 94%, 96%, 98%, 99% sequence identity with the reference variable region nucleic acid or amino acid sequence.
  • Corresponding human germline sequences can be determined, for example, through the publicly available international ImMunoGeneTics database (IMGT) (on the worldwide web at imgt.cines.fr/) and V-base (on the worldwide web at vbase.mrc- cpe.cam.ac.uk).
  • IMGT international ImMunoGeneTics database
  • V-base on the worldwide web at vbase.mrc- cpe.cam.ac.uk.
  • the antibodies or binding agents with a particular binding specificity bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample.
  • Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to have been selected for its specificity for a particular protein. This selection may be achieved by subtracting out antibodies that cross-react with, e.g., DR5 molecules from other species.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective binding reaction will produce a signal at least twice over the background signal and more typically at least than 10 to 100 times over the background.
  • Equilibrium dissociation constant (K D , M) refers to the dissociation rate constant (k d , time "1 ) divided by the association rate constant (k a , time "1 , M “1 ). Equilibrium dissociation constants can be measured using any known method in the art.
  • the antibodies of the present invention generally will have an equilibrium dissociation constant of less than about 10 "8 M, for example, less than about 10 "9 M or 10 "10 M, in some embodiments, less than about 10 "11 M, 10 "12 M or 10 "13 M.
  • the term "antigen-binding region” refers to a domain of the DR5- binding molecule of this invention that is responsible for the specific binding between the molecule and DR5.
  • An antigen-binding region includes at least one antibody heavy chain variable region and at least one antibody light chain variable region. There are at least one such antigen-binding regions present in each DR5 -binding molecule of this invention, and each of the antigen-binding regions may be identical or different from the others. In some embodiments, at least one of the antigen-binding regions of a DR5 -binding molecule of this invention acts as an agonist of DR5.
  • agonist refers to an agent that is capable of specifically binding and activating a receptor to induce a full or partial response mediated by the receptor.
  • an agonist of DR5 specifically binds to the receptor and induces DR5-mediated signaling.
  • a DR5 agonist can be identified by its ability to bind to DR5 and induce apoptosis in Jurkat cells.
  • An "antibody agonist” refers to the situation where the agonist is an activating antibody.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., MoI. Cell. Probes 8:91-98 (1994)).
  • polypeptide As used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ -carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g. , norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i. e. , gaps) as compared to the reference sequence (e.g. , a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same sequences.
  • Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the invention provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein (e.g., the CDRs exemplified in any one of SEQ ID NOS: 1-22, 54-56 and 66-72).
  • the identity exists over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence.
  • identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence.
  • shorter amino acid sequences e.g., amino acid sequences of 20 or fewer amino acids
  • substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. ScL USA 85:2444, by computerized implementations of these algorithms (GAP,
  • BESTFIT FASTA
  • TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. MoI. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et ah, supra).
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. ScL USA 90:5873- 5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • link when used in the context of describing how the antigen-binding regions are connected within a DR5-binding molecule of this invention, encompasses all possible means for physically joining the regions.
  • the multitude of antigen-binding regions are frequently joined by chemical bonds such as a covalent bond (e.g. , a peptide bond or a disulfide bond) or a non-covalent bond, which can be either a direct bond (i.e., without a linker between two antigen-binding regions) or indirect bond (i.e., with the aid of at least one linker molecule between two or more antigen-binding regions).
  • SMAC refers to a mitochondrial polypeptide, which is released together with cytochrome c from the mitochondria in response to apoptotic stimuli. SMAC promotes caspase activation by binding and neutralizing the IAPs. See, e.g., Du et al, Cell 102:33-42 (2000); Verhagen et al, Cell 102:43-53 (2000).
  • terapéuticaally acceptable amount refers to an amount sufficient to effect the desired result (i.e., apoptosis of a target cell). Preferably, a therapeutically acceptable amount does not effect undesirable side effects.
  • a therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved.
  • Figure 1 illustrates a dilution ELISA comparison of the sub-cloned V-regions in a Fab' format (DR5-1) ( ⁇ ) and the original NVP-LCR211 Mab (A) binding to Thio-DR5 antigen. No significant binding signal is observed in the PBS control sample (T).
  • Figure 2 depicts a schematic depiction of the structural units that comprise the V- regions of an antibody.
  • the heavy chain is encoded by three gene families (Heavy-V, D and J) and the light chain is encoded by two gene families (Kappa or Lambda V and J). The recombination of these genes results in the intact V-region.
  • the CDR3 sequences are at the recombination sites of the heavy V, D and J genes, in the case of the heavy chain, and the kappa or lambda V and J genes in the case of the light chain.
  • Figure 3 illustrates V-region amino acid sequence alignments of partial framework 3 regions (FR3) and the complete CDR3 and framework 4 regions (FR4) (SEQ ID NOS:76-78 and 80-82) of the murine and optimized reference Fab's.
  • the residues changed from the original murine sequence are highlighted in red and underlined.
  • the CDR3 BSD residues are highlighted in bold and the CDR3 regions are boxed.
  • the human germline J-segments JH4 (SEQ ID NO:75) and JK2 (SEQ ID NO:79) are also aligned.
  • Figure 4 illustrates a dilution ELISA comparison of the sub-cloned V-regions in a Fab' format DR5-1 ( ⁇ ) and the optimized reference controls DR9-1 (A) and DRlO-I (T) binding to Thio-DR5 antigen.
  • Figure 5 illustrates a V-heavy CDR2 mutagenic library comprising reference (SEQ ID NO:4) or human germline (VHl -46) (SEQ ID NO: 107) amino acids at each position. Amino acids that are common between reference and human were kept constant and at two positions extra amino acids were possible due to degenerate codon usage.
  • Figure 6 illustrates the V-heavy CDR2 sequences (SEQ ID NOS:6, 4 and 83-91, respectively) selected according to DR5 antigen binding activity from a Fab' library. Mutations with reference to human germline sequence are underlined.
  • Figure 7 illustrates the selected V-heavy CDR2 mutants binding to DR5 antigen in a dilution ELISA. Specific binding activity can be seen in comparison to the reference control and a clone negative for antigen binding (DR83-D3).
  • Figure 8 illustrates V-light CDR3 sequences (8A) (SEQ ID NOS: 19, 66, 20 and 67- 72) and V-heavy CDR3 sequences (8B) (SEQ ID NOS:7, 8 and 54-56) that were selected from CDR3 affinity maturation libraries based on their DR5-antigen binding activity.
  • the amino acids that differ from the reference CDR3 sequence are shaded blue and underlined.
  • Figure 9 illustrates the DR5-antigen binding activity of the selected CDR3 affinity maturation mutants.
  • the ELISA assay shows significant antigen binding of the selected clones when compared to a negative control Fab' (background).
  • Figure 10 illustrates a kinetic analysis of reference Fab' or selected, fully optimized Fab's binding to recombinant DR5 antigen by bio layer interferometry using ForteBio Octet biosensor technology.
  • Figure 11 illustrates aligned V-region amino acid sequences of improved Fab's DR106-2 (SEQ ID NOS:39 and 40), DRl 12 (SEQ ID NOS:39 and 41) and DRl 14 (SEQ ID NOS :39 and 42) amino acid sequences and comparison with the closest single human germline V-gene and J-segment (Human VH1-46/JH4 (SEQ ID NO:43) and VkIV-B3/Jk2 (SEQ ID NO:45) or VK1-L4/JK2 (SEQ ID NO:47)).
  • CDRs are boxed and residues that differ from the corresponding position in the germline sequence (excluding the CDR3 "BSD" sequence) are shaded red. Affinity maturation changes to the CDR3s are shaded blue.
  • DR5 is a so-called death receptor expressed on the surface of a wide variety of cancer cells ⁇ see, Chaudhary, et al., Immunity 7:821-830 (1997) and GenBank Accession number AF016268). Upon binding of its ligand, TRAIL or Apo2L, DR5 triggers a cascade of signaling events via its cytoplasmic death domain, ultimately leading to apoptosis.
  • the present invention provides antibodies that specifically bind DR5, contain minimum binding determinant sequences derived from a non-human anti-DR5 reference monoclonal antibody (e.g., mouse), and variable region amino acid sequences encoded by corresponding human germline variable region sequences.
  • the antibodies of the present invention specifically bind to DR5.
  • the antibodies may block the binding of a native ligand, act as an antagonist or act as an agonist.
  • the anti-DR5 antibodies of the present invention act as agonists of a DR5 receptor.
  • a DR5 antibody agonist is an antibody that specifically binds DR5 and activates DR5 -mediated signaling.
  • the anti-DR5 antibodies optionally can be multimerized (e.g., trimerized) and used according to the methods of this invention.
  • the anti-DR5 antibodies can be a full-length tetrameric antibody (i.e., having two light chains and two heavy chains), a single chain antibody (e.g., a ScFv), or a molecule comprising antibody fragments that form one or more antigen-binding sites and confer DR5-binding specificity, e.g., comprising heavy and light chain variable regions (for instance, Fab' or other similar fragments).
  • Anti-DR5 antibody fragments can be produced by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies can be obtained by, e.g., hybridoma or recombinant production.
  • Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.
  • the constant regions of the anti- DR5 antibodies can be any type or subtype, as appropriate, and can be selected to be from the species of the subject to be treated by the present methods (e.g., human, non-human primate or other mammal, for example, agricultural mammal (e.g., equine, ovine, bovine, porcine), domestic mammal (e.g., canine, feline) or rodent (e.g., rat, mouse, hamster, rabbit).
  • human, non-human primate or other mammal for example, agricultural mammal (e.g., equine, ovine, bovine, porcine), domestic mammal (e.g., canine, feline) or rodent (e.g., rat, mouse, hamster, rabbit).
  • variable regions of the anti-DR5 antibodies of the present invention are derived from a reference monoclonal antibody known to bind DR5 with high affinity, and acts as an agonist.
  • the antibodies are improved or optimized by reducing the amino acid sequence segments corresponding to a non-human species (e.g., mouse) and increasing the amino acid sequence segments corresponding to human germline amino acid sequences. In this way, sequences that could potentially induce an immune response in a human host against the anti- DR5 antibodies are reduced, minimized or eliminated.
  • Methods for engineering human antibodies have been described. See, e.g., U.S. Patent Publication No. 2005/0255552 and U.S. Patent Publication No. 2006/0134098, the disclosures of both of which are hereby incorporated herein by reference in their entirety for all purposes.
  • the improved anti-DR5 antibodies of the invention are engineered human antibodies with V-region sequences having substantial amino acid sequence identity to human germ-line V-region sequences while retaining the specificity and affinity of a reference antibody. See, U.S. Patent Publication No. 2005/0255552 and U.S. Patent
  • the process of improvement identifies minimal sequence information required to determine antigen-binding specificity from the variable region of a reference antibody, and transfers that information to a library of human partial V-region gene sequences to generate an epitope-focused library of human antibody V-regions.
  • a microbial- based secretion system can be used to express members of the library as antibody Fab' fragments and the library is screened for antigen-binding Fab's, for example, using a colony- lift binding assay. See, e.g., U.S. Patent Publication No. 2007/0020685. Positive clones can be further characterized to identify those with the highest affinity.
  • the resultant engineered human Fab's retain the binding specificity of the parent, reference anti-DR5 antibody, typically have equivalent or higher affinity for antigen in comparison to the parent antibody, and have V-regions with a high degree of sequence identity compared with human germ-line antibody V-regions.
  • the minimum binding specificity determinant (BSD) required to generate the epitope-focused library is typically represented by a sequence within the heavy chain CDR3 ("CDRH3") and a sequence within the light chain of CDR3 ("CDRL3").
  • the BSD can comprise a portion or the entire length of a CDR3.
  • the BSD can be comprised of contiguous or non-contiguous amino acid residues.
  • the epitope-focused library is constructed from human V-segment sequences linked to the unique CDR3-FR4 region from the reference antibody containing the BSD and human germ-line J-segment sequences (see, Figure 2 and U.S. Patent Publication No. 2005/0255552).
  • the human V-segment libraries can be generated by sequential cassette replacement in which only part of the reference antibody V-segment is initially replaced by a library of human sequences. The identified human "cassettes" supporting binding in the context of residual reference antibody amino acid sequences are then recombined in a second library screen to generate completely human V-segments (see, U.S. Patent Publication No. 2006/0134098).
  • paired heavy and light chain CDR3 segments, CDR3-FR4 segments, or J-segments, containing specificity determinants from the reference antibody are used to constrain the binding specificity so that antigen-binders obtained from the library retain the epitope-specificity of the reference antibody. Additional maturational changes can be introduced in the CDR3 regions of each chain during the library construction in order to identify antibodies with optimal binding kinetics.
  • the resulting engineered human antibodies have V-segment sequences derived from the human germ-line libraries, retain the short BSD sequence from within the CDR3 regions and have human germ-line framework 4 (FR4) regions.
  • the anti-DR5 antibodies contain a minimum binding sequence determinant (BSD) within the CDR3 of the heavy and light chains derived from the originating monoclonal antibody.
  • the heavy chain CDR3 comprises the BSD
  • the light chain CDR3 comprises the BSD QXHXXTP (SEQ ID NO:50), wherein X is any amino acid. See, e.g., Figures 3, 8 and 11.
  • the remaining sequences of the heavy chain and light chain variable regions (CDR and FR), e.g., V-segment and J-segment, are from corresponding human germline amino acid sequences.
  • the V- segments can be selected from a human V-segment library. Further sequence refinement can be accomplished by affinity maturation.
  • the heavy and light chains of the anti-DR5 antibodies contain a human V-segment from the corresponding human germline sequence (FRl-CDRl- FR2-CDR2-FR3), e.g., selected from a human V-segment library, and a CDR3-FR4 sequence segment from the originating monoclonal antibody.
  • the CDR3-FR4 sequence segment can be further refined by replacing sequence segments with corresponding human germline sequences and/or by affinity maturation.
  • the FR4 and/or the CDR3 sequence surrounding the BSD can be replaced with the corresponding human germline sequence, while the BSD from the CDR3 of the originating monoclonal antibody is retained.
  • the corresponding human germline sequence for the heavy chain V-segment is VH 146. In some embodiments, the corresponding human germline sequence for the heavy chain is J-segment is JH4.
  • the variable region genes are referenced in accordance with the standard nomenclature for immunoglobulin variable region genes. Current immunoglobulin gene information is available through the worldwide web, for example, on the ImMunoGeneTics (IMGT), V-base and PubMed databases. See also, Lefranc, Exp Clin lmmuno genet. 2001; 18(2): 100- 16; Lefranc, Exp Clin Immuno genet. 2001;18(3):161-74; Exp Clin Immuno genet.
  • the heavy chain J-segment has the amino acid sequence YFD(Y/K)WGQGT(L/T)(V/L)TVSS (SEQ ID NO:73).
  • the corresponding human germline sequence for the light chain V-segment is VKIV or VKIL4.
  • the corresponding human germline sequence for the heavy chain J-segment is JK2.
  • the light chain J-segment has the amino acid sequence (Y/F)TFG(Q/S)GTKLEIK (SEQ ID NO:74).
  • the heavy chain V-segment shares at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence
  • the light chain V-segment shares at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity an amino acid sequence selected from
  • the heavy chain CDR3 comprises amino acid sequence motif HEEGIYFX 1 X 2 (SEQ ID NO:51), wherein X 1 is D, T or K and X 2 is Y, K or V; and ii) the light chain CDR3 comprises amino acid sequence motif QX 3 HX 4 X 5 TP (SEQ ID NO:52), wherein X 3 is Q or H, X 4 is Y, L or K, and X 5 is T, Q, I, E, H or G.
  • the heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of HEEGIYFDY (SEQ ID NO:7), HEEGIYFDK (SEQ ID NO:8), HEEGIYFDV (SEQ ID NO:54), HEEGIYFTY (SEQ ID NO:55) AND HEEGIYFKY (SEQ ID NO:56); and ii) the light chain CDR3 comprises an amino acid sequence selected from the group consisting of QQHYTTP (SEQ ID NO:57), QQHYQTP (SEQ ID NO:58), QQHYITP (SEQ ID NO:59), QQHYETP (SEQ ID NO:60), QQHYHTP (SEQ ID NO:61), QQHYGTP (SEQ ID NO:62), QQHLTTP (SEQ ID NO:63), QQHKTTP (SEQ ID NO:64) and QHHYTTP (SEQ ID NO: 65); or ii) the light chain CDR3 comprises
  • the antibodies of the invention comprise a heavy chain variable region comprising a CDRl comprising an amino acid sequence SY(YZT)MH (SEQ ID NO:95); a CDR2 comprising an amino acid sequence
  • the antibodies of the invention comprise a light chain variable region comprises a CDRl comprising an amino acid sequence selected from KSSQS(V/F)L(Y/G)SSN(N/G)KNY(L/V)A (SEQ ID NO:97) and
  • RASQ(G/D)(I/V)S(S/G)ALA (SEQ ID NO:98); a CDR2 comprising an amino acid sequence (W/D)AS(S/T/M)(R/L)(E/V)S (SEQ ID NO:99); and a CDR3 comprising an amino acid sequence of Q(Q/H)(Y/H/F)(Y/N/L/K)(S/I/E/H/G/Q/T)(T/Y)P(Y/F)T (SEQ ID NO: 100).
  • antibodies do not comprise all of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NO: 18.
  • the heavy chain variable region comprises a FRl comprising the amino acid sequence (E/Q)VQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 101); a FR2 comprising SEQ ID NO:25; a FR3 comprising the amino acid sequence RVTMT(R/G)DTSTSTVYMELSSLRSEDTA VYYCAR (SEQ ID NO: 102); and a FR4 comprising SEQ ID NO: 28.
  • the identified amino acid sequences may have one or more substituted amino acids (e.g., from affinity maturation) or one or two conservatively substituted amino acids.
  • the light chain variable region comprises a FRl comprising an amino acid sequence selected from DIVMTQSPDSLA(V/A)SLGE(R/K)ATINC (SEQ ID NO: 103) or (A/D)IQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 104); a FR2 comprising the amino acid sequence WYQQKPG(Q/K)(P/A)PKLLIY (SEQ ID NO: 105); a FR3 comprising the amino acid sequence GVP(D/S)RFSGSGSGTDFTLTISSLQ(A/P)ED(V/F)A(V/T)YYC (SEQ ID NO: 106); and a FR4 comprising SEQ ID NO:37.
  • the identified amino acid sequences may have one or more substituted amino acids (e.g., from affinity maturation) or one or two conservatively substituted amino acids.
  • variable regions of the anti-DR5 antibodies of the present invention generally will have an overall (e.g., FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) amino acid sequence identity of at least about 90%, for example, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to the corresponding human germline variable region amino acid sequence.
  • heavy chain of the anti-DR5 antibodies can share at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the human germline variable region VH146/JH4.
  • the light chain of the anti-DR5 antibodies can share at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the human germline variable region VKIV/JK2 or VKIL4/JK2.
  • the anti-DR5 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region selected from SEQ ID NOS:39 and 43 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region selected from SEQ ID NOS:40, 41, 42, 45 and 47.
  • the anti-DR5 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:39 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region selected from SEQ ID NOS:40, 41 and 42.
  • the anti-DR5 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:43 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region selected from SEQ ID NOS: 45 and 47.
  • the anti-DR5 antibodies of the present invention generally will bind DR5 with an equilibrium dissociation constant (K D ) of less than about 10 "8 M or 10 "9 M, for example, less than about 10 "10 M or 10 ⁇ M, in some embodiments less than about 10 "12 M or 10 "13 M.
  • K D equilibrium dissociation constant
  • Anti-DR5 antibodies have been described previously in, e.g., U.S. Patent Application No. 10/723,383, published as US 2005/0079172, PCT WO 01/83560 (antibody TRA-8; ATCC PTA- 1428) and PCT WO 02/079377.
  • the variable regions of the heavy and light chains of an exemplary anti-DR5 antibody agonist have been published in US 2005/0079172.
  • the DR5-binding molecule of this invention contains at least one DR5 -binding region that competes with the antibody exemplified in US 2005/0079172 for binding to DR5.
  • the DR5-binding molecule of this invention contains at least one DR5-binding region that has CDRs substantially similar in amino acid sequence to the CDRs exemplified in US 2005/0079172.
  • any type of DR5 antibody agonist may be used according to the methods of the present invention.
  • the antibodies used are monoclonal antibodies, which can be generated by any one of the methods known in the art (e.g., hybridomas and recombinant expression).
  • antibody fragments comprising heavy and light chain variable regions, rather than full-length antibodies, are used to construct the DR5-binding molecule of this invention.
  • the antigen-binding region(s) of the DR5-binding molecule are single chain antibodies (ScFv). Techniques useful for producing ScFv and antibodies are described in, e.g., U.S. Pat. Nos.
  • a DR5 antibody that specifically binds to DR5 can be identified using techniques well known in the art, for example, ELISA, Surface Plasmon Resonance, interferometry (e.g., using ForteBio Octet biosensor system).
  • a DR5 antibody agonist can be identified by testing for antibody's ability to trigger a DR5-mediated events, e.g., inducing apoptosis in a DR5- expressing cancer cell.
  • a variety of assays known in the art can be used to detect induction of apoptosis.
  • Jurkat cells are contacted with a DR5 antibody that is a candidate agonist.
  • the cells are then monitored for viability as a function of antibody concentration. Reduced cell viability caused by increased apoptosis corresponding to increased antibody concentration indicates that the antibody is an agonist.
  • Cell viability can be assayed by adding Alamar blue to the cell culture. The dye fluoresces in the presence of living, but not dead, cells.
  • DR5 antibody agonists can also be identified by screening hybridomas raised against DR5 and then screening the hybridoma supernatant for the ability to induce apoptosis in Jurkat cells. Appropriate positive and negative controls can be used to confirm the results. For example, a cell line that does not undergo DR5-mediated, TRAIL-induced apoptosis should not undergo apoptosis in response to a candidate DR5 antibody agonist. 3. METHODS OF INDUCING APOPTOSIS
  • the antibodies of the present invention can be used to induce apoptosis in a target cell, if they can act as an agonist of a DR5 receptor.
  • one or more anti-DR5 antibodies of the invention are contacted with one or more DR5 receptors on a target cell ⁇ e.g., a cancer cell), in vivo or in vitro, in an amount sufficient to induce apoptosis in the target cell.
  • the antibodies of the invention are contacted with a DR5 receptor on a target cell concurrently with a second apoptosis-inducing agent.
  • the present invention provides for the improved effect of anti-DR5 antibody agonists with a second apoptosis-inducing agent.
  • Apoptosis-inducing agents include any agent that induces apoptosis in cells.
  • the apoptosis-inducing agent preferentially induces apoptosis in cancer cells compared to non-cancer cells.
  • the apoptosis-inducing agents are agonists or activators of apoptosis or antagonists of inhibitors of apoptosis.
  • Exemplary apoptosis-inducing agents include, e.g., agonists or mimetics of the following: SMAC, Bax, Bik, Bok, Bim, Bak, Bid, Noxa, Puma, Hrk, or Bad; BH3, p53, TRAIL, Fadd, Myc, and Mekkl, signal recognition particle 72kD (SRP72), Caspase-8, Bid, B lymphoid tyrosine kinase (BLK), gene product similar to Pyruvate kinase, M2 isozyme (LOC148283), glycogen synthase kinase 3 alpha (GSK3A), hypothetical protein FLJ32312 (FLJ32312), mitogen- activated protein kinase 10 (MAPKlO), TCF4: transcription factor 4, v- abl Abelson murine leukemia viral oncogene homolog 2 (arg, Abelson-related gene) (AB L2),
  • NLK e.g., accession no. NM_016231
  • GRAF e.g., accession no. NM_015071
  • GCK e.g., accession no. NM_000162
  • ERK5 e.g., accession no. NM_002749
  • FGR e.g., accession no. NM_005248
  • ACVRLl e.g., accession no. NM_000020
  • MEKK5 e.g., accession no. NM_002757
  • PIP5K1C e.g., accession no. XM_047620
  • MAPKAPK2 e.g., accession no. NM_004759
  • RFTl e.g., accession no. NM_052859
  • MKNKl e.g., accession no.
  • Additional exemplary apoptosis- inducing agents include, e.g., agents that enhance DR5 expression and/or stability, agents that enhance caspase activity or stability, and agents that induce or enhance a DNA damage response.
  • Agonist or mimetics in the above list include the gene products themselves, e.g., p53 is a p53 agonist; TRAIL is a TRAIL agonist.
  • Antagonists include agents that directly inhibit activity and agents that indirectly inhibit activity through decreasing expression or stability of target molecule mRNA (e.g., siRNAs) or protein.
  • Apoptosis-inducing agents that can be identified by targeting these gene products include compounds of various chemical natures.
  • modulators of these gene products can be screened with libraries of polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, polypeptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • the apoptosis-inducing agent is a polynucleotide.
  • it can be an siRNA targeting a gene that inhibits TRAIL- induced apoptosis (e.g., UbcHIO, plexin Bl (PLXNBl), SET domain containing protein 7 (SET7), mitogen- activated protein kinase kinase kinase 5 (MAP3K5); STE20-like kinase (JIK), endoplasmic reticulum multispan transmembrane protein (RFTl), MAP kinase-interacting serine/threonine kinase 1 (MKNKl), mitogen- activated protein kinase-activated protein kinase 2 (MAPKAP K2), phosphatidylinositol-4-phosphate 5-kinase, type 1, gamma (PIP5K1C), MAP2k5,
  • the apoptosis-inducing agent is a small molecule compound (e.g., a molecule with a molecular weight of less than 1500 Daltons and in some cases, less than 1000 Daltons).
  • the apoptosis-inducing agent can be a small molecule compound that inhibits expression or activity of a gene product that inhibit TRAIL-induced apoptosis (e.g., UbcHIO, plexin Bl (PLXNBl), SET domain containing protein 7 (SET7), mitogen- activated protein kinase kinase kinase 5 (MAP3K5); STE20-like kinase (JIK), endoplasmic reticulum multispan transmembrane protein (RFTl), MAP kinase-interacting serine/threonine kinase 1 (MKNKl), mitogen- activated protein kinase-activated protein kinase 2 (MAPKAPK2), phosphatidylinositol-4-phosphate 5-kinase, type 1, gamma (PIP5K1C), MAP2k5, cyclin-dependent kinase 6 (CDK
  • the apoptosis-inducing agent can also be a small molecule compound that enhanced expression or activity of a gene product that promote TRAIL-induced apoptosis (e.g.,
  • the apoptosis-inducing agent is conjugated to the anti-DR5 antibody agonist. In other embodiments, the apoptosis-inducing agent is not conjugated to the anti-DR5 antibody agonist.
  • the apoptosis-inducing agent is SMAC/Diablo or a SMAC mimetic or agonist.
  • SMAC/Diablo promotes caspase activity by binding Inhibitor of Apoptosis Proteins (IAPs). See, e.g. , Du et al. , Cell 102:33-42 (2000); Verhagen et al. , Cell 102:43-53, 2000; U.S. Patent Application No. 2002/0110851.
  • Administration or expression of SMAC in cells is encompassed by the present invention.
  • SMAC fragments such as the N- terminal peptides of SMAC (e.g., the N-terminal tetra or heptapeptides (Guo et al., Blood 99(9):3419-3426 (2002); Srinivasula et al, J. Biol. Chem. 275:36152-36157 (2000)), can also be expressed or administered. See, also, U.S. Patent Application 2002/0132786.
  • SMAC mimetic compounds can also be used according to the present invention. These compounds can have useful pharmaceutical properties and as such can be more efficient for administration with anti-DR5 antibodies.
  • Exemplary SMAC mimetics include, e.g., peptides comprising a tetrapeptide that binds the surface groove within the BIR domain of an IAP, including tetrapeptides of the formula X 1 X 2 X 3 X 4 , wherein X] is A, X 2 is V, T, or I, X 3 is P or A and X 4 is F, Y, I or V or other SMAC peptides, agonists or peptidiomimetics described in PCT WO 02/26775.
  • Other exemplary SMAC mimetics include LBP672. See, e.g., Figure 15 from U.S. Patent Publication No. 2005/0079172
  • the apoptosis-inducing agent is a 26S Proteasome inhibitor.
  • Proteasome inhibitors are agents that inhibit the proteasome-ubiquitin pathway, thereby preventing degradation of IKB and subsequent nuclear localization of IKB'S partner, NFKB.
  • the proteasome has two functional components: the 2OS core catalytic subunit and the 19S regulatory subunit.
  • the 2OS and 19S subunits form a 26S complex that degrades proteins targeted for degradation with the addition of ubiquitin.
  • Exemplary proteasome inhibitors include, e.g., lactacystin, PS-341 (NSC no. 681239) and analogs (Adams, Cur. Opin. Chem Biol. 6:493-500 (2002)), PS-273 (NSC no. 681226); PS-293 (NSC no. 681227); PS-296
  • NSC no. 681228 PS-303 (NSC no. 681229); PS-305 (NSC no. 681231); PS-313 (NSC no. 681234); PS-321 (NSC no. 681236); PS-334 (NSC no. 681237); PS-364 (NSC no. 681242); PS-325 (NSC no. 683086); PS-352 (NSC no. 683094); PS-383 (NSC no. 683098), YUlOl (ac-hFLFL-epoxide) (see, e.g. , Elofsson, et al , Chem. Biol.
  • proteasome inhibitor N-benzyloxycarbonyl-Ile-Glu(O-tert-butyl)-Ala- leucinal Assays for identifying proteasome inhibitors are commercially available from, e.g., Disco verx (Fremont, CA).
  • DR5 agonists e.g., anti-DR5 antibodies of the present invention
  • the combination can be particularly effective against cancer cells with Bax or mitochondrial defects.
  • colon cancer patients frequently have tumor cells that are Bax defective. Therefore, proteasome inhibitor combined with a DR5 antagonist is particularly effective to treat colon cancers involving Bax or mitochondrial defects.
  • the proteasome inhibitor is a compound of formula I
  • Rl is unsubstituted or substituted aryl; arylalkylcarbonyl, wherein the aryl moiety is unsubstituted or substituted; unsubstituted or substituted heterocyclyl; or heterocyclylalkylcarbonyl, wherein the heterocyclyl moiety is unsubstituted or substituted;
  • R 2 is unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl;
  • R 3 is hydrogen, unsubstituted or substituted aryl or alkyl which is unsubstituted or substituted by unsubstituted or substituted cycloalkyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl comprising at least one nitrogen atom;
  • R 4 is a moiety of the formula IA,
  • A] and A 2 are hydroxy or substituted hydroxy, or together with the binding boron atom and the two binding oxygen atoms form a ring of the formula IA*,
  • W is alkylene, substituted alkylene, unsubstituted or substituted cycloalkylene, unsubstituted or substituted bicycloalkylene or unsubstituted or substituted tricycloalkylene; and R 5 is unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, or unsubstituted or substituted cycloalkyl; or salts thereof.
  • Aryl preferably has a ring system of not more than 20 carbon atoms, especially not more than 12 carbon atoms, is preferably mono-, bi- or trie-cyclic, and is unsubstituted or substituted, preferably in each case unsubstituted or substituted phenyl or (especially 1- or 2- )naphthyl, one or more substituents preferably being independently selected from the group consisting of an aliphatic radical; free, etherified or esterified hydroxy; free or esterified carboxy; formyl; alkanoyl; unsubstituted, mono- or di-substituted amino; mercapto; sulfo; alkyl-thio; carbamoyl; N-alkyl-carbamoyl; N,N-di-alkyl-carbamoyl; phenyl; naphthyl; heterocyclyl
  • halogen-alkyl e.g., trifluoromethyl
  • phenyl (especially 1- or 2-)-naphthyl, and heterocyclyl, especially as defined below, especially pyridyl, e.g., 3-, 4- or especially 2-pyridyl, each of which is unsubstituted or substituted with one or more, especially up to three, substituents, especially independently selected from the other aryl substitutents just mentioned.
  • Aryl R] is more preferably biphenylyl, especially 2-, 4- or preferably 3-biphenylyl, pyridylphenyl, especially 4-, 3- or most especially 2-pyridyl-(2-, 4- or preferably 3-)phenyl, or lower alkyl-phenyl, especially propyl-phenyl, such as 2-, 4- or especially 3-isopropylphenyl.
  • Arylalkylcarbonyl R] (with unsubstituted or preferably substituted aryl) is preferably aryl-lower alkylcarbonyl with aryl as defined above, more preferably phenyl-lower alkyloxy-phenyl-lower alkylcarbonyl, especially 2-, 4- or preferably 3-benzyloxy-phenyl-acetyl or -propionyl, pyridyl-lower alkyloxyphenyl-lower alkylcarbonyl, especially 2-, 4- or preferably 3-(pyridin-2-, -4- or preferably -3-)-acetyl or -propionyl, or phenyl-lower alkylcarbonyl, especially phenyl-2- or preferably 3-phenyl-propionyl or phenylacetyl, wherein phenyl is unsubstituted or substituted by up to three substitutents independently selected from lower alkoxy, especially methoxy, halogen
  • Unsubstituted or substituted aryl R 2 or (independently) R 3 is preferably mono-, di- or trisubstituted phenyl, especially substituted by up to four substituents independently selected from the substitutents mentioned for aryl, especially from hydroxy, lower alkoxy (most preferred), preferably methoxy, halogen, preferably fluoro or chloro, and halogen-lower alkyl, preferably trifluoromethyl, especially phenyl substituted by up to three lower alkoxy, preferably methoxy, substituents, or in case of R 3 unsubstituted phenyl, or further unsubstituted or substituted napthyl, especially 1- or 2-naphthyl that is unsubstituted or substituted by up to four substituents independently selected from the substitutents mentioned for aryl, especially from hydroxy, lower alkoxy (most preferred), preferably methoxy, halogen, preferably fluoro or chloro, and hal
  • Unsubstituted heterocyclyl is preferably a heterocyclic radical that is unsaturated, saturated or partially saturated in the bonding ring and is preferably monocyclic or in a broader sense bicyclic or tricyclic ring; has 3 to 24, more preferably 4 to 16 ring atoms; wherein at least in the ring bonding to the radical of the molecule of formula I one or more, preferably one to four, especially one or two carbon atoms of a corresponding aryl radical are substituted by a heteroatom selected from the group consisting of nitrogen, oxygen and sulfur, the bonding ring preferably having 4 to 12, especially 5 to 7 ring atoms; heteroaryl being unsubstituted or substituted by one or more, especially 1 to 3, substitutents independently selected from the group consisting of the substituents defined above as substituents of substituted aryl; and especially being a heteroaryl radical selected from the group consisting of imidazolyl, thienyl, furyl, tetrahydr
  • ⁇ eterocyclyl is unsubstituted or substituted by one or more, preferably up to three, substitutents independently selected from those mentioned above for aryl (where heterocyclyl as substituent of heterocyclyl carries no further heterocyclyl substituent other than pyridyl or indolyl) and from aryl as defined above, especially phenyl, especially those mentioned as being preferred. Unsubstituted heterocyclyl is preferred.
  • heterocyclylalkylcarbonyl Ri the heterocyclyl moiety is preferably substituted or especially unsubstituted heterocyclyl as mentioned above; preferred is substituted or preferably unsubstituted heterocyclyl-lower alkyl, especially with terminal substituted or preferably unsubstituted heterocyclyl, with heterocyclyl as described above; preferred is pyridyl-lower alkylcarbonyl, such as -acetyl or -propionyl.
  • R 1 unsubstituted or substituted aryl or substituted aryl-lower alkylcarbonyl is preferred.
  • Heteroaryl R 2 is preferably unsubstituted or substituted heteroaryl as mentioned above, especially indolyl that is unsubstituted or substituted by one or more, especially up to three, substitutents independently selected from those mentioned above for substituted aryl, especially from hydroxy, lower alkoxy (most preferred), preferably methoxy, halogen, preferably fluoro or chloro, and halogen-lower alkyl, preferably trifluoromethyl.
  • R 2 is preferably substituted aryl.
  • An aliphatic radical preferably has up to 12 carbon atoms, preferably up to 7 carbon atoms, most preferably up to 4 carbon atoms, and is an aliphatic hydrocarbon radical, such as an unsubstituted or substituted alkynyl, alkenyl or preferably alkyl radical, more preferably lower alkyl, especially methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl or tert-butyl.
  • Alkyl which may be branched or linear, preferably has up to 12 carbon atoms, and is more preferably lower alkyl.
  • Alkyl R 3 is preferably lower alkyl, especially isobutyl.
  • Lower alkyl is, preferably, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert- butyl, n-pentyl, isopentyl, neopentyl, n-hexyl or n-heptyl, preferably isobutyl, sec-butyl, tert- butyl, isopropyl, ethyl or methyl, most preferably isopropyl, ethyl or methyl.
  • Etherified hydroxy is, for example, alkoxy, especially lower alkoxy, such as ethoxy or methoxy, aryloxy, especially phenyloxy, aryl-lower alkoxy, especially phenyl-lower alkoxy, heterocyclyloxy, especially pyridyloxy, or heterocyclyl-lower alkoxy, especially pyridyl-lower alkoxy (aryl and heterocyclyl preferably have the meanings given above).
  • Esterified hydroxy is preferably hydroxy esterified by an organic carboxylic acid, such as an alkanoic acid, for example lower alkanoyloxy.
  • Esterified carboxy is, for example, alkoxycarbonyl, especially lower alkoxycarbonyl, such as methoxycarbonyl.
  • Mono- or di-substituted amino is, preferably, N-alkylamino or N,N-dialkylamino, especially N-lower alkylamino or lower N,N-di-lower alkylamino, such as N-methylamino or N,N-dimethylamino.
  • Halogen is fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
  • Unsubstituted or substituted cycloalkyl preferably has up to 12, more preferably 3 to 8 ring carbonyl atoms and is substituted by one or more, especially up to three, substitutents independently selected from those mentioned for substituted aryl, or preferably unsubstituted. Preferred is cyclopentyl, cyclohexyl or cycloheptyl.
  • alkyl R 3 substituted with unsubstituted or substituted cycloalkyl alkyl is preferably as defined above, more preferably lower alkyl, especially isopropyl, and is (preferably terminally) substituted by cycloalkyl as defined above.
  • alkyl R 3 substituted with unsubstituted or substituted aryl alkyl is preferably as defined in the last paragraph, and aryl is defined as above and is substituted by one or more, especially up to three, substitutents independently selected from those mentioned for substituted aryl, or unsubstituted; especially aryl is phenyl substituted by one or more, especially up to three, substitutents independently selected from halogen, especially fluoro, hydroxy or lower alkoxy, especially methoxy, or it is unsubstituted phenyl.
  • alkyl R 3 substituted with unsubstituted or substituted heterocyclyl alkyl is preferably as defined for alkyl R 3 substituted with cycloalkyl, and heterocyclyl is defined as above and is substituted by one or more, especially up to three, substitutents independently selected from those mentioned for substituted heterocyclyl, or unsubstituted.
  • substituted hydroxy is preferably alkyloxy, especially lower alkyloxy, aryloxy, especially with unsubstituted or substituted aryl as defined above, or cycloalkyloxy with unsubstituted or substituted cycloalkyl as defined above.
  • W preferably carries the two oxygen atoms bound to the boron atom on two different carbon atoms that are spatially nearby or neighbouring carbon atoms, especially in vicinal ("1,2-") or in "l,3"-position (relatively to each other).
  • Alkylene is preferably an unbranched C 2 -C 12 -, preferably C 2 -C 7 alkylene moiety, e.g.
  • ethylene, or propylene in a broader aspect butylene, pentylene or hexylene, bound via two different carbon atoms as just described, preferably vicinal or in "l,3"-position.
  • One or more, especially one, of the carbon atoms not bound to the oxygen atoms binding to the boron atom may be replaced by a heteroatom selected from O, S or preferably N (carrying the required number of H atoms, respectively), for example in l,5-(3-aza-pentylene).
  • Substituted alkylene is preferably an unbranched lower alkylene moiety as defined above which is subsituted or unsubstituted by one or more, especially up to three, substituents preferably independently selected from lower alkyl, such as methyl or ethyl, e.g. in 1- methylethylene, 1 ,2-dimethylethylene, hydroxy, e.g. in 2-hydroxy -propylene, or hydroxy- lower alkyl, such as hydroxymethyl, e.g. in 1-hydroxymethyl-ethylene.
  • substituents preferably independently selected from lower alkyl, such as methyl or ethyl, e.g. in 1- methylethylene, 1 ,2-dimethylethylene, hydroxy, e.g. in 2-hydroxy -propylene, or hydroxy- lower alkyl, such as hydroxymethyl, e.g. in 1-hydroxymethyl-ethylene.
  • Unsubstituted or substituted cycloalkylene is preferably C 3 -C] 2 -, more preferably C 3 -Cs-cycloalkylene bound via two different carbon atoms as described for W, preferably vicinal or in "l,3"-position, such as cyclohexylene or cyclopentylene, in which one or more, especially one, of the carbon atoms not bound to the oxygen atoms binding to the boron atom may be replaced by a heteroatom selected from O, S or N (carrying the required number of H atoms, respectively), for example in tetrahydrofurylene or tetrahydropyranylene, and may be unsubstituted or substituted by one or more, especially up to three substituents independently selected from lower alkyl, such as methyl or ethyl, hydroxy, hydroxy-lower alkyl, such as methoxy, or mono- or oligosaccharidyl bound via an oxy
  • Unsubstituted or substituted Bicycloalkylene is preferably Cs-C -bicycloalkylene bound via two different carbon atoms as described for W, preferably vicinal or in "1,3"- position, in which one or more, especially one, of the carbon atoms not bound to the oxygen atoms binding to the boron atom may be replaced by a heteroatom selected from O, S or N (carrying the required number of H atoms, respectively), and may be unsubstituted or substituted by one or more, especially up to three substituents independently selected from lower alkyl, such as methyl or ethyl, hydroxy and hydroxy-lower alkyl, such as methoxy.
  • lower alkyl such as methyl or ethyl, hydroxy and hydroxy-lower alkyl, such as methoxy.
  • Unsubstituted or substituted tricycloalkylene is preferably bound via two different carbon atoms as described for W, preferably vicinal or in "1,3"- position, in which one or more, especially one, of the carbon atoms not bound to the oxygen atoms binding to the boron atom may be replaced by a heteroatom selected from O, S or N (carrying the required number of H atoms, respectively), and may be unsubstituted or substituted by one or more, especially up to three substituents independently selected from lower alkyl, such as methyl or ethyl, hydroxy and hydroxy-lower alkyl, such as methoxy.
  • R 4 is -B(OH) 2 or 2,9,9-trimethyl-3,5-dioxa-4-bora- tricyclo[6.1.1.0 2 ' 6 ]dec-4-yl, especially (IS, 2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora- tricyclo[6.1.1.0 2 ' 6 ]dec-4-yl.
  • alkyl which may be branched or linear, preferably has up to 12 carbon atoms, and is more preferably lower alkyl.
  • Alkyl R 5 is preferably lower alkyl, especially isopropyl.
  • Substituents are independently selected from unsubstituted or substituted aryl (especially phenyl or hydroxyphenyl), unsubsituted or substituted heterocyclyl (especially imidazolyl or indolyl), unsubstituted or substituted cycloalkyl, each as defined above; hydroxy (preferred), carboxy (preferred), carbamoyl, mercapto, lower alkylthio, e.g. methylthio, phenyl, hydroxyphenyl, indolyl, imidazolyl, amino, tri-lower alkylamino, e.g.
  • R 5 may be methyl, isopropyl, isobutyl, sec-butyl, mercaptomethyl, 2-methylthio- ethyl, phenylmethyl, hydroxyphenylmethyl, indol-3-ylmethyl, hydroxymethyl, 1- hydroxyethyl, 2-hydroxyethyl, carbamoylmethyl, 2-carbamoylethyl, 4-aminobutyl, 3- guanidinopropyl, 5-imidazolylmethyl, carboxymethyl or 2-carboxyethyl.
  • Asymmetric carbon atoms of a compound of formula I that are present may exist in the (R), (S) or (R,S) configuration, preferably in the (R) or (S) configuration, most preferably in the configuration indicated in formula I* below.
  • the compounds may thus be present as mixtures of isomers or preferably as pure isomers.
  • Salt-forming groups in a compound of formula I are groups or radicals having basic or acidic properties.
  • Compounds having at least one basic group or at least one basic radical, for example amino, a secondary amino group not forming a peptide bond or a pyridyl radical may form acid addition salts, for example with inorganic acids, such as hydrochloric acid, sulfuric acid or a phosphoric acid, or with suitable organic carboxylic or sulfonic acids, for example aliphatic mono- or di-carboxylic acids, such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acid or oxalic acid, or amino acids such as arginine or lysine, aromatic carboxylic acids, such as benzoic acid, 2-phenoxy-benzoic acid, 2-acetoxy-benzoic acid, salicylic acid, 4-
  • Compounds of formula I having acidic groups may form metal or ammonium salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts, or ammonium salts with ammonia or suitable organic amines, such as tertiary monoamines, for example triethylamine or tri-(2- hydroxyethyl)-amine, or heterocyclic bases, for example N-ethyl-piperidine or NN- dimethylpiperazine. Mixtures of salts are possible.
  • Compounds of formula I having both acidic and basic groups can form internal salts.
  • Exemplary compounds of formula I include those wherein Ri is either substituted aryl- lower alkylcarbonyl or unsubstituted or substituted aryl; R 2 is substituted aryl or unsubstituted or substituted heterocyclyl; R 3 is lower alkyl, unsubstituted or substituted aryl or lower alkyl which is substituted by unsubstituted or substituted aryl; R 4 is a moiety of the formula IA given above wherein Ai and A 2 are hydroxy, lower alkyloxy, aryloxy with unsubstituted or substituted aryl or cycloalkyloxy with unsubstituted or substituted cycloalkyl, or wherein Ai and A 2 , together with the binding boron atom and the two binding oxygen atoms form a ring of the formula IA* given above wherein W is unsubstituted or substituted
  • Exemplary compounds of formula I include those wherein Ri is phenyloxyphenyl- lower alkylcarbonyl; phenyl-lower alkoxyphenyl-lower alkylcarbonyl; pyridyloxyphenyl- lower alkylcarbonyl; phenyl-lower alkylcarbonyl substituted by lower alkoxy, especially methoxy, halogen, especially fluoro or chloro, or halogen-lower alkyl, especially trifluoromethyl; or preferably unsubstituted or substituted phenyl or naphthyl, wherein in both cases the substituents if present are independently one or more, especially one to three, substituents selected from the group consisting of lower alkyl, hydroxy, lower alkoxy, lower alkanoyloxy, carboxy, lower alkoxycarbonyl, formyl, lower alkanoyl, amino, N-lower alkylamino, N,N-di-lower alkylamin
  • Exemplary compounds of formula I include those wherein Ri is phenyloxyphenylacetyl, benzyloxyphenylacetyl, pyridyloxyphenylacetyl, biphenylyl, pyridylphenyl, lower alkylphenyl or substituted phenylpropionyloxy wherein the phenyl substituents are up to three substituents independently selected from the group consisting of methoxy, fluoro, chloro and trifluoromethyl; R 2 is phenyl substituted with up to three methoxy substituents, especially 2,3,4-trimethoxyphenyl or 3,4,5-trimethoxyphenyl; R 3 is isobutyl or phenyl that is unsubstituted or substituted with up to three moieties independently selected from hydroxy, fluoro and methoxy; R 4 is (lS,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-
  • Exemplary compounds of formula I include those wherein R] is biphenylyl, lower alkyl-phenyl, phenyl-lower alkyl-carbonyl, phenoxy-phenyl-lower alkyl-carbonyl, phenyl- lower alkoxy-phenyl-lower alkyl-carbonyl or pyridyl-phenyl; R 2 is either phenyl substituted by 1 to 3 lower alkoxy radicals or phenyl-lower alkoxy-phenyl; R 3 is lower alkyl or phenyl- lower alkyl; R 4 is 4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl, (lS,2S,6R,8S)-2,9,9- trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0 2 ' 6 ]dec-4-yl or -B(OH) 2 ; and R 5 is lower alkyl-
  • R 3 , R 4 and R 5 have the meanings given under formula I, with an amino acid of the formula III,
  • Ri and R 2 have the meanings given under formula I, functional groups present in a compound of formula II and/or III, with the exception of the groups participating in the reaction, being protected if necessary by readily removable protecting groups, and any protecting groups present are removed; or b) for the production of a compound of the formula I wherein R] is arylalkylcarbonyl or heterocyclylalkylcarbonyl and the other moieties R 2 to R 5 have the meanings given under formula I, reacting an amino compound of the formula IV,
  • R 2 , R 3 , R 4 and R 5 have the meanings given under formula I, with a carbonic acid of the formula V,
  • Ri is arylalkylcarbonyl or heterocyclylalkylcarbonyl
  • functional groups present in a compound of formula IV and/or V with the exception of the groups participating in the reaction, being protected if necessary by readily removable protecting groups, and any protecting groups present are removed, and, if desired, converting a compound of formula I obtained by process a) or b) into another compound of formula I, converting an obtained free compound of formula I into a salt, converting an obtained salt of a compound of formula I into a different salt or into its free form, and/or separating a mixture of isomeric compounds of formula I into the individual isomers.
  • compounds of formula I can be prepared by using educts with the appropriate configuration.
  • compounds of formula I* or salts thereof can be prepared by a) reacting a dipeptide analogue of the formula II*,
  • R 3 , R 4 and R 5 have the meanings given under formula I, with an amino acid of the formula III*,
  • Ri and R 2 have the meanings given under formula I, functional groups present in a compound of formula II* and/or III*, with the exception of the groups participating in the reaction, being protected if necessary by readily removable protecting groups, and any protecting groups present are removed; or b) for the production of a compound of the formula I* wherein R] is arylalkylcarbonyl or heterocyclylalkylcarbonyl and the other moieties R 2 to R 5 have the meanings given under formula I, reacting an amino compound of the formula IV*,
  • R 2 , R 3 , R 4 and R 5 have the meanings given under formula I, with a carbonic acid of the formula V, R ' ⁇ OH ⁇ V > or a reactive derivative thereof, wherein Ri is arylalkylcarbonyl or heterocyclylalkylcarbonyl, functional groups present in a compound of formula IV* and/or V, with the exception of the groups participating in the reaction, being protected if necessary by readily removable protecting groups, and any protecting groups present are removed, and, if desired, converting a compound of formula I* obtained by process a) or b) into another compound of formula I*, converting an obtained free compound of formula I* into a salt, or converting an obtained salt of a compound of formula I* into a different salt or into its free form.
  • the end products of formula I may contain substituents that can also be used as protecting groups in starting materials for the preparation of other end products of formula I, e.g., in the case of R 4 other than -B(OH) 2 .
  • R 4 other than -B(OH) 2 .
  • a characteristic of protecting groups is that they can be removed readily (i.e. without the occurrence of undesired secondary reactions) for example by solvolysis, reduction, photolysis or alternatively under physiological conditions (e.g. by enzymatic cleavage).
  • Removal of a protecting group for the -B(OH) 2 -group preferably takes place with an acid, e.g., hydrogen chloride, in an appropriate solvent, e.g., a lower alkanol, such as methanol, or a lower alkane, such as hexane, or a mixture thereof, at temperatures of 0 to 50 0 C, e.g., at room temperature.
  • an acid e.g., hydrogen chloride
  • process "a" the reaction is carried out by dissolving the compounds of formulae II and III in a suitable solvent, for example N,N-dimethylformamide, NN- dimethylacetamide, N-methyl-2-pyrrolidone, methylene chloride, or a mixture of two or more such solvents, and by the addition of a suitable base, for example triethylamine, diisopropylethylamine (DIEA) or N-methylmorpholine and a suitable coupling agent that forms a preferred reactive derivative of the carbonic acid of formula III in situ, for example dicyclohexylcarbodiimide/l-hydroxybenzotriazole (DCC/ HOBT); O-(l,2-dihydro-2-oxo-l- pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU);
  • a suitable solvent for example N,N-dimethylformamide, NN- dimethylacet
  • the reaction mixture is preferably stirred at a temperature of between approximately -20 and 50 0 C, especially between 0 0 C and room temperature, to yield a compound of formula I.
  • the reaction is preferably carried out under an inert gas, e.g., nitrogen or argon.
  • reaction is preferably carried out under conditions analogous to those described for process a).
  • Salts of a compound of formula I with a salt-forming group may be prepared in a manner known per se. Acid addition salts of compounds of formula I may thus be obtained by treatment with an acid or with a suitable anion exchange reagent.
  • Salts can usually be converted to free compounds, e.g., by treating with suitable basic agents, for example with alkali metal carbonates, hydrogencarbonates, or hydroxides, typically potassium carbonate or sodium hydroxide.
  • suitable basic agents for example with alkali metal carbonates, hydrogencarbonates, or hydroxides, typically potassium carbonate or sodium hydroxide.
  • Stereoisomeric mixtures e.g., mixtures of diastereomers
  • Diastereomeric mixtures for example may be separated into their individual diastereomers by means of fractionated crystallization, chromatography, solvent distribution, and similar procedures. This separation may take place either at the level of one of the starting compounds or in a compound of formula I itself.
  • Enantiomers may be separated through the formation of diastereomeric salts, for example by salt formation with an enantiomer-pure chiral acid, or by means of chromatography, for example by HPLC, using chromatographic substrates with chiral ligands.
  • the solvents from which those can be selected which are suitable for the reaction in question include for example water, esters, typically lower alkyl-lower alkanoate, e.g., diethyl acetate, ethers, typically aliphatic ethers, e.g., diethylether, or cyclic ethers, e.g., tetrahydrofuran, liquid aromatic hydrocarbons, typically benzene or toluene, alcohols, typically methanol, ethanol or 1- or 2-propanol, nitriles, typically acetonitrile, halogenated hydrocarbons, typically dichloromethane, acid amides, typically dimethylformamide, bases, typically heterocyclic nitrogen bases, e.g., pyridine, carboxylic acids, typically lower alkanecarboxylic acids, e.g., acetic acid, carboxy
  • New starting materials and/or intermediates, as well as processes for the preparation thereof, are likewise the subject of this invention.
  • such starting materials are used and reaction conditions selected such as to allow the manufacture of the preferred compounds.
  • the starting materials of formulae II - V or their precursors are known, can be prepared according to known processes, or are commercially obtainable; in particular, they can be prepared using processes identical or in analogy to those described in the Examples.
  • R 3 has the meanings given above for compounds of formula I and R 4 has the meanings other than -B(OH) 2 mentioned above for compounds of formula I, especially is (lS,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0 2 ' 6 ]dec-4-yl, or an acid addition salt thereof, especially the salt thereof with trifluoroacetic acid, is condensed with an amino acid of the formula VII
  • R 5 has the meanings given above for compounds of the formula I and Pri is a protected amino group, preferably tert-butoxycarbonylamino, under reaction conditions analogous to those described for reaction a) above (also a condensation reaction, also preferably with in situ formation of active carbonic acid derivatives), thus yielding a compound of formula II in N-protected form which is then N-deprotected, e.g., using conditions described in the standard textbooks mentioned above, in the case of tert- butoxycarbonylamino e.g., with hydrochloric acid in an appropriate solvent, e.g.
  • boronic acids of the formula VI are known, commercially available and/or can be synthesized according to known procedures.
  • compounds of the formula VI wherein R 3 is lower alkyl, especially isobutyl and R 4 is as described for compounds of the formula VI, preferably (lS,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora- tricyclo[6.1.1.0 2 ' ]dec-4-yl can be prepared by reacting a compound of the formula VIII,
  • R 4 has the meanings just described, in an appropriate solvent, e.g. methylene chloride, with n-lower alkyl lithium, especially n-butyllithium, and subsequently with zinc chloride, yielding a compound of the formula IX,
  • R 4 has the meanings given under formula VI, which is a compound of the formula VI and can then be used directly for reaction with the compound of formula VII as shown above.
  • a compound of the formula III is known, commercially available and/or can be obtained according to standard procedures.
  • a compound of the formula III wherein Ri is aryl, especially biphenylyl may be prepared by reacting a compound of the formula XI,
  • R 2 has the meanings given for a compound of the formula I, which is known, commercially available or obtainable according to standard procedures, with a compound of the formula XII,
  • an alkali metal carbonate e.g., potassium carbonate
  • Amino acid derivatives of the formula VII are known, commercially available or obtainable according to standard procedures. They are preferably used in the amino protected form, e.g., with tert-butoxycarbonylamino, instead of the free amino group.
  • Compounds of the formula IV can be obtained e.g. by reacting a compound of the formula II comprising for example the configuration as indicated in formula II*, as defined in process a), with an N-protected amino acid of the formula XIII,
  • the apoptosis-inducing agent is a proteasome inhibitor from the 2,4-diamino-3-hyroxycarboxylic acid family of compounds. See, PCT WO 00/64863.
  • the proteasome inhibitor is a 2,4-diamino-3- hydroxycarboxylic acids of formula XIV,
  • a and B independently represent a bond or an unsubstituted or substituted aminoacyl moiety;
  • R] represents hydrogen; an amino protecting group; or a group of formula R 5 Y- wherein R 5 represents hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkinyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl or heterocyclylalkyl group; and Y represents -CO-; -NH-CO-; -NH-CS-; -SO 2 -; -O-CO-; or -O-CS-; R 2 represents the side chain of a natural amino acid; an alkyl, arylalkyl, heteroarylalkyl or cycloalkylalkyl group; or trimethylsilylmethyl, 2-thienylmethyl or styrylmethyl; R 3 represents halogen, alkyl, alkoxy or hydroxyalkoxy; and R 4 represents 2(R)
  • Unsubstituted or substituted alkyl preferably is alkyl of 1 to 5 carbon atoms, preferably of 1 to 4 carbon atoms; e.g., methyl, ethyl, isopropyl or tert-butyl; it is especially of 1 or 4 carbon atoms.
  • the substituent is e.g., phenoxy, hydroxy or unprotected or protected amino.
  • Unsubstituted or substituted arylalkyl is, e.g., phenylalkyl of altogether 7 to 10 carbon atoms, such as benzyl or 2-phenylethyl. It is unsubstituted or substituted in the aryl or alkyl moiety by, e.g., hydroxy, such as in benzyl-CH(OH)- or phenyl -CH(CH 2 OH)-, by alkyl, amino or alkylamino; or is, e.g., naphthylalkyl of 1 to 4 carbon atoms in the alkylene part, especially naphthylmethyl.
  • An amino protecting group preferably is benzyloxycarbonyl, cycloalkyialkoxycarbonyl, especially cyclohexylmethoxycarbonyl, or tert-butoxycarbonyl.
  • Unsubstituted or substituted heteroarylalkyl preferably is pyridylalkyl, especially 2- pyridylmethyl and 4-pyridylmethyl.
  • Aryl, heteroaryl and the aryl parts of arylalkyl and heteroarylalkyl may be mono- or polycyclic, such as pyridyl, naphthyl, 9-fluorenylmethoxycarbonyl (FMOC) or benz- imidazolyl.
  • the alkylene part of arylalkyl or heteroarylalkyl may be substituted by e.g. hydroxy.
  • a heterocyclyl group, and the heterocyclyl part of a heterocyclylalkyl group is a saturated heterocyclic group having one or more heteroatoms selected from nitrogen, oxygen and sulfur. It preferably has 5 or 6 ring constitutent atoms, and preferably up to 3 heteroatoms.
  • Cycloalkylalkyl preferably is cyclohexylalkyl; it preferably is of 1 to 4 carbon atoms in the alkylene part.
  • Halogen is fluorine, chlorine, bromine or iodine, preferably chlorine or bromine.
  • Alkyl and alkoxy preferably are of 1 to 4 carbon atoms, especially of 1 or 2 carbon atoms, more especially methyl or methoxy.
  • Hydroxyalkoxy preferably is ⁇ -hydroxyalkoxy of 2 to 4 carbon atoms, especially 2- hydroxyethoxy.
  • a salt is, e.g., an acid addition salt such as a hydrochloride.
  • the compounds of formula I have several chiral centers and can therefore exist in a variety of stereoisomers.
  • the invention provides all stereoisomers as well as racemic mixtures unless specified otherwise.
  • the isomers may be resolved or separated by conventional techniques, e.g., chromatographically. As appears from formula I the configuration at the carbon atom in the 2 position is R, in the 3 and 4 positions it is S.
  • Ri preferably is hydrogen, pyridylalkoxycarbonyl, naphthylalkoxycarbonyl, naphthylalkylcarbonyl, benzyl-CH(OH)-carbonyl, phenoxymethylcarbonyl, phenylalkylcarbonyl or an amino protecting group such as tert.-butoxycarbonyl, cycloalkylalkoxycarbonyl, especially cyclohexylmethoxycarbonyl, or benzyloxycarbonyl which is unsubstituted or substituted by alkyl or amino; it especially is naphthylmethoxycarbonyl, naphthylmethylcarbonyl, pyridylmethoxycarbonyl, phenylpropionyl, aminophenylpropionyl, tert.-butoxycarbonyl, aminobenzyfoxycarbonyl, alkylbenzyloxycarbonyl, dialkylbenzyloxycarbonyl or benzyloxy
  • A is an unsubstituted or substituted aminoacyl moiety
  • it preferably is an unsubstituted or substituted ⁇ -aminoacyl moiety such as alanine, leucine, isoleucine, asparagine, valine, tert-butylglycine, tert-leucine or histidine.
  • It preferably is the protected or unprotected moiety of a natural ⁇ -amino acid, preferably of an amino acid which is a normal constitutive part of proteins, or tent leucine. It preferably has the L configuration.
  • A is especially glycine, L- valine, L-tert- leucine or a bond, even more preferably L-tert- leucine.
  • R 2 preferably is the side chain of a natural amino acid, preferably of an ⁇ -amino acid, preferably of an amino acid which is a normal constitutive part of proteins. It is, e.g., isopropyl, aminocarbonylmethyl, methyl, 1-methylpropyl, benzyl, 4-hydroxybenzyl or isobutyl, preferably benzyl.
  • B is an unsubstituted or substituted aminoacyl moiety
  • it preferably is an unsubstituted or substituted ⁇ -aminoacyl moiety, such as phenylalanine, valine, leucine, isoleucine, alanine or asparagine.
  • It preferably is the unsubstituted or substituted moiety of a natural ⁇ -amino acid, preferably of an amino acid which is a normal constitutive part of proteins.
  • ⁇ -Amino acids with a second carboxyl group e.g., glutaminic acid
  • Q-C 3 alcohol especially methanol. It preferably has the L-configuration.
  • B especially is L- valine, L-glutaminic acid methyl ester or a bond, even more preferably L- valine.
  • R 3 preferably is halogen, methyl or methoxy, especially methoxy.
  • R 4 preferably is 2(R)-hydroxyindan-l(S)-yl or 2-hydroxybenzyl unsubstituted or substituted as defined above, especially 2-hydroxy-4-methoxy-benzyl.
  • Y preferably is -CO- or -O-CO-, especially -O-CO-.
  • R 5 preferably is an unsubstituted or substituted alkyl, arylalkyl or heteroarylalkyl group, especially alkyl; when it is unsubstituted or substituted heteroarylalkyl it preferably is pyridylalkyl, especially 2-pyddylmethyl; when it is unsubstituted or substituted arylalkyl it preferably is benzyl-CH(OH)-; when it is substituted alkyl it preferably is phenoxymethyl.
  • the proteasome inhibitor is a 2-amino-3-hydroxy-4-tert- leucyl-amino-5-phenyl-pentanoic acid amide derivative. See, e.g., PCT 01/89282.
  • the proteasome inhibitors of the invention relate to compounds of formula XV
  • R] and R 2 are independently of the other an aliphatic radical, or an aromatic, aromatic- aliphatic, cycloaliphatic, cycloaliphatic-aliphatic, heterocyclic or heterocyclic-aliphatic radical, each radical having not more than 20 carbon atoms;
  • R 3 is hydrogen, oxa-alkyl, an aliphatic radical or a radical with up to 20 carbon atoms of the formula -(Y) 1n -R 6 , wherein Y is alkyl, m is 0 or 1 and R 6 is an unsubstituted or substituted monocyclic radical with 5 or 6 ring members containing up to 3 hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur, wherein said monocyclic radical can also be fused to a benzo ring;
  • R 4 and R 5 are independently selected from the group consisting of hydrogen; an aliphatic radical ; free, etherified or esterified hydroxy; free or esterified carb
  • n is 0 or 1, preferably 0.
  • An aliphatic radical has up to 12 carbon atoms, preferably up to 7 carbon atoms, most preferably up to 4 carbon atoms, and is such an unsubstituted or substituted aliphatic hydrocarbon radical, that is to say such an unsubstituted or substituted alkynyl, alkenyl or preferably alkyl radical, one or more substituents preferably being independently selected from the group consisting of free, etherified or esterified hydroxy; free or esterified carboxy; formyl; alkanol; unsubstituted, mono-or di- substituted amino; guanidino; mercapto; sulfo; alkyl-thio; carbamoyl; N-alkyl-carbamoyl; N, N-di-alkyl-carbamoyl; cyano and nitro.
  • An aliphatic radical Rj is preferably lower alkyl, such as especially tert-butyl.
  • An aliphatic radical R 3 is preferably unsubstituted lower alkyl or lower alkyl substituted by hydroxy, carboxy, amino, carbamoyl, guanidino, mercapto or alkyl-thio, most preferably a side chain of the amino acids alanine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamin, aspartate, glutamate, lysine or arginine, especially valine.
  • An aliphatic radical R 4 is preferably methoxy.
  • An aromatic radical R] or R 2 has not more than 20 carbon atoms, especially not more than 12 carbon atoms, and is unsubstituted or substituted, preferably in each case unsubstituted or substituted phenyl or naphthyl, especially 1-naphthyl, one or more substituents preferably being independently selected from the group consisting of an aliphatic radical; free, etherified or esterified hydroxy; free or esterified carboxy; formyl; alkanol; unsubstituted, mono-or di-substituted amino; mercapto; sulfo; alkyl-thio; carbamoyl; N-alkyl-carbamoyl; N, N-di-alkyl-carbamoyl; cyano and nitro, more preferably being selected from alkyl, e.g., methyl, ethyl or propyl; alkoxy, e.g., methoxy or e
  • an aromatic-aliphatic radical R] or R 2 having not more than 20 carbon atoms the aromatic moiety is as defined above and the aliphatic moiety is preferably lower alkyl, such as especially Ci-C 2 alkyl, which is substituted preferably as defined for the aromatic radical or preferably unsubstituted.
  • An aromatic-aliphatic radical Ri is preferably benzyl or naphthalen-1-ylmethyl.
  • An aromatic-aliphatic radical R 2 is preferably benzyl substituted in the benzene moiety by 1-5, preferably by 1-3 methoxy groups; benzyl substituted in the benzene moiety, preferably in position 4, by a dimethyl- amino group; or naphthalen-1- ylmethyl.
  • an aromatic-aliphatic radical R2 is 2,3,4- or 3,4,5-trimethoxy- benzyl.
  • a cycloaliphatic radical R] or R 2 has up to 20, especially up to 10 carbon atoms, is mono-or poly-cyclic and is substituted preferably as defined for the aromatic radical or preferably unsubstituted, for example such a cycloalkyl radical, especially such a 5 -or 6- membered cycloalkyl radical, such as preferably cyclohexyl.
  • a cycloaliphatic-aliphatic radical R] or R 2 having not more than 20 carbon atoms the cycloaliphatic moiety is as defined above and the aliphatic moiety is preferably lower alkyl, such as especially Ci-C 2 alkyl, which is substituted preferably as defined for the aromatic radical or preferably unsubstituted, for example cyclohexyl-methyl.
  • a heterocyclic radical Ri or R 2 contains up to 20 carbon atoms, generally up to 12 carbon atoms, and is substituted preferably as defined for the aromatic radical or unsubstituted and is preferably a saturated or unsaturated monocyclic radical having 5 or 6 ring members and 1 to 3 hetero atoms which are preferably selected from the group consisting of nitrogen, oxygen and sulfur, for example, thienyl or pyridyl, or a bi-or tri-cyclic radical wherein, for example, a benzene radical is fused to the mentioned monocyclic radical, especially, for example, indolyl, such as 5-indolyl, or chinolyl, such as 8-chinolyl.
  • heterocyclic-aliphatic radical Ri or R 2 having not more than 20 carbon atoms the heterocyclic moiety is as defined above and the aliphatic moiety is preferably lower alkyl, such as especially Ci-C 2 alkyl, which is substituted preferably as defined for the aromatic radical or preferably unsubstituted.
  • a heterocyclic-aliphatic radical Ri or R 2 is for example indolyl-methyl, especially 5-indolyl-methyl, or chinolyl-methyl, especially 8-chinolyl- methyl.
  • Oxa-alkyl R 3 is a radical of the formula -G(O-CH 2 -CH 2 ) t -R 7 , in which G and R 7 are alkyl, preferably lower alkyl, and t is 1 to 3, preferably 2, and is especially 2-(l,4-dioxa- hexyl) -ethyl.
  • Y is alkyl, preferably lower alkyl
  • m is 0 or 1
  • the radical R 6 is a saturated or unsaturated monocyclic radical having 5 or 6 ring members and up to 3 hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur and alternatively containing a fused benzo ring, such a radical being substituted preferably as defined for the aromatic radical or preferably unsubstituted.
  • a radical R 6 is preferably bound to Y via a ring carbon atom and is for example an unsubstituted or substituted member selected from the group consisting of cyclopentyl, cyclohexyl, cyclopentadienyl, phenyl, pyrrolidyl, pyrazolidyl, imidazolidyl, tetrahydrofuryl, piperidyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, imidazolyl, furyl, thienyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, indenyl, naphthyl, indolyl and chinolyl.
  • a radical of the formula -(Y) 1n -Ro is piperidyl, especially 4- piperidyl, piperazin-ethyl, especially piperazin-1-ylethyl, morpholinyl-ethyl, especially morpholin-4-ylethyl, pyridyl-methyl, such as 2-, 3-or 4-pyridyl-methyl, or a side chain of the amino acids phenylalanine, tyrosine, tryptophane or histidine.
  • X is preferably oxygen (-O-).
  • Alkyl is preferably lower alkyl.
  • the prefix “lower” denotes a radical having up to and including 7, preferably up to and including 4, carbon atoms.
  • Lower alkyl is, for example, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, n- hexyl or n-heptyl, preferably isobutyl, sec-butyl, tert-butyl, isopropyl, ethyl or methyl, most preferably isobutyl, ethyl or methyl.
  • Etherified hydroxy is, for example, alkoxy, especially lower alkoxy, such as ethoxy or methoxy.
  • Esterified hydroxy is preferably hydroxy esterified by an organic carboxylic acid, such as alkanoic acid, or a mineral acid, such as a hydrohalic adic, for example lower alkanoyloxy or especially halogen, such as iodine or especially fluorine, chlorine or bromine.
  • Esterified carboxy is, for example, alkoxycarbonyl, especially lower alkoxycarbonyl, such as methoxycarbonyl.
  • Alkanol is, for example, alkylcarbonyl, especially lower alkylcarbonyl, such as e. g. acetyl.
  • Mono-or di-substituted amino is, for example, N-alkylamino or N, N-dialkylamino, especially N-lower alkylamino or lower N,N-di-lower alkylamino, such as e. g. N- methylamino or N,N-dimethylamino.
  • Halogen is fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
  • the structure of Formula XV as shown above indicates the absolute configuration.
  • Salt-forming groups in a compound of Formula XV are groups or radicals having basic or acidic properties.
  • Compounds having at least one basic group or at least one basic radical may form acid addition salts, for example with inorganic acids, such as hydrochloric acid, sulfuric acid or a phosphoric acid, or with suitable organic carboxylic or sulfonic acids, for example aliphatic mono-or di-carboxylic acids, such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acid or oxalic acid, or amino acids such as arginine or lysine, aromatic carboxylic acids, such as benzoic acid, 2-phenoxy-benzoic acid, 2-acetoxy-benzoic acid, salicylic acid, 4-aminosalicylic acid, aromatic-aliphatic carboxylic acids, such as mandelic acid or cinn
  • Compounds of Formula XV having acidic groups may form metal or ammonium salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts, or ammonium salts with ammonia or suitable organic amines, such as tertiary monoamines, for example triethyl- amine or tri-(2-hydroxyethyl)-amine, or heterocyclic bases, for example N-ethyl-piperidine or N,N'-dimethyl-piperazine.
  • metal or ammonium salts such as alkali metal or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts
  • ammonium salts with ammonia or suitable organic amines such as tertiary monoamines, for example triethyl- amine or tri-(2-hydroxyethyl)-amine, or heterocyclic bases, for example N-ethyl-piperidine or N,N'-dimethyl-piperazine.
  • the antibodies and agents of the invention can be administered directly to the mammalian subject for treatment, e.g., of hyperproliferative disorders including cancer such as, but not limited to: carcinomas, gliomas, mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer, cancer of the gallbladder, cancer of the small intestine, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pan
  • mammalian subject humans and non-human primates, and other mammals including agricultural mammals (e.g., equines, bovines, ovines, and porcines, etc.) and domestic or companion mammals (e.g., canines and felines), as well as laboratory mammals (e.g., rodents including rabbits, rats, hamsters and mice, etc.).
  • agricultural mammals e.g., equines, bovines, ovines, and porcines, etc.
  • domestic or companion mammals e.g., canines and felines
  • laboratory mammals e.g., rodents including rabbits, rats, hamsters and mice, etc.
  • compositions of the present invention is by any of the routes normally used for introducing a chemotherapeutic compound into ultimate contact with the tissue to be treated.
  • the antibodies and agents are administered in any suitable manner, optionally with pharmaceutically acceptable carriers. Suitable methods of administering such antibodies and agents are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • compositions of the present invention are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 21st edition, University of the Sciences in Philadelphia (USIP), Lippincott, Williams & Wilkins (2005).
  • the antibodies and agents, alone or in combination with other suitable components can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, by orally and parenterally, including topically, transdermally, transmucosally intravenously, subcutaneously, intratumorally, intramuscularly, intraperitoneally, intravesically or intrathecally.
  • the compositions are administered nasally or inhalationally.
  • the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the modulators can also be administered as part of a prepared food or drug.
  • the compounds of the present invention can also be used effectively in combination with one or more additional active agents (e.g., chemotherapeutics) depending on the desired therapy or effect.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in a therapeutically effective amount.
  • the amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • an efficacious or effective amount of one or more anti-DR5 antibodies is determined by first administering a low dose or small amount of an anti-DR5 antibody and then incrementally increasing the administered dose or dosages, and/or adding an apoptosis agent as needed, until a desired effect of inducing apoptosis in a target cell population is observed in the treated subject, with minimal or no toxic side effects.
  • Applicable methods for determining an appropriate dose and dosing schedule for administration of a pharmaceutical composition of the present invention is described, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., Brunton, et ah, Eds., McGraw-Hill (2006), and in Remington: The Science and Practice of Pharmacy, supra, both of which are hereby incorporated herein by reference.
  • typical dosages can range from about 0.1 ⁇ g/kg body weight up to and including about lgm/kg body weight, preferably between about 1 ⁇ g/kg body weight to about 500 mg/kg body weight. More preferably, about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg/kg body weight.
  • typical dosages can range from about 0.1 mg/kg body weight up to and including about 100 mg/kg body weight, preferably between about 1 mg/kg body weight to about 50 mg/kg body weight. More preferably, about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mg/kg body weight.
  • typical dosages can range from about 0.1 ⁇ g/kg body weight up to and including about lgm/kg body weight, preferably between about 1 ⁇ g/kg body weight to about 500 mg/kg body weight. More preferably, about 0.1, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg/kg body weight.
  • the exact dose will depend on a variety of factors as discussed supra, including the particular antibody or apoptosis inducing agent, severity of the disease, and route of administration. Determining the exact therapeutically effective dose can be determined by a clinician without undue experimentation and can include any dose included within the ranges disclosed above.
  • the antibody agonist and the apoptosis-inducing agent can be administered together in a mixture or each can be administered separately.
  • the antibody agent and the apoptosis inducing agent can be, but need not, administered concurrently.
  • Example 1 The following example provides the construction and screening of anti-DR5 antibodies with minimized immunogenicity in humans.
  • the murine V-regions were sub-cloned from the plasmids pBW212 and pBW214. See, U.S. Patent Publication No. 2005/0079172 for the murine V-region sequences. PCR was used to amplify the V-genes of the V-heavy and V-Kappa regions and incorporate restriction enzyme sites suitable for cloning into expression vectors. V-regions were cloned as Fab' fragments with human IgGl constant regions in a pBR322 vector system. Fab's were expressed from pTAC promoters in E. coli.
  • the purified Fab' protein (Clone DR5-1) was shown to bind DR5 antigen in an ELISA assay. Nucleotide sequence optimization of these V-regions resulted in Fab' fragments with human amino acid sequences in the portion of framework 3 adjacent to CDR3. This allowed for subsequent human V-segment library subcloning.
  • These optimized reference Fab's also had CDR3 BSD sequences and human J- chain / Framework 4 sequences provided by JK2 and JH4 germlines. The optimized Fab's were tested for DR5-antigen binding and are referred to as reference sequences DR9-1 and DRlO-I.
  • Fab' fragments were expressed by secretion from E. coli using expression vectors. Cells were grown in 2xYT medium to an optical density measured at 600 nm wavelength (OD ⁇ oo) of 0.6. Expression was induced using isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3 hours at 33 0 C. Assembled Fab' was obtained from periplasmic fractions and purified by affinity chromatography using Streptococcal Protein G (HiTrap Protein G HP columns; GE Healthcare) according to standard methods. Fab's were eluted in pH 2.0 buffer, immediately adjusted to pH 7.0 and dialyzed against PBS pH7.4 (PBS is without calcium and magnesium). ELISA
  • DR5 antigen typically, 100 ng/well of DR5 antigen was bound to a 96 well microtiter plate by overnight incubation at 4 0 C. The plate was blocked with a solution of 5% milk in phosphate- buffered saline containing 0.1% Tween20 ("PBST") for one hour at 33 0 C. The purified Fab's or the reference Fab' (DR9-1) were diluted in PBS and 50 ⁇ l was added to each well. After one hour incubation at 33 0 C, the plate was rinsed three times with PBST.
  • PBST phosphate- buffered saline containing 0.1% Tween20
  • HRP horseradish peroxidase
  • V-regions were cloned, sequenced and expressed. V-regions were cloned as Fab' fragments with human IgGl constant regions and expressed in E. coli.
  • the cloned Fab' and original Mab NVP-LCR211 produced binding curves that were dependent on antibody concentration.
  • the Mab is seen to have a stronger binding signal than the Fab', this is likely to be due to avidity effects and differences in top-layer ELISA detection. See, Figure 1.
  • the Fab' DR5-1 has intact murine V-regions whereas the reference Fab's DR9-1 and DRlO-I are optimized for subsequent library construction. Nucleotide changes made within the portion of framework 3 adjacent to CDR3 on both the heavy and light chains allow for subsequent library construction and result in human germline amino acid sequence.
  • Both DR9-1 and DRlO-I have a heavy chain CDR3 BSD of sequence HEEGI (SEQ ID NO:49) and the human germline J-segment sequence (JH4).
  • DR9-1 has a light chain CDR3 sequence including the BSD of QQHYTTP (SEQ ID NO: 57) and a framework 4 sequence conferred by human JK2.
  • DRlO-I has the light chain BSD sequence QQHYTTP (SEQ ID NO:57) and a human germline J-segment (human JK2). See Figure 3. Both optimized reference Fab's DR9-1 and DRlO-I were seen to have DR5 antigen binding activity. See Figure 4.
  • Epitope-focused libraries were constructed from a library of human V-segment sequences linked to the unique CDR3-FR4 region of reference Fab'DR9-l. These "full- length” libraries were used as a base for construction of "cassette” libraries in which only part of the murine V-segment is initially replaced by a library of human sequences.
  • the cassettes for both V-heavy and V-kappa chains were made by bridge PCR with overlapping common sequences within the framework 2 (FR2) region. In this way "front-end” and “middle” human cassette libraries were constructed for human V-heavy 1 and 3 and V-kappa I, III and IV isotypes.
  • Human cassettes which supported binding to DR5-antigen were identified by colony-lift binding assay and ranked according to affinity in ELISA and Forte Octet biosensor analysis. Pools of the highest affinity cassettes were then recombined in a second library screen to generate completely human V-segments. Cassette screening completed in this way identified multiple human V-light chains and human V-heavy "front-end" cassettes that had DR5-antigen binding activity.
  • a mutagenic library was constructed in which each residue within this CDR2 was mutated to either the murine sequence or the corresponding residue from a single human germline sequence (VH 1-46 was the closest human germline sequence to the identified "front-end” cassettes) (see, Figure 5).
  • This mutagenic library was coupled to selected "front ends” and human framework 3 (FR3) sequences that were seen to support antigen binding. All members of this library had the common CDR3 / FR4 sequence of the DR9-1 reference Fab'.
  • FR3 human framework 3
  • Table 1 shows an analysis of Fab' binding to recombinant DR5 antigen by bio-layer interferometry using ForteBio Octet biosensor technology, showing association rate constant (k a ), dissociation rate constant (k d ) and calculated affinity (K D ).
  • Kinetic analysis of the improved clones DR106-2, DRl 14-1, DRl 12-1 and the reference clone DR9-1 demonstrated all to have low to sub-nanomolar affinities for DR5 antigen.
  • Figure 11 shows an amino acid sequence alignment of the V-region sequences of optimized Fab's DR106, DRl 12 and DRl 14 compared with a human germline sequence. The percentage amino acid sequence identities of the reference and optimized V-region sequences to corresponding human germline amino acid sequences are shown in Table 2.

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Abstract

La présente invention concerne des anticorps anti-DR5 avec une immunogénicité minimisée chez les humains et des procédés d'utilisation de tels anticorps.
PCT/US2008/066202 2007-06-08 2008-06-06 Procédés et compositions pour induire une apoptose dans des cellules cancéreuses WO2008154439A1 (fr)

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EP2819697A4 (fr) * 2012-02-29 2016-03-09 Gilead Biologics Inc Anticorps dirigés contre la métalloprotéinase 9 de matrice
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US11634505B2 (en) 2012-02-29 2023-04-25 Gilead Sciences, Inc. Antibodies to matrix metalloproteinase 9
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CN106397594B (zh) * 2016-10-25 2019-06-04 中国药科大学 一种全人源的抗人死亡受体5的激动剂单链抗体及应用

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