TW200911835A - Methods and compositions for inducing apoptosis in cancer cells - Google Patents

Abstract

The present invention provides anti-DR5 antibodies with minimized immunogenicity in humans and methods of using such antibodies.

Description

200911835 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD The present invention relates to an antibody that specifically binds to death receptor 5 ("DR5"). The present application claims the benefit of U.S. Provisional Application Serial No. 60/942,862, filed on Jun. [Prior Art] Apoptosis is a highly conserved cellular suicide procedure necessary for the development of all metazoan organisms and tissue homeostasis. Changes in apoptotic pathways that prevent or delay normal cell renewal are as important in disease pathogenesis as abnormalities in cell cycle regulation. Like cell division controlled by complex interactions between cell cycle regulatory proteins, under normal circumstances, apoptosis is similarly regulated by the interaction of gene products that prevent or induce cell death. (TNF)-associated apoptosis-inducing ligand (trail, also known as /^〇21^ is a member of the TNF cytokine family. Once DR5 binds to tRAIL, cell death is induced by apoptosis. See, for example, Pan Et al., 277: 815-8 (1997); Sheridan et al. 'Ken Gae 277: 818-21 3 (1997); Walczak et al., vol. 16.5386-97 4 (1997). In vitro, TRAIL has been shown to kill broad-spectrum tumor cells that exhibit DR5, but is relatively non-toxic to normal cells. Given the high prevalence of cancer in human populations, there is always a new and more effective therapeutic strategy for cancer treatment. The present invention will address this and other related needs. 131613.doc 200911835 SUMMARY OF THE INVENTION In a first aspect, the invention provides an antibody that binds to death receptor 5 (DR5). In some embodiments, such The body comprises (a) a heavy chain variable region comprising a human heavy chain V-segment, a heavy chain complementarity determining region 3 (CDR3) and a heavy chain framework region 4 (FR4), and (b) comprising a human light chain V segment a light chain variable region of the light chain CDR3 and the light chain FR4, wherein i) the heavy chain CDR3 comprises an amino acid sequence 歹ij HEEGI (SEQ ID NO: 49); and ii) the light chain CDR3 variable region comprises an amino acid Sequence QXHXXTP (SEQ ID NO: 50), wherein X represents any amino acid. In another aspect, the invention provides for binding to death receptor 5 (DR5) comprising a heavy chain variable region and a light chain variable region An antibody, wherein the heavy chain variable region and the light chain variable region each comprise the following three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3; wherein: i) the CDR1 of the heavy chain variable region comprises SEQ ID NO: 1. Amino acid sequence of the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3; ii) CDR2 of the heavy chain variable region comprising SEQ ID NO: 4, SEQ ID NO: 5 And the amino acid sequence 歹 ij of the group consisting of SEQ ID NO: 6; iii) the CDR3 of the heavy chain variable region comprises a group selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: Amino acid sequence 歹 ij ; iv) CDR1 of the light chain variable region An amino acid sequence comprising a group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; 131613.doc 200911835 V) the light chain The CDR2 of the 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 selected from the group consisting of SEQ ID NO : 19 'Amino acid sequence of the group consisting of SEQ ID NO: 20 and SEQ ID NO: 21, provided 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. In some embodiments, the antibodies comprise a heavy chain V-segment that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical in sequence to SEQ ID NO:44. In some embodiments, the antibodies comprise a light chain that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical in sequence to SEQ ID NO: 46 or SEQ ID NO: 48 V-section. In some embodiments of the antibodies: i) the heavy chain CDR3 comprises the amino acid sequence motif HEEGIYFX1X2 (SEQ ID NO: 51), wherein X is D, T or K and the X2 is Y, K or V; Ii) the light chain CDR3 comprises an amino acid sequence motif QX3HX4X5TP (SEQ ID NO: 52) or QX3HX4X5TPFT (SEQ ID NO: 53), wherein X3 is Q or H, X4 is Y, L or K, and X5 is T, Q, I, E, H or G. In some embodiments of the antibodies: i) 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 131613. doc 200911835 ϋ) 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) &gt QQHLTTP (SEQ ID NO: 63) > QQHKTTP (SEQ ID NO: 64) and QHHYTTP (SEQ ID NO: 65); or iii) 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 N: 20), QQHYETPFT (SEQ ID NO: 67), QQHYHTPFT (SEQ ID NO: 68), QQHYGTPFT (SEQ ID NO: 69), QQHLTTPFT (SEQ ID NO: 70), QQHKTTPFT (SEQ ID NO: 71) and QHHYTTPFT (SEQ ID NO: 72). In some embodiments, the heavy chain FR4 is a human germline FR4. In some embodiments, FR4 is a human germline JH4 (SEQ ID NO: 28). In some embodiments, the heavy chain comprises a human germline J-segment. In some embodiments, the heavy bond human germline J-segment is JH4. In some embodiments, the heavy chain human germline J-segmental amino acid sequence YFD(Y/K)WGQGT(L/T)(V/L)TVSS (SEQ ID NO:73) ° in some embodiments Medium, light chain FR4 is a human germline FR4. In some embodiments, FR4 is a human germline JK2 (SEQ ID NO: 38).

In some embodiments, the light chain comprises a human germline J-segment. In some embodiments, the light chain human germline J-segment is JK2. In some embodiments, the light chain human germline J-segment is an amino acid sequence, !(Y/F)TFG(Q/S)GTKLEIK 131613.doc 200911835 (SEQ ID NO: 74). In some embodiments of the antibodies: i) the CDR1 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 selected from the group consisting of SEQ ID NO: 5 and the amino acid sequence of the group consisting of SEQ ID NO: 6; iii) CDR1 of the light chain comprises SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID: And 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. In some embodiments of the antibodies: i) 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 selected from the group consisting of SEQ ID NO :20. The amino acid sequence of the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22. In some embodiments of the antibodies: i) CDR1 of the heavy chain V-segment comprises SEQ ID NO: 2; ii) CDR2 of the heavy chain V-segment comprises SEQ ID NO: 5; iii) heavy chain CDR3 comprises SEQ ID NO:8; iv) CDR1 of the light chain V-segment comprises SEQ ID NO:1; v) CDR2 of the light chain V-segment comprises SEQ ID NO:16; and vi) the light chain CDR3 comprises SEQ ID NO: 20. In some embodiments of such antibodies: 131613.doc -10- 200911835 i) CDR1 of the heavy chain V-segment comprises SEQ ID NO: 2; ii) CDR2 of the heavy chain V-segment comprises SEQ ID NO: 5 Iii) the heavy chain CDR3 comprises SEQ ID NO:8; iv) the CDR1 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. In some embodiments of the antibodies: i) CDR1 of the heavy chain V-segment comprises SEQ ID NO: 2; ii) CDR2 of the heavy chain V-segment comprises SEQ ID NO: 5; iii) heavy chain CDR3 comprises SEQ ID NO:8; iv) CDR1 of the light chain V-segment comprises SEQ ID NO: U; v) CDR2 of the light chain V-segment comprises SEQ ID NO: 17; and vi) light chain CDR3 comprises SEQ ID NO: . In some embodiments, the heavy chain variable region shares at least 90%, 93%, 95%, 96%, 97%, 98°/ of the variable region of SEQ ID NO:43. Or 99% amino acid sequence identity. In some embodiments, the light chain variable region shares at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% amine with the variable region of SEQ ID NO:45 or SEQ ID NO:47 Base acid sequence identity. In some embodiments, the antibody binds to DR5 with an equilibrium dissociation constant (KD) of less than 1 X 1 〇 '8 Μ. In some embodiments, the anti-system FAb' fragment. In some embodiments, the anti-system IgG. In some embodiments, the anti-system single chain antibody (scFv). In some embodiments, the antibody comprises a human constant region. In some embodiments, the anti-system DR5 agonist. In some embodiments 131613.doc 200911835, the anti-system DR5 antagonist. 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 are selected from SEQ ID NO: 40 The amino acid sequence of the group consisting of SEQ ID NO: 41 and SEQ ID NO: 42 has at least 95%, 96%, 97%, 98% or 99°/. Light chain of sequence identity. In some embodiments, the antibodies comprise heavy and light chains, the heavy chain comprising SEQ ID NO: 39, the light chain comprising the selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: The group of amino acid sequences. In some embodiments, the light chain comprises SEQ ID NO:40. In some embodiments, the light chain comprises SEQ ID NO:41. In some embodiments, the light chain comprises SEQ ID NO:42. In a related aspect, the invention provides a pharmaceutically acceptable composition comprising an anti-DR5 antibody of the invention and a physiologically compatible excipient. Examples of antibodies in such compositions are as described above and throughout the application. In still another aspect, the invention provides a method of inducing apoptosis in cancer cell cells comprising contacting the cell with an anti-DR5 antibody of the invention, wherein the anti-system DR5 is an agonist. Examples of antibodies in such methods are as described above and throughout the application. In a related aspect, the invention provides a method of inducing apoptosis of cancer cell cells in an individual comprising administering to the individual a therapeutically effective amount of an anti-DR5 antibody of the invention, wherein the anti-system DR5 is an agonist. In some embodiments of the methods, the cell is further contacted with a cell apoptosis inducing agent. 131613.doc -12- 200911835 [Embodiment] The definition of ''Xi's anti-injection body') is sufficient for non-covalent soils to reversibly and in a special way with corresponding anti-immunoglobulin steroid polypeptides or immunoglobulins. A polypeptide of a fragment. Exemplary antibody structural units comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains joined by disulfide bonds, each pair having one light chain 1 (about 25 kD) and - ,, heavy chain" (about 5〇_7〇kD). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, £ and _ site genes as well as numerous immunoglobulin variable region genes. The light chain is divided into κ*λ. The heavy chain is divided into γ, μ, α, δ or ε, which respectively define the immunoglobulin classes igG, IgM, IgA, IgD& IgED. The N_-terminus of each chain is mainly responsible for antigen recognition and has about 100 to 110 or More variable regions of amino acids. The terms variable light chain (VL) and variable heavy chain (vH) refer to these regions of the light and heavy chains, respectively, and the "antibody" as used in this specification encompasses specific binding with specificity (eg, for DR5). All variants of antibodies and fragments thereof. Thus, within the scope of this concept are full length antibodies, occluded antibodies, single chain antibodies (ScFv), Fab, Fab' and multimeric forms of such fragments (eg, F(ab')2 having the same binding specificity. ). The "complementarity determining domain" or "complementarity determining area"("CDR") is interchangeable, and both refer to the hypervariable region of VL&VH. The CDRs secrete a protein binding site for the antibody chain specific for the target protein. There are three CDRs in each human vL or vH (CDR1-3 'numbered sequentially from the N-terminus), accounting for approximately 15-20% of the variable domain. The CDRs are structurally complementary to the epitope of the target protein and are therefore the direct cause of binding specificity. The remaining fragments of VL or VH (so-called box 131613.doc -13 · 200911835 shelf area) show minor changes in the amino acid sequence (Kuby, Immunology, 4th edition 'Chapter 4' WH Freeman & Co., New York , 2000). The positions of the CDRs and framework regions are determined using a variety of definitions well known to those skilled in the art, for example, Kabat, Chothia, International ImMuno GeneTics database (IMGT) (on the World Wide Web site imgt.cines.fr) / above) and AbM (see, for example, Johnson et al, Nucleic Acids Res., 29: 205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); Chothia et al. Human, Nature, 342: 877-883 (1989); Chothia et al, J. Mol. Biol., 227: 799-817 (1992); Al-Lazikani et al, J. Mol. Biol., 273:927- 748 (1997)). The definition of antigen binding sites is also described in the following literature: Ruiz et al, Nucleic Acids Res., 28: 219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29: 207-209 (2001); MacCallum et al, J. Mol. Biol,, 262:732-745 (1996); and Martin et al, Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al.' Methods Enzymol 203:121-153 (1991); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). The term "binding specificity determinant" or "BSD" is used interchangeably and refers to the minimally continuous amino acid sequence necessary for determining the binding specificity of an antibody within a complementary determining region. The minimal binding specificity determinant can be within one or more CDR sequences. In some embodiments, the minimal binding specificity determinant is present in one or both of the antibody heavy and light bonds (i.e., 'by only its determination). 131613.doc 14 200911835 As used herein, "antibody light chain |, or, antibody heavy bond VH polypeptide. Endogenous Vl is made "variable by gene segment" and "joined: encoded, and endogenous VH With v, D (diversity) and ang code. Each VH includes a CDR and a frame positive. In this application, antibody light or antibody heavy chains are sometimes collectively referred to as "antibody chains". Those skilled in the art will readily recognize that such terms encompass mutations that do not destroy the basic knot of WH: Antibody chain.

The antibody is present as a complete immunoglobulin or as a plurality of fragments which are sufficiently characterized by digestion with a plurality of peptidases. Thus, for example, pepsin digests antibodies under the disulfide bond in the hinge region to produce Η讣), 2 (Fab, dimer) 'Fab, which is itself a light chain linked to Vh_Ch1 by a disulfide bond. . F(ab)'2 can be reduced under moderate conditions to cleave the disulfide bond in the hinge region, thereby converting the F(ab), 2 dimer to a Fab, monomer. Fab, the monomer is essentially a Fab and part of the hinge region. Paul, A"(10) (iv) 〇/〇灯3rd edition (1993). While various antibody fragments are defined for intact antibody digestion, those skilled in the art will recognize that such fragments can be synthesized de novo in a chemical manner or by using recombinant DNA methods. Accordingly, the term "antibody" as used herein also includes antibody fragments produced by modification of all antibodies or those identified by the use of recombinant DNA methods from de novo synthesizers (eg, single-chain Fv) or those using the sputum display library. (See, for example, McCafferty et al, 348:552-554 (1990)). For the preparation of single or multiple antibodies, any technique known to those skilled in the art can be used (see 'for example ' Kohler & Milstein, jVaiwre 256.495-497 (1975) 'Kozbor et al., 4:72 131613.doc - 15-200911835 (1983) 'Cole et al., Monoclonal Antibodies and Cancer Therapy' page 77_96. Aiari r Liss, 1985). The technique for producing a single bond antibody (U.S. Patent No. 4,946,778) can be modified to produce a polypeptide antibody of the present invention. Moreover, transgenic mice or other organisms such as other mammals can be used to express humanized antibodies. Alternatively, sputum display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to the selected antigen (see, for example, McCafferty et al., supra; Marks et al., valence. (10) 1〇: 779_783, (1992)). Methods for humanizing or primatizing non-human antibodies are well known to those skilled in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. Such non-human amino acid residues are often referred to as introduced residues' which are typically taken from the introduction of variable domains. Humanization can be carried out by replacing the corresponding sequence of the human antibody with a rodent CDR sequence in accordance with the method of Winter and colleagues (see, for example, J〇nes et al ' Nature, 321:522-525 (1986); Riechmann et al. , TVaiwre, 332:323-327 (1988); Verhoeyen et al., 239: 1534-1 536 (1988) and Presta, Cwrr. (9/?. β;·〇/· 2:593-596 (1992)) Thus, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,81,567) in which substantially less than one intact human variable domain has been replaced by a corresponding sequence from a non-human species. Humanized antibodies are generally human antibodies in which some of the complementarity determining regions ("CDR") residues and possibly some framework ("FR") residues are replaced by residues from analogous sites in rodent antibodies. "Chimeric antibody" is an antibody molecule in which: (a) a constant region or a portion thereof 131613.doc 16 200911835 Red, k, substituted or substituted to make the antigen binding site (variable region) different or altered Species, effector functions, and/or constant regions of species, or conferion = Molecules (eg, enzymes, toxins, hormones, growth factors, and drugs) that are completely different in the novel properties of the antibody; or (b) variable regions or portions thereof that are altered by variable regions with different or altered antigen specificities The term "variable region" or "v_region" is used interchangeably and refers to a heavy or light chain comprising FR2-CDR2-FR3-CDR3-FR4. See Figure 2. Endogenous The variable region is encoded by the immunoglobulin heavy chain v_D_j gene or the light chain v_j gene. The V-region may be naturally occurring, recombinant or synthetic. The term "variable segment" or "V-segment" is used interchangeably herein. , which all refer to the variable region sequences including FR1-CDR1-FR2-CDR2-FR3. See Figure 2. The endogenous V-segment is encoded by the immunoglobulin v_gene. The v_ segment can be naturally occurring, recombined. Or synthesis. As used herein, the term "J-segment" refers to a sequence comprising the C-terminal portion of CDR3 and the variable region of FR4. The endogenous j-segment is encoded by an immunoglobulin; See Figure 2. The J-segment can be naturally occurring, recombined or synthesized. "Humanization" Anti-system retains the anti-human antibody Rather in humans has a lower immunogenicity of the antibody. This may be by (e.g.) preservation of non-human CDR regions and replacing the remaining parts of the antibody with human counterparts obtained. See, e.g.,

Morrison et al., Proc. AW. 5W. tAS^, 81:6851-6855 (1984); Morrison and Oi, di/v. 7mm(d)〇/., 44:65-92 (1988); Verhoeyen et al.' Sczewce, 239: 1534-1536 (1988); Padlan, Molec. Immun., 28: 489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994). 131613.doc 200911835 The corresponding nuclear sequence of human variable region amino acid sequence or subsequence, corresponding to human germline immunoglobulin variable region sequence coding Other

Compared with the amino acid sequence of the parent region, the variable amino acid sequence or subsequence and the reference variable region amino acid sequence = the subsequence (4) have the highest determining of the seric acid sequence - pure. The corresponding human sequence may also refer to a human variable region amine group having the highest amino acid sequence identity to the reference variable region amino acid sequence or subsequence compared to all other valence (4) amino acid sequences. Acid sequence or subsequence. The corresponding human species sequence may be only the framework region, only the complementarity determining region, the framework region and the complementarity determining region, the variable segment (as defined above) or other sequence or subsequence combination containing the variable region. The alignment of the two sequences can be performed using the methods described herein, for example, using BLAST, ALIGN, or another alignment algorithm known to those skilled in the art. The corresponding human germline nucleic acid or amino acid sequence can be at least about 9% identical to the reference variable region nucleic acid or amino acid sequence. Corresponding to the human germline genetic genetics database 92〇/〇, 94%, 96%, 98%, 99% of the sequence – the sequence can be obtained by, for example, the publicly available country (IMGT) (in the world information network imgt.cines .fr/top) and v_library (on the World Wide Web vbase.mrc-cpe.cam.ac.uk). When used to describe the interaction between an antigen (eg, a protein) and an antibody or antibody-derived binding agent, the phrase "specific (or selective) binding refers to a heterogeneous population that is determined in proteins and other biological agents. There is an antigen binding reaction. Thus, under specified immunoassay conditions, an antibody or binding agent having a specific E. coli specificity is specifically bound by at least twice the background amount and substantially does not bind substantially to other antigens present in the sample. 131613.doc -18- 200911835 Under these conditions with antibodies && people -,,,. 5蜊specific binding may require selection of the specificity of the antibody or binding agent for a particular peptone, baibai. This selection can be achieved by subtracting antibodies from, for example, DR5 molecules from other species; The western immune analysis format can be used to select antibodies that specifically immunoreact with a specific protein by 丨 联 联 联. For example, solid phase ELISA immunoassays are commonly used to select antibodies that are specific for a protein-specific immune response (see, for example, Plus (10) & Lane, Using Antibodies, A Laboratory Manual (1998) - for the determination of specific immune responses. Immunoassay form and conditions). - In general, a specific or selective binding reaction will produce at least two times the letter of the Moon Hall, the K number and, more generally, at least 丨〇〇 to the background.

The term "equilibrium dissociation constant (κ〇, M)" refers to the dissociation rate constant (kd, time ' divided by the association rate constant (ka, time 'M.) The equilibrium dissociation constant can be any of those known to those skilled in the art. Method Measurements. The antibodies of the invention will typically have less than about 1 〇-8 Μ (e.g., less than about 1 〇·9 Μ or 1 〇 1 〇Μ, in some embodiments, less than about 1 〇-丨丨Μ, 1 The equilibrium dissociation constant of 〇-12 乂 or 1〇-13(4). The term "antigen binding region" as used herein refers to the domain of the molecule which causes specific binding between the DR5_binding molecule of the present invention and DR5. The antigen binding region includes At least one antibody heavy chain variable region and at least one antibody light chain variable region. At least one of said antigen binding regions is present in each DR5-binding molecule of the invention and each antigen binding region may be identical or different from each other. In some embodiments, at least one of the antigen binding regions of the DR5-binding molecules of the invention functions as an agonist of DR5. 131613.doc 19 200911835 The term "literature agent" as used herein refers to the ability to specifically bind and activate. Subject to

do not. ''Antibody agonists•' specifically bind to this receptor and induce Dr5-mediated 'DR5 agonists' ability to recognize apoptosis with DR5-k cells (jurkat ceU) This agonist is the case where the antibody is activated. The term "nucleic acid," or "polynucleic acid," refers to deoxyribonucleic acid (D N A) or ribonucleotide I (RNA) and its multimers in single or double stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties to 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, homologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and/or a deoxyinosine residue (Batzer et al.) Person, 19:5081 (1991); Ohtsuka et al., price/. 260:2605-2608 (1985); and Rossolini et al., Mo/· Ce//_ Proves 8:91-98 (1994)) "Polypeptides""peptides" and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues correspond to an artificial chemical mimetic of a naturally occurring amino acid, as well as naturally occurring amino acid multimers and non-naturally occurring amines. A base acid multimer. 131613.doc -20- 200911835 The term "amino acid" refers to naturally occurring and synthetic amino acids and amino acid analogs and amino acid simulations that function in a manner similar to naturally occurring amino acids. Things. Naturally occurring amino acids are those encoded by the genetic code and their later modified amino acids, for example, hydroxyproline, "carboxy mate, and guanidine-phosphoric acid. Amine An acid analog refers to a compound having the same basic chemical structure as a naturally occurring amino acid (ie, an a-carbon bonded to a hydrogen, a slow group, an amine group, and an R group), for example, homoserine, orthraamine Acid, methionine sulfoxide, methyl methionine methyl. These analogs have a modified R group (such as 'positive leucine) or a modified peptide backbone, but retain the naturally occurring amine The basic chemical structure is the same as the base acid. The amino acid mimetic refers to a chemical compound having a structure different from the usual chemical structure of the amino acid but acting in a manner similar to the naturally occurring amino acid.

Subgen (AUG (which is usually methionine-to, every cryptocodon in a nucleic acid) and butyl GG (which usually encodes more than one nucleic acid sequence also describes the silent variation of the nucleic acid. The skilled artisan will recognize that 核酸, the nucleic acid 131613.doc •21 - 200911835 is the only one for the tryptophan-codon) can be modified to produce a knife of the same function. Accordingly, each silent variation of the nucleic acid encoding the polypeptide is implicit in each of the sequences. For amino acid sequences, those skilled in the art will recognize that the core: a change in the sequence of a purine, peptide, polypeptide or protein f, addition or deletion of a single amino acid or a small portion of an amino acid in the coding sequence. Substituting, deleting or adding alone is a "conservatively modified variant, wherein the alteration results in the replacement of the amino acid by a chemically similar amino acid. Providing a functionally similar amino acid is representative of this. The conservatively modified variants also include, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention. Each of the following eight groups contains amino acids that are conservatively substituted with each other. : 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) aspartame (N), face amidoxime (q) 4) arginine (R), lysine (κ); 〇 5) isoleucine (1), leucine (L), thiol amide (M), valine (v); 6) phenylalanine (F), tyrosine (γ), tryptophan (w); • 7) serine (S), threonine (T); and, 8) cysteine (C) ), methionine (M) (see, for example, Creight〇n, /Voie/as (1984)). Sequence Consistency Percentage is determined by comparing two optimal alignment sequences within a comparison window, wherein the comparison of portions of the polynucleotide sequence within the window with references that do not contain additions or deletions Sequences (eg, 'polypeptides of the invention) may include additions or deletions (ie, gaps) to optimally align the two sequences. 131613.doc • 22- 200911835 The percentage is determined by the following steps: ~ The number of positions of the same nucleic acid test or amino acid residues in the two sequences in the two sequences is (four) mm (four) quantity 'will match the position The number is divided by the total number of positions in the comparison window, and the result is multiplied by 100 to get a sequence percentage. In the case of two or more nucleic acid or polypeptide sequences, the term "consistent

Percent or caustic percentage refers to two or more sequences or subsequences that are the same sequence. When comparing and aligning in the comparison window or specified area to obtain the maximum correspondence, such as using the following sequence comparison algorithm - or by manual comparison and visual measurement, if the two sequences are identical amino acid residues Or the nucleotide is a specified percentage (ie, within the specified region or when not specified within the entire sequence of the reference sequence, 70%, 75%, 80%, 85%, 9%, 95%, 96%, 97°/., 9 8% or 99% sequence identity), the two sequences are substantially identical. The invention provides polypeptides or polynucleic acids as exemplified herein (eg, 'in SEQ ID NOS: l- 22. The CDRs exemplified in any of 22, 54-56 and 66-72, respectively, are substantially identical polypeptides or polynucleotides. As the case may be, the identity is present at a length of at least about 5, 25 or 50 nucleosides. Within the acid region, or better within a region of 10〇 to 5〇〇 or 1〇〇〇 or more in length or within the full-length reference sequence. Consistency or substantially uniform for amino acid sequences Sex may be present in a length of at least 5, 1 〇, 15 or 20 amino acids, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids, optionally in the region of at least about 15 Å, 200 or 250 amino acids or in the full length reference sequence. For shorter amino acid sequences (eg 'with 2 〇 or Less amino acid amino acid sequence) 'When one or two amino acid residues are conservatively substituted according to conservative substitutions as defined herein 131613.doc • 23- 200911835 There is substantial agreement. For sequences of reference sequences - Percentage/column comparison, a sequence will be compared as a reference sequence to the test sequence. When the Corioli (4) method is used, the test sequence and the reference sequence are input to the computer, and if necessary, the subsequence is specified. (4), and specify the sequence program parameter 1 to use the default program parameters, or you can specify other parameters. Then the 'sequence comparison algorithm will calculate the test sequence based on the program parameters

As used herein, "comparison window" includes any of a plurality of consecutive positions selected from the group consisting of 2 to 6 inches, typically from about 50 to about 200, and usually from about 1 to about 15 positions. a reference to a segment of the position in which the sequence can be compared to a reference sequence having the same number of consecutive positions after optimal alignment. The sequence alignment method for comparison is familiar to those skilled in the art. Well known. The best sequence alignment for comparison can be implemented by the following. For example, Smith and Waterman (1970) Wv, stealing 2_482〇 local homology algorithm, chemistry 6 (116111 mad 11 and '\^11115 ( :11 (1970)1/ Mo/·仏0/. 48:443 homology alignment algorithm, Pears〇n and Upman (1988) Proc. jVa〆/· dead C/iSJ 85:2444 Computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, in the Wisconsin Genetics Software Package, Genetics Computer

Group, 5 75 Science Dr., Madison, WI) or manual alignment and visual inspection (see, for example, ' Ausubel et al., Cwrreni Proioco/s Mo/ecw/ar (1995 Supplement)). Two examples of algorithms suitable for determining sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschu et al 131613.doc • 24-200911835, respectively (1977) n 25:3389_34〇 uAitschui et al., (靡于财心/. 215:4〇3_41〇. Software for performing blast analysis is publicly available through the National Center for Biotechnology Information (4) such as Biotechnology Inf()_iGn). The algorithm first involves identifying a high-score sequence pair by identifying the code length abbreviation W in the query sequence. (10) When the code is compared with a code of the same length in a database sequence, it will be associated with a certain positive threshold. The score τ matches or satisfies its condition. Τ refers to the adjacent code score threshold (Ahschui et al., supra). These initial adjacent repeat codes will be used as primers to initiate a search to find the longer ones containing them. The repetition code is extended in two directions of each _sequence, and S can increase the convolution ratio. For the nucleotide sequence, the cumulative score is calculated using the parameter Μ (the reward score for the matching residue pair; always greater than 〇) and Ν (the penalty score for the unmatched residue; always small (four)). For amino acid sequences, a score matrix is used to calculate the cumulative score. The extension of the repeat code in each direction will stop at the following time: the cumulative alignment score is reduced from the maximum value it achieves; the cumulative score is changed due to the accumulation of multiple negative score residues. Zero or negative; or reach the end of either sequence. 'BLAST algorithm parameters w, Τ and X determine the sensitivity and speed of the alignment. The default values used for the BLASTNfe sequence (9) in the nucleus; g: acid sequence) are code length (w) 11, expected value (E) 10, M=5, N=_4, and comparison values of the two chains. For the amino acid sequence, the BLASTN program gives (iv) default values a gamma length 3 and an expected value (E) 10 and a BLOSUM62 score matrix (see Henikoff and

Hemk〇ff (1989) &,··· 89:10915) Comparison (B) 50, expected value (E) 1〇, M=5, N_4 and comparison values of the two chains. 131613.doc -25· 200911835 The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul 〇 993) households (10) fine / (10) (four) 73_5787) β The BLAST algorithm provides a similar The sex measurement is the smallest sum probability (P(N)), which indicates the probability of a match between the two nucleotide or amino acid sequences. For example, if you are testing nucleic acids and

The most J in the comparison of reference nucleic acids, such as φ, 玄 I, 乂, 敢, and the probability is less than about 〇·2, more preferably less than about 〇. (Η 'and optimally less than about ', the nucleic acid and the reference sequence are considered similar.

As described below, it is shown that the two cores are immunized by antibodies produced by the polypeptide encoded by the first nucleic acid. The first polypeptide is substantially identical, such as in other instances. As described below, one case is that under stringent conditions it is indicated that the two nucleic acid sequences are substantially the same as the ones to amplify the sequences. Where the acid sequence or polypeptide is substantially identical The peptide reacts with the resistance by the second nucleic acid encoding the parent. Thus, 'polypeptides generally differ from the two peptides in that only conservative substitutions are present, and the two other nucleic acid sequences are substantially identical to each other or their complements hybridize to each other. In still another case where the same reference can be used to illustrate how the antigen binding regions are linked within the DR5_binding molecule of the invention, the term "linking" encompasses all possible hands that physically bind the region. The binding region is often joined by a chemical bond, such as a covalent bond (eg, a peptide bond or a disulfide bond) or a non-covalent bond, which can be a direct bond (ie, no linker between the two antigen binding regions) or an indirect bond. (ie, by means of at least one linker molecule between two or more antigen-binding regions.) SMAC is a 4曰 mitochondrial polypeptide that, together with cytochrome, responds to mitochondrial release from apoptotic stimuli. SMAC by binding And neutralize lAp to promote 131613.doc -26- 200911835 caspase activation. See, for example, 'Du et al, 〇// 102:33-42 (2000), Verhagen et al, CW/102:43-53 (2000). The term "therapeutically acceptable amount" refers to an amount sufficient to achieve the desired result (ie, target cell cell death). Preferably, a therapeutically acceptable amount does not cause undesirable side effects. A therapeutically acceptable amount can be determined by first administering a low dose and then increasing the dose until the desired effect is achieved. 1. Introduction The DR5 is a so-called death receptor on the surface of a variety of cancer cells (see Chaudhary et al., /Jie. Shakespeare. 7:821.83 (1997) and GenBank Accession No. AF016268). Once bound to its ligand (TRAIL4Ap〇2L), DR5 triggers a series of signal transduction events via its cytoplasmic death domain that ultimately leads to apoptosis. The present invention provides a minimal binding determinant sequence that specifically binds to 〇115 and contains a non-human anti-DR5 reference monoclonal antibody (eg, a mouse) and a variable region amine encoded by a corresponding human germline variable region sequence. An antibody to a base acid sequence. 2. Improved anti-sense combined with death by Zhao 5 (DRS) a. Overview The antibodies of the invention specifically bind to DR5. Thus, such antibodies may block intrinsic ligand binding, acting as an antagonist or acting as an agonist. In some embodiments, an anti-DR5 antibody of the invention functions as a DR5 receptor agonist. The DR5 antibody agonist specifically binds to DR5 and activates DR5-mediated nicknamed antibodies. The anti-DR5 antibodies may optionally be poly(e.g., dimerized) and used in accordance with the methods of the invention. The anti-DR5 antibodies may be full length tetrameric antibodies (ie, having two light chains and two heavy chains), single chain antibodies (eg, 131613.doc -27-200911835, eg, ScFv) or contain formation-less binding specificity The antibody binding fragment of the octagonal solid antigen binding site and confers a DR5-variant region (for example, other classes 1; two ', including heavy and light chains, which can be learned by the skilled person Means can be produced, including but not limited to, performance, chemical synthesis and antibodies, antibodies can be obtained by, for example, hybridomas or heavy-dumen

Mammalian host cells, bacterial diploid recombination table 5 see, for example, host cells and the like. The constant region of the BN5 cell, the yeast host cell, the insect, and the anti-DR5 antibody may be of any type or subtype (optional) and may be selected from the group to be treated by the method of the present invention. Individual species (eg, humans, humans, human mammals, or other mammals, eg, agricultural mammals (^ equines, sheep, porcines), domesticated mammals (eg, dogs) Animals, tick animals, or rodents (eg, rats, mice, hamsters, rabbits) b. Anti-DR5 antibody variable regions The variable regions of the anti-DR5 antibodies of the invention are derived from known high Affinity binds to a reference monoclonal antibody of DR5 and acts as an agonist by reducing the amino acid sequence segment corresponding to a non-human species (eg, mouse) and increasing the amino group corresponding to the human germline amino acid sequence The acid sequence segments are modified or optimized for such antibodies, in such a way that sequences that potentially induce an immune response against the anti-DR5 antibody in the human host are reduced, minimized or eliminated. Method has been elaborated See, for example, U.S. Patent Publication No. 2005/0255552 and U.S. Patent Publication No. 131 613. doc -28-200911835 2006/0134098, the disclosures of each of each of The improved anti-DR5 anti-system of the present invention is designed to have a human antibody having a v_region sequence that has a substantially amino acid sequence identity to the human germline V-region sequence while retaining reference antibody specificity and affinity. U.S. Patent Publication No. 20〇5/0255552 and U.S. Patent Publication No. 2/6/〇134〇98

number. The improved process recognizes the minimal sequence information required for antigen-binding specificity from the variable region of the reference antibody and transmits this information to the human V-region gene sequence (4) to generate a pool of epitopes for the human antibody ¥_ region. A microbial based secretion system can be used to represent this library member as an antibody fragment and to screen for antigen binding Fab' for the library, for example, using colony transfer binding assays. See, for example, U.S. Patent Publication No. 2007/0020685. Positive pure lines can be further characterized to identify those with the most affinity. The resulting human Fab, which retains the binding specificity of the parent (reference anti-DR5 antibody), has an equal or higher affinity for the antigen as compared to the parent antibody and has a human germline antibody. Compared to the V_zone with a degree of sequence consistency. The generation of the antigenic sputum is concentrated in the cytoplasmic determinant (BSD), which is a sequence and light in the CDR3 ("CDRH3" A sequence representation within the chain CDR3 ("CDRL3"). Na may contain a portion or full length CDR3. BSD can be composed of continuous 踬 田 田 踬 or non-contiguous amino acid residues. In some cases, the epitope is constructed by linking a human V-segment sequence to a unique CDR3-FR4 region of a reference antibody containing BSD and human germline region only sequences (to bamboo ^ See Figure 2 and U.S. Patent Publication No. 131613.doc -29-200911835 2005/0255552). Alternatively, the human genus can be replaced by a sequence cassette, wherein only a portion of the reference 俨V F P I 'product is initially replaced by a human sequence library. After the sputum, the residual reference antibody amino acid sequence, the 汴 π τ fork symbol combined with the identification of "Yi" in the second library screening "and the & people' pk 丫, and in a few full Human ν_ segment (see National Patent Publication No. 2006/0 1 34098).

In each case, the paired heavy and light chain CDR3 segments, CDR3_FR4 segments or segments containing the specificity of the reference antibody are used to bind the binding specificity such that the antigen binding agent obtained from the library retains the reference antibody ^ epitope specificity. Other maturation changes can be introduced in the cd regions of each strand during the construction of the library to identify antibodies with optimal binding kinetics. The resulting human antibody has a short BSD sequence in the CDR3 region that is derived from the human germline library and has a human germline framework 4 (FR4) region. Thus, in some embodiments, the anti-〇115 antibodies comprise a minimal binding sequence determinant (BSD) within the heavy and light chain CDR3 derived from the original antibody. The heavy chain CDR3 comprises the BSD HEEGI (SEQ ID NO: 49)' light chain CDR3 comprises BSD QXHXXTP (SEQ ID NO: 50), wherein x is any amino acid. See, for example, Figures 3, 8 and 11. The remaining sequences (CDRs and FRs) of the heavy and light chain variable regions (e. g., V-segment and J-segment) are derived from the corresponding human germline amino acid sequences. The V-segment can be selected from a human V_segment library. Other sequence improvements can be accomplished by affinity maturation. In another embodiment, the heavy and light chains of the anti-DR5 antibodies comprise human V-segments (FR1-CDR1-FR2-CDR2-FR3) from corresponding human germline sequences (eg, selected from humans) V-segment library) and 131613.doc • 30· 200911835 CDR3-FR4 sequence segment from the original monoclonal antibody. The CDR3-FR4 sequence segment can be further improved by replacing the sequence segments with corresponding human germline sequences and/or by affinity maturation. For example, the FR4 and/or CDR3 sequences surrounding the BSD can be replaced with corresponding human germline sequences while retaining the BSD from the CDR3 of the original monoclonal antibody. In some embodiments, the corresponding human germline sequence of the heavy chain V-segment is VH1 46. In some embodiments, the corresponding human germline sequence of the heavy chain J-segment is JH4. The variable region gene line is referred to in accordance with the standard nomenclature of immunoglobulin variable region genes. Current immunoglobulin gene information is available through the World Wide Web, such as the ImMunoGeneTics (IMGT), V-Library, and PubMed databases. See also Lefranc, Clin Immunogenet. 2001; 18(2): 100-16; Lefranc, Exp Clin Immunogenet. 2001; 18(3): 161-74; Exp Clin Immunogenet. 2001; 18(4): 242-54; And Giudicelli et al., jYwc/ez'c iies. January 1, 2005; 33 (Database): D256-61. In some embodiments, the heavy chain J-segment has the amino acid sequence YFD(Y/K)WGQGT(L/T)(V/L)TVSS (SEQ ID NO: 73). In some embodiments, the corresponding human germline sequence of the light chain V-segment is VKIV or VKIL4. In some embodiments, the corresponding human germline sequence JK2 of the heavy chain J-segment. In some embodiments, the light chain J-segment has an amino acid sequence (Y/F)TFG(Q/S)GTKLEIK (SEQ ID NO: 74). In some embodiments, the heavy chain V-segment is associated with an amino acid sequence (E/Q) VQLV QSGAEVKKPGASVKVSCKASGYTFTSY(Y/T)MHWVRQAP GQGLEWMG(I/W)I(N/Y)P(S/G)GG( S/Y)T(S/K)YAQKF(Q/F) 131613.doc -31 - 200911835

GRVTMT (R/G) DTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 92) shares at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the light chain V-segment is selected from the group consisting of DIVMTQSPDSLA (V/A) SLGE (R/K) ATINCKSSQS (V/F) L (Y/G) SSN (N/G) KNY (L/V) AWYQQKPGQPPKLLIYWAS(T/M)R(E/V)SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:93) and (A/D)IQLTQSPSSLSASVGDRVTITCRAS Q(G/D)(I/V)S(S/G)ALAWYQQKPG(K/Q (A/P)PKLLIY(D/W) AS(S/T)(L/R)ESGVP(S/D)RFSGSGSGTDFTLTISSLQ(P/A)E 0(?/¥)八(17¥)丫丫( The amino acid sequence of 3 (8 £ (5 10 > 40:94) has a total of at least 90% '93% '95%, 96%, 97%, 98%, 99% or 100% sequence 歹>J identity. In some embodiments: i) the heavy chain CDR3 comprises an amino acid sequence motif HEEGIYFX X2 (SEQ ID NO: 5 1), wherein the oxime is T, T or K and the X2 is Y, K or V; The light chain CDR3 comprises the amino acid sequence motif QX3HX4X5TP (SEQ ID NO: 52), wherein X3 is Q or Η, X4 is Y, L or K, and X5 is T, Q, I, E, Η or G. In some embodiments: i) 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 131613.doc-32-200911835 column: 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 iii) 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: 68), QQHYGTPFT (SEQ ID NO: 69), QQHLTTPFT (SEQ ID NO: 70), QQHKTTPFT (SEQ ID NO: 71) and QHHYTTPFT (SEQ ID NO: 72). In some embodiments, the antibodies of the invention comprise a heavy chain variable region comprising CDR1, CDR2 and CDR3 comprising the amino acid sequence SY(Y/T)MH (SEQ ID NO :95); the CDR2 comprises an amino acid sequence (I/W)(I/F)(N/Y)P(S/G)GG(S/Y)(T/I)(S/K/R/ N) Y (A/N/S/T/D) (Q/E) KF (Q/K) (G/D) (SEQ ID NO: 96); the CDR3 comprises the amino acid sequence HEEGIYF (D/T) /K) (Y/K/V) (SEQ ID NO: 51). In some embodiments, the antibodies of the invention comprise a light chain variable region comprising CDR1, CDR2 and CDR3 comprising a 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) amino acid sequence The CDR2 comprises an amino acid sequence (W/D) AS(S/T/M)(R/L)(E/V)S (SEQ ID NO: 99); the CDR3 comprises an amino acid sequence Q (Q) /H)(Y/H/F) 131613.doc -33- 200911835 (Y/N/L/K)(S/I/E/H/G/Q/T)(T/Y)P(Y/ F) T (SEQ ID NO: 100). In some embodiments, 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: 18. In some embodiments, the heavy chain variable region comprises FR1, FR2' FR3 and FR4 comprising amino acid sequence (E/Q) VQLVQSGAEVKKPGAS VKVSCKASGYTFT (SEQ ID NO: 101); the FR2 comprising SEQ ID NO: The FR3 comprises the amino acid sequence RVTMT (R/G) DTSTSTVYME LSSLRSEDTAVYYCAR (SEQ ID NO: 102); the FR4 comprises SEQ ID NO:28. The identified amino acid sequence may have one or more substituted amino acids (e.g., from affinity maturation) or one or two conservatively substituted amino acids. In some embodiments, the light chain variable region comprises FR1, FR2, FR3, and FR4 comprising DIVMTQSPDSLA (V/A) SLGE (R/K) ATINC (SEQ ID NO: 103) or (A/D) An amino acid sequence of IQLTQSPSSLSASVGDRV TITC (SEQ ID NO: 104); the FR2 comprises an amino acid sequence WYQQKPG(Q/K)(P/A)PKLLIY (SEQ ID NO: 105); the FR3 comprises an amino acid sequence GVP (D/S) RFSGSGSGTDFTLTIS SLQ (A/P) ED (V/F) A (V/T) YYC (SEQ ID NO: 106); This FR4 comprises SEQ ID NO:37. The identified amino acid sequence may have one or more substituted amino acids (e.g., from affinity maturation) or one or two conservatively substituted amino acids. Within the full length of the variable region of an anti-DR5 antibody of the invention, it will typically have at least about 90% of the corresponding human germline variable region amino acid sequence (e.g., at least about 131613.doc-34-200911835 91%, 92) %, 93%, 94%, 95% '96%, 97%, 98%, 99% or 100%) of the total (eg 'FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) amino acid sequence consistent Sex. For example, the heavy chain of such anti-DR5 antibodies can share at least about 913⁄4, 92%, 93%, 94%, 95°/ with the human germline variable region VH146/JH4. , 96°/. , 97%, 98%, 99% or 100% amino acid sequence identity. The light chain of the anti-DR5 antibody may be at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% shared with the human germline variable region VKIV/JK2 or VKIL4/JK2. , 99% or 100% amino acid sequence identity. And X. a heavy chain variable region of 96%, 97%, 98%, 99% or 100% amino acid sequence identity and comprising a light chain variable selected from the group consisting of SEQ ID NOS: 40, 41, 42, 45 and 47 The zone has at least 90°/. , 91%, 92% '93% ' 94% ' 95% > 96% ' 97% ' 98% > 99%^ 100% Amino acid sequence-consistent light chain variable region. In some embodiments, an anti-DR5 antibody of the invention comprises at least 90 ❶/ of the heavy chain variable region of SEQ ID NO:39. a heavy chain variable region of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity and comprising and selected from SEQ ID NOS The light chain variable regions of 40, 41 and 42 have at least 90°/. 91%, 92%, 93%, 94%, 95%, 96%, 97°/. , 98%, 99°/. Or a light chain variable region with 100% amino acid sequence identity.

In some embodiments, the anti-DR5 antibody of the invention comprises at least 90%, 91%, 92%, 93%, 94%, 95 with the heavy chain variable region of SEQ ID 131613.doc-35-200911835 NO:43 %, 96%, 97%, 98%, 99% or 100% amino acid sequence 一致性 J consensus heavy chain variable region and comprising a light chain variable region selected from the group consisting of SEQ ID NOS: 45 and 47 At least 90%, 91%, 92%, 93%, 94%, 95%, 96°/. , 97%, 98%, 99% or 100% amino acid sequence consistent light chain variable region. For amino acid sequences identified to be less than 20 amino acids in length, one or two conservative amino acid residue substitutions can be tolerated while still retaining the desired specific binding and/or agonist activity. The anti-DR5 antibodies of the invention will generally be at a balance of less than about 10.8 Å or ΙΟ·9 Μ (e.g., less than about 10 μμM or ΗΤ11 Μ, in some embodiments less than about 10·12 Μ or 10 _13 Μ). The dissociation constant (KD) binds to DR5. c. DR5 antibody agonists and screening methods Anti-DR5 antibodies have been previously available, for example, in U.S. Patent Application Serial No. 10/723,383, U.S. Patent No. 2005/0079172, PCT WO 01/83560 (antibody TRA-8; ATCC PTA) -1428) and PCT WO 02/0793 77. The heavy and light chain variable regions of exemplary anti-DR5 antibody agonists are disclosed in U.S. Patent No. 2005/0079172. In some embodiments, the DR5-binding molecules of the invention comprise at least one DR5-binding region that competes with the antibodies exemplified in U.S. Patent No. 2005/0079 172 for binding to DR5. In other embodiments, the DR5-binding molecules of the invention comprise at least one DR5-binding region having a CDR substantially similar to the CDR amino acid sequence exemplified in U.S. Patent No. 2005/0079172. Any DR5 antibody agonist type can be used in accordance with the methods of the invention. </ RTI> 131613.doc -36- 200911835 often used anti-systemic monoclonal antibodies, which can be produced by any of the methods known to those skilled in the art (eg, hybridomas and recombinant expression) ^ in some embodiments, The DR5-binding molecules of the invention are constructed using antibody fragments comprising heavy and light chain variable regions rather than full length antibodies. In some embodiments, the antigen binding region of the DR5-binding molecule is a single chain antibody (ScFv). Techniques for the production of ScFv and antibodies are described in, for example, U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., 仏〇 k k 203:46-88 (1991); Shu et al. &quot;· dcW. 5W· 90:7995-7999 (1993); and Skerra et al.' *SWe«ce 240:1038-1040 (1988). DR5 antibodies that specifically bind to DR5 can be identified using techniques well known to those skilled in the art&apos; such as &apos; ELIS A, surface plasma resonance, interferometry (e. g. 'using the ForteBio Octet biosensor system). DR5 antibody agonists can be identified by the ability of a test antibody to trigger a DR5 mediated event, for example, to induce apoptosis in cancer cells expressing DR5. A variety of assays known to those skilled in the art can be used to detect the induction of apoptosis. In the cleavage, the Jurkat cells are contacted with a dr5 antibody that is a candidate agonist. The viability of these cells was then monitored as a function of antibody concentration. A decrease in cell viability caused by an increase in apoptosis corresponds to an increase in antibody orthotypic indicating an anti-system agonist. Cell viability can be analyzed by adding Alamar blue to the cell culture. The dye fluoresces in the presence of live, not dead cells. DR5 antibody agonists can also be identified by screening for hybridomas grown under dR5 and subsequently screening hybridoma supernatants for the ability to induce apoptosis in Jurkat cells 131613.doc -37- 200911835. Suitable for positive and negative controls can be used to confirm the results. For example, a cell line that has not undergone DR5-mediated TRAIL-induced apoptosis should not undergo apoptosis in response to a candidate DR5 antibody agonist. 3. Method of Inducing Apoptosis If the antibodies of the present invention are useful as agonists of the DR5 receptor, they can be used to induce apoptosis of target cells. To this end, one or more of the anti-DR5 antibodies of the invention are contacted in vivo or ex vivo with an DR5 receptor on one or more target cells (e.g., cancer cells) in an amount sufficient to induce apoptosis of the target cells. In some embodiments, an antibody of the invention is contacted with a second apoptosis activating agent and a DR5 receptor on a target cell. a. Apoptosis Inducing Agent The present invention provides an improved effect of an anti-DR5 antibody agonist and a second apoptosis inducing agent. Apoptosis-inducing agents include any agent that induces apoptosis in cells. In some embodiments, the apoptosis inducer preferentially induces apoptosis in cancer cells as compared to non-cancer cells. In general, an apoptosis inducing agent is an agonist or activator of apoptosis or an antagonist or inhibitor of apoptosis. Exemplary apoptosis inducing agents include, for example, the following agonists or mimetics: 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 lymph tyrosine kinase (BLK), gene product similar to pyruvate kinase, M2 isozyme (LOC148283), glycogen Enzyme Kinase 3 α (GSK3A), Hypothetical Protein FLJ32312 (FLJ3 2312), Mitogen Activation 131613.doc -38- 200911835 Protein Kinase 10 (MAPK10), TCF4: Transcription Factor 4, v_abl Abelson Murine Leukemia Virus Oncogene Homologous 2 (arg, Abels〇n related gene) (ABL2), V-ros bird UR2 sarcoma virus oncogene homolog j (ROS 1) and v-myc avian cytomegalovirus virus oncogene homologue and the following antagonists Or inhibitor: 26S proteasome inhibitor 'c_nip' NFkB pathway, 1AP family members (eg, XIAP, cIAP1, CIAP2, NAIP, MLIAP/Livin, survivin), proteasome pathway members (eg, μ, E2, and E3) 'kinase pi3, Aktl, 2 and 3, Rip, Nik; CD40; Bcl2 steroid members (eg 'Bcl2, Bcl-xl, A1, Mcll), ubiquitin-binding enzyme UbcH10 (polynucleotide encoding human UbcH1 〇 variant) The acid sequence includes (9) such as) accession number NM-181803, NM_181802, NM -181801, NM-181800, NM-181799, NM-007019 and BC050736), Osteoproteol, plexin B1 (PLXNB1), SET domain-containing protein 7 (SET7), mitogen-activated protein kinase Kinase kinase 5 (MAP3K5), STE20-like kinase (JIK), MAP kinase interaction, serine, prostaglandin/threonine kinase 1 (MKNK1), putative endoplasmic reticulum multi-span transmembrane protein (RFT1) ' 5-kinase, J-type, γ (PIP5K1C), mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), mitogen-activated protein kinase kinase 5 (MAP2K5), cell cycle regulatory protein-dependent kinase 6 (CDK6b activin A receptor Type II-1 (ACVRL1), Gardner-Rasheed Descriptive Animal Sarcoma Virus (v-fgr) Oncogene Homolog (FGR), False §More Protein FLJ21802 (FLJ21802), Muscle, Skeleton, Receptor Tyrosine Kinase (MUSK), chromosome 20 open reading frame 88 (C2〇orf88), budding 1 without benzommiasis (budding uninhibited by benzimidazoles 131613.doc -39- 200911835 1) (yeast homolog) (BUB1 ), ribosomal protein S6 kinase, 90kD, peptide 5 (RPS6KA5), v-yes -1 Yamaguchi Tumor virus-associated oncogene homolog (LYN), mitogen-activated protein kinase 7 (MAPK7), and v-akt murine thymoma virus oncogene homolog 1 (AKT1), ΡΑΚΙ (including, for example, any of the following Ρ21 ( CDKN1A) Activated kinase 1, 卩八反八,? 65-ΡΑΚ, Ρ68-ΡΑΚ, α-ΡΑΚ 'MUK2, ΡΑΚ1Β (ρ21-activated kinase IB), P21/Cdc42/Racl-activated kinase 1 (associated with yeast Ste20), Cdc42/Rac effector kinase PAK-A, protein kinase MUK2) , nsurf, stkl2 (including, for example, serine/threonine kinase 12, aurora-related kinase 2, aurora/IPL1-like kinase 2, AIK2, ARK2, AIM-1, and AIM1), apoptosis signal-regulated kinase 1 ( Askl), TLK1 (eg, accession number NM_012290), NLK (eg, accession number NM_016231), GRAF (eg, accession number NM_015071), GCK (eg, accession number NM_000162), ERK5 (eg, accession number NM_002749), FGR ( For example, login number NM_005248), ACVRL1 (eg, login number NM-000020), MEKK5 (eg, login number NM_002757), PIP5K1C (eg, login number XM_047620), MAPKAPK2 (eg, login number NM_004759), RFT1 (eg, login No. NM—052859), MKNK1 (for example, accession number NM—003684), PLXNB1 (for example, accession number NM_002673). Additional exemplary apoptosis inducing agents include, for example, agents that enhance DR5 expression and/or stability, agents that enhance caspase activity or stability, and agents that induce or enhance DNA damage responses. The agonist or mimetic listed above includes its own gene product, for example, a p53 line p53 agonist; TRAIL is a TRAIL agonist. Antagonists include agents that inhibit activity and agents that indirectly inhibit activity by reducing the performance or stability of target molecule mRNA (eg, siRNA) or protein. Apoptosis-inducing agents that can be recognized by stems to such gene products include compounds that are borne in winter. For example, modulators of such gene products can be screened using the following libraries: polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatics, heterocyclic compounds, benzodiazepines, oligomerization Ν_Substituted glycine, oligocarbamate, polypeptide, saccharide, fatty acid, steroid, purine, pyrimidine, derivative, structural analog or a combination thereof. In some embodiments, the apoptosis inducing agent is a polynucleotide. For example, it may be a siRNA (such as 'UbcH10, plexin (9) exin) B1 (PLXNB1), a SET domain-containing protein 7 (SET7), a mitogen-activated protein kinase kinase, which is a gene that inhibits TRAIL-induced apoptosis. Kinase 5 (MAP3K5); STE20-like kinase (JIK), endoplasmic reticulum multi-span transmembrane protein (RFT1), MAP kinase interaction with serine/threonine kinase! (MKNK1), mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) 'phospholipidinoinositol_4_phosphate 5_kinase, type 1, sputum (PIP5K1C), MAP2k5, cell cycle regulatory protein-dependent kinase 6 ( CDK6), muscle, bone, receptor tyrosine kinase (MUSK), activin A receptor type II-1 (acvRLI), Gardner-Rasheed feline sarcoma virus (v-fgr) oncogene homolog ( FGR), ribosomal protein S6 kinase, 90 kD 'polypeptide 5 (RPS6KA5) and mitogen-activated protein kinase 7 (MAPK7); see, Figure 28 of U.S. Patent Publication No. 2/5/79,172. 131613.doc 41 200911835 In some embodiments, the apoptosis inducing agent is a small molecule compound (e.g., a molecule having a molecular weight of less than 1500 Daltons and in some cases less than 1 Dodron). For example, the apoptosis inducing agent may be a small molecule compound that inhibits the expression or activity of a gene product that inhibits TRAIL-induced apoptosis (for example, UbcH10, plexin b 1 (PLXNB 1), contains SET domain protein 7 (SET7), mitogen-activated protein kinase stimulates S# 5 (MAP3K5), STE20-like kinase (jik), endoplasmic reticulum multi-span transmembrane protein (RFT1), MAP kinase interactions Acid/threonine kinase 1 (MKNK1), mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), phospholipid inositol-4-phosphate 5-kinase, type 1, gamma (PIP5K1C), MAP2k5, cells Cyclin-dependent kinase 6 (CDK6), muscle, bone, receptor tyrosine kinase (MUSK) 'activin A receptor type II-1 (ACVRL1), Gardner-Rasheed feline sarcoma virus (v- Fgr) Oncogene homolog (FGR) 'ribosomal protein S6 kinase, 90 kD, peptide 5 (RPS6KA5) and mitogen-activated protein kinase 7 (MAPK7); see 'US Patent Publication No. 2〇〇5/〇〇79172 Figure 28) The apoptosis inducing agent may also be a small molecule compound that enhances the expression or activity of a gene product that promotes TRAIL-induced apoptosis (eg, 'UbcHIO, plexin B1 (PLXNB1), contains a SET domain) Protein 7 (SET7), mitogen-activated protein kinase kinase kinase $ (MAP3K5); STE20-like kinase (jik), endoplasmic reticulum multi-span transmembrane protein (RFT1), MAP kinase interaction with serine/threonine kinase 1 (MKNK1), mitogen-activated protein kinase-activated protein kinase 2 131613.doc •42- 200911835 (MAPKAPK2), phospholipid creatinine _4_phosphate 5_kinase, type 1, sputum (PIP5K1C), MAP2k5, Cell cycle regulatory protein-dependent kinase 6 (CDK6), muscle, bone, receptor tyrosine kinase (MUSK), activin steroidal steroid type _1 (ACVRL1), Gardner-Rasheed feline sarcoma virus (v- Fgr) oncogene homolog (FGR), ribosomal protein S6 kinase, 90 kD 'polypeptide 5 (RPS6KA5) and mitogen-activated protein kinase 7 (MAPK7); see, Figure 28 of US Patent Publication No. 2005/00791 72 ). Methods for screening for genes and modulators thereof, including small molecule modulators, and methods for polymerizing siRNA or other genes for inhibiting the detection of polynucleotides are well known to those skilled in the art. See, for example, U.S. Patent Nos. 6,573,099 and 苐 6,506,559; /Vkcz.p/e·? Practice of High Throughput Screening, K. Murray (ed.), CRC Press (2003); Throughput Screening:

MeMoA and /Voiocok, W. Janzen (ed.), Humana Press (2002); PCT Publications W〇95/35503, WO 95/30642 and WO 91/18980; Schultz et al., Bioorg Med Chem Lett 8: 2409-2414 , 1998; and Weller et al. 'Mol Divers. 3: 61-70, 1997. Additional methods that can be used to screen for modulators of such genes and their products are disclosed, for example, in Sambrook et al., Mo/ecw/ar CVomig JJ, Cold Spring Harbor Press ' Ν.Υ·, 3rd edition (2001) ); and Ausubel Temple, Current Protocols in Molecular Biology,

John Wiley &amp; Sons, New York (1987-2006). In some embodiments, the apoptosis inducing agent is combined with an anti-DR5 antibody agonist 131613.doc-43-200911835. In other embodiments, the apoptosis inducing agent does not bind to an anti-DR5 antibody agonist.

1. SMAC In some embodiments, the apoptosis inducing agent is a SMAC/Diablo or SMAC mimetic or agonist. SMAC/Diablo promotes caspase activity by binding to an inhibitor of apoptosis protein (IAP). See, for example, Du et al.' CeU 102: 33-42 (2000); Verhagen et al., Ce//102 43-53, 2000; U.S. Patent Application Serial No. 2002/01 10851. The present invention contemplates administering or expressing SMAC in a cell. SMAC fragments can also be expressed or administered, such as the N-terminal peptide of SMAC (eg, N-terminal tetrapeptide or heptapeptide (Guo et al. '99(9): 3419_3426 (2〇〇2); human '乂' /. C/zem. 275:36152-36157 (2000)). See also U.S. Patent Application Serial No. 2002/0 132,786. Further, 'SMAC mimetic compounds can also be used in accordance with the present invention. These compounds can have useful medicines. Characterization and therefore administration with an anti-DR5 antibody may be more effective. Exemplary SMAC mimetics include, for example, a tetrapeptide (including a tetrapeptide of the formula, wherein 乂) line A comprising a surface groove in the BIR domain of IAP , Χ 2 is a peptide of V, Τ or I, Χ 3 Ρ or Α and Χ 4 is F, Υ, I or V) or other SMAC peptide, agonist or peptidomimetic set forth in PCT WO 02/26775. The SMAC mimetic includes LBP 672. See, for example, Figure 15 from U.S. Patent Publication No. 2005/0079172. 2. 26S Proteasome Inhibitor In some embodiments, the apoptosis inducing agent is 26S proteasome inhibitor 131613. Doc •44- 200911835. Proteasome inhibitors inhibit the proteasome-ubiquitin pathway Reagents for the degradation of ΙκΙ and subsequent NFkB nuclear localization of ΙκΒ. The proteasome has two functional components: the 20S core catalytic subunit and the 19S regulatory subunit. The 20S and 19S subunits form a protein that degrades the target when ubiquitin is added. Degraded 26S complexes are implemented. Exemplary proteasome inhibitors include, for example, 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. 68123 1), 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), YU101 (ac-hFLFL-epoxide) (see, for example, Elofsson et al., C/zem. 5ζ·ο /·6:811-822 (1999)), MG262, MG132, MG115, PSI (proteasome inhibitor N-benzyloxycarbonyl_iie_Glu (0-t-butyl)-Ala-leucine). Analytes for screening protein inhibitors are commercially available, for example, from Disc 〇 verx (Fremont, Calif.). While the combination of a 26S proteasome inhibitor with a DR5 agonist (e.g., an anti-DR5 antibody of the invention) can be an effective treatment for a variety of hyperproliferative disorders, the combination is particularly effective against cancer cells having beta-like or mitochondrial defects. For example, colon cancer patients typically have tumor cells with defective Bax. Thus, proteasome inhibitors in combination with DR5 antagonists are particularly effective in the treatment of colon cancer including beta alpha or mitochondrial defects. 131613.doc -45- 200911835 In some embodiments, the egg

Leukosome inhibitor system

W is an unsubstituted or substituted aryl group; aryl ketone _, an anthracene moiety unsubstituted or substituted; un, a base; or a hydrazine hydrazine quinone * 士# substituted heterocyclic ring, 1 Alkyl-reactive group in which the far heterocyclyl moiety is unsubstituted; R2 is unsubstituted or substituted heteroaryl..., unsubstituted or substituted by two ==: unsubstituted or substituted by: Unsubstituted or substituted: ring: a substituted or substituted aryl group, or an unsubstituted or substituted heteroaryl group containing a Si atom to the earth; &amp; U portion, , '

B--A2 A1 (ΙΑ): ΑΑΑ2 is a hydrazone group or a substituted hydrazine group or a combination of a deleted atom and two bonded oxygen atoms to form a ΙΑ* ring,

〇~—w (ΙΑ*) wherein w is alkyl, substituted alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted cycloalkyl or unsubstituted or via Substituted tricycloalkyl; and Rs is unsubstituted or substituted alkyl, not 1316l3.doc • 46· 200911835 disubstituted or substituted aryl, unsubstituted or substituted heterocyclic, ge Unsubstituted or substituted cycloalkyl; or a salt thereof. In the case of Formula 1, the general terms used have the following meanings: the aryl group preferably has a ring system of not more than 20 carbon atoms, particularly preferably not more than 12 carbon atoms, and is more monocyclic or bicyclic or Tricyclic, and unsubstituted or substituted, in each case preferably unsubstituted or substituted phenyl or (Ultrab or) fluorenyl- or a plurality of substituents are preferably independently selected from the following Group of constituents: aliphatic group; free, etherified or esterified hydroxyl group; free or esterified carboxyl group; formazan group, alkanoyl group; unsubstituted, mono- or di-substituted amine group; , a base of 'sulfuryl group; an amine fluorenyl group; an alkyl-amine fluorenyl group; a di-alkyl-aminocarboxamidine group; a phenyl group; a naphthyl group; a heterocyclic group, particularly preferably a pyridyl group; a cyano group and a nitrate More preferably, selected from alkyl, for example, methyl, ethyl or propyl; alkoxy's such as 'decyloxy or ethoxy; di-substituted amines such as 'monomethylamino"; _', such as chlorine or bromine; haloalkyl, for example, difluoromethyl; and phenyl, (or preferably 丨 or 2)-naphthyl, and especially heterocyclic groups as defined below Pyridyl', for example, 3-, 4- or especially 2-pyrimidinyl, each of which is unsubstituted or one or more, especially up to three, is especially independently selected from the other aryl substituents just mentioned. Substituent substitution. The aryl group &amp; is more preferably a biphenyl group, particularly preferably a 2-, 4- or preferably 3-biphenyl group; an acridinylphenyl group is particularly preferably 4-, 3- or most preferably 2-pyridyl-( 2-, 4- or preferably 3-)phenyl' or a low carbon number-phenyl-p-ethylidene phenyl group, such as 2-, 4- or especially 3-isopropylphenyl. The arylalkylcarbonyl group Ri (aryl unsubstituted or preferably substituted) is preferably an aryl group as defined above, an aryl-lower alkylcarbonyl group, more preferably a phenyl-lower alkyloxy group. Phenyl-phenyl-lower alkylcarbonyl, I31613.doc -47- 200911835 especially preferably 2-, 4- or (preferably) 3-benzyloxy-phenyl-ethenyl or propylpropanyl; Pyridyl-lower alkyloxyphenyl-lower alkyl group, especially preferably 2, 4- or (preferably) 3-(pyridine-2-, -4- or (preferably ethyl) Or -propenyl; or phenyl-lower alkylcarbonyl, especially phenyl or (preferably) 3-phenyl-propyl or benzyl thiol' wherein phenyl is unsubstituted or at most Substituted by three substituents independently selected from the group consisting of a lower alkoxy group (preferably alkoxy group), a south group (partially fluorine or chlorine) or a halogen-lower alkyl group (for example, a trifluoromethyl group). The substituted or substituted aryl &amp; or (independently) &amp; is preferably a mono-, di- or tri-substituted phenyl group, particularly preferably up to four independently selected from those mentioned for the aryl group a substituent substituted phenyl group, the substituent is preferably selected from a hydroxyl group, a lower alkoxy group (best (preferably methoxy), _ (preferably fluorine or chlorine) and halogenated-lower alkyl (preferably trifluoromethyl), especially preferably up to three lower alkyl a phenyl group substituted with an oxy group (preferably a methoxy group) or an unsubstituted phenyl group or other unsubstituted or substituted naphthyl group in the case of &amp; preferably unsubstituted or at most The tetraindole is independently selected from the group consisting of a 1- or 2-naphthyl group substituted with a substituent of the aryl group, and the substituent is preferably selected from the group consisting of a reduced or a low carbon number (the preferred K is preferably a methoxy group). , _ (preferably fluorine or chlorine) and dentate _ lower carbon number (preferably trifluoromethyl). The unsubstituted heterocyclic group is preferably a heterocyclic group as follows: the bonding ring is not Saturated, saturated or partially saturated and preferably monocyclic or more broadly bicyclic or -%%, having from 3 to 24, more preferably 4 to one ring atom; wherein at least One of the corresponding aryl groups in the ring of one molecule group or more than one car, one or two carbon atoms are substituted by a hetero atom selected from the group consisting of nitrogen, oxygen and sulfur, Bonding ring is preferred There are 4 131613.doc -48- 200911835 to 12, preferably 5 to 7 ring atoms; heteroaryl unsubstituted or one or more, especially preferably 1 to 3 independently selected from the above Substituent substituents defining a group consisting of substituents of a substituent of a substituted aryl group; and particularly preferred

Free heteroaryl groups of the following composition: imidazolyl, thienyl,. 'Farmanyl' tetrahydrofuranyl, pyranyl, thioxyl, isobenzofuranyl' benzofuranyl, nonenyl, 2/7-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl , imidazolidinyl, benzimidazolyl, pyrazolyl, π-pyrazolidine, pyranyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, Hexahydropyridyl, hexahydropyrazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, pyridazinyl, isodecyl, 3-decyl, decyl, oxazolyl, triazolyl, Tetrazolyl, fluorenyl, 4 quinolinazinyl, isoquinolinyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, benzo Furanyl, benzothiophene, pyridazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, quinazolinyl, porphyrinyl, acridinyl, oxazolyl, β-° morpholinyl, Pentidyl, acridinyl, naphthalene: diazaphenyl, phenanthrenyl, Df-decyl, phenanthrenyl, phendazine, phenanthrenyl, iso-bite, and blister The groups are unsubstituted or selected from one or two Substituted by groups of the following groups: lower alkyl (preferably methyl or tert-butyl) 'lower alkoxy (especially methoxy), and ^ (excellent Or chlorine); biting base (especially 2 or 3_ bite base or skillful base), in a wider range of states, low carbon number base (9) base, mouth bite base or low number of turns Alkylpyrimidinyl, halo-lower alkyl pyridyl, lower carbon: alkoxy-pyridyl, di-lower alkyl-pyridyl or deuterated-pyridinium. Heterocyclyl unsubstituted or Substituted one or more (preferably up to three) independently selected 131613.doc -49- 200911835 from the substituents described above, which are mentioned above for the aryl group (wherein the heterocyclic filamentary group is heterozygous) a cyclyl group as defined above or a cyclyl group as defined above, and especially preferably a phenyl group, especially as a preferred one. Unsubstituted heterocyclic ring. Preferably, in the heterocyclylalkylcarbonyl core, the heterocyclic moiety is preferably a substituted or especially unsubstituted heterocyclic group as mentioned above; preferably substituted or preferably not Replace the miscellaneous a cycloalkyl-low carbon number, especially a terminally substituted or unsubstituted heterocyclic group (heterocyclyl as described above) preferably acridinyl-lower alkylcarbonyl (eg _ Ethyl aryl or propyl hydrazide. Preferably, R is an unsubstituted or substituted aryl or substituted aryl-lower alkyl carbonyl group. Heteroaryl I is preferably as mentioned above. And an unsubstituted or substituted heteroaryl group, particularly preferably unsubstituted or substituted by one or more Z (especially up to three) independently selected from those mentioned above for the substituted aryl group Substituted, the silk, the substituent is especially selected from the group consisting of secret, lower carbon alkoxy (optimal) (preferably methoxy), dentate (preferably fluorine or phantom: generation - lower alkyl) (preferably trifluoromethyl). &amp; preferably is a substituted group. π is entangled into the soil of the earth, preferably up to 4 carbon atoms +' and is an aliphatic hydrocarbon group, for example, a substituted or substituted alkynyl group, an alkenyl group or preferably an alkyl group, more preferably a low carbon number &amp; base group, particularly preferably a methyl group, an ethyl group, a n-propyl group, a hetero-diyl group, a n-butyl group, a Dibutyl, isobutyl Or a third butyl group. The base which may be branched or linear preferably has up to 12 carbon atoms, and more preferably a lower alkyl group. The alkyl group &amp; is preferably a lower alkyl group, particularly preferably isobutyl 131613.doc -50- 200911835. The prefix "low carbon number" means a group having up to and including 7, preferably up to and including 4 carbon atoms. The lower alkyl group is preferably n-propyl, isopropyl, n-butyl or isobutyl. Base, second butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl or n-heptyl' is preferably isobutyl, second butyl 't-butyl, isopropyl a group, an ethyl group or a decyl group, preferably an isopropyl group, an ethyl group or a methyl group. The etherified hydroxy group (for example) alkoxy group, particularly preferably a lower alkoxy group such as an ethoxy group or a decyloxy group; Aryloxy, especially phenyloxy; aryl-lower alkoxy, especially phenyl-lower alkoxy; heterocyclyloxy, especially pyridyloxy, or heterocyclic a benzyl-lower alkoxy group, especially a acridine-lower alkoxy group (the aryl group and the heterocyclic group preferably have the meanings given above). The acetinyl group is preferably organic A hydroxy group esterified with a carboxylic acid (e.g., alkanoic acid), such as a lower alkyl alkoxy group. A thiol group, for example, an alkoxycarbonyl group, particularly preferably a lower alkoxycarbonyl group (e.g., methoxy) Carbocarbonyl). Single- or two-take The amine group is preferably an alkylamino group or a dialkylamino group, particularly preferably a lower alkylamino group or a lower carbon number, see a di-low carbon number alkyl group, such as 7V-methyl. Amino or dimethylamino. Halogen is fluorine, gas, bromine or iodine, preferably fluorine, gas or bromine. The unsubstituted or substituted cycloalkyl group preferably has up to 2, more preferably 3 Up to 8 ring carbon atoms and one or more (especially up to three) independently selected from the substituents of the substituted aryl group, or preferably unsubstituted. Preferred ring Amyl, cyclohexyl or cycloheptyl. 131613.doc -51 · 200911835 In the alkyl group of the unsubstituted or substituted cycloalkyl group, the alkyl car is as defined above. a lower alkyl group, more preferably an isopropyl group, and (preferably a terminal group) is substituted with a cycloalkyl group as defined above. In an unsubstituted or substituted aryl group substituted group, the alkyl group is more ^ as defined in the most recent paragraph, and the aryl group is as defined above and is independently selected from one or the other (especially to the eve) of the substitution of those mentioned for the substituted aryl group. Substituted, or unsubstituted; especially aryl is - or a plurality (U) - independently selected from dentate (especially gas), trans- or low-carbon alkoxy (especially A Substituted by a substituent of oxy) or it is an unsubstituted phenyl group. ^ In the alkyl group R3 substituted by an unsubstituted or substituted heterocyclic group, the alkyl group is further defined by the radically substituted alkyl group R3, and the heterocyclic group is as defined above and is - or more The individual (especially up to three) are independently selected from the substituents substituted or unsubstituted by the substituents mentioned in the U-based hetero group. ^1 and 八2 are each a substituted hydroxy group, and the substituted hydroxy group is preferably an alkane oxime lower alkoxyalkyloxy group; an aryloxy group, particularly preferably having an unsubstituted or defined as defined above a substituted aryl group; or a cycloalkyloxy group having a t-substituted or substituted cyclodole as described above. „a2 combines with a boron atom and an oxygen atom to form the above-mentioned formula JW. # has two ϋ combined with a stone friend atom on two different carbon atoms; § 2 _ mountain, son, The two different carbon atoms are spatially adjacent or adjacent slave atoms 'especially in the C'1,2-") or '3"-position (relative to each other). 8. The elementary base is preferably non-supported. Chain C2-Ci2-, car-crossing is c2_c7 stretching base 1 stretching ethyl or stretching propyl 'in the broader aspect is butyl, extension 1316I3.doc • 52 · 200911835 pentyl or hexyl, its grade It is bonded by two different carbon atoms as described just now (preferably adjacent or at the "1, 3, - position"). One or more (especially one) carbon atoms not bonded to an oxygen atom (in combination with a boron atom) may be replaced by a hetero atom selected from hydrazine, s or preferably N (respectively with a desired number of H atoms), for example at , 5-(3-aza-exetyl). The 'disubstituted alkylene group is preferably a non-branched lower carbon number extending alkyl knives as defined above, which are unsubstituted or independently selected by one or more (especially up to three) Substituting a lower alkyl group (for example, a decyl group or an ethyl group) from the following substituents, for example, in i-methyl-extended ethyl, anthracene, 2-dimethylexene ethyl; hydroxy^, for example, at 2_ Or a hydroxy-lower alkyl group, such as a hydroxymethyl group, for example, in a 1-hydroxyindenyl-extended ethyl group. The unsubstituted or substituted cycloalkylene group is preferably via C3_k' which is bonded to two different carbon atoms as described for the product (preferably adjacent or at the &quot;1,3,,-position). For the c3-c8_cycloalkyl group, for example, a cyclohexyl group or a ring-shaped ring, 2 or 8 (especially one) is not bonded to an oxygen atom (bonded to a boron atom), and the carbon atom may be selected from the group consisting of ruthenium , s or Ν (min (4) has the required number of pristine: the hetero atom replacement 'for example in the tetrahydrogen bite. South base or tetrahydrogen ^ south base: can be unsubstituted or one or more (especially up to three) Independently selected from the group consisting of a substituent substituted with a low carbon number (eg, methyl or ethyl), a di- or di-carbo-lower alkyl group (eg, methoxy) or a mono- or a sugar-based group ( It is preferred to include up to five glycosyl moieties. The = substituted or substituted bicyclic alkyl group is preferably via the same carbon atom as w (preferably in the '3' heart % pit) a carbonaceous material in which one or more (especially one) is not combined with an oxygen atom (in combination with 131613.doc •53·200911835 deleted atoms) may be selected from the group consisting of ruthenium, (with a demand, an atom, respectively) Atomic substitution, and may be unsubstituted or substituted by one or more hydrazines: two up to three) substituents independently selected from the group consisting of lower carbon number alkane (such as methyl or ethyl), trans- and trans-base _ low carbon number hospital base (such as indoyloxy) is preferred to be an alkyl group (pinanyiene;) (2,3-(2,6,6-trimethyl-bicyclo[3.1.1]heptane" The earth-, unsubstituted or substituted three (four) base is preferably via, for example, W

a C12-extension ring-ring compound in which two different carbon atoms (preferably adjacent or at the "丨, 3," position) are combined, wherein one or more (especially one) are not associated with an oxygen atom (in combination with a butterfly atom) The combined carbon atom may be replaced by a hetero atom selected from the group consisting of ruthenium, s or N (with the desired number of ruthenium atoms, respectively), and may be unsubstituted or one or more (especially up to three) independently selected from the following Substituent substitution: lower carbon number (eg methyl or ethyl), hydroxyl and hydroxy-lower alkyl (eg, decyloxy). Preferably, r4 is 2(〇Η) 2 or 2, 9,9_Trimethyl_3,5_dioxa_4,_%% [6.1.1.〇2'6]癸_4_ base, especially (18,25, this, 88)_2,9 , 9_trimethyl _ 3,5-dioxa-4-boron_tricyclo[611〇2,6]癸_4_yl. In the unsubstituted or substituted alkyl Rs 'may be The branched or straight chain alkyl ruthenium preferably has up to 12 carbon atoms, and more preferably a lower alkyl group. The alkane, 5 is preferably a low singly burnt group, particularly preferably an isopropyl group. A plurality of (especially at most two) substituents are independently selected from unsubstituted or substituted aromatic earths (especially stupid or prepared) Phenyl), unsubstituted or substituted heterocyclic (undomendazole or fluorenyl), unsubstituted or substituted cycloalkyl, each as defined above; hydroxy (preferably), carboxy (preferred), amidoxime, hydrazine 131613.doc -54- 200911835 yl, lower alkylalkylthio (eg thiol), phenyl, hydroxyphenyl, decyl, miso, amine , a tri-lower alkylalkyl group (eg, a tridecylamino group), a low nuenyl aminyl group (eg, an ethoxylated amino group), a fluorenyl group, and an N-lower alkyl alkyl group ( For example, N-methylindenyl) or any other substituent which is complete to contain the amino acid of the heart. Preferably, R5 may be an anthracenyl group, an isopropyl group, an isobutyl group, a first butyl group, a decyl fluorenyl group. , 2 曱 thioethyl, phenyl fluorenyl, hydroxyphenyl methyl, 丨 朵-3-ylmethyl, hydroxy fluorenyl, hydrazine hydroxyethyl, 2 hydryl ethyl, amine formazan Methyl, 2-aminomethylaminoethyl, 4-aminobutyl, 3-mercaptopropyl, 5-imidazolylmethyl, carboxyindenyl or 2-carboxyethyl. Compounds of formula I present The asymmetric carbon atom can be (R), (s) or (R , s) The configuration exists, preferably in the (R) or (S) configuration, preferably in the configuration shown in the following formula: The substituent on the double bond or ring may be cis_(=z_) Or in the form of trans (-E-). Thus, the compounds may exist as a mixture of isomers or preferably as pure isomers. The salt forming groups in the compounds of formula I are basic Or a group or group having an acidic character. A compound having at least one basic group or at least one basic group (for example, an amine group, a secondary amine group which does not form a peptide bond or a specific base group) can form an acid addition salt. , for example, with inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or with suitable organic carboxylic acids or sulfonic acids 'eg aliphatic mono- or di-carboxylic acids, such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, Maleic acid, fumar I, Zhaoji maleic acid, malic acid, tartaric acid, citric acid or oxalic acid; or an amino acid such as arginine or lysine; aromatic carboxylic acid such as benzoic acid, 2-phenoxy Benzo-benzoic acid, 2-ethyloxy-benzoic acid, salicylic acid, 4-amino water 131613.doc •55· 200911835 « H aliphatic An acid such as mandelic acid or cinnamic acid; a heteroaromatic citric acid such as nicotinic acid or isonicotinic acid; an aliphatic acid such as methyl acetonide, ethane- or 2/sulfenyl sulfonic acid; or an aromatic sulfonate An acid such as benzene, p-nonyl- or naphthalene-2-3⁄4 acid. When a plurality of basic groups are present, a mono- or poly-acid addition salt can be formed. Compounds of the formula r having an acidic group (for example a free boronic acid group (_b(〇h) 2, ie, each of AjA2 in the formula IA*) or a tracing group) can form a metal or ammonium salt such as an alkali metal or Alkaline earth metal salts (such as sodium, potassium, magnesium or calcium salts) or with ammonia or a suitable organic amine (such as a tertiary monoamine such as triethylamine or tris-(2-pyridylethyl)-amine or a heterocyclic ring An ammonium salt formed by, for example, ethyl-hexahydrogen, specific or dimercaptohexahydropyrazine. It can be a mixture of salts. It has the formula of both acidic and inspectable groups! Compounds can. Forming internal salts. Examples of compounds include those in which: substituted aryl _ lower carbon number or unsubstituted or substituted aryl; fluorene substituted aryl or unsubstituted or Substituted heterocyclic group; fluorene-lower alkyl group, unsubstituted ortho-substituted U group or county (four) substituted aryl substituted lower carbon number base; R4 is a part of formula ια given above, wherein And Α2 are a benzyl group, a lower carbyloxy group, a aryl group having an unsubstituted or substituted aryl group or a cycloalkyloxy group having an unsubstituted or substituted cyclodole' or Wherein Al and Α2 are combined with a butterfly atom and two bonded oxygen atoms to form a ΙΑ* ring of the formula given above, wherein w is an unsubstituted or substituted low carbon number extension county, which is spatially adjacent to ^ Two different carbon atoms in the vicinity (especially in the field or adjacent to each other at the Ά position); recognize a low-order alkyl group, or a salt thereof. 131613.doc • 56- 200911835 Examples of compounds of formula I include those wherein R1 is phenyloxyphenyl-lower alkylcarbonyl; phenyl-lower alkoxyphenyl_low carbon a number of alkyl &amp; base '"pyridyloxyphenyl-lower alkylalkyl; phenyl-lower alkyl group' which has a lower alkoxy group (especially methoxy), Halogen (preferably fluorine &quot; or chlorine) or a halo-lower alkyl (preferably trifluoromethyl) substitution; or preferably unsubstituted or substituted phenyl or naphthyl, in two Wherein the substituent-group (if present) is independently one or more (particularly one to three) substituents selected from the group consisting of: lower alkyl, hydroxy, lower alkoxy C, yl, Lower alkyl alkoxyoxy, carboxy, lower alkoxycarbonyl, fluorenyl, lower carbon decyl, amine, fluorene-lower alkylalkyl, hydrazine, fluorene-di-low Carbon number alkylamino group, mercapto group, sulfo group, lower alkylalkylthio group, amine mercapto group, lower alkyl group-amine fluorenyl group; 7^, bis-lower alkyl group Phenyl, naphthyl, acridinyl, cyano a nitro group, more preferably a lower alkoxy alkoxy group (preferably a decyloxy group or an ethoxy group); the r2 group is independently selected from the group consisting of one or more (particularly one to three) selected from the group consisting of Substituted phenyl I groups: transbasic, lower alkoxy (especially methoxy), halogen (excellent fluorine or chlorine) and halogenated-low carbon number (especially trifluoromethyl); R3 a lower carbon number (preferably isobutyl), a phenyl group or a phenyl group substituted with one or more (especially up to three) substituents independently selected from the group consisting of: hydroxy, lower alkyl -oxy (especially methoxy), _ (preferably fluorine or gas) and _-lower alkyl (especially trifluoromethyl); rule 4 series-8 (〇11) 2 (especially佳) or 2,9,9-trimethyl-3,; 5-dioxa-4-boron-tricyclo[6.1.1.〇2,6]癸-4-yl, especially good (13,23 , 6 feet, 88)-2,9,9-trimethyl-3,5-dioxa-4-boron-tricyclo[6,1·1·〇2,6]癸-4-yl;

Rs is a lower alkyl group, particularly preferably an isopropyl group; or a salt thereof. 131613.doc -57- 200911835 Exemplary compounds of formula i include those in which the phenyloxyphenylethyl fluorenyl group, the f-oxyphenylphenyl fluorenyl m-oxyphenyl group, and the Phenyl, acridinylphenyl, lower alkylphenyl or substituted phenylpropenyloxy, wherein the phenyl substituent is up to three independently selected from the group consisting of methoxy, fluoro, chloro and trifluoro a substituent of a group consisting of fluorenyl groups; 1 is a phenyl group substituted with up to three decyloxy substituents, preferably 2,3,4-trimethoxyphenyl or 3,4,5-dimethoxybenzene a phenyl group which is unsubstituted or substituted with at most two moieties which are independently selected from the group consisting of hydroxy, fluoro and methoxy; dent 4 (lS, 2S, 6R, 8S)-2,9 , 9-trimethyl-3,5-dioxa-4-penpen-tricyclo[6.1.1.02'6]non-4-yl or Yujia-B(〇H)2; and R5 is isopropyl Or a exemplified compound of the formula I includes those wherein R1 is biphenyl, lower alkyl-phenyl, phenyl-lower alkyl-carbonyl, phenoxy-phenyl Lower alkyl-carbonyl, strepyl-lower alkoxy-phenyl-lower alkyl-carbonyl or pyridyl-phenyl; R2 a phenyl or phenyl-lower alkoxy-phenyl substituted with 1 to 3 lower carbon alkoxy groups; R3 lower alkyl or phenyl-lower alkyl; R4 4,4,5,5-tetradecyl-[1,3,2]dioxaboroin-2-yl, (18,28,611,88)-2,9,9-trimethyl-3,5 -dioxa-4-boron-tricyclo[6.1.1_02'6]non-4-yl or -8(〇1^)2; and 115 is a low carbon number&gt;base; or other exemplary The compounds of the formula I or their salts include those in which the stereochemistry is as depicted in the formula I*: I31613.doc -58- 200911835

The configuration shown therein represents an absolute configuration and wherein R, R2, r3, R4 and 5, especially in the above, are described as having a stupid, better meaning as defined for the compound of formula I. Or a salt thereof, including those in the stereochemistry, other exemplary compounds of formula I, as depicted in formula I**:

R2, R3, ft. 4 and 115 have the meanings set forth above for the absolute configuration and wherein R1 has the meaning as defined for the compound of formula I, particularly preferred. Other exemplary compounds of formula I or soy 4 1 a include mixtures of diastereomers wherein the stereochemistry is as depicted in formula I***:

The configuration shown therein represents the absolute structure 33 and its tR, R2, R3, ~ and ^ have the meanings as defined for the compounds of the formula I, in particular, which are described above as being of a preferred meaning. Most preferably, the compound of formula I as set forth in the examples, or a pharmaceutically acceptable salt thereof, is available from 131613.doc-59 to 200911835. The compound of the formula 1 or a salt thereof is prepared according to a conventional method and comprises: a) a dipeptide analog of the formula II;

Wherein R3, R4 and R5 have the meaning given under formula I and the amino acid of formula III

〇

Or a reactive derivative I 'eight eight Z points 匁 1 1 1 1 1 1 1 1 1 1 1 1 , , , , , , , , , , , , , , , , , , 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物 化合物) as needed, by easy removal of the 徂 1 blade sand Chen Zhibao 4 group protection, and shift

Except for any protecting groups present; 4b) is a heterocyclic alkylcarbonyl group and the other parts r2 to R

The meaning of the compound of formula I is such that the amine compound of formula IV produces the Ri arylalkylcarbonyl group 5 having the formula

Wherein R2, R·3, R4 and Rs have the meaning given by the formula τ and the carbonic acid of the formula V

Ri

(V) or a reactive derivative thereof, wherein Ri is an arylalkylcarbonyl group or a heterocyclic group 131613.doc-60-200911835 alkylcarbyl group, a functional group present in the compound of the formula IV and/or V (except for participation*) The group of the reaction is protected by an easily removable protecting group, and any protecting groups present are removed, and if desired, the formula obtained by method a) or b) Conversion of the compound to another hydrazine compound, conversion of the obtained free compound of formula I to a salt, conversion of the salt of the obtained compound to a different salt or conversion to its free form, and/or the isomerization of the compound The mixture is separated into individual isomers. The different possible stereoisomers of the compounds of formula I can be prepared by using educts having the appropriate configuration. For example, a compound of formula p or a salt thereof can be prepared by 4: a) a dipeptide analog of formula II*

Wherein R3, R4 and R5 have the meanings given under formula I, and the amino acid of formula ΠΙ*

"-一, "久^...' 官能u all the functional groups present in the official body (except for the protection of the easy removal of a certain _ _ reaction group) as needed by easy to move In addition to the burden

1316I3.doc 61 200911835

(ΙΝΛ) where r2

R3, chemistry and Han 5 have the meaning of Rl\ OH given under formula I, and are opposite to the carbonic acid (V) of formula V or its reactive derivative

Dan TK is a arylalkyl group or an alkylcarbonyl group, and the functional group present in the formula IV* and the human genus by the hexazone X-coating kiln compound (except the group participating in the reaction) can be borrowed as needed. Protected by a protecting group that is easily removed: and removes any protecting groups present, and if desired, the compound of formula I* known by method a) or b) is converted to another compound of formula p, The obtained free compound of formula I* is converted to a salt, or the salt of the compound of the formula obtained is converted to a different salt or converted to its free form.

The final product of formula I may contain substituents which may also be used as a protecting group in the starting materials used to prepare other end products of the formula, for example, where 1 is not Β(ΟΗ)2. Therefore, within the scope of this document, unless otherwise stated herein, the groups that are readily removed, which are not particularly desirable for the formation of the final product of Formula I, are referred to as &quot;protecting groups.&quot; The protection, the protecting group itself and its excision reactions are described, for example, in standard reference works such as j. F. w. Mc〇mie, 'Protective Groups in Organic Chemistry', Plenum Press, London and New York 1973; TW Greene and PGM

Wuts, &quot;Protective Groups in Organic Synthesis&quot;, 3rd ed., Wiley, New York 1999;,, The Peptides” ’ vol. 3 (Editor E. 131613.doc -62- 200911835

Gross and J. Meienhofer), Academic Press, London and New York 1981; &quot;Methoden der organischen Chemie&quot; (Methods of organic chemistry), Houben Weyl, 4th edition, Vol. 15/1, Georg Thieme Verlag, Stuttgart 1974; H.-D. Jakubke and Η· Jescheit, &quot;Aminosauren, Peptide, Proteine&quot; (Amino acids, Peptides, Proteins), Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982; and Jochen Lehmann, &quot;Chemie der Kohlenhydrate: Monosaccharide und Derivate&quot; (Chemistry of Carbohydrates: Monosaccharides and Derivatives), Georg Thieme Verlag, Stuttgart 1974 ° The protective group is characterized by, for example, solvolysis, reduction, photolysis or another selection under physiological conditions. It is easily removed (eg, by enzymatic excision) (ie, no undesirable side reactions occur). - Β (ΟΗ) 2, removal of the group protecting group (to obtain a compound of formula I in which the quaternary 4 series _b(〇h) 2 ) is preferably acid (for example, hydrochloric acid) in a suitable solvent (for example, low carbon) The calcination of an alcohol (for example, a decyl alcohol) or a lower alkane (for example, hexane) or a mixture thereof occurs at a temperature of from 0 to 50 ° C (for example, room temperature). At least two methods can be used to synthesize a proteasome inhibitor of guanidine. In the method &quot;a&quot;, the reaction is carried out by dissolving the compound of formula m in a suitable solvent (e.g., dimethyl decylamine, dimethyl acetamide, methyl-2-hydantoin). , dichloromethane or a mixture of two or more such solvents) and adding a suitable base (such as triethylamine, diisopropylethylamine (DIEA) or iV-mercaptomorpholine) and Suitable coupling reagents for in situ formation of preferred reactive derivatives of m-carbonic acid, such as dicyclohexylcarbodiimide _ 131613.doc • 63 - 200911835 Hydroxybenzotriazole (DCC/HOBT); tetrafluoroborate II Hydrogen 2_oxo-pyridyl)-HA^', A^-tetramethylurea Τ (Τρτυ); 四 tetrafluoroborate _ (benzotriazol-1-yl)-N,N,N' , N'-tetradecyl adenine (TBTU); or 丨 (3-didecylaminopropyl)-3-ethylcarbodiimide hydrochloride (edC). For other comments on possible coupling reagents, see, for example, Klauser; Bodansky, ICP, 1972, 453-463. Preferably, the reaction mixture is stirred at a temperature between about _2 〇〇 c and 5 〇〇 c, especially between 〇 ° C and room temperature to produce a compound of formula I. The reaction is preferably carried out in an inert gas such as nitrogen or argon. In the process "b", the reaction is preferably carried out under conditions similar to those described for the process a). The salt of the compound of the formula I having a salt-forming group can be prepared in a manner known per se. Thus, an acid addition salt of a compound of formula J can be obtained by treatment with an acid or with a suitable anion exchange reagent. The salt can generally be converted, for example, by treatment with a suitable basic reagent to the free compound, for example with a base. Treatment with metal carbonates, bicarbonates or hydroxides (generally s is carbonic acid or sodium hydroxide). Suitable separation methods are the isomers which are known per se. For example, diastereomers Method chromatography, /column distribution method and similar procedures stereoisomeric mixtures (such as mixtures of diastereomers) can be divided into their corresponding parts by means of a suitable method

131613.doc •64- 200911835 Help chromatography (eg by HPLC) using a chromatography matrix and a palmitic ligand. Isobutyl-boric acid (i_BuB(〇H)2) can be used according to standard procedures (for example) in the presence of an acid (preferably hydrohalic acid) in a water/methanol/hexane mixture, preferably between 0 C and 50 C. A compound of formula I in which &amp; is not -B(OH)2 is converted to a compound of formula &lt;b(〇h)2, at a temperature within the range (e.g., at room temperature). In both methods a) and b), for the conversion or synthesis of the intermediate or starting material, unless otherwise stated in the method description, the solvent suitable for the reaction is selected therefrom (for example) Water, g| (usually a lower alkyl-lower alkanoate such as diethyl acetate), an ether (usually an aliphatic ether such as diethyl ether, or a cyclic ether such as tetrahydrofuran), a liquid An aromatic hydrocarbon (usually benzene or toluene), an alcohol (usually decyl alcohol, ethanol or _ or 2-propanol), a nitrile (usually acetonitrile), a halogenated hydrocarbon (usually dioxane), acid bismuth Amine (monomethylamine), test (usually a heterocyclic nitrogen test, for example, pyridine), carboxylic acid (usually a low carbon number alkane carboxylic acid, such as acetic acid), carboxylic anhydride (usually low carbon) Alkane anhydrides such as acetic anhydride), cyclic, linear or branched hydrocarbons (usually cyclohexane, hexane or isopentane), or mixtures of such solvents (for example aqueous solutions). These solvent mixtures can also be used, for example, in chromatography or distribution processing. Novel starting materials and/or intermediates and methods for their preparation are also within the purview of the present invention. In a preferred embodiment, the starting materials are used and the reaction conditions are selected to permit preparation of the preferred compound. The starting materials of formula II-V or precursors thereof are known to us, which may be prepared according to the method known from the prior art, or are commercially available; in particular, they may be used with them 131613.doc -65- 200911835 The methods described in the examples were prepared in the same or similar manner. The compound of the formula π wherein the substituent is as defined in the above formula I can be obtained, for example, by the following reaction: First, the boronic acid analog of the amino acid of the formula VI is used.

It contains, for example, the configuration represented by the formula VI*

(VI*) wherein R3 has the meaning given above for the compound of formula I and has the meaning of _Β(〇Η)2 as mentioned above for the compound of formula I, especially preferably (lS, 2S) ,6R,8S)-2,9,9-tridecyl-3,5.dioxa-4-boron_tricyclo[6.1.1 ·02'6]癸_4_yl, or its acid addition a salt (especially trifluoroacetate), with an amino acid of formula VII

(VII) it comprises, for example, the configuration represented by formula VII*

Rc (VII*) or a reactive derivative thereof (wherein Rs has the meaning given above for the compound and the Pri-protected amine group, preferably the third butoxycarbonylamine 1316l3.doc-66 - 200911835 Bases) condensed under reaction conditions similar to those described above for reaction a) above (also preferably condensation reaction, preferably in situ formation of an active carbonic acid derivative) resulting in an N-protected form A compound of the formula (which is subsequently deprotected) 'for example' uses the conditions set forth in the standard textbooks mentioned above 'in the case of a second butoxycarbonylamine group, for example with hydrochloric acid in a suitable solvent (eg Compounds of formula II which can be used directly in process a) are given in dioxane and/or dichloromethane. The rotten acid of formula VI is known to be 'commercially available and/or can be synthesized according to known procedures. For example, 'where R3 is a lower alkyl (preferably isobutyl)) and Rule 4 is as described for the compound of formula VI (preferably (1S, 2S, 6R, 8S)_2, 9, 9-trimethyl A compound of formula VI wherein _ 3,5-dioxa-4-boron-tricyclo[61102'6]癸_4_yl) can be prepared by making a compound of formula VIII

R4 (viii)

Wherein I has the meaning just described, reacting with n-lower alkyl lithium (preferably n-butyllithium) and subsequently with zinc chloride in a suitable solvent (e.g., methylene chloride) to produce a compound of formula IX,

(IX) wherein R4 has the meaning given under formula VI above. The compound is then reacted with LiN(SiCH3)2 and the resulting compound is reacted in the presence of trifluoroacetic acid 131613.doc •67- 200911835 to give the salt of formula x

Wherein R4 has the meaning given under formula VI, which is a compound of formula VI and which can be used directly in the reaction with a compound of formula VII as indicated above. The compound of formula III is known, commercially available and/or can be cleaved according to standard procedures. For example, a compound of the formula hi wherein R is a phenyl group (preferably biphenyl group) can be prepared by making a compound of formula XI 2\

R

(XI) It contains, for example, the configuration represented by Formula XI*

(XI*) ^ has a compound for the formula!

R.-X wherein R 1 is a aryl (XII) 131613.doc group and X is an imine (youjia Mo), in a suitable solution such as ^ 68 - 200911835 methyl carbamide) The salt, for example, potassium carbonate, is preferably reacted in an inert gas (e.g., nitrogen or argon) at a temperature between 50 C and 100 C (e.g., at 9 t). This directly produces the corresponding compound of formula III. Amino acid derivatives of formula VII are known, are commercially available and/or can be obtained according to standard procedures. It is preferably used in the form of a protected amine group, for example, a third butoxycarbonylamino group in place of the free amine group. The oxime conjugate can be obtained, for example, by a compound of the formula n as defined in (e.g., method a) as defined in the formula (e.g., method A) and an N-protected amino acid of formula XIII.

(XIII) which comprises, for example, the configuration represented by the formula XIII*

(XIII*) or a reactive derivative thereof is reacted under the conditions as specified in the above method a), wherein the rule 2 is as defined in the formula: the condensed anti-amino group (or preferably the third butoxycarbonylamino group) Subsequent removal of the N-group from the protected amine group in a protected form to form a hydrazine compound (wherein the N-group, for example, in the third butoxylamine 5) removes N - End protecting the alkane. Hydrochloric acid in the form of cesium in the shape of cesium 131613.doc •69- 200911835

2,4-Diamino _3_monoamino-3-hydroxycarboxylic acid, in other examples &amp; carboxylic acid compound % 00/64863. For example, XIV 2,4-diamine | See also PCT WO proteasome inhibitor system

Wherein A and B independently represent a bond or an unsubstituted or substituted aminoalkyl moiety: R, representing hydrogen; an amine protecting group; or a RsY group wherein ^ represents hydrogen or unsubstituted or substituted Alkyl, alkenyl, alkynyl, aryl, arylalkyl, (tetra), heteroarylalkyl, heterocyclyl or heterocyclyl; and -S02-; -O-CO-; or _〇 _ Y represents -CO-; -NH-CO-; -NH-CS-; CS-; R2 represents a side chain of a natural amino acid; alkyl, arylalkyl, heteroarylalkyl or cycloalkylane Or a trimethylmethanylmethyl group, a 2-thienyl fluorenyl group or a styrylmethyl group; R3 represents a halogen, an alkyl group, an alkoxy group or a hydroxyalkoxy group; and R_4 represents a 2(R)-hydroxy group. Indoline-1(S)-yl; (s)-2-hydroxy-1·phenylethyl; or 2-hydroxy-benzyl which is unsubstituted or substituted with a methoxy group at the 4-position; The 2,4-diamino-3-hydroxycarboxylic acid is in a free form and is a pharmaceutically acceptable salt or pharmaceutical composition thereof. The unsubstituted or substituted alkyl group is preferably an alkyl group having 1 to 5 carbon atoms, preferably 1 to 4 carbon atoms; for example, an anthracenyl group, an ethyl group, an isopropyl group or a tert-butyl group It is particularly preferred to have one or four carbon atoms. Substituents such as phenoxy, hydroxy or unprotected or protected amine groups. 131613.doc -70- 200911835 Unsubstituted or substituted arylalkyl is, for example, a phenylalkyl group having a total of from 7 to 10 carbon atoms, such as benzyl or 2-phenylethyl. It is unsubstituted or in the aryl or alkyl moiety via, for example, a hydroxyl group (for example, in benzyl-CH(OH)- or phenyl-CH(CH2OH)-), via an alkyl group, an amine group or an alkylamine. Substituent; or, for example, a naphthylalkyl group having from 1 to 4 carbon atoms in the alkylene moiety, and more preferably a naphthylmethyl group. The amino protecting group is preferably a benzyloxycarbonyl group, a cycloalkylalkoxycarbonyl group, particularly preferably a cyclohexyloxycarbonyl group or a third butoxycarbonyl group. The unsubstituted or substituted heteroarylalkyl group is preferably a pyridylalkyl group, particularly preferably a 2-pyridylmethyl group and a 4-&quot;pyridylmethyl group. The aryl moiety of the aryl, heteroaryl and arylalkyl and heteroarylalkyl groups may be monocyclic or polycyclic, such as pyridinyl, naphthyl, 9-fluorenylmethoxycarbonyl (FMOC) or Benzimidazolyl. The alkyl moiety of the arylalkyl or heteroarylalkyl group can be substituted with, for example, a hydroxyl group. The heterocyclic group of the heterocyclic group and the heterocyclic alkyl group is a saturated heterocyclic group having one or more hetero atoms selected from nitrogen, oxygen and sulfur. It preferably has 5 or 6 ring-constituting atoms, and preferably up to 3 hetero atoms. The cycloalkylalkyl group is preferably a cyclohexylalkyl group; the alkylene moiety preferably has from 1 to 4 carbon atoms. Halogen II, gas, desert or block, preferably chlorine or desert. The alkyl group and the alkoxy group preferably have 1 to 4 carbon atoms, more preferably 1 or 2 carbon atoms, still more preferably a mercapto group or a decyloxy group. The hydroxyalkoxy group is preferably an ω-hydroxyalkoxy group having 2 to 4 carbon atoms, and particularly preferably a 2-hydroxyethoxy group. 131613.doc 200911835 Salt is an acid addition salt such as hydrochloride. The compounds of formula I have several pairs of palmar centers and can therefore exist in a variety of stereoisomers. Unless otherwise stated, the invention provides all stereoisomers as well as racemic mixtures. The isomer can be resolved or separated by conventional techniques such as stratification. It is seen from the formula I that the carbon atom at the 2-position has a configuration of R and at the 3 and 4 positions is S. 1 preferably hydrogen, acridinyl alkoxycarbonyl, naphthyl alkoxycarbonyl, naphthylalkylcarbonyl, benzyl-CH(OH)-carbonyl, phenoxymethylcarbonyl, phenylalkylcarbonyl or An amino protecting group such as a third butoxycarbonyl group, a cycloalkylalkoxycarbonyl group (preferably cyclohexylmethoxycarbonyl group) or a benzyloxycarbonyl group which is unsubstituted or substituted with an alkyl group or an amine group; More preferably, it is a naphthyl methoxycarbonyl group, a naphthylmethylcarbonyl group, a pyridyl methoxycarbonyl group, a phenyl propyl fluorenyl group, an aminophenyl phenyl fluorenyl group, a tert-butoxycarbonyl group, an amine benzyl group. Alkoxycarbonyl group, alkylbenzyloxycarbonyl group, dialkylbenzyloxycarbonyl group or benzyloxycarbonyl group, even more preferably a benzyloxy group. When A is an unsubstituted or substituted aminomercapto moiety, it is preferably an unsubstituted or substituted a-aminoindenyl moiety, such as alanine, leucine, isoleucine, day Winter amide, valine, t-butylglycine, third leucine or histidine. It is preferably a protected or unprotected portion of the native alpha-amino acid, preferably a protected or unprotected portion of the amino acid of the normal constituent of the protein, or a third leucine. It preferably has an L configuration. It is preferably glycine, L-proline, L-third leucine or a bond, and even more preferably L-third leucine. R2 is preferably a side chain of a natural amino acid, preferably a side chain of an α-amino acid, and 131613.doc-72-200911835 is preferably a side chain of an amino acid which is a normal constituent of the protein. It is, for example, isopropyl, aminocarbonylmethyl, methyl, 1-mercaptopropyl, benzyl, 4-hydroxybenzyl or isobutyl, preferably benzyl. When B is an unsubstituted or substituted amino fluorenyl moiety, it is preferably an unsubstituted or substituted a-amino fluorenyl moiety, such as phenylalanine, amidinoic acid, leucine, a heterologous white Amine acid, alanine or aspartame. Preferably, the substituted or unsubstituted portion of the natural a-amino acid is preferably a substituted or unsubstituted portion of the amino acid which is a normal constituent of the protein. The α-amino acid having the first carboxyl group (e.g., glutamic acid) is preferably esterified with a C|_C3 alcohol (or preferably decyl alcohol). It preferably has an L_ configuration. B is preferably L-proline, L- face amine decyl or a bond, and even more preferably L-proline. R3 is preferably halogen, fluorenyl or decyloxy, and particularly preferably methoxy. R4 is preferably an unsubstituted or substituted 2(R)-hydroxyindoline-l(S)-yl or 2-hydroxybenzyl group as defined above, and more preferably 2-hydroxyl-4-methoxy Base - benzyl. The soil γ is preferably -CO- or -O-CO-, and more preferably -〇_c〇_. Preferably, the unsubstituted or substituted alkyl, arylalkyl or heteroarylalkyl group is preferably an alkyl group; when it is an unsubstituted or substituted heteroaryl burnt soil, it is preferably each 0 ratio. a base group, particularly preferably a 2 - ° ratio of stildenylmethyl; when it is an unsubstituted or substituted arylalkyl group, it is preferably a benzyl group; when it is a substituted alkyl group, It is preferably a phenoxymethyl group. In some embodiments, the proteasome inhibitor is 2-amino-3_hydroxy-4_first whitefee-amino-amino-5-phenyl-pentanoic acid amine derivative. See, for example, 131613.doc -73- 200911835, for example, PCT 01/89282. For example, in some embodiments, the present invention is directed to a compound of formula XV.

Where η is. Or 1;...2 is independent of the other side as a fat: base, or aryl:: fatty ΓΓ::, _, alicyclic-aliphatic, heterocyclic or heterocyclic-monthly aliphatic group's bases are not much At 20 carbon atoms; R3 is hydrogen, oxaalkyl, aliphatic or a group of formula (YVR6 having up to 2G carbon atoms, wherein Y is a group having 5 or 6 ring members Up to three unsubstituted or substituted monocyclic groups selected from heteroatoms consisting of nitrogen, oxygen and sulfur, wherein the monocyclic group may also be slightly synthesized into a benzo ring; RAR5 is independently selected from the group consisting of Group of constituents: A; aliphatic group; free, etherified or esterified hydroxyl group; free or esterified carboxyl group; mercapto group; alkanol; unsubstituted, mono- or di-substituted amine group; mercapto group; ^; 2 thio; amine fluorenyl; N-alkyl-amine carbaryl; N, N_: _alkyl-amine fluorenyl; cyano and nitro; a carbon atom having at most ,, if the conditions are: ^, i, &amp; and Rs are not all hydrogen, &amp; benzyl or tert-butyl, R2 benzyl or 4- methoxy-benzyl, R3 is isopropyl and lanthanide oxygen and if η is 0 or 1, R4 is not 曱The base 2 is 4-methoxy-benzyl, which is hydrogen and X-based oxygen; and X is nitrogen, oxygen or sulfur; or a salt thereof. 131613.doc -74- 200911835 in 2-amino-3- In the case of a hydroxy-4_third-leucine-indenyl-amino-5-phenyl-pentanoic acid amine derivative, the general terms used above and below preferably have the following meanings: η-system 〇 or 1, Preferably, the aliphatic group has up to 12 carbon atoms, preferably up to 7 carbon atoms, optimally up to 4 carbon atoms, and is an unsubstituted or substituted aliphatic hydrocarbon group, in other words, one. The substituted or substituted alkynyl, dilute or (preferably) alkyl group's one or more substituents are preferably independently selected from the group consisting of: free, shouted or esterified hydroxyl groups; free or esterified carboxyl groups; Mercapto group; alcohol; unsubstituted, mono- or di-substituted amine group; fluorenyl group; fluorenyl group; sulphate base; sulphur-sodium group; amine carbaryl group; Ν-alkyl-amine fluorenyl group; Ν, Ν_di-alkyl-amine hydrazino; cyano group and nitro group. The aliphatic group R, preferably a lower alkyl group, for example, a tributyl group is preferred. The aliphatic group R3 is preferably Unsubstituted low carbon number a lower alkyl group substituted with a hydroxyl group, a carboxyl group, an amine group, an amine methyl sulfonyl group, a decyl group, a decyl group or an alkylthio group, preferably an amino acid alanine, leucine, isoleucine, or silk a side chain of an amine, a sulphate, a cysteine, a sulphate, an aspartame, a glutamine, an aspartate, a glutamate, an lysine or a arginine, especially a valeric acid side chain. The aliphatic group &amp; preferably is a methoxy group. The aromatic group core or 1 has no more than 20 carbon atoms, especially no more than 12 carbon atoms, and is unsubstituted or Substituted, preferably in each case an unsubstituted or substituted phenyl or naphthyl group, particularly preferably a fluorenyl-naphthyl group, preferably one or more substituents independently of the group consisting of: aliphatic groups Free, etherified or esterified hydroxyl group; free or esterified carboxyl group; formyl group; alkanol; unsubstituted, mono- or mono-substituted amine group; mercapto group; sulfo group; alkylthio group; Base; N_131613.doc -75- 200911835 alkyl-amine-mercapto; N,N-di-alkyl-amine-methylcarbonyl; cyano and nitro, more preferably selected from alkyl, for example, methyl, Ethyl or propyl; Alkoxy, e.g., methoxy or ethoxy; by two - the substituted amine, e.g., di-Yue-yl group; a halogen, e.g., chlorine or bromine; and a halogenated alkyl group, e.g., methyl three gas. -

In an aromatic-aliphatic core or core having no more than 20 carbon atoms, the aromatic moiety is as defined above and the aliphatic moiety is preferably a lower carbon number alkyl group, such as, in particular, a CrC2 alkyl group, Preferably, it is substituted or preferably unsubstituted as defined for the aromatic group. The aromatic-aliphatic core is preferably a benzyl group or a naphthalene-1-ylindenyl group. The aromatic-aliphatic group R2 is preferably a phenyl moiety substituted by 丨5, preferably 1-3 oxiranyl; the benzene moiety (preferably at the * position) is substituted with a dimethyl-amino group Base group; or naphthalene-丨-ylmethyl. Most preferably, the aromatic-aliphatic R.2 is 2,3,4- or 3,4,5-trimethoxy-benzyl. The alicyclic base or caliper 2 has up to 20, particularly preferably up to 1 carbon atoms, which are monocyclic or polycyclic and preferably have a silk substitution or preferably unsubstituted, such as a ring. The alkyl group is, for example, preferably a 5- or 6-membered cycloalkyl group, for example, preferably a cyclohexyl group. In the alicyclic aliphatic group having not more than 20 carbon atoms, the 'alicyclic moiety is as defined above and the aliphatic (four) moiety is preferably a low carbon (tetra) group, for example, preferably a Cl_C group. Preferably, it is substituted or preferably unsubstituted as defined for the aromatic group, such as cyclohexyl-methyl. The heterocyclic group contains up to 2 carbon atoms, usually up to 12 carbon atoms, and is preferably substituted or unsubstituted as defined by the aromatic group, and preferably has 5 or 6 ring members and i Up to three saturated or unsaturated monocyclic groups (for example, thienyl or acridinyl), or two or three, preferably selected from the group consisting of nitrogen, oxygen: 131613.doc • 76· 200911835 sulfur a cyclic group wherein, for example, a phenyl group is condensed to a monocyclic group as mentioned, especially, for example, a fluorenyl group such as a 5-nonyl group or a quinolyl group such as an 8-quinolyl group. : in a heterocyclic ring-aliphatic group 1 or Han 2 having not more than 20 carbon atoms; the heterocyclic moiety is as defined above and the aliphatic moiety is preferably a lower alkyl group. For example, Ci-C2 alkyl, preferably ρ substituted or preferably unsubstituted as defined for the aromatic group. Heterocyclic-aliphatic base or stalk 2 (for example) D-xyl-methyl (preferably 5-nonyl-methyl) or quinolinyl fluorenyl (preferably 8 quinolinyl-A) base). An oxa-alkyl group of the formula -CKO-CHrCHA-R7, wherein G&amp;R7 is an alkyl group, preferably a lower alkyl group, and 1 is 丨 to 3, preferably 2, and particularly preferably 2-(1,4-Dioxa-hexyl)-ethyl. In the formula of the formula -(Y)m_R6 having up to 20 carbon atoms, a γ-based alkyl group, preferably a lower alkyl group, a quinone oxime or i and a group I having 5 or 6 ring members and a plurality of three or more saturated or unsaturated monocyclic groups selected from the group consisting of nitrogen, oxygen and sulfur, or containing a fused benzo ring, preferably substituted or as defined for the aromatic group Good is not replaced. The group I is preferably bonded to γ via a ring carbon atom and is, for example, an unsubstituted or substituted member selected from the group consisting of cyclopentyl, cyclohexylcyclopentanyl, phenyl, 吼σ Each bite base'. ratio. Sitting on the base, _. The thiol group, tetrahydrofuranyl group, hexahydropyridyl group, hexahydropyrazinyl group, morpholinyl group, pyridinium group, succinyl group, glutinous rice sulphonyl group, tertylene group, and trexyl group. More than hydrazine, η is 嗓 、, σ 秦 秦, pyrimidinyl, benzyl, naphthyl, anthracenyl and quinolyl. 131613.doc -77- 200911835 Finally, the formula _(Y)m_R6 is hexahydropyridinyl (preferably 4-hexahydro.pyridyl)' /, hydropyrazine-ethyl (or better) Is hexahydro D-pyrazine-丨-ylethyl); morpholinyl-ethyl (preferably morpholin-4-ylethyl); pyridyl-methyl (eg 2, 3 or 4 pyridine) - mercapto) or amino acid phenylalanine, tyrosine, tryptophan or a group of amino acids. X is preferably oxygen. The alkyl group is preferably a lower alkyl group. The prefix, low carbon number, means a group having up to and including - 7, preferably up to and including 4 carbon atoms. Lower alkyl ('e.g., n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, first butyl, n-pentyl, isopentyl, neopentyl, hexyl Or n-heptyl, preferably isobutyl, t-butyl, tert-butyl, isopropyl, ethyl or methyl' is optimally isobutyl 'ethyl or decyl. (for example) an alkoxy group, particularly preferably a lower alkoxy group such as an ethoxy group or a methoxy group. The esterified hydroxyl group is preferably an organic carboxylic acid (for example, an alkanoic acid) or a mineral acid (for example, a hydrogen-acid a hydroxyl group esterified, such as a lower alkyl alkoxy group or a particularly preferred halogen (for example, iodine or especially fluorine, chlorine or bromine). V a condensed slow base such as an alkoxycarbonyl group, particularly preferably a low carbon number Alkoxycarbonyl 'e.g. methoxycarbonyl. Alkanol is, for example, alkylcarbonyl, especially preferably lower alkylcarbonyl, such as ethyl-indenyl. I mono- or di-substituted amine system (eg N-alkylamino or N,N-dialkylamino 'Ultra N_lower alkylamino or lower carbon N,N-di-low carbon alkylamino' such as N- Mercaptoamine or ν,Ν-didecylamino. Fluorine, gas, bromine or iodine&apos; is preferably fluorine, gas or bromine. The structure of formula XV as shown above represents the absolute configuration. 131613.doc -78- 200911835

The salt forming group in the compound of the formula xv is a group or group having a basic or acidic character. A compound having at least one basic group or at least one basic group (for example, a free amine group, a pyrazinyl group or a pyridyl group) can form an acid addition salt, for example, with a mineral acid such as hydrochloric acid, sulfuric acid or acid, or Suitable organic carboxylic acids or acid anhydrides, such as aliphatic mono- or di-acids, such as trifluoroacetic acid, acetic acid, propionic acid, trans-acetic acid, succinic acid, maleic acid, fumaric acid, kiramic acid , malic acid, tartaric acid, citric acid or oxalic acid; or an amino acid such as arginine or lysine; an aromatic carboxylic acid such as benzoic acid, 2-phenoxy-benzoic acid, 2-ethyloxine Benzo-benzoic acid, salicylic acid, 4-aminosalicylic acid; aromatic aliphatic carboxylic acid, such as mandelic acid or cinnamic acid; heteroaromatic acid, such as niacin or isonicotinic acid; aliphatic sulfonate An acid such as tomazan _, ethene _ or 2 - light basal acid; or an aromatic acid such as benzene, p-toluene-bite 2-sulfonic acid. When a plurality of basic groups are present, a mono- or poly-acid addition switch can be formed which has an acidic group (for example, a free carboxyl group in the base Rio). The compound of the formula ν can form a metal or ammonium salt, such as an alkali metal or an alkaline earth. a metal salt (for example a sodium, potassium, magnesium or calcium salt) or with ammonia or a suitable organic amine (for example a tertiary monoamine such as triethylamine or tris-(2-hydroxyethyl)-amine or a heterocyclic base, for example An ammonium salt formed by ethyl·hexahydropyridine or ν,π-dimethylhexahydropyrazine. The compound of the formula ’, which has both 'ϋ and a basic group, can form an internal salt. A pharmaceutically unacceptable salt may also be used for the purpose of leaving or sealing the body, and for further use of the compound as a middle door. θ However, only pharmaceutically acceptable non-toxic salts are useful for therapeutic purposes, and thus their salts are preferred. 1 because 131613.doc -79- 200911835 in view of the close relationship between the free forms of the novel compounds and their salts, including the salts which they can be used as intermediates, for example in the purification or identification of such novel compounds, Any reference to such free compounds above and below is to be understood to include the corresponding salt, if appropriate and convenient. 4. Administration and pharmaceutical compositions

The antibodies and agents of the invention can be administered directly to a mammalian subject for the treatment of, for example, hyperproliferative disorders including cancer, such as, but not limited to, carcinoma, glioma, mesothelioma, melanoma, lymphoma , leukemia, adenocarcinoma... breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer and Burkitu lymphoma (Burkiu, s yymphoma), head and neck cancer, colon cancer, colorectal cancer, non- Small cell lung cancer small cell lung cancer, appetite cancer, gastric cancer, adenocarcinoma, hepatobiliary carcinoma, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer , vaginal cancer, uterine cancer, Indian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, septic endocrine cancer, carcinoid, bone cancer, skin cancer, retinoblastoma, multiple myeloma, He Jiejin Lymphoma (the brain's lymph surface) and non-Hodgkin's lymphoma surface _ H〇dgkinis lymph0 brew (see other cancers, cancer: principles and PRACTICE (DeVita, ν τ et al., Μ)). Mammalian individuals mean humans and non-human primates and include agricultural mammals (eg 'family snails, snails, snails, sheep, and porcines, etc.) and residual or Other mammals with low mammals (eg, canines and I seedlings) and experimental mammals (eg, rodents including rabbits, rats, hamsters, and mice) 131613.doc •80- 200911835 The administration of the compositions of the present invention is any route that is commonly used to introduce a chemotherapeutic person to make final contact with the tissue to be treated. Such anti-limb agents (as appropriate) and pharmaceutically acceptable The carrier is administered in any suitable manner. Suitable methods of administering such antibodies and agents can be utilized and such methods are well known to those skilled in the art' and although more than one route can be used to administer a particular combination 'but a specific way to provide a more direct and more efficient response than the other way.

The portion of the carrier that is pharmaceutically acceptable for administration is determined by the particular composition being administered and by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions of the present invention (see, for example, ... such as Sighing Heart Pkr Hall (10) &quot; W Coffee, 21st Edition, (5)

Sciences in Philadelphia (USIP), Lippincott. Williams &amp; Wilkins (2005)). The antibodies and agents of the invention (alone or in combination with other suitable components) can be formulated into aerosol formulations (i.e., they can be "foamed") for administration via inhalation. The aerosol formulation can be placed in a pressurized acceptable propellant (e.g., difluoromethane, propyl, nitrogen, and the like). Suitable formulations include aqueous and non-aqueous solutions, isotonic sterile solutions (which may contain antioxidants, buffers, bacteriostats, and isotonic) and aqueous and non-aqueous sterility Suspensions (which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives). In the practice of the present invention, the composition can be administered as follows, for example, orally and parenterally, including topical, transdermal, transmucosal, intravenous, subcutaneous, intratumoral, intramuscular, intraperitoneal, intravesical or Intrathecal. These compositions are administered nasally or by inhalation 131613.doc -81- 200911835, as appropriate. These compound formulations may be present in a unit dose of 5| φ, and the 丨i a piece of 丨 役 役 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷 谷Solutions and suspensions may be prepared from sterile powders, granules and lozenges of the type described. The conditioning agent can also be administered in part as a food or drug. The present invention is also effective in combination with one or more additional active agents (e.g., chemotherapeutic agents). The pharmaceutical composition of the present invention comprises a composition wherein the active ingredient is contained in a therapeutically effective amount. Of course, the amount of composition administered will depend on the individual being treated, the weight of the individual, the severity of the pain, the mode of administration, and the judgment of the physician. The effective amount is well known to those skilled in the art and can be determined in particular from the detailed disclosure provided herein. Generally, a qualitative or effective amount of one or more anti-DR5 antibodies is determined by first administering a low dose or a small amount of an anti-DR5 antibody and then increasing the dose (dose or dose) and/or It is desirable to add an apoptotic agent until the desired effect of inducing apoptosis in the target cell population is observed in the treated individual with minimal or no toxic side effects. Suitable methods for determining the appropriate dosage and dosage schedule for administration of the pharmaceutical compositions of the present invention are set forth in (You, Ru, Goodman and Gilman's The Pharmacological Basis of, nth edition, edited by Brunt〇n et al., McGraw-Hill ( 2006, anti-Remington: The Science and Practice of Pharmacy, supra, 'both of which are incorporated herein by reference. In an embodiment wherein the agent is a polypeptide or antibody, a typical dose may be between about 0.1 pg/kg The body weight is up to and including about i gm / kg body weight, preferably between about 1 pg / kg body weight to about 500 mg / kg body weight. More preferably 131613.doc -82 - 200911835 ground, about 1, 2, 3 , 4, 5, 10, 20, 30, 40, 50, 6〇, 7〇80' 90 or 100 mg/kg body weight. In the embodiment in which the reagent is a nucleic acid, the typical dose may be between about 〇·1 The mg/kg body weight is 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.

In embodiments wherein the agent is a small organic compound, typical dosages may range from about 0.1 pg/kg body weight up to and including about 1 gm/kg body weight, preferably from about 1 pg/kg body weight to about 500 mg. /kg body weight. More preferably, about 0.1, 1, 2, 3, 4, 5, 10, 20, 30, 40, 5, 60, 70, 80, 90 or 100 mg/kg body weight. The exact dose will depend on a number of factors discussed above, including the specific antibody or apoptosis inducing agent, the severity of the disease, and the route of administration. The determination of a therapeutically effective dose can be determined by the clinician without undue experimentation and can include any of the dosages included within the scope disclosed above. The antibody agonist and the cell apoptosis inducer may be administered together in a mixture or may be administered separately. The antibody reagent and apoptosis induction-, (but not required) simultaneous administration. The following examples are provided to illustrate, but not limit, the claimed invention Example 1: The following examples provide a method for the construction and screening of μ r with minimal immunogenic frog DR5 antibody in humans. 131613.doc -83 - 200911835 Sub-selection of murine v_regions from plasmids PBW212 and PBW214. The murine v-region sequence is described in U.S. Patent Publication No. 2/5/79i72. PCR is used to amplify the v-gene of the V-heavy chain and the ν-κ region and to incorporate restriction enzyme sites suitable for selection into expression vectors. The v_ region was cloned in a pBR322 vector system with a Fab&apos; fragment having a human IgGl constant region. From the PTAC promoter in E. coli (five (3)/〇. The purified Fab is shown in the EUSA* assay, and the protein (pure line DR5_1} binds to the DR5 antigen. The nucleotide sequence optimization of these V-regions A Fab fragment that results in a human amino acid sequence in the framework 3 portion adjacent to CDR3. This allows for subsequent colonization of the human V-segment. The optimized reference Fab also has a cdr3 BSD sequence and The human;_chain/frame 4 sequences provided by the JK2 and JH4 lines. These optimized Fabs were tested against DR5_antigen binding and referred to as the reference sequences DR9-1 and DR10-1.

Fab' purification

Fab fragments are expressed by expression vectors derived from secretions of E. coli. The cells were grown in 2xYT medium so that the optical density (〇D6〇〇) measured at a wavelength of 60 〇 nm was 0.6. The use of isopropyl-β-D-galactose sulfopyranoside (IPTG) will be demonstrated at 33. (: induction for 3 hours. The assembled Fab' was obtained from the cytoplasmic fraction and purified by affinity chromatography using streptococcal peptone (HiTrap protein G HP column; GE Healthcare) according to standard methods. Fab' at pH It was dissolved in 2.0 buffer, immediately adjusted to pH 7.0 and dialyzed against PBS pH 7.4 (PBS without calcium and magnesium).

The ELISA is generally at 4th. (: Incubate overnight to bind 100 ng/well of DR5 antigen 131613.doc -84- 200911835 to a 96-well microtiter plate. Under 33, use 5% milk in a solution containing 〇·1% Tween20 ("PBST") The plate was blocked for 1 hour with a solution in phosphate buffered saline. The purified Fab, or reference Fab, (1) was diluted in PBS and 5 μl was added to each well. It lies in 33. After incubation for a few hours under the armpit, the plate was rinsed three times with PBST. 5 μl of anti-human-κ chain horseradish peroxidase (HRp) conjugate (Sigma; diluted to 0.1 ng/ml in pBsT) was added to each well and the plate was incubated at 33 for 3 min. . The plate was washed three times with PBST and once with PBS. I〇〇y 3,3',5,5,-tetramethylbenzidine (7^^ receptor (34〇^) was added to each well and the plate was incubated at room temperature for about 5 min. The reaction was stopped and 丨〇〇μ1 〇2 N HjO4 was added to each well. The plate was read at 45 〇 11111 in a spectrophotometer.

The screening of the Fab' fragment library was carried out using a nitrocellulose-coated granule coated with a recombinant DR5 antigen as described in U.S. Patent Publication Nos. 2005/0255552 and 2006/0134098. The disclosures of both of these patent publications are hereby incorporated by reference in their entirety for all purposes for all purposes. Affinity test

The binding kinetics of the Fab' fragment was analyzed using a porteBi(R) 〇ctet biosensor. The recombinant antigen was biotinylated using the EZ-link biotinylated kit (Pierce) according to the manufacturer's protocol. The antigen was then bound to a sensor coated with Neutravidin (ForteBio). The biofilm interference measurement analysis and the software provided by the manufacturer are used to monitor the Fab in real time. Affinity was calculated from the association and dissociation constants determined. Results 131613.doc -85- 200911835 Selection and performance of V-region EL1SA confirmed the DR5 antigen-binding activity of the sub-selected V-region. Colonization, sequencing and performance of the V-region of the murine. The V_ region was cloned with a Fab' fragment having a human IgG1 constant region and expressed in E. coli. In the dilution ELISA test for DR5 antigen binding, the selected Fab' and the original Mab NVP-LCR21 1 produced a binding curve depending on the antibody concentration. - Mab was found to have a stronger binding signal than Fab'; this may be due to differences in the avidity of the antibody and the top ELISA assay. See Figure O i. Murine v-region amino acid sequence

Fab' DR5-1 has a complete murine V-region, while the reference Fab' DR9-1 and DR 1 0-1 are optimized for subsequent library construction. Nucleotide changes made within the framework 3 portion adjacent to the CDR3 on both the heavy and light chains allow subsequent construction of the library and result in the production of human germline amino acid sequences. Both DR9-1 and DR10-1 have the heavy chain/CDR3 BSD of the sequence HEEGI (SEQ ID NO: 49) and the human germline J-segment sequence (JH4). DR9-1 has a light chain CDR3 sequence comprising BSD of QQHYTTP (SEQ ID NO: 57) and a framework 4 sequence conferred by human JK2. DR10-1 has a light chain BSD sequence · QQHYTTP (SEQ ID NO: 57) and human germline J-segment (human JK2). See Figure 3. Both of the optimized reference Fab1 DR9-1 and DR10-1 were found to have DR5 antigen binding activity. See Figure 4. Library Construction and V-boxes A pool of epitopes was constructed from a human V-segment sequence library linked to the unique CDR3-FR4 region of the reference Fab, DR9-1. These "full-length libraries are used as the basis for the 131613.doc -86-200911835: two-box" library, in which only a portion of the murine v-segment was initially replaced by a column library. The cassettes of both the V-rekey and the V-K bond are made by bridge PCR with overlapping common sequences within the framework 2/. Construct a V-heavy chain in this way! And 3 and V_K!, job IV|5] type of "front" &quot; &&quot; medium spine: class box library. Human cassettes that support binding to the trace antigen are identified by colony analysis and analyzed by affinity in ELISA and Forte 〇ctet biosensor assays. The episode of the highest affinity cassette was then recombined into the =screen to fully generate the human V. segment. In this box, a box of A, ‘not to have a plurality of human v-type V-heavy chains with DR5_antigen binding activity&quot; front end, box. In alternative pathways, the key and human construct a mutant library in which each residue in the cDR2 is mutated into a murine sequence or from a single human germline sequence (the _46 line with the identified &quot;front end&quot; Corresponding residues of the closest human germline sequence (see, Figure 5). The mutant library is ligated to the selected discovery support antigen, the front end &quot; and human framework 3 (FR3) sequences. All members of the library have a common CDR3/FR4 sequence of the DR9-1 reference Fab. Therefore, the designed human "intermediate" box library was established, and the colony transfer binding analysis was used to screen and select the antigen-binding pure line and further analysis by EUSA&amp;F〇rte 〇ctet. In this way, more CDR sequences that support DR5 antigen binding to human germline amino acid sequences (see Figure 6). EUS octa binding activity is shown in Figure 7. After identifying high-affinity Fabs close to human germline sequences, set up, Affinity maturity library. A pool of optimized Fab's pure lineage CDR3 sequences was mutated to generate a library using the merging. The mutated library was screened using colony transfer junction 131613.doc-87.200911835. And Forte analysis ranks the selected Fab' for affinity. Identify mutations that support equal or improved affinity for the antigen compared to the DR9-1 reference Fab'. See Figure 8. As can be seen in Figure 9, ELIS A Analysis showed that the selected Fab' has binding activity to the DR5-antigen. The affinity of FabU human DR5 antigen analyzed by Forte Octet was analyzed by colony transfer binding assay and ELISA self-boxing and mutant sieving The isolated fully optimized Fab' was further characterized by comparison with the kinetics of the reference Fab' DR9-1. The binding kinetics were analyzed by real-time label-free monitoring of protein-protein interactions using the ForteBio Octet system. Kinetic analysis is shown in Figure 10. The calculated association and dissociation and total affinity constants are shown in Table 1. Table 1 Antibody Reference DR9-1 DR106-2 DR114-1 DR112-1 Molar Concentration (M) IE- 7 IE-7 IE-7 7.5E-8 Dissociation rate constant (kd[l/s]) 9.63E-5 8.52E-5 1.24E-4 2.25E-4 kd error 2.99E-6 2.63E-6 2.63E -6 4.54E-6 Association rate constant (ka[l/Ms]) 1.39E5 9.21E4 1.38E5 1.41E5 equilibrium dissociation constant (KD [Μ]) 6.91E-10 9.25E-10 9.02E-10 1.59E- 9 Table 1 shows Fab' binding analysis with recombinant DR5 antigen by biolayer interference measurement using ForteBio Octet biosensor technology, showing association rate constant (ka), dissociation rate constant (kd), and calculated affinity ( Kinetic analysis of modified pure lines DR106-2, DR1 14-1, DR1 12-1 and reference pure line DR9-1 showed that all had low to sub-nan 131131 for DR5 antigen .doc -88- 200911835 Moer's affinity. Figure 11 shows an amino acid sequence alignment of the optimized Fab' DR 106, DR112 and DRll4iV_ region sequences compared to human germline sequences. The percent identity of the reference and the most deuterated V-region sequence to the amino acid sequence of the corresponding human germline amino acid sequence is shown in Table 2. Table 2

All percent sequence identity in Table 2 represents the identity to a single human germline sequence spanning the v_region and excludes CDR3 BSD sequences. can watch

Out, the v_ regions of all two modified Fabs are extremely close to the corresponding human germline sequence, and the amino acid sequence identity percentage is about 9 〇〇/〇. The foregoing invention has been described in considerable detail by way of illustration and example of the embodiments of the present invention, and it will be apparent to those skilled in the <RTIgt; Certain modifications and variations of the present invention are possible in the context of the scope. All publications, databases, GenBank sequences, patents, and patents cited in the present specification are hereby incorporated by reference in their entirety herein in their entirety in the extent of the disclosure of the disclosure. 131613.doc -89- 200911835 [Simple description of the diagram] Figure 1 shows the sub-selection V-region (DR5-1) (·) of the Fab' form and the original NVP-LCR21 1 Mab (▲) versus thio -Dilute ELISA comparison of DR5 antigen binding. No significant binding signal (▼) was observed in the PBS control samples. Figure 2 depicts a schematic representation of a structural unit comprising an antibody V-region. The heavy chain is encoded by three gene families (heavy-V, D and J) and the light chain is encoded by two gene families (κ or λ V and J). Recombination of these genes results in the production of a complete V-region. In the case of heavy chain, the CDR3 sequence is located at the recombination sites of the heavy V, D and J genes, and in the case of the light chain at the recombination sites of the kappa or lambda V and J genes. Figure 3 depicts the V-region amino acid sequence alignment of the partial framework 3 region (FR3) and the complete CDR3 and framework 4 region (FR4) of the murine animal and optimized reference Fab' (SEQ ID NOS: 76) -78 and 80-82). Residues that have changed from the original murine sequence are highlighted in red and underlined. The CDR3 BSD residues are highlighted in bold and the CDR3 region is in the box. Human germline J-segments JH4 (SEQ ID NO: 75) and JK2 (SEQ ID NO: 79) were also aligned. Figure 4 depicts the dilution of the sub-selected V-region DR5-1 () with the optimized reference control DR9-1 (▲) and DR10-1 (▼) for thio-DR5 antigen binding in Fab' form. ELISA comparison. Figure 5 depicts a V-heavy chain CDR2 mutant library comprising a reference (SEQ ID NO: 4) or human germline (VH1-46) (SEQ ID NO: 107) amino acid at each position. The common amino acid between the reference and the human remains constant and additional amino acids may be present at both positions due to the use of degenerate codons. Figure 6 depicts the V-heavy chain selected from the Fab&apos; library according to DR5 antigen binding activity 131613.doc-90-200911835 CDR2 sequence 歹 ij (SEQ ID NOS: 6, 4 and 83-91, respectively). Mutations relative to human germline sequences are underlined. Figure 7 depicts selected V-heavy chain CDR2 mutants that bind to the DR5 antigen in a dilution ELISA. The specific binding activity can be seen by comparison with the reference control and the pure line (DR83-D3) which is negative for antigen binding. Figure 8 depicts the V-light chain CDR3 sequence (8A) (SEQ ID NOS: 19, 66, 20 and 67-72) and V-heavy chain CDR3 sequences selected from the CDR3 affinity mature library based on the sequence of DR5-antigen binding activity. (8B) (SEQ ID NOS: 7, 8 and 54-56). Amino acids different from the reference CDR3 sequence are shaded in blue and underlined. Figure 9 depicts the DR5-antigen binding activity of selected CDR3 affinity matured mutants. ELISA analysis showed significant antigen binding of the selected pure lines compared to the negative control Fab&apos; (background). Figure 10 depicts the kinetic analysis of Fab&apos; or selected fully optimized Fab&apos; binding to recombinant DR5 antigen by biolayer interference measurement using the ForteBio Octet biosensor technology. Figure 11 shows an alignment of modified Fab' DR106-2 (SEQ ID NOS: 39 and 40), DR112 (SEQ ID NOS: 39 and 41) and DR114 (SEQ ID NOS: 39 and 42) amino acid sequences. V-region amino acid sequence and closest to 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 a comparison of VK1-L4/JK2 (SEQ ID NO: 47)). Residues of the CDRs within the box and corresponding to the corresponding positions in the germline sequence (excluding the CDR3 &quot;BSD" sequence are shaded in red. Affinity maturity changes of CDR3 are marked with blue shading. 131613.doc -91 - 200911835 Sequence Listing &lt;110&gt;Aim&lt;120&gt; Method and composition for inducing apoptosis of cancer cell cells &lt;130&gt; P1281 &lt;140&gt; TW 097121278 &lt;141&gt; 2008-06-06 &lt;150&gt; 60/ 942,862 &lt;151&gt; 2007-06-08 &lt;160&gt; 107 &lt;170&gt; Patentln version 3.3 &lt;210&gt; 1 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400〉 1

Asp Tyr Thr lie His &lt;210&gt; 2 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Ser Tyr Thr Met His &lt;210&gt; 3 &lt;211> 5 &lt;212&gt; PRT &lt;213&gt; Homo sapiens V, &lt;400&gt; 3

Ser Tyr Tyr Met His &lt;210&gt; 4 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Trp Phe Tyr Pro Gly Gly Gly Tyr He Lys Tyr Asn Glu Lys Phe Lys 15 10 15

Asp &lt;210&gt; 5 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence 200911835 &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Trp lie Tyr Pro Gly Gly Gly Tyr Thr Lys Tyr Ala Gin Lys Phe Lys 15 10 15

Gly &lt;210&gt; 6 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 6

He lie Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gin Lys Phe Gin 15 10 15

Gly &lt;210> 7 &lt;211> 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

His Glu Glu Gly lie Tyr Phe Asp Tyr &lt;210&gt; 8 &lt;211> 9 &lt;212> PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

His Glu Glu Gly lie Tyr Phe Asp Lys &lt;210> 9 &lt;211> 4 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Tyr Phe Asp Tyr &lt;210&gt; 10 &lt;211&gt;11 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Lys Ala Ser Gin Asp Val Asn Thr Ala lie Ala 1 5 10 -2- 200911835 &lt;210&gt; 11 &lt;211&gt; 17 &lt;212> PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Artificial Sequence Description: Synthetic peptide &lt;400&gt; 11

Lys Ser Ser Gin Ser Phe Leu Gly Ser Ser Asn Gly Lys Asn Tyr Val 15 10 15

Ala &lt;210&gt; 12 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Arg Ala Ser Gin Asp Val Ser Gly Ala Leu Ala 1 5 10 &lt;210> 13 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 13

Lys Ser Ser Gin Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu 15 10 15

Ala &lt;210&gt; 14 &lt;211> 11 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 14

Arg Ala Ser Gin Gly lie Ser Ser Ala Leu Ala 1 5 10 &lt;210&gt; 15 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;&gt;15

Trp Ala Ser Thr Arg His Thr &lt;210&gt; 16 &lt;211> 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide 200911835 &lt;400> 16

Trp Ala Ser Met Arg Val Ser &lt;210&gt; 17 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Trp Ala Ser Thr Arg Glu Ser &lt;210&gt; 18 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 18

Asp Ala Ser Ser Leu Glu Ser &lt;210> 19 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

20: lt; 211 &gt; 9 &lt

Gin Gin His Tyr lie Thr Pro Phe Thr &lt;210&gt; 21 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 21

Gin Gin Tyr Tyr Ser Thr Pro Tyr Thr &lt;210&gt; 22 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Gin Gin Phe Asn Ser Tyr Pro Phe Thr

&lt;210&gt; 23 &lt;211&gt; 30 &lt;212> PRT 200911835 ^久丄二&gt; y乂丄 &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 15 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 &lt;210&gt; 24 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 15 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 &lt;210&gt; 25 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met Gly &lt;210&gt; 26 &lt;211&gt; 32 &lt;212> PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt; 26

Arg Val Thr Met Thr Gly Asp Thr Ser Thr Ser Thr Val Tyr Met Glu 1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 &lt;210&gt; 27 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu 15 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 &lt;210&gt; 28 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt;Artificial Sequence&lt;220> 200911835 Description: Synthetic Peptide &lt;;400&gt; 28

Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 1 5 10 &lt;210&gt; 29 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt; 29

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Ala Ser Leu Gly 1 5 10 15

Glu Lys Ala Thr lie Asn Cys 20

&lt;210&gt; 30 &lt;211&gt; 23 &lt;212> PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt; 30

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Ala Ser Leu Gly 15 10 15

Glu Arg Ala Thr lie Asn Cys 20 &lt;210&gt; 31 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Asp lie Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

Asp Arg Val Thr He Thr Cys 20 &lt;210&gt; 32 &lt;211> 23 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 32

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 15 10 15

Glu Arg Ala Thr lie Asn Cys 20

&lt;210> 33 &lt;211&gt; 23 &lt;212&gt; PRT 200911835, H 曰 &lt;400> 33

Ala lie Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

Asp Arg Val Thr lie Thr Cys 20 &lt;210&gt; 34 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu lie Tyr 15 10 15

&lt;210&gt; 35 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 35

Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr 15 10 15 &lt;210&gt; 36 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Artificial Sequence Description: Synthetic peptide &lt;400&gt; 36

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 15 10 15

Leu Thr lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys 20 25 30 &lt;210&gt; 37 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400> 37

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 15 10 15

Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 &lt;210> 38 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Artificial Sequence Description: Synthetic peptide &lt;400〉 38

Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 10 10200911835 &lt;210&gt; 39 &lt;211&gt; 118 &lt;212&gt; PRT &lt;213&gt;Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic polypeptide &lt;400&gt; 39

Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 15 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30

Thr Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45

Gly Trp lie Tyr Pro Gly Gly Gly Tyr Thr Lys Tyr Ala Gin Lys Phe 50 55 60

Lys Gly Arg Val Thr Met Thr Gly Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg His Glu Glu Gly lie Tyr Phe Asp Lys Trp Gly Gin Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 &lt;210> 40 &lt;211&gt; 113 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic polypeptide &lt;400&gt; 40

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Ala Ser Leu Gly 15 10 15

Glu Lys Ala Thr lie Asn Cys Lys Ser Ser Gin Ser Phe Leu Gly Ser 20 25 30

Ser Asn Gly Lys Asn Tyr Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu lie Tyr Trp Ala Ser Met Arg Val Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 200911835 丄丄. Bcu u!n Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin 85 90 95

His Tyr lie Thr Pro Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu lie 100 105 110

Lys &lt;210&gt; 41 &lt;211&gt; 113 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic polypeptide &lt;400&gt;

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Ala Ser Leu Gly 15 10 15

Glu Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Ser Phe Leu Gly Ser 20 25 30

Ser Asn Gly Lys Asn Tyr Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu lie Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin 85 90 95

His Tyr lie Thr Pro Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu lie 100 105 110

Lys &lt;210&gt; 42 &lt;211&gt; 107 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic polypeptide &lt;400&gt;

Asp He Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp Val Ser Gly Ala 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu lie 35 40 45 -9- 200911835

Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Ala 65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin His Tyr lie Thr Pro Phe 85 90 95

Thr Phe Gly Gin Gly Gly Thr Lys Leu Glu lie Lys 100 105 &lt;210&gt; 43 &lt;211&gt; 113 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 15 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30

Tyr Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45

Gly lie lie Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gin Lys Phe 50 55 60

Gin Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser 100 105 110

Ser &lt;210&gt; 44 &lt;211&gt; 98 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 44

Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 15 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30

Tyr Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45

Gly lie lie Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gin Lys Phe 50 55 60 •10· 200911835

Gin Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg &lt;210&gt; 45 &lt;211&gt; 113 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 45

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 15 10 15

Glu Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Ser Val Leu Tyr Ser 20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu lie Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin 85 90 95

Tyr Tyr Ser Thr Pro Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu lie 100 105 110

Lys &lt;210&gt; 46 &lt;211&gt; 94 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 15 10 15

Glu Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Ser Val Leu Tyr Ser 20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu lie Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr -π - 200911835 70 75 80 lie Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys 85 90 &lt;210&gt; 47 &lt;211&gt; 107 &lt ;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400〉 47

Ala lie Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Gly lie Ser Ser Ala 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu He 35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Phe Asn Ser Tyr Pro Phe 85 90 95

Thr Phe Gly Gin Gly Gly Thr Lys Leu Glu He Lys 100 105 &lt;210&gt; 48 &lt;211&gt; 88 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Ala lie Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Gly lie Ser Ser Ala 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys &lt;210&gt; 49 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Artificial sequence-12-200911835 &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt; 49

&lt;211&gt (2) &lt Acid &lt;400&gt; 50

Gin Xaa His Xaa Xaa Thr Pro &lt;210&gt; 51 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;220&gt;&lt;221 〉 MOD RES &lt;222&gt; (8)..(8) &lt;223&gt; Asp, ihr or Lys &lt;220&gt;&lt;221&gt; MOD.RES &lt;222&gt; (9)..(9) &lt;223&gt ; Tyr, Lys or Val &lt;400&gt; 51

His Glu Glu Gly lie Tyr Phe Xaa Xaa &lt;210&gt; 52 &lt;211&gt; 7 &lt;212> PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;220&gt;&lt;;221&gt; M0D_RES &lt;222&gt; (2)..(2) &lt;223&gt; Gin or His &lt;220&gt;&lt;221> MOD-RES &lt;222&gt; (4)..(4) &lt;223&gt; Tyr, Leu or Lys 200911835 &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (5)..(5) &lt;223&gt; Thr, Gin, lie, Glu, His or Gly &lt;400&gt;

Gin Xaa His Xaa Xaa Thr Pro &lt;210&gt; 53 &lt;211> 9 &lt;212> PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;220&gt;&lt;221&gt; M0DJRES &lt;222&gt; (2)..(2) &lt;223> Gin or His &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; (4)..(4) &lt;223&gt; Tyr, Leu or Lys &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; (5)..(5) &lt;223&gt; Thr, Gin, lie, Glu, His or Gly &lt;400&gt; 53

Gin Xaa His Xaa Xaa Thr Pro Phe Thr &lt;210&gt; 54 &lt;211&gt; 9 &lt;212&gt; PRT &lt;2Π&gt;Artificial Sequence&lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide / &lt;400&gt; 54

His Glu Glu Gly lie Tyr Phe Asp Val &lt;210> 55 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

&lt;211&gt 200911835 ms uiu uiu uiy ne Tyr Phe Lys Tyr &lt;210&gt; 57 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt; 57

Gin Gin His Tyr Thr Thr Pro &lt;210> 58 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt;

Fin &lt;212&gt

Gin Gin His Tyr lie Thr Pro 1 5 &lt;210> 60 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt; 60

Gin Gin His Tyr Glu Thr Pro &lt;210&gt; 61 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

62, &lt;211&gt 62

Gin Gin His Tyr Gly Thr Pro 1 5 &lt;210&gt; 63 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt; 63

Gin Gin His Leu Thr Thr Pro &lt;210&gt; 64 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt;

65, &lt;211&gt

66: &lt;211&gt

Gin Gin His Tyr Gin Thr Pro Phe Thr &lt;210&gt; 67 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt;

Gin Gin His Tyr Glu Thr Pro Phe Thr &lt;210&gt; 68 &lt;211&gt; 9 &lt;212&gt; PRT 200911835 &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt;

69, lt; 211 &gt; 9 &lt

70, &lt;211&gt; 9 &lt

71, &lt;211&gt;&lt

Gin Gin His Lys Thr Thr Pro Phe Thr &lt;210&gt; 72 &lt;211&gt; 9 &lt;212&gt; PRT &lt;2i3&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt;

Gin His His Tyr Thr Thr Pro Phe Thr &lt;210&gt; 73 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;220&gt;&lt;221> MOD-RES &lt;222&gt; (4).. (4) &lt;223&gt; Tyr or Lys &lt;220&gt;

&lt;221&gt; MOD RES 200911835 &lt;223&gt; Leu or Thr &lt;220&gt;

&lt;221&gt; MOD-RES &lt;222&gt; (11)..(11) &lt;223&gt; Val or Leu &lt;400&gt; 73

Tyr Phe Asp Xaa Trp Gly Gin Gly Thr Xaa Xaa Thr Val Ser Ser 15 10 15 &lt;210&gt; 74 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (1).. (1) &lt;223&gt; Tyr or Phe &lt;220〉

&lt;221> MOD-RES &lt;222&gt; (5)..(5) &lt;223&gt; Gin or Ser &lt;400> 74

Xaa Thr Phe Gly Xaa Gly Thr Lys Leu Glu lie Lys 1 5 10 &lt;210&gt; 75 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 15 10 15 &lt;210> 76 &lt;211&gt; 28 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence 1 - &lt;220&gt;&lt;223&gt; Description of the sequence: synthetic peptide &lt;400&gt; 76 . Ser Ala Val Tyr Phe Cys Ala Arg His Glu Glu Gly lie Thr Phe Asp 15 10 15

Tyr Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser 20 25 &lt;210&gt; 77 &lt;211&gt; 28 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400〉 77

Thr Ala Val Tyr Tyr Cys Ala Arg His Glu Glu Gly lie Tyr Phe Asp 15 10 15

Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser -18- 25 200911835 &lt;210&gt; 78 &lt;211&gt; 28 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence : synthetic peptide &lt;400&gt; 78

Thr Ala Val Tyr Tyr Cys Ala Arg His Glu Glu Gly He Tyr Phe Asp 15 10 15

Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 20 25 &lt;210〉 79 . &lt;211&gt; 12 &lt;212> PRT &lt;213&gt; Homo sapiens &lt;400〉 79 I &quot; Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 1 5 10 &lt;210&gt; 80 &lt;211&gt; 25 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt; 80

Leu Ala Leu Tyr Tyr Cys Gin Gin His Tyr Thr Thr Pro Phe Thr Phe 15 10 15

Gly Ser Gly Thr Lys Leu Glu lie Lys 20 25 &lt;210> 81 &lt;211&gt; 25 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt;; 81

Phe Ala Thr Tyr Tyr Cys Gin Gin His Tyr Thr Thr Pro Phe Thr Phe 15 10 15

Gly Gin Gly Thr Lys Leu Glu lie Lys 20 25 &lt;210&gt; 82 &lt;211&gt; 25 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt;; 82

Phe Ala Thr Tyr Tyr Cys Gin Gin His Tyr Thr Thr Pro Tyr Thr Phe 15 10 15 -19- 200911835

Gly Gin Gly Thr Lys Leu Glu lie Lys 20 25 &lt;210&gt; 83 &lt;211&gt; 17 &lt;212> PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;400&gt;; 83

Trp lie Tyr Pro Gly Gly Gly Tyr lie Arg Tyr Asn Gin Lys Phe Lys 15 10 15

Asp &lt;210&gt; 84 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt; 84

Trp lie Tyr Pro Gly Gly Gly Tyr lie Lys Tyr Asn Gin Lys Phe Lys 15 10 15

Asp &lt;210&gt; 85 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide

&lt;400&gt; 85

Trp lie Tyr Pro Gly Gly Gly Tyr Thr Lys Tyr Asn Glu Lys Phe Lys 15 10 15

Gly &lt;210&gt; 86 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Trp Phe Tyr Pro Gly Gly Gly Tyr Thr Lys Tyr Asn Gin Lys Phe Lys 15 10 15

Asp &lt;210&gt; 87 -20- 200911835, 厶i i〆 1 / &lt;212> PRT &lt; 213 &gt; artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Trp lie Tyr Pro Gly Gly Gly Tyr lie Lys Tyr Ala Gin Lys Phe Lys 15 10 15

Asp &lt;210&gt; 88 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Trp Phe Tyr Pro Gly Gly Gly Tyr Thr Arg Tyr Asn Gin Lys Phe Lys 15 10 15

Gly &lt;210&gt; 89 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Trp Phe Tyr Pro Gly Gly Gly Tyr lie Lys Tyr Ser Glu Lys Phe Lys 15 10 15

Asp &lt;210&gt; 90 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;400&gt;

Trp Phe Tyr Pro Gly Gly Gly Tyr Thr Lys Tyr Ala Gin Lys Phe Lys 15 10 15

Asp &lt;210&gt; 91 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide -21 - 200911835 &lt;s4uu^ yi

Trp Phe Tyr Pro Gly Gly Gly Tyr Thr Lys Tyr Asn Gin Lys Phe Lys 15 10 15

Asp &lt;210&gt; 92 &lt;211&gt; 98 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;220&gt;

&lt;221&gt; MOD-RES &lt;222&gt; (1)..(1) &lt;223&gt; Glu or Gin &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (33)..(33) &lt;;223&gt; Tyr or Thr &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (50)..(50) &lt;223&gt; lie or Trp &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; 52)..(52) &lt;223&gt; Asn or Tyr &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (54)..(54) &lt;223&gt; Ser or Gly &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (57)..(57) &lt;223&gt; Ser or Tyr &lt;220&gt;&lt;221> MOD-RES &lt;222&gt; (59)..(59) &lt;223&gt ; Ser or Lys &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; (65T..(65) &lt;223&gt; Gin or Phe &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (72). .(72) &lt;223&gt; into rg or Gly &lt;400&gt; 92

Xaa Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 15 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30

Xaa Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45

Gly Xaa lie Xaa Pro Xaa Gly Gly Xaa Thr Xaa Tyr Ala Gin Lys Phe 50 55 60 •22- 200911835

Xaa Gly Arg Val Thr Met Thr Xaa Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg &lt;210&gt; 93 &lt;211&gt; 94 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; (13)..(13) &lt;223&gt; Val or Ala &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; (18)..(18) &lt;223&gt; Arg or Lys &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; (297..(29) &lt;;223&gt; Val or Phe &lt;220&gt;&lt;221> MOD-RES &lt;222&gt; (3lT..(31) &lt;223&gt; Tyr or Gly &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (357..(35) &lt;223&gt; Asn or Gly &lt;220&gt;&lt;221&gt; MOD.RES &lt;222&gt; (39)..(39) &lt;223&gt; Leu or Val &lt;220&gt;&lt;221&gt; MOD_RES &lt;222&gt; (59)..(59) &lt;223&gt; Thr or Met &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (61)..(61) &lt;223&gt; Glu Or Val &lt;400&gt; 93

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Xaa Ser Leu Gly 15 10 15

Glu Xaa Ala Thr lie Asn Cys Lys Ser Ser Gin Ser Xaa Leu Xaa Ser 20 25 30

Ser Asn Xaa Lys Asn Tyr Xaa Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45

Pro Pro Lys Leu Leu lie Tyr Trp Ala Ser Xaa Arg Xaa Ser Gly Val 50 55 60 -23- 200911835

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

He Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys 85 90 &lt;210&gt; 94 &lt;211&gt; 88 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (1)..(1) ' &lt;223&gt; Ala or Asp &lt;220> &lt;221&gt; MOD-RES &lt;222&gt; (28)..(28) &lt;223&gt; Gly or Asp f, &lt;220> &lt;221&gt; MOD-RES &lt;222&gt; (29)..(29) &lt;223&gt; lie or Val &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (31).. (31) &lt;223&gt; Ser or Gly &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (42)..(42) &lt;223&gt; Lys or Gin &lt;220&gt;&lt;221&gt; MOD_RES &lt;;222&gt; (43j..(43) &lt;223&gt; Ala or Pro &lt;220&gt;

&lt;221&gt; M0D_RES f &lt;222&gt; (5〇T..(50) \ &lt;223&gt; Asp or Trp &lt;220> &lt;221&gt; M0D_RES &lt;222&gt; (53)..(53) &lt;223&gt; Ser or Thr. &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; (54)..(54) &lt;223&gt; Leu or Arg &lt;220&gt;&lt;221> MOD-RES &lt;222&gt; 60)..(60) &lt;223&gt; Ser or Asp &lt;220&gt;&lt;221> MOD-RES &lt;222&gt; (80)..(80) &lt;223&gt; Pro or Ala &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; (83)..(83) &lt;223> Phe or Val -24- 200911835 &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (85)..(85) &lt;;223&gt; Thr or Val &lt;400&gt; 94

Xaa lie Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Xaa Xaa Ser Xaa Ala 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Xaa Xaa Pro Lys Leu Leu He 35 40 45

Tyr Xaa Ala Ser Xaa Xaa Glu Ser Gly Val Pro Xaa Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Xaa 65 70 75 80

Glu Asp Xaa Ala Xaa Tyr Tyr Cys 85 &lt;210&gt; 95 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Description of artificial sequence artificial sequence: synthetic peptide &lt;220&gt;&lt;221&gt; MOD.RES &lt;222&gt; (3)..(3) &lt;223&gt; Tyr or Thr &lt;400&gt; 95

Ser Tyr Xaa Met His &lt;210&gt; 96 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;220&gt;&lt;221&gt; MOD .RES &lt;222&gt; (1)..(1) &lt;223&gt; lie or Trp &lt;220&gt;&lt;221> MOD-RES &lt;222&gt; (2)..(2) &lt;223&gt; tie or Fhe &lt;220&gt;&lt;221> MOD-RES &lt;222&gt; (3)..(3) &lt;223&gt; Asn or Tyr -25 - &lt;220> 200911835 &lt;ll\&gt;MUU_Kbi&gt;&lt;222&gt; (5)..(5) &lt;223&gt; Ser or Gly &lt;220&gt;

&lt;221> MOD-RES &lt;222&gt; (8)..(8) &lt;223&gt; Ser or Tyr &lt;220> &lt;221> MOD-RES &lt;222&gt; (9)..(9) &lt;;223&gt; Thr or He &lt;220&gt;

&lt;221&gt; M0D_RES &lt;222&gt; (10)..(10) &lt;223&gt; Ser, Lys, Arg or Asn &lt;220〉

&lt;221&gt; MOD-RES &lt;222&gt; (12)..(12) &lt;223&gt; Ala, Asn, Ser, Thr or Asp &lt;220〉

&lt;221&gt; MOD-RES f &lt;222&gt; (13)..(13) &lt;223&gt; Gin or Glu &lt;220&gt;&lt;221&gt; MOD_RES &lt;222&gt; (16)..(16) &lt;;223&gt; Gin or Lys &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (17)..(17) &lt;223&gt; Gly or Asp &lt;400&gt; 96

Xaa Xaa Xaa Pro Xaa Gly Gly Xaa Xaa Xaa Tyra Xaa Xaa Lys Phe Xaa 1 5 10 15

Xaa

&lt;210&gt; 97 &lt;211&gt; 17 &lt;212> PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt;220&gt;&lt;221>MOD-RES&lt;222&gt; (6)..(6) &lt;223&gt; Val or Phe &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (8)..(8) &lt;223&gt; Tyr or Gly &lt;220&gt;&lt;221> MOD-RES &lt;222&gt; (12)..(12) &lt;223&gt; Asn or Gly &lt;220&gt;

&lt;221> M〇D_RES &lt;222&gt; (16T..(16) -26- 200911835 &lt;22ό&gt; Leu or vai &lt;400&gt; 97

Lys Ser Ser Gin Ser Xaa Leu Xaa Ser Ser Asn Xaa Lys Asn Tyr Xaa 15 10 15

Ala &lt;210&gt; 98 &lt;211&gt; 11 &lt;212> PRT &lt;213&gt; Description of artificial sequence human X sequence: synthetic peptide &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (5),. (5) &lt;223&gt; Gly or into sp &lt;220> &lt;221&gt; MOD-RES &lt;222&gt; (6)..(6) &lt;223&gt; lie or Val &lt;220&gt;&lt;221&gt; MOD RES &lt;222&gt; (8)..(8) &lt;223&gt; Ser or Gly &lt;400&gt; 98

Arg Ala Ser Gin Xaa Xaa Ser Xaa Ala Leu Ala 1 5 10 &lt;210&gt; 99 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Description of artificial sequence containing Si human 1 sequence: synthetic peptide (^ &lt;220&gt;;&lt;221> M0D-RES &lt;222&gt; (1)..(1) &lt;223&gt; Trp or Asp &lt;220&gt;&lt;221&gt; M0D.RES &lt;222> (4)..(4) &lt;223&gt; Ser, Thr or Met &lt;220&gt;&lt;221&gt; M0D-RES &lt;222&gt; (5)..(5) &lt;223&gt; Arg or Leu &lt;220&gt;

&lt;221> MOD-RES &lt;222&gt; (6)., (6) &lt;223&gt; Glu or Val &lt;400&gt; 99

Xaa Ala Ser Xaa Xaa Xaa Ser &lt;210> 100 •27- 200911835, heart xx〆-y &lt;212&gt; PRT &lt;213&gt;Artificial sequence &lt;220&gt;&lt;223&gt; Description of artificial sequence: synthetic peptide &lt; 220> &lt;221&gt; MOD-RES &lt;222&gt; (2)..(2) &lt;223&gt; Gin or His &lt;220&gt;&lt;221&gt; M0D_RES &lt;222> (3),.(3) &lt;;223&gt; Tyr, His or Phe &lt;220&gt;&lt;221&gt; MOD-RES &lt;222> (4)., (4) &lt;223&gt; Tyr, Asn, Leu or Lys &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; (5)..(5) &lt;223&gt; Ser, lie, Glu, His, Gly, Gin or Thr &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (6).. (6) &lt;223&gt; Thr or Tyr &lt;220&gt;&lt;221&gt; M0D_RES &lt;222&gt; (8)..(8) &lt;223&gt; Tyr or Phe &lt;400> 100

Gin Xaa Xaa Xaa Xaa Xaa Pro Xaa Thr &lt;210&gt; 101 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;220&gt;;221> MOD-RES &lt;222&gt; (1)..(1) &lt;223&gt; Glu or Gin &lt;400&gt; 101

Xaa Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 15 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 &lt;210&gt; 102 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence : synthetic peptide 28- 200911835 &lt;221&gt; M0D_RES &lt;222&gt; (6)..(6) &lt;223&gt; Arg or Gly &lt;400&gt;

Arg Val Thr Met Thr Xaa Asp Thr Ser Thr Ser Thr Val Tyr Met Glu 15 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 &lt;210&gt; 103 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Artificial Sequence Description: Synthetic peptide &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (13)..(13) &lt;223&gt; Val or Ala &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (18)..(18) &lt;223&gt; Arg or Lys &lt;400&gt; 103

Asp lie Val Met Thr Gin Ser Pro Asp Ser Leu Ala Xaa Ser Leu Gly 15 10 15

Glu Xaa Ala Thr lie Asn Cys 20 &lt;210&gt; 104 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Description of Artificial Sequence: Synthetic Peptide &lt;220> &lt; 221> MOD-RES &lt;222&gt; (1)..(1) &lt;223&gt; Ala or Asp &lt;400> 104

Xaa He Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

&lt;211&gt 〉 200911835 丄;&gt; MlJU— &lt;222> (8)..(8) &lt;223&gt; Gin or Lys &lt;220> &lt;221> MOD-RES &lt;222> (9)..(9) &lt;223&gt; Pro or into la &lt;400> 105

Trp Tyr Gin Gin Lys Pro Gly Xaa Xaa Pro Lys Leu Leu lie Tyr 15 10 15 &lt;210> 106 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Artificial Sequence Description: Synthetic peptide &lt;220&gt;&lt;221> M0D-RES &lt;222&gt; (4)..(4) &lt;223&gt; Asp or Ser &lt;220> &lt;221> MOD-RES &lt;222&gt; 24)..(24) &lt;223&gt; Ala or Pro &lt;220&gt;&lt;221&gt; MOD-RES &lt;222&gt; (27)..(27) &lt;223&gt; Val or Phe &lt;220&gt;&lt; 221> MOD-RES &lt;222&gt; (29)..(29) &lt;223&gt; Val or Thr &lt;400&gt; 106

Gly Val Pro Xaa Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 15 10 15

Leu Thr lie Ser Ser Leu Gin Xaa Glu Asp Xaa Ala Xaa Tyr Tyr Cys 20 25 30 &lt;210&gt; 107 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;220&gt;&lt;221&gt; MOD_RES &lt;;222&gt; (10*). .(10) &lt;223&gt; Ser, Asn or Arg &lt;220&gt;&lt;221> MOD-RES &lt;222&gt; (12)..(12) &lt;223&gt; Ala, Thr or Asp &lt;400&gt; 107 lie lie Asn Pro Ser Gly Gly Ser Thr Xaa Tyr Xaa Gin Lys Phe Gin 15 10 15

Gly -30-

Claims (1)

200911835 X. Patent Application Range: 1. An antibody that binds to death receptor 5 (DR5), wherein the antibody comprises (a) a human heavy chain V-segment, a heavy chain complementarity determining region 3 (CDR3) and a heavy chain a heavy chain variable region of framework region 4 (FR4), and (b) a light bond variable region comprising a human light chain V-segment, a light chain CDR3, and a light chain FR4, wherein the heavy chain CDR3 comprises an amine group Acid sequence HEEGI (SEQ ID NO: 49); and Γ) U) The light chain CDR3 variable region comprises the amino acid sequence QXHXXTP (SEQ ID NO: 50), wherein X represents any amino acid. 2. The antibody of claim 1, wherein the heavy chain v_ segment shares at least 90% sequence identity with SEq m NO:44, wherein the light chain v_ segment and SEq ID NO: 46 or SEQ ID NO: 48 has at least 90% sequence identity. 3. The antibody of claim 1, wherein: i) the heavy chain CDR3 comprises an amino acid motif motif HEEGIYFXA2 (SEQ ID NO: 51), wherein XjD, T or K, X2 i„^ is Y, K or V; and ii) the light chain CDR3 comprises an amino acid sequence motif qx3hX4X5TP (SEQ ID NO: 52), wherein X3 is Q or Η, X4 is Y, L or K, and X5 is T, Q, I, E, H or G. k. The antibody of claim 1, wherein: i) the heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: J: HEEGIYFDY (SEQ ID NO: 7) &gt HEEGIYFDK (SEQ ID NO: 8), HEEGIYFDV (SEQ ID NO: 54), 131613.doc 200911835 HEEGIYFTY (SEQ ID NO: 55) and HEEGIYFKY (SEQ ID NO: 56); and ii) the light chain CDR3 comprises a The amino acid sequence of the following composition is selected: 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), QQHKT TP (SEQ ID NO: 64), and QHHYTTP (SEQ ID NO: 65). The antibody of any one of 3 or 4, wherein the heavy chain FR4 is a human germline (germli) 6. The antibody of claim 5, wherein the FR4 is a human germline JH4 (SEQ ID NO: 28). The antibody of any one of claims 1, 3 or 4, wherein the heavy chain The human germline segment is included. 8. The antibody of claim 7, wherein the heavy chain human germline J-segment is JH4. 9. The antibody of any one of claims 1, 3 or 4 wherein the light The FR4 is a human germline FR4. 10. The antibody of claim 9, wherein the FR4 is a human germline JK2 (SEQ ID NO: 38) ° 1 1. The antibody of any one of claims 1, 3 or 4 Wherein the light chain comprises a human germline segment. 1 2. The antibody of claim 1 wherein the light chain human germline J-segment is JK2. 1 3. The antibody of claim 1, wherein the heavy The chain V-segment and the light chain V-segment each comprise a complementarity determining region 1 (CDR1) and a complementarity determining region 2 (CDR2); 131613.doc 200911835 wherein: i) the CDR1 of the heavy chain v-segment comprises An amino acid sequence selected from the group consisting of *seqid NO: 2 and SEQ ID NO: 3; ii) the CDR2 of the heavy chain V-segment comprises a population selected from the group consisting of SEQ m NO: 5 and SEQ ID NO: Amino acid sequence; in) the light chain V-region The CDR1 of the segment comprises an amino acid sequence selected from the group consisting of SEq ID 11:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID N〇:14; and iv) side light chain V-region The CDR2 of the segment comprises an amino acid sequence selected from the group consisting of sEQ NO: 16 and SEQ ID NO: 17. 14. The antibody of claim 13, wherein: i) 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 a The amino acid sequence of the group consisting of SEQ ID NO: 20 and SEQ ID NO: 21 is selected. 1 5. The antibody of claim 13 wherein: i) the CDR1 of the heavy chain V-segment comprises SEQ ID NO: 2; ii) the CDR 2 of the heavy chain V-segment comprises SEQ ID NO: 5; iii) The heavy chain CDR3 comprises SEQ ID NO:8; iv) the CDR1 of the light chain V-segment comprises SEQ ID ΝΟ:11; v) the CDR2 of the light chain V-segment comprises SEQ ID NO:16; The light chain CDR3 comprises SEQ ID NO:20. 16. The antibody of claim 13, wherein: i) the CDR1 of the heavy chain V-segment comprises SEQ ID NO: 2; 131613. doc 200911835 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 CDR1 of the light chain V-segment comprises SEQ ID NO:12; and v) the CDR2 of the light chain V-segment comprises SEQ ID NO: 17; vi) The light chain CDR3 comprises SEQ ID NO:20. The antibody of claim 13 wherein: i) the CDR1 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 The heavy chain CDR3 comprises SEQ ID NO:8; iv) the CDR1 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 antibody of claim 1, wherein the heavy chain variable region shares at least 9 % amino acid sequence identity with the variable region of SEQ ID NO: 43, the light chain variable region and SEQ ID NO: 45 Or the variable region of SEQ ID NO: 47 shares at least 90% amino acid sequence identity. 1 9. The antibody of claim 1, wherein the antibody binds to DR5 with an equilibrium dissociation constant (KD) of less than 1 X 1 ο·8 。. 20. The antibody of claim 1, wherein the anti-system FAb, fragment. 2 1. The antibody of claim 1, wherein the anti-system IgG. 22. The antibody of claim 1, wherein the anti-system single chain antibody (scFv). 23. The antibody of claim 1, wherein the antibody comprises a human constant region. The antibody of claim 1, wherein the anti-system DR5 agonist (ag〇nist). The antibody of claim 1, wherein the antibody comprises a heavy chain having at least 95% amino acid sequence identity to SEQ ID NO: 39 with 131613.doc 200911835 and a SEQ ID NO: 40, SEQ The amino-based Ssl sequence of the group consisting of ID NO: 41 and SEQ ID NO: 42 has a light chain of at least 5% sequence identity. 26. The antibody of claim 25, wherein the antibody comprises a heavy chain comprising seq mountain NO: 39 and an amine comprising a group selected from the group consisting of SEQ id NO: 40, SEQ ID NO: 41 and SEQ ID NO: The light chain of the base acid sequence. 27. The antibody of claim 26, wherein the light chain comprises SEq ID N〇: 4〇. 28. The antibody of claim 26, wherein the light chain comprises seq ID NO: 41. 29. The antibody of claim 26, wherein the light chain comprises SEq ID N〇:42. 30. A pharmaceutically acceptable composition comprising the antibody of any one of claims -28, and a physiologically compatible excipient. A method of inducing apoptosis of cancer cell cells, which comprises contacting the cell with an antibody according to any one of claims 1 to 28, wherein the anti-system DR5 is an agonist. 3. The method of claim 31, further comprising contacting the cell with an apoptosis inducing agent. . 33. A method of inducing apoptosis of cancer cell cells in an individual, comprising administering to the individual a therapeutically effective amount of an antibody according to any one of claims 1 to 28, wherein the anti-system DR5 is an agonist. 3. The method of claim 3, further comprising contacting the cell with a cell death inducer. 35. An antibody that binds to death receptor 5 (DR5), wherein the antibody comprises 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 Complementarity determining regions (CDRs) - CDR1, CDR2 131613.doc 200911835 and CDR3; wherein: i) the CDR1 of the heavy chain variable region comprises a SEQ ID ΝΟ: 1, SEQ ID NO: 2 and SEQ ID NO: 3 a group of amino acid sequences; - ii) the CDR2 of the heavy chain variable region comprising an amino acid selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: a sequence, iii) the CDR3 of the heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID (.' NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9; iv) The CDR1 of the 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; 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; (V vi) the light chain variable region The CDR3 contains an option Free amino acid sequence of the group consisting of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, but the antibody does not comprise all SEQ ID ΝΟ: 1, SEQ ID NO: 4, SEQ ID NO: 7. SEQ ID NO: 10, SEQ ID NO: 15 and SEQ ID NO: 19. 131613.doc