CN101072581A - Therapeutic agents with decreased toxicity - Google Patents
Therapeutic agents with decreased toxicity Download PDFInfo
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Abstract
The present invention relates to therapeutic agents with reduced toxicity comprising a serum albumin binding peptide (SABP), a targeting agent and a cytotoxic agent. The present invention also relates to methods for reducing the toxicity agents and methods of treatment using the therapeutic agents with reduced toxicity.
Description
The application is the non-provisional application of submitting to according to 37CFR1.53 (b) (1), require the provisional application the 60/641st of submission on January 5th, 2005 according to 35USC119 (e), the provisional application the 60/616th that No. 534 and on October 5th, 2004 submit to, No. 507 priority, their content is incorporated herein by reference at this.
Invention field
The present invention relates to have the novel treatment of the toxicity in vivo of minimizing, comprise the compositions of novel treatment, be used for reducing the method for therapeutic agent toxicity in vivo and comprising the method that is used for the treatment of the patient of using novel treatment.
Background of invention
Attempt using antibody-drug conjugates (ADC) to come local delivery cytotoxic agent or cytostatic agent, promptly in treatment of cancer, killed or suppressed medicine (Payne, G. (2003) the CancerCell 3:207-212 of tumor cell; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv.Drug Del.Rev.26:151-172; US4975278).In theory, can produce under the unacceptable toxic level normal cell at these not link coupled pharmaceutical agents of systemic administration, drug moiety is answered target tumor and by internalization (Baldwin etc., (1986) Lancet pp. (Mar.15,1986): 603-05; Thorpe, (1985) " Antibody Carriers OfCytotoxic Agents In Cancer Therapy:A Review; " in Monoclonal Antibodies ' 84:Biological And Clinical Applications, A.Pinchera etc. (editor), pp.475-506).
Polyclonal antibody and monoclonal antibody all be used to make ADC (Rowland etc., (1986) Cancer Immunol.Immunother., 21:183-87).The medicine that uses in these methods comprises daunomycin, doxorubicin, methotrexate and vindesine (Rowland etc., (1986) are the same).The toxin that uses in antibody-toxin conjugated thing comprises the bacteriotoxin such as diphtheria toxin, diphtherotoxin, such as the phytotoxin of ricin, such as geldanamycin (Mandler etc. (2000) J. of the Nat.Cancer Inst.92 (19): 1573-1581; Mandler etc. (2000) Bioorganic ﹠amp; Med.Chem.Letters10:1025-1028; Mandler etc. (2002) Bioconjugate Chem.13:786-791), CHROMATOGRAPHIC FRACTIONATION AND MASS (EP1391213; Liu etc., (1996) Proc.Natl.Acad.Sci USA 93:8618-8623) and calicheamicin (Lode etc. (1998) Cancer Res.58:2928; Hinman etc. (1993) CancerRes.53:3336-3342) micromolecule toxin.Recently, with the synthetic analogues (WO02/088172) of auristatin peptide, auristatin E (AE) and monomethylauristatin (MMAE) and dolastatin and full length antibody coupling (as Klussman, Deng (2004), Bioconjugate Chemistry15 (4): 765-773; Doronina etc. (2003) Nature Biotechnology 21 (7): 778-784; Francisco etc. (2003) Blood 102 (4): 1458-1465; US2004/0018194; WO04/032828; Mao waits (2004) Cancer Res.64 (3): 781-788; Bhaskar etc. (2003) Cancer Res.63:6387-6394; WO03/043583; Mao etc. (2004) Cancer Res.64:781-788).At US5767237; Also disclosed the variant of auristatin E among the US6124431.
ZEVALIN (ibritumomab tiuxetan (ibritumomab tiuxetan), Biogen Idee Inc.) is a kind of antibody-radiosiotope conjugate, by at the antigenic Mus IgGl of the CD20 κ monoclonal antibody that sees normal and virulent bone-marrow-derived lymphocyte surface and
111In or
90The Y radiosiotope connects to form (Wiseman etc. (2000) Eur.J. Nucl.Med.27 (7): 766-77 by thiourea linker-chelating agen; Wiseman etc. (2002) Blood 99 (12): 4336-42; Witzig etc. (2002) J. Clin. Oncol.20 (10): 2453-63; Witzig etc. (2002) J. Clin.Oncol. 20 (15): 3262-69).Though ZEVALIN has the activity at B-cell non-Hodgkin's (NHL), in Most patients, use and cause serious and persistent cytopenia.MYLOTARG
TM(Gemtuzumab Ozogamicin (gemtuzumab ozogamicin), Wyeth Pharmaceuticals), a kind of antibody drug conjugates of being made up of the CD33 antibody that connects calicheamicin went through to be used for the treatment of acute myeloid leukemia (Drugs of the Future (2000) 25 (7): 686 by injection in 2000; The US patent No. 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001).Cantuzumab mertansine (Immunogen, Inc.), a kind of antibody drug conjugates of forming by the huC242 antibody that connects CHROMATOGRAPHIC FRACTIONATION AND MASS drug moiety DM1 by disulphide joint SPP (Xie etc. (2004) J. of Pharm.and Exp. Ther.308 (3): 1073-1082), just entering the II phase that is used for the treatment of cancer such as the colon cancer of expressing CanAg, cancer of pancreas, gastric cancer or the like to test.MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), a kind of antibody drug conjugates of being made up of anti-prostate specific membrane antigen (PSMA) monoclonal antibody that connects CHROMATOGRAPHIC FRACTIONATION AND MASS drug moiety DM1 is being developed the potential treatment that is used for tumor of prostate.
Attempted several different methods already to improve the half-life of micromolecule or biopharmaceuticals.For example, glycosylation site has been imported (Keyt etc., 1994, PNAS USA 91:3670-74) in the molecule, and with molecule and PEG coupling (Clark etc., 1996, J. Biol.Chem., 271:21969-77; Lee etc., 1999, Bioconjugate Chem.10:973-981; Tanaka etc., 1991, CancerRes.51:3710-14) eliminate the half-life (elimination half-time) to increase bulk of molecule and to increase.Some have attempted the therapeutic use that end user's serum albumin improves medicine.For example, albumin had connected micromolecule (Syed etc., 1997, Blood 89:3243-3252 already; Burger etc., 2001 Int.J. Cancer92:718-724; Wosikowski K, etc., Clin Cancer Res.2003 May 9 (5): 1917-26); CD4 (Yeh etc., 1992, PNASUSA 89:1904-1908); Fc part (Ashkenazi etc. (1997) the Curr.Opin in Immunol.9:195-200 of IgG; IL-2 (Yao, Z etc., (2004 May) CancerImmunol Immunother.53 (5): 404-10) and bridge joint (Affleck, K etc., (1992) Br J Cancer.65 (6): 838-44) between anti--gp72 antibody and methotrexate molecule.
Also studied the purposes of albumin in conjunction with polypeptide.Reported the interior half-life prolongation of body (Makrides etc., (1996) J. Pharmacol.Exptl.Ther.277:532-541) that combines the people's 1 type soluble complement receptor (sCR1) that merges in the territory with albumin from streptococcal protein G.The proteic albumin of G of having described labelling is in conjunction with territory (EP0486,525).The applicant has described the deutero-albumin binding peptide of some phage displays.Referring to WO01/45746, U.S. Patent Publication No. 2004/0001827, and Dennis, MS, etc., (2002) JBC 277 (38): 35035-43.In theory, the serum albumin binding peptide combines with serum albumin is non-covalent in vivo.Like this, the serum albumin binding peptide is that serum albumin self step of removing from body-internal-circulation mechanism is necessary.
What be described below the present invention proposes in the conjugate of context and targeting agent/cytotoxic agent the unexpected favourable purposes of albumin binding peptide.
Summary of the invention
The present invention relates to comprise the conjugate molecule of the combination of covalently bound at least one serum albumin bound fraction (SABM), targeting agent (TA) and cytotoxic agent (CA).According to an embodiment, the conjugate molecule comprises 2 or more a plurality of CA.According to an embodiment, the conjugate molecule comprises 2 or more a plurality of TA.
According to one embodiment of the invention, it is at least 50% same as the aminoacid sequence of DICLPRWGCLW (SEQ ID NO:8) sequence that SABM comprises, and wherein aminoacid sequence has two Cys residues, has 5 amino acid residues between the Cys residue.According to an embodiment, aminoacid sequence and SEQ ID NO:8 have the homogeneity percent that is selected from least 60% homogeneity, at least 70% homogeneity, at least 80% homogeneity, at least 85% homogeneity, at least 90% homogeneity, at least 95% homogeneity, at least 98% homogeneity and at least 99% homogeneity.
According to another embodiment, SABM comprises the variant of DICLPRWGCLW (SEQ ID NO:8) aminoacid sequence, and wherein except that the Cys residue, the 1-5 of SEQ ID NO:8 any residue replaced by different amino acid residues.
According to another embodiment, SABM comprises linearity or cyclic amino acid sequence, is selected from:
Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa
Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys[SEQ ID NO：1]
Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe[SEQ ID NO：2]
Cys-Tyr-Xaal-Pro-Gly-Xaa-Cys[SEQ ID NO：3]
Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp[SEQ ID NO：4]
Trp-Cys-Asp-Xaa-Xaa-Leu-Xaa-Ala-Xaa-Asp-Leu-Cys[SEQ ID NO：5]；
Asp-Leu-Val-Xaa-Leu-Gly-Leu-Glu-Cys-Trp[SEQ ID NO：6]；
CXXGPXXXXC[SEQ ID NO：21]
XXXXCXXGPXXXXCXXXX[SEQ ID NO：22]
CXXXXXXCXXXXXXCCXXXCXXXXXXC[SEQ ID NO：23]
CCXXXCXXXXXXC[SEQ ID NO：24]
CCXXXXXCXXXXCXXXXCC[SEQ ID NO：25]
CXCXXXXXXXCXXXCXXXXXX[SEQ ID NO：26]
XXXXXDXCLPXWGCLWXXXX[SEQ ID NO：155]
XXXXDXCLPXWGCLWXXX[SEQ ID NO：156]
DXCLPXWGCLW[SEQ ID NO：423]
XXXXDICLPRWGCLWXXX[SEQ ID NO：424]，
XXXXXDICLPRWGCLWXXXX[SEQ ID NO：425]
XXEMCYFPGICWMXX[SEQ ID NO：426]
XXDLCLRDWGCLWXX[SEQ ID NO：427]
Wherein X is an arbitrary amino acid residue.
According to an embodiment preferred, the SABM sequence of above-mentioned general formula, particularly SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 are at N-end (Xaa)
xWith C-end (Xaa)
zThe place comprises extra aminoacid, and wherein Xaa is a seed amino acid, and x and z are the integers more than or equal to 0 (zero), are generally less than 100, preferably less than 10 and more preferably 0,1, and 2,3,4 or 5, more preferably 4 or 5, Xaa
1Be selected from Ile, Phe, Tyr and VaI.In one embodiment, the present invention relates to comprise sequence D ICLPRWGCLW[SEQ ID NO 8] the purposes of albumin binding peptide.According to an embodiment, SABM comprises the aminoacid sequence of the arbitrary SEQ of being selected from ID NOs:7-20,27-154 and 157-421.According to an embodiment preferred, SABM comprises and is selected from SEQID NOs:7,8,9,10,11,12,13,14,15,16,17,18,19 and 20 aminoacid sequence.
According to another embodiment, SABM comprises following amino acid sequences: Xaa
i-Cys-Xaa
j-Cys-Xaa
k, wherein i, j and k's and be about 25 or still less, Xaa is an arbitrary amino acid residue.According to an embodiment preferred, i, j and k's and be about 18 residues or still less.According to another embodiment preferred, i, j and k's and be about 11 residues or still less.
According to another embodiment, SABM comprises the peptide sequence of table any one described in the 1-9.
According to one embodiment of the invention, all above-mentioned SABM sequences combine with serum albumin, have about 100 μ M or lower K
dAccording to another embodiment, K
dBe selected from about 10 μ M or littler, about 1 μ M or littler, about 500nM or littler, about 100nM or littler, about 50nM or littler and about 10nM or littler.
According to another embodiment, TA be comprise can with the polypeptide of the bonded aminoacid sequence of target cell surface protein, wherein TA comprise cell surface protein part, adhesin or antibody or can with the bonded above-mentioned arbitrary segmental aminoacid sequence of cell surface protein.According to an embodiment, be the B cell surface marker by the cell surface protein of targeting.According to another embodiment, be selected from HER2, CD20, EGFR, PDGFR, BR3, Flt-1, KDR and EphB2 by the receptor of targeting.According to another embodiment, TA is the antibody at arbitrary those receptors.According to an embodiment preferred, antibody exists with arbitrary following form: Fab, F (ab)
2, scFv and double antibody.According to another embodiment, TA comprises described VH or VL sequence (as comprise anti--HER2 antibody of the antigen-binding portion thereof of SEQ ID NO:428 and 429) herein.
According to an embodiment, anti--HER2 antibody comprises the variable region of SEQ ID NO:428 and 429.According to an embodiment, anti--HER2 antibody comprises the variant of the light chain variable region sequence of SEQ ID NO:428, wherein at least one or a plurality of Q27 (V that is selected from according to Kabat numbering system numbering
L), D28 (V
L), N30 (V
L), T31 (V
L), A32 (V
L), Y49 (V
L), F53 (V
L), Y55 (V
L), R66 (V
L), H91 (V
L), Y92 (V
L) and T94 (V
L) aminoacid replaced by any aminoacid except that alanine.According to an embodiment, anti--HER2 antibody comprises the variant of the light chain variable region sequence of SEQ ID NO:428, and wherein at least one of variable region or a plurality of aminoacid have the D28 of being selected from (V
L) Q, D28 (V
L) G, N30 (V
L) S, T31 (V
L) S, A32 (V
L) G, Y49 (V
L) W, Y49 (V
L) D, Y49 (V
L) V, F53 (V
L) W, F53 (V
L) V, F53 (V
L) Q, Y55 (V
L) W, R66 (V
L) N, H91 (V
L) F, H91 (V
L) Y, Y92 (V
L) W and T94 (V
L) replacement of S.According to an embodiment, anti--HER2 antibody comprises the variant of the light chain variable region sequence of SEQ ID NO:428, and wherein the variable region comprises at least three and replaces Y49 (V
L) D, F53 (V
L) W and Y55 (V
L) W.According to an embodiment, anti--HER2 antibody comprises the variant of the light chain variable region sequence of SEQ ID NO:428, and wherein the variable region comprises at least three and replaces N30 (V
L) S, H91 (V
1) F and Y92 (V
L) W.
According to an embodiment, anti--HER2 antibody comprises the variant of the weight chain variabl area sequence of SEQ ID NO:429, wherein at least one or a plurality of W95 (V that is selected from according to Kabat numbering system numbering
H), D98 (V
H), F100 (V
H), Y100a (V
H) and Y102 (V
H) aminoacid replaced by any aminoacid except that alanine.According to an embodiment, anti--HER2 antibody comprises the variant of the weight chain variabl area sequence of SEQ IDNO:429, and wherein the variable region comprises at least one or a plurality of W95 of being selected from (V
H) Y, D98 (V
H) W, D98 (V
H) R, D98 (V
H) K, D98 (V
H) H, F100 (V
H) P, F100 (V
H) L, F100 (V
H) M, F100 (V
H) W, Y100a (V
H) F, Y102 (V
H) V, Y102 (V
H) K and Y102 (V
H) replacement of L.According to an embodiment, anti--HER2 antibody comprises the variant of the weight chain variabl area sequence of SEQ ID NO:429, and wherein the variable region comprises F100 (V at least
H) P and Y102 (V
H) replacement of K.According to an embodiment, anti--HER2 antibody comprises the variant of the weight chain variabl area sequence of SEQ ID NO:429, and wherein the variable region comprises F100 (V at least
H) P and Y102 (V
H) replacement of L.
According to an embodiment, anti--HER2 antibody comprises the variant of the weight chain variabl area sequence of the light chain variable region sequence of SEQ ID NO:428 and SEQ ID NO:429, wherein at least one or a plurality of D28 (V that is selected from according to Kabat numbering system numbering
L), N30 (V
L), T31 (V
L), A32 (V
L), Y49 (V
L), F53 (V
L), Y55 (V
L), R66 (V
L), H91 (V
L), Y92 (V
L), T94 (V
L), W95 (V
H), D98 (V
H), F100 (V
H), Y100a (V
H) and Y102 (V
H) aminoacid replaced by any aminoacid except that alanine.According to an embodiment, anti--HER2 antibody comprises the variant of the weight chain variabl area sequence of the light chain variable region sequence of SEQ ID NO:428 and SEQ ID NO:429, comprises at least one or a plurality of following replacement: D28 (V
L) Q, D28 (V
L) G, N30 (V
L) S, T31 (V
L) S, A32 (V
L) G, Y49 (V
L) W, Y49 (V
L) D, Y49 (V
L) V, F53 (V
L) W, F53 (V
L) V, F53 (V
L) Q, Y55 (V
L) W, R66 (V
L) N, H91 (V
L) F, H91 (V
L) Y, Y92 (V
L) W, T94 (V
L) S, W95 (V
H) Y, D98 (V
H) W, D98 (V
H) R, D98 (V
H) K, D98 (V
H) H, F100 (V
H) P, F100 (V
H) L, F100 (V
H) M, Y100a (V
H) F, Y102 (V
H) V, Y102 (V
H) K and Y102 (V
H) L.According to an embodiment, anti--HER2 antibody comprises the variant of the weight chain variabl area sequence of the light chain variable region sequence of SEQ ID NO:428 and SEQ ID NO:429, comprises following at least replacement: Y49 (V
L) D, F53 (V
L) W, Y55 (V
L) W, F100 (V
H) P and Y102 (V
H) K.According to an embodiment, anti--HER2 antibody comprises the variant of the weight chain variabl area sequence of the light chain variable region sequence of SEQ ID NO:428 and SEQ ID NO:429, comprises following at least replacement: Y49 (V
L) D, F53 (V
L) W, Y55 (V
L) W, F100 (V
H) P and Y102 (V
H) L.According to another embodiment, described resisting-HER2 antibody is any U.S. Patent Publication No. 2003/0228663A1 that submitted on April 9th, 2003 that is disclosed in; WO03/087131; Carter etc., anti--HER2 antibody of (1992) PNAS89:4285-4289, these publications are incorporated herein by reference especially at this.
According to an embodiment, except that with cell outer surface on the ability of protein bound, TA has extra biological activity.According to another embodiment, described other biological activity is the ability that the cell of block ligand mediation signals by cell.According to another embodiment, described other biological activity is the apoptotic ability of inducing by targeting.According to another embodiment, TA be with the cell of being considered on protein bound, have the polypeptide that is selected from 10uM or littler, 1uM or littler, 500nm or littler, 100nm or littler and 10nm or littler Kd.
According to an embodiment, to compare with normal cell, the protein on the bonded cells of interest of TA is crossed in cancerous cell and is expressed.According to another embodiment, be pathogenic cell by the cell of TA targeting, such as tumor cell.
According to an embodiment preferred, cytotoxic agent is monomethylauristatin (MMAE).
According to another embodiment preferred, the conjugate molecule comprises the blank area between described SABM and targeting agent or cytotoxic agent.In one embodiment, blank area comprises aminoacid sequence: GGGS (SEQ ID NO:422).
According to another embodiment, SABM combines with people's albumin.According to another embodiment, SABM is coupled to the N-or the C-end region of heavy chain or the variable region of light chain of TA.
The invention provides the compositions that comprises with the blended conjugate molecule of pharmaceutical carrier.The present invention also provides the application of conjugate molecule in medication preparation.
The present invention also provides the method that is used to reduce therapeutic agent toxicity, and comprising with coupling has the serum albumin bound fraction (SABM) of therapeutic agent to produce the step of therapeutic agent.This method can further comprise therapeutic agent that relatively has SABM and the toxic step that does not have the therapeutic agent of SABM.According to an embodiment, this method further comprises the mensuration therapeutic agent: the toxic step of SABM conjugate.
The invention provides the method that reduces therapeutic agent toxicity in the mammal, comprise to administration treatment effective dose according to conjugate molecule of the present invention.According to an embodiment, this method further comprises the mensuration therapeutic agent: the toxic step of SABM conjugate.According to an embodiment preferred, mammal suffers from autoimmune disease or cancer.
The invention provides the method for tumor in the treatment mammal, comprise with the bonded conjugate molecule of the present invention of the vascular system with around tumor cell or the tumor of treatment effective dose and handle the mammiferous step of suffering from tumor.The present invention also provides the method for autoimmune sexual disorder in the treatment mammal, comprises with the conjugate molecule of the present invention of treatment effective dose and handles the mammiferous step of suffering from the autoimmune sexual disorder.According to an embodiment preferred, the conjugate molecule with help or cause that the B-cell of autoimmune sexual disorder combines.The present invention also provides the method for cell proliferative disorders in the treatment mammal, comprises with the conjugate molecule of the present invention of treatment effective dose and handles the mammiferous step of suffering from cell proliferative disorders.According to another embodiment, the invention provides the method that is used for subduing mammal B cell, comprise with the treatment effective dose handle mammiferous step with the bonded conjugate molecule of the present invention of B cell.
According to an embodiment, Therapeutic Method of the present invention further comprises the toxic step of measuring conjugate molecule in the mammal.
According to an embodiment, toxicity be shown as be selected from lose weight, the committed stem cell (hemopoietic progenitor cell) of hemopoietic toxicity, nephrotoxicity, liver toxicity, gastrointestinal toxicity, minimizing by bone marrow to each of mobilization, anemia, bone marrow depression, pancytopenia, thrombocytopenia, neutrophil cell minimizing, lymphopenia, leukopenia, stomatitis, alopecia, headache and the myalgia of peripheral blood.
The present invention also provides the product of package insert that comprises container, the compositions that comprises conjugate molecule of the present invention in container, contains the operation instruction of administering therapeutic effective dose.
The accompanying drawing summary
Fig. 1 shown at injection contrast media (circle), Herceptin -vc-Pab-MMAE (square), Ab.Fab4D5-H-vc-PAB-MMAE (rhombus), Fab3D4-vc-PAB-MMAE (triangle) and Ab.Fab contrast-vc-PAB-MMAE (hollow circle) afterwards, along with the gross tumor volume in past of time.
Fig. 2 has shown and is using Herceptin -vc-MMAE (square), Herceptin -F (ab ')
24D5-vc-MMAE (cross), free MMAE (circle) afterwards, the group of body weight changes (groupchange).
Fig. 3 has shown and is using Herceptin -vc-MMAE (rhombus), Fab4D5-vc-MMAE (triangle), AB.Fab4D5-H-vc-MMAE (circle) and PBS (square) afterwards that the group of body weight changes.
Fig. 4 shown humanization anti--variable region of light chain [SEQ ID NO:428] and the humanization of HER2 antibody resist-aminoacid sequence of the variable region of heavy chain [SEQ ID NO:429] of HER2 antibody.
Description of Preferred Embodiments
I. definition
Term " serum albumin binding peptide " or " serum albumin bound fraction " (" SABM ") refer to comprise chemical compound or the polypeptide in conjunction with the aminoacid sequence of serum albumin.According to an embodiment preferred, SABM is in conjunction with the human serum albumin.According to an embodiment, SABM comprises the arbitrary sequence in conjunction with rabbit, rat, mice or human serum albumin described at least one sequence table.According to another embodiment, SABM comprises the arbitrary sequence in conjunction with the serum albumin of multiple species described at least one sequence table.According to an embodiment, SABM comprise at least one table described in 1-9 in conjunction with arbitrary sequence any among rabbit, rat, mice or the human serum albumin or combination.According to another embodiment, SABM comprises the arbitrary sequence in conjunction with the serum albumin of multiple species of at least one table described in 1-9.The example of multiple species conjugate comprises that those are at least in conjunction with people and the albuminised SABM of rat blood serum; Those are at least in conjunction with people, rat and rabbit serum albumin; Those are at least in conjunction with people and rabbit serum albumin; And those are albuminised in conjunction with people and mice serum at least.
According to an embodiment preferred, the SABM peptide is can be in conjunction with the aminoacid sequence of albuminised non-natural existence.SABM in the context of the invention can be less than about 50 amino acid residues, preferably (that is to say less than the affined of about 40 amino acid residues, have some structural details, for example, the aminoacid that has initial β-corner or beta-pleated sheet, perhaps for example, the existence of the Cys residue by becoming disulfide bond and cyclisation) or unconfined (linearity) aminoacid sequence.Less than the SABM of about 40 amino acid residues preferably about 10 to the SABM between about 30 amino acid residues, the SABM of particularly about 20 amino acid residues.But, reading disclosure postscript, those skilled in the art will be appreciated that the length that is not specific SABM, but itself and the bonded ability of albumin just can be used to distinguish SABM of the present invention.
" targeting agent " of the present invention or " TA " can be with enough affinitys and specificity in conjunction with the target molecule on the cell surface, if TA is external and " go back to the nest " preferably in vivo to, " combination " or " targeting " target molecule, such as the words of the particular cell types of carrying target molecule (referring to for example, Pasqualini and Ruoslahti, 1996 Nature, 380:364-366 and Arap etc., 1998 Science, the application of term among the 279:377-380 " go back to the nest to ", " going back to the nest " and " targeting ").Generally speaking, TA can be to be characterized as dissociation constant K
dLess than about 10 μ M, preferably less than about 100nM with less than the affinity binding target molecule of about 10nM.But, in the context of the invention, target molecule had less than the polypeptide or the micromolecule of about 1nM and preferred affinity between about 1pM and 1nM may be competent at TA equally.Preferably, TA is polypeptide (as an antibody).Generally speaking, can by any many technical points known in the art from and identify above-mentioned and the bonded TA of certain target molecules.
TA is the above-mentioned the aminoacid sequence natural and amino acid residue that non-natural exists that contains, such as the deutero-antibody of phage display.In the context of the invention, so-calledly comprise that " peptide mimics " and " peptide analogues " of the non-amino acid chemical structure of simulation specific amino acids or peptide can be TA.Such analogies or analog are characterized by usually and represent in the peptide homologue that sees them, are present in the similar physical characteristic in the suitable spatial orientation, such as size, electric charge or hydrophobicity.An object lesson of peptide simulated compound be between wherein one or more aminoacid amido link for chemical compound that for example carbon-carbon bond or other keys well-known in the art replaced (referring to for example Sawyer, 1995, In:PeptideBased Drug Design pp.378-422, ACS, Washington DC).
" B cell surface marker " or " B cell surface antigen " refers to express on the B cell surface in this article, available its antigen of antagonist targeting in conjunction with it.Exemplary B cell surface marker comprises CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 leukocyte surface sign, and (description is referring to " The Leukocyte Antigen Facts Book ", the 2nd edition, 1997, people such as Barclay compile, AcademicPress, Harcourt Brace ﹠amp; Co., New York).Other B cell surface marker comprises RP105, FcRH2, CD79A, C79B, CR2, CCR6, CD72, P2X5, HLA-DOB, CXCR5, FCER2, BR3, BTLA, NAG14 (aka LRRC4), SLGC16270 (ala LOC283663), FcRH1, IRTA2, ATWD578 (aka MGC15619), FcRH3, IRTA1, FcRH6 (akaLOC343413) and BCMA (aka TNFRSF17).
Interested especially B cell surface marker is preferentially expressed than mammiferous other non-B cell tissue on the B cell, and can express on the two at precursor B cell and mature B cell.Preferred herein B cell surface marker is CD20 and CD22.
" CD20 " antigen is the non-glycosylated phosphoprotein of finding on 90% the B cell surface from peripheral blood or lymphoid organ surpassing.CD20 expresses in the pre B lymphocyte growth course in early days, and remains to the plasma cell differentiation.CD20 is present in normal B cell and Malignant B cell on the two.CD20 other title in the literature comprises " bone-marrow-derived lymphocyte limited antigen " B1 and " Bp35 ".CD20 antigen is recorded in for example Clark et al., PNAS (USA) 82:1766 (1985).The aminoacid sequence of people CD20 is shown in " The Leukocyte Antigen Facts Book ", and people such as Barclay see above, the 182nd page; And EMBL gene bank numbering X12530 and Swissprot P11836.
" CD22 " antigen is also referred to as BL-CAM or Lyb8, is the 1 type conformity membrane glycoproteins of about 130 (shortenings) of molecular weight to 140kD (not shortening).It is expressed in the two at the Cytoplasm of bone-marrow-derived lymphocyte and cell membrane.CD22 antigen is in the early stage appearance of B cell lymphocyte differentiation, and is big about the stage identical with CD19 antigen.Different with other B cell sign, the CD22 film expression is confined to differentiation late period of comprising between mature B cell (CD22+) and the plasma cell (CD22-).CD22 antigen is recorded in for example Wilson et al., J. Exp.Med. 173:137 (1991) and Wilson et al., J.Immunol.150:5013 (1993).
" CD19 " antigen refers to the antigen (Kiesel etal., Leukemia Research II 12:1119 (1987)) by for example HD237-CD19 or the evaluation of B4 antibody.CD19 sees on pro B lymphocyte, pre B lymphocyte, immature and sophisticated, the activated and memory B cell, that time before not being divided into plasma cell eventually.CD19 and CD20 neither express on hematopoietic stem cell or plasma cell.Antagonist can cause the antigenic internalization of CD19 to the combination of CD19.The aminoacid sequence of people CD19 is shown in " The Leukocyte Antigen Facts Book ", and people such as Barclay see above, the 180th page; And EMBL gene bank numbering M28170 and Swissprot P11836.
When being used for this paper, " the B cell is subdued " refers to after medicine or antibody treatment, compares the reduction of b cell level among the animal or human with the level before handling.B cell level can use known algoscopy to measure, such as by obtaining complete blood count (cbc), and by to the painted facs analysis of known B cell marking, and by such as the method for describing in the EXPERIMENTAL EXAMPLE.The B cell subdue can be the part or completely.In one embodiment, the subduing of B cell of expression CD20 is at least 25%.Subdue among the patient of medicine accepting the B cell, the persistent period that the B cell is subdued normally medicine in the time of patient's body-internal-circulation and the time of B cellular-restoring.
Therefore, term " aminoacid " uses with its broad sense within the scope of the invention, and intention comprises naturally occurring L a-amino acid or residue.Natural exist amino acid whose single-letter commonly used and trigram abbreviation (Lehninger, A.L., 1975, " Biochemistry ", the 2nd edition, 71-92 page or leaf, Worth Publishers, New York) have been used herein.Corresponding relation between standard single-letter code and the standard trigram code is that those of skill in the art are well-known, repeats in this: A=Ala; C=Cys; D=Asp; E=Glu; F=Phe; G=GIy; H=His; I=Ile; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=VaI; W=Trp; Y=Tyr.This term comprise D-aminoacid and through the aminoacid of chemical modification such as amino acid analogue, do not mix the proteinic natural aminoacid that exists usually such as nor-leucine, and have the chemosynthesis chemical compound of the characteristic for amino acid characteristics known in the art.For example, allow that the Phe of identical with crude benzene alanine or the proline conformation restriction to peptide compounds or Pro analog or analogies are included in the amino acid whose definition.Such analog and analogies are referred to herein as amino acid whose " functional equivalent ".Amino acid whose other example is listed in Roberts and Vellaccio, and 1983, at " The Peptides:Analysis, Synthesis, Biology " in, Gross and Meiehofer compile, the 5th volume, the 341st page, Academic Press, Inc., N.Y. is collected herein by reference.
Be not limited to the aminoacid of gene code by for example synthetic SABM of standard solid-phase synthetic technology and TA.Usually run into, be not that the genetic code amino acids coding for example comprises and putting down in writing among those international publication numbers WO90/01940, such as for example for the 2-aminoadipic acid (Aad) of Glu and Asp; 2-diaminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric acid (Abu) for Met, Leu and other aliphatic amino acid; 2-aminoheptylic acid (Ahe) for Met, Leu and other aliphatic amino acid; 2-aminoisobutyric acid (Aib) for Gly; Cyclohexylalanine (Cha) for Val, Leu and Ile; Homoarginine (Har) for Arg and Lys; For 2 of Lys, Arg and His, 3-diaminopropionic acid (Dpr); Ethylglycocoll (EtGly) for Gly, Pro and Ala; Ethylglycocoll (EtGly) for Gly, Pro and Ala; N-ethyl asparagine (EtAsn) for Asn and Gln; Oxylysine (Hyl) for Lys; Allohydroxylysine (AHyl) for Lys; For 3-(and 4-)-hydroxyproline of Pro, Ser and Thr (3Hyp, 4Hyp); Alloisoleucine (AIle) for Ile, Leu and Val; For Ala to Amidinophenylalaninederivatives; Sarcosine (MeGly, sarcosine) for Gly, Pro and Ala; N-methyl isoleucine (MeIle) for Ile; Norvaline (Nva) for Met and other aliphatic amino acid; Nor-leucine (Nle) for Met and other aliphatic amino acid; Ornithine (Orn) for Lys, Arg and His; Citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn and Gln; N-methylbenzene alanine (MePhe), trimethylbenzene alanine, halo (F, Cl, Br and I) phenylalanine, three fluorenyl phenylalanine for Phe.
SABM and TA can be " (engineering) transformed " in linguistic context of the present invention, promptly can right and wrong TA natural or that non-natural exists." non-natural " or " non-natural exists " means that the aminoacid sequence of specific SABM can not find at occurring in nature.That is to say that TA that non-natural or non-natural exists or the aminoacid sequence of SABM need not corresponding to natural protein or the amino acid sequence of polypeptide of existing.This TA or SABM can use multiple technologies to generate or select, and comprise that those those of skill in the art are well-known.For example, utilize this area standard technique, Lowman et al. for example, 1998, Biochemistry 37:8870-8878 can produce at random on phage and show and is tied or unconfined peptide library.
When SABM and TA and cytotoxic agent use in linguistic context of the present invention " coupling " each other.Term " coupling " uses with its broad sense, contains all methods of covalent attachment known in the art or connection.For example, in a typical embodiment, SABM is a kind of protein, and TA is that the C-of SABM or one section aminoacid of N-end extend.In addition, lacking the aminoacid joint sequence can be between protein therapeutic agent and SABM.In this case, SABM, optional joint and TA will be by such nucleic acid coding, and it comprises the sequence of the SABM that encodes and the sequence of its joint sequence and TA that encodes that can be operatively connected (be successive and be in the reading frame with regard to DNA sequence) optional coding short polypeptide as described below.In this typical case, think that SABM and TA are optional through joint sequence " coupling ".In a related embodiment, the SABM aminoacid sequence can interrupt or replace the part of TA aminoacid sequence, certainly, if the insertion of the SABM aminoacid sequence words of the function of interferencing protein therapeutic agent not.In another typical embodiment, SABM will choose wantonly with TA or other therapeutic agent and be connected by for example chemical coupling through joint sequence.Be typically, according to this embodiment, SABM will not disturb the amino acid whose side chain in somewhere of the active ability of TA identification target to be connected with TA through the TA middle part.Once more, think SABM and TA " coupling ".
Term " antibody " uses with broad sense, specifically covers monoclonal antibody (comprising full length monoclonal antibodies), polyclonal antibody, multi-specificity antibody (as bi-specific antibody) and antibody fragment, as long as they represent the expectation biologic activity.
" functional fragment " of antibody of the present invention comprises the part of complete antibody, generally includes the antigen binding domain of complete antibody or the antibody Fc district of variable region or reservation FcR binding ability.The example of antibody fragment comprises linear antibody; The single-chain antibody molecule; With the multi-specificity antibody that forms by antibody fragment.
Term " monoclonal antibody " refers to that when being used for this paper each antibody that promptly constitutes colony is identical from a group antibody that obtains of the antibody of homogeneity basically, except may be with indivisible possible natural the existence the sudden change that exists.Monoclonal antibody is a high special, and every kind at one or both antigenic sites, normally a kind of site.In addition, and make several antibody at routine (polyclone) the antibody preparations difference of different determinants (epi-position) at a kind of antigen by animal derived, every kind of monoclonal antibody is at the single determinant on the antigen.Outside their specificity, the advantage of monoclonal antibody is that they are synthetic by the hybridoma cultivation, are not subjected to the pollution of other immunoglobulin.Modifier " monoclonal " indication antibody should not be construed as requirement and generates antibody by any ad hoc approach from the feature that the antibody population of homogeneity basically obtains.For example, will can pass through at first by Kohler et al. according to the monoclonal antibody that the present invention uses, the hybridoma method of Nature 256:495 (1975) record prepares, and perhaps can prepare (referring to as United States Patent (USP) 4,816,567) by recombinant DNA method." monoclonal antibody " also can use for example Clackson et al., and the technology of record is separated from phage antibody library among Nature 352:624-628 (1991) and the Marks et al., J. Mol. Biol.222:581-597 (1991).
Monoclonal antibody clearly comprises " chimeric " antibody (immunoglobulin) in this article, wherein the part of heavy chain and/or light chain with derived from specific species or belong to the identical or homology of corresponding sequence in the antibody of specific antibodies class or subclass, and the remainder of chain with derived from another species or belong to the identical or homology of corresponding sequence in the antibody of another antibody class or subclass, and the fragment of such antibody, as long as they represent expectation biologic activity (United States Patent (USP) 4,816,567; Morrison et al., Proc.Natl.Acad.Sci.USA, 81:6851-6855 (1984)).The method for preparing chimeric antibody is known in the art.
" humanization " form of inhuman (as Mus) antibody is to comprise gomphosis immunoglobulin, immunoglobulin chain or its fragment derived from the minimum sequence of non-human immunoglobulin (such as Fv, Fab, Fab ', F (ab ')
2Or other antigen zygote sequence of antibody).Largely, humanized antibody is such human normal immunoglobulin's (receptor antibody), and wherein the residue of receptor complementary determining region (CDR) is replaced by the residue of inhuman species (donor antibody) such as the CDR of mice, rat or rabbit with expectation specificity, affinity and ability.In some situation, human normal immunoglobulin's Fv framework region (FR) residue is replaced by corresponding inhuman residue.In addition, humanized antibody can be included in the residue that does not all find in receptor antibody and input CDR or the framework sequence.Carrying out these modifies with further improvement and maximization antibody performance.Generally speaking, humanized antibody will comprise at least one, common two whole basically variable regions, wherein all or basically all hypermutation rings corresponding to those of non-human immunoglobulin, and all or basically all FR districts are those of human normal immunoglobulin's sequence, although the FR district can comprise the amino acid replacement that binding affinity is improved in a place or many places.The number of these amino acid replacements is no more than 6 usually among the FR in the H chain, is no more than 3 in the L chain.Humanized antibody preferably also will comprise at least a portion of constant region for immunoglobulin (Fc), normally the human normal immunoglobulin.More details are referring to Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); Presta, Curr.Op.Struct.Biol.2:593-596 (1992).Humanized antibody comprises PRIMATIZED
Antibody, wherein the antigen binding domain of antibody is derived from the antibody by for example producing with purpose antigen immune macaque.The method for preparing humanized antibody is known in the art.
Also can use multiple technologies known in the art to generate people's antibody, comprise the phage display storehouse.Hoogenboom and Winter，J. Mol. Biol.，227：381(1991)；Marks et al.，J.Mol.Biol.，222：581(1991)。People's such as people such as Cole and Boerner technology also can be used for preparing human monoclonal antibodies.People such as Cole, " Monoclonal Antibodies and Cancer Therapy ", Alan R.Liss, the 77th page, 1985; Boerner et al., J. Immunol.147 (1): 86-95 (1991).
When being used for this paper, term " immunoadhesin " refers to antibody sample molecule that the effector function of the binding specificity of heterologous protein (" adhesin ") and constant region for immunoglobulin is joined together.Structurally, immunoadhesin comprises the antigen recognition that is different from antibody and binding site (promptly being " allos "), has the aminoacid sequence of expectation binding specificity and the fusions of constant region for immunoglobulin sequence.The adhesin part of immunoadhesin molecule normally comprises the continuous amino acid sequence of the binding site of receptor or part at least.Constant region for immunoglobulin sequence in the immunoadhesin can obtain from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 hypotype, IgA (comprising IgA-1 and IgA-2), IgE, IgD or IgM.
The polypeptide that " fusion rotein " and " fused polypeptide " refers to have at least two covalently bound parts, wherein each part is the polypeptide with different qualities.Described characteristic can be a biological characteristics, such as external or activity in vivo.Described characteristic can also be simple chemistry or physical characteristic, such as with the combining of target molecule, catalytic reaction etc.Described part can directly connect by single peptide bond, perhaps via the peptide linker that comprises one or more amino acid residues.Usually, described part and joint will be in the reading frame each other.
" isolating " polypeptide or antibody refer to identify and with/separate and/or reclaim by a kind of composition of its natural surroundings.The contaminative composition of its natural surroundings refers to disturb the diagnosis of this polypeptide or antibody or the material of therapeutic use, can comprise the solute of enzyme, hormone and other protein properties or nonprotein character.In preferred embodiments, with antibody purification to (1) mensuration according to the Lowry method, antibody weight surpasses 95%, most preferably weight surpasses 99%, (2) be enough to by using the rotary-cup type sequenator to obtain the N-end of at least 15 residues or the degree of internal amino acid sequence, or (3) reach homogeneity according to the SDS-PAGE that uses under Coomassie blue or preferred silver-colored painted reproducibility or the irreducibility condition.Since at least a composition of antibody natural surroundings will can not exist, isolated antibody comprises the original position antibody in the reconstitution cell so.Yet isolated antibody will prepare by at least one purification step usually.
Humanization anti-ErbB (HER2) antibody comprises as United States Patent (USP) 5,821, huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and the huMAb4D5-8 (HERCEPTIN ) of record clearly are collected herein by reference in 337 tables 3; Humanization 520C9 (WO93/21319) and humanization 2C4 antibody as record in the common pending application serial number 09/811115, and comprise in WO03/087131 and the U.S. Patent Publication No. 2003/0228663 antibody of the variable region of the anti-HER2 variant that discloses, be collected herein by reference.In the open book of entire chapter, term " huMAb4D5-8 " and " hu4D5-8 " are used interchangeably.
Term " cytotoxic agent " refers to suppress when being used for this paper or prevents the function of cell and/or cause the material of cytoclasis.Cytotoxic agent should internalization and/or can be need not in conjunction with cell surface from the extracellular cell growth inhibiting.According to an embodiment preferred, this medicament is a micromolecule.According to another embodiment, the active part of cytotoxic agent is 1100 dalton or littler.This term intention comprises: radiosiotope, and as At
211, I
131, I
125, y
90, Re
186, Re
188, Sm
153, Bi
212, Bi
213, P
32Radiosiotope with Lu; Chemotherapeutics, for example methotrexate (methotrexate), amycin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorambucil), daunorubicin (daunorubicin) or other intercalator; Enzyme and fragment thereof are such as nucleolytic enzyme; Antibiotic; And toxin, such as the enzyme of micromolecule toxin or antibacterial, fungus, plant or animal origin toxin (as MMAE) alive, comprise its fragment and/or variant; And the various antitumor or anticarcinogen or the growth inhibitor that hereinafter disclose.Other cytotoxic agent has hereinafter been described.According to an embodiment preferred, cytotoxic agent is not a radiosiotope.
" chemotherapeutics " refers to can be used for treating the chemical compound of cancer.The example of chemotherapeutics comprises alkylating agent class (alkylating agents), replaces such as plug and sends (thiotepa) and CYTOXAN cyclophosphamide (cyclophosphamide); Sulfonic alkyl esters (alkyl sulfonates) is such as busulfan (busulfan), an improsulfan (improsulfan) and piposulfan (piposulfan); Aziridines (aziridines) is such as benzodepa (benzodepa), carboquone (carboquone), U.S. appropriate in sending (meturedepa) and uredepa (uredepa); Ethylenimine class (ethylenimines) and methylmelamine class (methylamelamines) comprise altretamine (altretamine), triethylenemelamine (triethylenemelamine), phosphoric acid triethyleneimide (triethylenephosphoramide), TESPA (triethylenethiophosphoramide) and trimethylolmelamine (trimethylolomelamine); Annonaceousacetogenicompounds (acetogenin) (especially bullatacin (bullatacin) and bullatacin ketone (bullatacinone)); Camptothecine (camptothecin) (comprising synthetic analogues hycamtin (topotecan)); Bryostatin (bryostatin); Callystatin; CC-1065 (comprising its adozelesin (adozelesin), carzelesin (carzelesin) and bizelesin (bizelesin) synthetic analogues); Latent algin class (cryptophycins) (particularly latent algin 1 and latent algin 8); Dolastatin (dolastatin); Duocarmycin (comprising synthetic analogues, KW-2189 and CB1-TM1); Eleutherobin. (eleutherobin); Pancratistatin; Sarcodictyin; Sponge chalone (spongistatin); Nitrogen mustards (nitrogen mustards) is such as chlorambucil (chlorambucil), chlornaphazine (chlornaphazine), gallbladder phosphamide (cholophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine), mustron (mechlorethamine oxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard (uracil mustard); Nitrosourea (nitrosoureas) is such as carmustine (carmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and Ranimustine (ranimustine); Antibiotics, such as enediyne class antibiotic (enediyne) (as calicheamicin (calicheamicin), especially calicheamicin γ 1I and calicheamicin ω I1 (referring to as Agnew, Chem.Intl.Ed.Engl.33:183-186 (1994)); Anthracycline antibiotics (dynemicin) comprises dynemicin A; Diphosphonates (bisphosphonates) is such as clodronate (clodronate); Ai Sibo mycin (esperamicin); And Neocarzinostatin (neocarzinostatin) chromophore and related color albumen enediyne class antibiotic chromophore), aklavine (aclacinomycin), D actinomycin D (actinomycin), anthramycin (anthramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), carabicin, carminomycin (carminomycin), cardinophyllin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), detorubicin (detorubicin), 6-phenodiazine-5-oxygen-L-nor-leucine, ADRIAMYCIN doxorubicin (doxorubicin) (comprises the morpholino doxorubicin, cyano group morpholino doxorubicin, 2-pyrroles is for doxorubicin and deoxidation doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycins) is such as ametycin, mycophenolic acid (mycophenolic acid), nogalamycin (nogalamycin), Olivomycin (olivomycin), peplomycin (peplomycin), potfiromycin, puromycin (puromycin), triferricdoxorubicin (quelamycin), rodorubicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), zinostatin (zinostatin), zorubicin (zorubicin); The antimetabolite class is such as methotrexate (methotrexate) and 5-fluorouracil (5-FU); Folacin is such as 9,10-dimethylpteroylglutamic acid (denopterin), methotrexate (methotrexate), pteroyltriglutamic acid (pteropterin), trimetrexate (trimetrexate); Purine analogue is such as fludarabine (fludarabine), Ismipur (mercaptopurine), ITG (thiamiprine), thioguanine (thioguanine); Pyrimidine analogue is such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytosine arabinoside (cytarabine), two BrdU (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); Androgens is such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), mepitiostane (mepitiostane), testolactone (testolactone); Anti-adrenal gland's class is such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); Folic acid supplement is such as folinic acid (folinic acid); Aceglatone (aceglatone); Aldophosphamide glucosides (aldophosphamide glycoside); Amino-laevulic acid (aminolevulinic acid); Eniluracil (eniluracil); Amsacrine (amsacrine); Bestrabucil; Bisantrene (bisantrene); Edatrexate (edatraxate); Defosfamide (defosfamide); Demecolcine (demecolcine); Diaziquone (diaziquone); Elfornithine; Elliptinium acetate (elliptinium acetate; Epothilones (epothilone); Etoglucid (etoglucid); Ganite (Fujisawa).; Hydroxyl urea (hydroxyurea); Lentinan (lentinan); Lonidamine (lonidamine); Maytansinoid class (maytansinoids) is such as maytansine (maytansine) and maytansinol (maytansinol); Ansamitocin (ansamitocin); Mitoguazone (mitoguazone); Mitoxantrone (mitoxantrone); Mopidamol (mopidamol); C-283 (nitracrine); Pentostatin (pentostatin); Phenamet (phenamet); Pirarubicin (pirarubicin); Losoxantrone (losoxantrone); Podophyllinic acid (podophyllinic acid); 2-ethyl hydrazides (ethylhydrazide); Procarbazine (procarbazine); PSK polysaccharides compound (JHS Natural Products, Eugene, OR); Razoxane (razoxane); Rhizomycin (rhizoxin); Sizofiran (sizofiran); Spirogermanium (spirogermanium); Tenuazonic acid (tenuazonic acid); Triaziquone (triaziquone); 2,2 ', 2 " RA3s; Trichothecin class (trichothecenes) (especially T-2 toxin, verrucarine (verrucarin) A, roridin (roridin) A and the rhzomorph (anguidin) that crawls); Urethane (urethan); Vindesine (vindesine); Dacarbazine (dacarbazine); Mannomustin (mannomustine); Mitobronitol (mitobronitol); Mitolactol (mitolactol); Pipobroman (pipobroman); Gacytosine; Cytosine arabinoside (arabinoside) (" Ara-C "); Cyclophosphamide (cyclophosphamide); Plug is for sending (thiotepa); Taxoid (taxoids), as TAXOL paclitaxel (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANETM does not contain nano-particle dosage form paclitaxel (the American Pharmaceutical Partners of cremophor (Cremophor), albumin transformation, Schaumberg, Illinois) and the many Xi Tasai of TAXOTERE (doxetaxel) (Rh ne-Poulenc Rorer, Antony, France); Chlorambucil (chlorambucil); GEMZAR gemcitabine (gemcitabine); 6-thioguanine (thioguanine); Purinethol (mercaptopurine); Methotrexate (methotrexate); Platinum analogs is such as cisplatin (cisplatin) and carboplatin (carboplatin); Vinblastine (vinblastine); Platinum; Etoposide (etoposide) (VP-16); Ifosfamide (ifosfamide); Mitoxantrone (mitoxantrone); Vincristine (vincristine); NAVELBINE vinorelbine (vinorelbine); NSC-279836 (novantrone); Teniposide (teniposide); Edatrexate (edatrexate); Daunomycin (daunomycin); Aminopterin (aminopterin); Xeloda (xeloda); Ibandronate (ibandronate); CPT-11; Topoisomerase enzyme inhibitor RFS 2000; Er Fujiajiniaoansuan (DMFO); Class tretinoin (retinoids) is such as tretinoin (retinoic acid); Capecitabine (capecitabine); Oxaliplatin (oxaliplatin) comprises oxaliplatin treatment scheme (FOLFOX); The inhibitor of PKC-α, Raf, H-Ras and EGFR is (as erlotinib (Tarceva
TM)); And the acceptable salt of pharmacopedics, acid or the derivant of any above-mentioned substance.
" growth inhibitor " refers to when being used for this paper in external and/or cytostatic in vivo chemical compound or compositions.Therefore, growth inhibitor can be the medicament that significantly reduces the cell percentage ratio that is in the S phase.The example of growth inhibitor comprises the advance medicament of (be in S phase beyond position) of blocking-up cell cycle, such as inducing the medicament that G1 stagnates and the M phase stagnates.Classical M phase blocker comprises Changchun medicine class (vincas) (vincristine (vincristine) and vinblastine (vinblastine)), TAXOL paclitaxel (paclitaxel) and topoisomerase II inhibitor such as doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide) and bleomycin (bleomycin).Medicaments of those retardances G1 also overflow and enter the S phase and stagnate, for example DNA alkylating agent class such as tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), chlormethine (mechlorethamine), cisplatin (cisplatin), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil) and ara-C.More information can be referring to " The Molecular Basis ofCancer) ", Mendelsohn and Israel compile, the 1st chapter is entitled as " Cell cycle regulation, oncogenes; and antieioplastic drugs ", people such as Murakaini, WB Saunders, Philadelphia, 1995, especially the 13rd page.
The example of " growth inhibitor " comprises that EGF-R ELISA (EGFR) antagonist (as tyrosine kinase inhibitor), HER1/EGFR inhibitor are (as erlotinib (Tarceva
TM)), the platelet derived growth factor inhibitor is (as Gleevec
TM(imatinib mesylate (Imatinib Mesylate))), cox 2 inhibitor (as celecoxib (celecoxib)) and other biological activity and organic chemistry agent etc.
Term " treatment effective dose " refers to disease or disorderly amount among conjugate molecule effective " alleviating " or " disposal " experimenter.Can prevent existing growth of cancer cells and/or kill with regard to it that with regard to the conjugate molecule it can be to suppress cell and/or cytotoxinic (Cytotoxic).
" disposal " or " treatment " refers to disease or disorderly improvement or alleviation.Need the experimenter who disposes to comprise suffering from disorderly experimenter for a long time and will prevent disorderly experimenter.If behind the conjugate according to the method amount of receiving treatment of the present invention, the patient demonstrates in one of specified disease or multinomial sign and symptom and can observe and/or measurable reduction or disappearance, so experimenter's success " treatment " cancer or autoimmune disease.For example, for cancer, the cancerous cell number reduces or cancerous cell disappears; Gross tumor volume dwindles; Neoplasm metastasis is suppressed (promptly slowing down to a certain degree preferably stops); Tumor growth is subjected to inhibition to a certain degree; Prolong paracmasis; And/or one or more symptoms relevant with particular cancers obtain alleviating to a certain degree; M ﹠ M reduces; And quality of life improves.Alleviating of the sign of disease or symptom can also be experienced by the patient.Disposal can realize responding fully, and all signs that are defined as cancer disappear, perhaps partial response, and wherein gross tumor volume dwindles, and preferably surpasses 50%, and more preferably 75%.If conditions of patients is stable, this patient also is considered as obtaining medical treatment so.In a preferred embodiment, the cancer patient is after 1 year, and preferably cancer is not still made progress after 15 months.These parameters that are used for the successful treatment of assess disease and improve are easy to have by this area the old process that the doctor the was familiar with measurement of suitable technical ability.In a preferred embodiment, the experimenter who disposes with the identical conjugate molecule that can accept to lack SABM compares, and the experimenter shows uncomfortable improvement, and the side effect that stands simultaneously still less.
Term " cancer " and " carcinous " refer to or describe typically to grow with cell in the mammal be not adjusted to the physiological situation of feature.The example of cancer includes but not limited to cancer, lymphoma, blastoma, sarcoma and leukemia or lymph sample malignant tumor.The more specifically example of this type of cancer comprises squamous cell carcinoma (as the epithelium squamous cell carcinoma), pulmonary carcinoma comprises small cell lung cancer, nonsmall-cell lung cancer, the adenocarcinoma of lung and the scale cancer of lung, peritoneal cancer, hepatocarcinoma, gastric cancer comprises human primary gastrointestinal cancers, cancer of pancreas, glioblastoma, cervical cancer, ovarian cancer, hepatocarcinoma, bladder cancer, the urinary tract cancer, hepatoma (hepatoma), breast carcinoma, colon cancer, rectal cancer, colorectal carcinoma, endometrium or uterus carcinoma, salivary-gland carcinoma, renal carcinoma, carcinoma of prostate, carcinoma vulvae, thyroid carcinoma, the cancer of liver, anus cancer, carcinoma of penis, melanoma, multiple myeloma and B cell lymphoma, the brain cancer, and head and neck cancer, and associated transitions.
Term " cell proliferative disorders " refers to the disorder relevant with abnormal cell proliferation to a certain degree with " proliferative disorders ".In one embodiment, cell proliferative disorders is a cancer.
" tumor " refers to growth of all tumor sexual cells and propagation when being used for this paper, no matter be virulent or benign, and (pre-cancerous) and cancerous cells and tissue before all cancers.
The autoimmune disease that the B cell is regulated comprises arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, and dermatitis comprises atopic dermatitis; Chronic autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic sclerosis and sclerosis, with relevant the replying of inflammatory bowel (IBD) (Crow engler (Crohn) disease, ulcerative colitis), respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, allergic rhinitis, encephalitis, uveitis, colitis, glomerulonephritis, the allergia situation, eczema, asthma, involve the situation that T cellular infiltration and chronic inflammatory are replied, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (sle) (SLE), lupus (comprises lupus nephritis, non-kidney lupus, discoid lupus, the lupus alopecia), children's hair style diabetes, multiple sclerosis, allergic encephalitis, the relevant immunne response of acute and delayed hypersensitivity (DH) with cytokine and T-cell mediated, tuberculosis, sarcoidosis, granulomatosis comprises Wei Genashi (Wegener) granulomatosis, agranulocytosis, vasculitis (comprising ANCA), aplastic anemia, the positive anemia of Ku Musishi (Coombs), wear-go on foot Er Shi (Diamond Blackfan) anemia, immune hemolytic anemia comprises autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor IX lacks, hemophilia A, the autoimmunity neutrophil cell reduces disease, pancytopenia, leukopenia, involve the disease that leukocyte oozes out, the disorder of CNS inflammatory, multiple organ injury's syndrome, myasthenia gravis, the disease of antigen-antibody complex mediation, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bei Qieteshi (Bechet) disease, Ka Siermanshi (Castleman) syndrome, Gu Depasiqiushi (Goodpasture) syndrome, Lambert-Eton (Lambert-Eaton) myasthenic syndrome, Lei Nuoshi (Reynaud) syndrome, Si Yegelunshi (Sjogren) syndrome, Shi-Yue Er Shi (Stevens-Johnson) syndrome, solid organ transplantation repels and (comprises that pretreatment is at high response antibody titer group, IgA deposition in the tissue etc.), graft versus host disease (GVHD), bullous pemphigoid, pemphigus (all comprises homeliness type, defoliation), autoimmune polyendocrine disease, Lay Te Shi (Reiter) disease, stiff man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, the neuropathy of IgM polyneuropathy or IgM mediation, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), AT, the autoimmune disease of testis and ovary comprises autoimmunity orchitis and oophoritis, primary hypothyroidism disease; Autoimmune endocrinopathy, comprise autoimmune thyroiditis, chronic thyroiditis (Hashimoto disease (Hashimoto) thyroiditis), subacute thyroiditis, the special property sent out hypothyroidism, A Disenshi (Addison) disease, Ge Leifusishi (Graves) disease, autoimmune polyglandular syndrome (or polyadenous body endocrinopathy syndrome), type i diabetes is also referred to as insulin-dependent diabetes (IDDM) and seat Han Shi (Sheehan) syndrome; Autoimmune hepatitis, LIP (HIV), bronchiolitis obliterans (Nonimplantation) is than NSIP, Ge-Ba Er Shi (Guillain-Barre) syndrome, trunk vasculitis (comprising polymyalgia rheumatica and giant cell (high iS-One (Takayasu)) arteritis), medium vessels vasculitis (comprising Chuan Qishi (Kawasaki) disease and polyarteritis nodosa), ankylosing spondylitis, Bei Geershi (Berger) sick (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, sprue (gluten enteropathy), cryoglobulinemia, ALS, coronary artery disease.
The B glucagonoma comprises positive He Jiejinshi (Hodgkin) disease of CD20, comprises the Hokdkin disease (LPHD) of lymphocytic predominance; Non_hodgkin lymphoma (NHL); FCC (FCC) lymphoma; Acute lymphoblastic leukemia (ALL); Chronic lymphocytic leukemia (CLL); Hairy cell leukemia.Non_hodgkin lymphoma comprises rudimentary/folliculus non_hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, middle rank/folliculus NHL, intermediate diffusivity NHL, senior immunoblast NHL, senior lymphoblast NHL, senior small non-cleaved cell NHL, thesaurismosis (bulky disease) NHL, Plasmacytoid lymphocytic lymphoma, lymphoma mantle cell, the relevant lymphoma of AIDS and Walden Si Telunshi (Waldenstrom) macroglobulinemia.Also imagined the disposal of these cancer returns.LPHD is a class still is tending towards frequent recurrence after radiation or chemotherapy disposal a Hokdkin disease, is characterized as the male malignant cell of CD20.CLL is one of leukemic four kinds of main types.A kind of cancer that is called lymphocytic mature B cell, CLL shows as cell progressive accumulation in blood, bone marrow and lymphoid tissue.Indolent lymphoma is a kind of incurable disease of slow development, and wherein at a plurality of alleviations and recurrence after date, patient's average survival is 6-10.
For the purpose of disposing, " mammal " aim is gone into mammiferous any animal, comprises the people, and domestic animal and domestic animal, and zoo, motion or pet animals are such as dog, horse, cat, cattle etc.Preferably, mammal refers to the people.The experimenter that will dispose according to the present invention is a mammal.
" disorder " refers to and will benefit from any situation of the disposal of the compositions that comprises conjugate molecule of the present invention.This comprises chronic and acute disorder or disease, comprises that those make mammal easily suffer from the disorderly pathological condition of discussing.
" eliminate the half-life " and use with its common implication, as " Goodman and Gillman ' s ThePharmaceutical Basis of Therapeutics ", the 21-25 page or leaf, Alfred Goodman Gilman, Louis S.Goodman and Alfred Gilman compile, the 6th edition, described in 1980.In brief, this term intention contains the quantitative measurement that medicine is eliminated time-histories.Because it is desired that drug level does not reach those saturated elimination processes usually, so the elimination of most drug is exponential (promptly following first order kinetics).The speed of exponential process can be expressed as its speed constant, k, and its expression time per unit mark changes, and perhaps is expressed as its half-life, t
, described process finished for 50% needed time.The unit of these two constants is respectively the time
-1And the time.The first order rate constant and the half-life of reaction are simple correlation (kxt
=0.693), therefore interchangeable.Because the constant mark of first order kinetics indication time per unit medicine forfeiture, so the logarithm of drug level is linear to the curve of time in initial distribution after the stage (promptly after drug absorption and distribution are finished) always.Can accurately measure the half-life that medicine is eliminated according to such figure.According to a preferred embodiment of the invention, compare with the conjugate molecule that lacks SABM, conjugate molecule of the present invention has longer half-life and lower toxicity.
" transfection " refers to the picked-up of host cell to expression vector, no matter whether any coded sequence in fact expresses.Those of ordinary skills know multiple transfection method, for example CaPO
4Precipitation and electroporation.When occurring any sign of this carrier-mediated transport in the host cell, it is generally acknowledged that transfection is successful.
" conversion " refers to that DNA is imported organism makes DNA reproducible, or forms part as extra-chromosomal element or as chromosome.According to used host cell, use the standard technique that is suitable for this type of cell to transform.As people such as Sambrook, 1989, " Molecular Cloning ", and the 2nd edition, Cold Spring Harbor Laboratory, 1.82 joints of NY are described, adopt the calcium of calcium chloride to handle other cell that is generally used for prokaryote or has firm cell wall barrier.As Shaw et al., Gene 23:315 (1983) and on June 29th, 1989 disclosed WO89/05859 described, use the infection of Agrobacterium tumefaciems (Agrobacterium tumefaciens) to be used to transform the certain plants cell.For the mammalian cell that does not have this type of cell wall, people such as preferred Sambrook, the calcium phosphate precipitation method described in the 16.30-16.37 that sees above saves.The ordinary circumstance that the mammalian cell host system transforms has been recorded in the United States Patent (USP) 4,399,216 of the Axel of bulletin on August 16 nineteen eighty-three.Zymic conversion is usually according to VanSolingen et al., J. Bact.130:946 (1977) and Hsiao et al., and the method for Proc.Natl.Acad.Sci. (USA) 76:3829 (1979) is carried out.Yet, also can use other method, such as merging by nuclear injection, electroporation or protoplast with the DNA transfered cell.
When being used for this paper, term " pulmonary's dispenser " refers to use preparaton of the present invention by sucking through lung.When being used for this paper, term " suction " refers to take in air to alveolar.In concrete example, absorption can the oneself of preparaton of the present invention be used when air-breathing, perhaps by taking place as the using of patient that gives on the respirator through respirator.When using about preparaton of the present invention, term " suction " and " pulmonary's dispenser " synonym.
When being used for this paper, term " parenteral " refers to by the approach except that intestinal chemical compound of the present invention be imported in the body, particularly in intravenous (i.v.), intra-arterial (i.a.), intraperitoneal (i.p.), intramuscular (i.m.), the ventricle and subcutaneous (s.c.) path.
When being used for this paper, term " aerosol " refers to airborne suspension.Particularly, aerosol refers to the granulating (particlization) and the aerial suspension thereof of preparaton of the present invention.According to the present invention, aerosol dosage forms is a kind of chemical compound of the present invention that comprises, and being suitable for aerosolization in air (aerosolization) is granulating and suspension, for the dosage form of suction or pulmonary's dispenser.
II. implement mode of the present invention
A.
SABM
SABM in the linguistic context of the present invention is in conjunction with albumin.Preferably the SABM in conjunction with serum albumin comprises linear peptides and cyclic peptide, preferably comprises the cyclic peptide chemical compound of following formula or for combine the peptide of the serum albumin of specific mammalian species with the peptide competition of following formula:
Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa
Phe-Cys-Xaa-Asp-Typ-Pro-Xaa-Xaa-Xaa-Ser-Cys[SEQ ID NO：1]
Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe[SEQ ID NO：2]
Cys-Tyr-Xaa1-Pro-Gly-Xaa-Cys[SEQ ID NO：3]
Know Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp[SEQ ID NO:4]
Preferably at N-end (Xaa)
xWith C-end (Xaa)
zThe peptide compounds that comprises extra amino acid whose aforementioned formula, wherein Xaa is an aminoacid, x and z are the integers more than or equal to 0 (zero), be generally less than 100, preferably less than 10, more preferably 0,1,2,3,4 or 5, also preferred 4 or 5, and wherein Xaa is selected from Ile, Phe, Tyr and Val.
Preferred SABM in conjunction with serum albumin is defined as the following general formula described in the linguistic context of this paper:
Trp-Cys-Asp-Xaa-Xaa-Leu-Xaa-Ala-Xaa-Asp-Leu-Cys(SEQ ID NO：5)and Asp-Leu-Val-Xaa-Leu-Gly-Leu-Glu-Cys-Trp[SEQ ID NO：6]
Wherein extra aminoacid can be positioned at N-end (Xaa)
xAnd C-end (Xaa)
z, wherein Xaa is an aminoacid, x and z are the integers more than or equal to 0, are generally less than 100, and preferably less than 10, more preferably 0,1,2,3,4 or 5, also preferred 4 or 5.
According to this aspect of the present invention, selecting can be with reference to hereinafter embodiment, the particularly form that is wherein comprised in conjunction with the albuminised peptide part of mammalian blood serum, and they have shown especially exemplary peptide and suitable aminoacid.One preferred aspect, but select the SABM reference table 7 of the serum albumin of several species of cross coupled.
According to this aspect of the present invention, preferred chemical compound comprises:
DLCLRDWGCLW (SEQ ID NO：7)
DICLPRWGCLW (SEQ ID N0：8)
MEDICLPRWGCLWGD (SEQ ID NO：9)
QRLMEDICLPRWGCLWEDDE (SEQ ID NO：10)
QGLIGDICLPRWGCLWGRSV (SEQ ID NO：11)
QGLIGDICLPRWGCLWGRSVK (SEQ ID NO：12)
EDICLPRWGCLWEDD (SEQ ID NO：13)
RLMEDICLPRWGCLWEDD (SEQ ID NO：14)
MEDICLPRWGCLWEDD (SEQ ID NO：15)
MEDICLPRWGCLWED (SEQ ID NO：16)
RLMEDICLARWGCLWEDD (SEQ ID NO：17)
EVRSFCTRWPAEKSCKPLRG (SEQ ID NO：18)
RAPESFVCYWETICFERSEQ (SEQ ID NO：19)
EMCYFPGICWM (SEQ ID NO：20)
In a preferred embodiment, SABM of the present invention is in conjunction with the human serum albumin, and can in having the external test of SABM of general formula as follows, use identify in conjunction with human serum albumin's ability that by its competition extra amino acid residue can be present in N-end (Xaa) in described general formula
xWith C-end (Xaa)
z:
DXCLPXWGCLW (SEQ ID NO：4)
FCXDWPXXXSC (SEQ ID NO：1)
VCYXXXICF (SEQ ID NO：2)
CYX1PGXCX (SEQ ID NO：3)
Wherein Xaa is an aminoacid, and x and z preferred 4 or 5, and Xaa is selected from Ile, Phe, Tyr and Val.
In specific embodiment, SABM of the present invention can be with arbitrary in the SABM shown in this above-mentioned SEQ IDNO:7-20 competition, and preferably can with the combining of SEQ ID NO:10 competition and human serum albumin.
To be understanded by preamble, term " competition " is relevant term with " ability of competition ".Therefore, above-mentioned term is when being used to describe SABM of the present invention, when finger exists with 50 μ M in standard competition assay described herein, when preferably existing with 1 μ M, more preferably 100nM, and preferably with 1nM or when still less existing, to the peptide of for example SEQ ID NO:10 representative in conjunction with producing 50% SABM that suppresses.Yet, serum albumin is had less than about 1nM, preferably the SABM of the affinity of about 1pM-1nM may be the SABM in the linguistic context of the present invention equally.
Determine for being used for whether peptide or other chemical compound have the external test system that combines " ability " of serum albumin described herein with the SABM competition, and those skilled in the art can adopt any in the multiple standards competition assay.The competitive binding assay method depends on the ability that combines limited amount part through the labelling standard substance with the competition of specimen analyte.The amount of analyte is inversely proportional to the amount that becomes with the bonded standard substance of part in the specimen.
Therefore, those skilled in the art can adopt following flow process to determine whether peptide or other chemical compound have and combine albuminised ability with SABM competition, include but not limited to use the competitive assay system of following technology, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) preferred enzyme linked immunosorbent assay (ELISA), " sandwich style " immunoassay, immunoradiometry, fluorescence immunoassay and immunoelectrophoresis algoscopy, differ for this reason and one enumerate.
For these purposes, but the SABM of selection will and be used for detection module labelling (after this SABM through detectable label is called " tracer ") combining albumin with the candidate compound competition in competition assay.Can utilize multiple detectable, they can preferably be divided into following a few class:
(a) radiosiotope, such as
35S,
14C,
125I,
3H and
131I.SABM for example can use people such as Coligen to compile, and 1991, the technology labelled with radioisotope described in " the Current Protocols in Immunology ", the 1st and 2 volumes, Wiley-Interscience, New York, N.Y..Radioactivity can use scinticounting to measure.
(b) can utilize fluorescent marker, such as rare-earth chelates (europium chelate) or fluorescein and derivant, rhodamine (rhodamine) and derivant thereof, dansyl (dansyl), Liz amine (lissamine), phycoerythrin and Texas red (Texas Red).Fluorescent marker for example can use technology and the peptide compounds coupling disclosed in " CurrentProtocols in Immunology " (seeing above).Fluorescence can use exometer quantitative.
(c) can utilize multiple enzyme-substrate label, United States Patent (USP) 4,275,149 provide the summary of some of them.The enzyme preferred catalytic can be used the chemical modification of the chromogenic substrate of multiple technologies measurement.For example, but the change color of the substrate that enzyme catalysis can be by metric measurement.Perhaps, enzyme can change the fluorescence or the chemiluminescence of substrate.The technology that quantitative fluorescence changes that is used for has above been described.Chemical luminous substrate becomes by chemical reaction that electronics is excited, and can send measurable light (for example using chemiluminescence photometer (chemiluminometer)) then or award fluorescent receptor with energy.The example of enzyme labelling thing comprises that luciferase is (as Lampyridea luciferase and bacteriofluorescein enzyme; United States Patent (USP) 4,737,456), luciferin, 2,3-dihydro phthalazine diketone (dihydrophthalazinedione), malic dehydrogenase, urase, peroxidase such as horseradish peroxidase (HRP), alkali phosphatase, beta galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (as glucoseoxidase, beta-Galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)), heterocycle oxidase (such as uricase and xanthine oxidase), lactoperoxidase, microperoxisome, or the like.
The example of enzyme-substrate combination for example comprises:
(i) horseradish peroxidase (HRP) and as the hydrogen peroxide of substrate, wherein the hydrogen peroxide oxidation dyestuff former (as ABTS, o-phenylenediamine (OPD) or 3,3 ', 5,5 '-tetramethyl benzidine hydrochloride (TMB));
(ii) alkali phosphatase (AP) and as the p-nitrophenyl phosphate ester of substrate; With
(iii) beta-D-galactosidase (β-D-Gal) and chromogenic substrate (as p-nitrophenyl-β-D-galactoside) or fluorogenic substrate 4-methyl umbrella shape base-β-D-galactoside.
According to a kind of particular assay method, tracer is with immobilization target thing incubation when having the unmarked candidate compound of variable concentrations.Improve the concentration effective competition tracer of successful candidate compound and combining of immobilization target thing.The concentration of unmarked candidate compound was called " IC when 50% of maximum combined tracer was replaced
50", it has reflected the target thing binding affinity of candidate compound.Therefore, IC
50For showing, the candidate compound of 1mM compares IC
50Be the candidate compound substance of 1 μ M weak with the interaction target thing.
In some phage display ELISA algoscopy, the binding affinity of sudden change (" mut ") sequence uses Cunningham et al., EMBO J.13:2508 the method described in (1994) directly with contrast (" con ") peptide relatively, and be characterized by parameter EC
50Algoscopy is at EC
50(con)/EC
50(mut) will be similar to K
d(con)/K
d(mut) carry out under the condition.
Therefore, the invention provides " competition of having the ability " bonded chemical compound of albumin such as human serum albumin in described external test method.Preferably, chemical compound has IC less than 1 μ M to target thing such as human serum albumin
50In these chemical compounds, preferably have less than about 100nM, preferably less than about 10nM or less than the IC of about 1nM
50Chemical compound.In the preferred embodiment of another aspect this according to the present invention, chemical compound shows less than about 100pM target molecule such as human serum albumin, is more preferably less than the IC of about 10pM
50
A kind of preferred external test method of ability that is used to measure candidate compound and SABM described herein competition is as follows, and more complete description is arranged in an embodiment.In preferred embodiments, candidate compound is a peptide.Candidate compound uses the ELISA monitoring with the ability that combines the human serum albumin through the competition of labelling SABM tracer.The diluent of candidate compound in buffer added to wrap by the microtitration plate of (as described in the embodiment part) with the human serum albumin with tracer reach 1 hour.Microtitration plate is cleaned with cleaning buffer solution, and the amount of measurement and the bonded tracer of human serum albumin.
B.
SABM:TA: cytotoxic agent combination
SABM and TA: cytotoxic agent is connected and forms the conjugate molecule that comprises at least one every kind component (i.e. at least three different components).Every kind of component can be chosen wantonly through the flexible joint territory interconnection.
According to the type and the production method thereof that connect, the SABM territory can be through its N-or terminal N-or the C-end that connects TA of C-.For example, divide the period of the day from 11 p.m. to 1 a.m preparing conjugate of the present invention by recombinant technique, the nucleic acid of coding SABM can be operatively connected the nucleic acid of coding TA sequence, and will be optional through the joint territory.Be typically the encode fusion rotein of N-end of the terminal TA of connection of C-of SABM wherein of construct.Yet especially when adopting synthetic technology, for example wherein the N-of the terminal connection of the N-of SABM TA or the fusion rotein of C-end also are possible.
In some situation, the TA intramolecularly can be inserted in the SABM territory, and the N-of disconnected TA or C-end.This structure can be used for putting into practice the present invention, as long as the function of SABM territory and TA obtains keeping.For example, SABM can insert the non-binding light chain CDR of immunoglobulin, and does not disturb immunoglobulin and the bonded ability of its target thing.Can identify by rule of thumb that the zone that can hold the SABM territory in the TA molecule and insert is (promptly by selecting to insert the site at random, the gained conjugate is measured the function of TA), perhaps by in the family of relevant TA, carry out sequence relatively (so as to proteinic TA) to locate the zone of low sequence homology.Compare with very conservative zone, the insertion in SABM territory is more likely tolerated in low sequence homology zone.For its three dimensional structure TA of known (as by X-radiocrystallgraphy or NMR research), this three dimensional structure can provide SABM to insert the guidance in site.For example, with the zone of higher level structure or relate to part in conjunction with or catalytic zone compare, the zone (being big temperature or " B " coefficient) of encircling or having high flexibility (mobility) more likely holds the SABM territory and inserts.
C.
The joint territory
The SABM territory is optional to connect TA through joint.The joint component of conjugate molecule of the present invention is not essential the participation, but has the function that helps the conjugate molecule.Therefore, the joint territory is defined as any molecular radical that the space bridge joint is provided between TA and SABM territory.
The joint territory can be different length and composition, yet, for setting up the space bridge joint, the importantly length in joint territory but not its structure.The SABM binding target molecule of conjugate molecule is preferably allowed in the joint territory, does not have the restriction of space and/or conformation basically.Therefore, the length in joint territory depends on that two " function " territories of conjugate molecule are the characteristic of SABM and TA.
Those skilled in the art will appreciate that based on the known distance between the different keys the homoatomic combination does not provide the different length molecule.Referring to for example Morrison and Boyd, 1997, " (OrganicChemistry ", the 3rd edition, Allyn and Bacon, Inc., Boston, MA.The joint territory can be the polypeptide of different length.The aminoacid of polypeptide is formed characteristic and the length that has determined joint.In a preferred embodiment, linkers comprises flexible, hydrophilic polypeptide chain.Exemplary joint territory comprises one or more Gly and/or Ser residue, such as those hereinafter described in the embodiment part.
D.
Be re-combined into
Composition of matter is contained in the present invention, and it comprises coding SABM described herein or comprises SABM and the isolating nucleic acid of the conjugate molecule of peptide T A, preferred DNA.The DNA of code book invention peptide can be by several different methods preparation known in the art.These methods include but not limited to the al. by Engels et, 1989, the chemosynthesis of any method described in the Agnew.Chem.Int.Ed.Engl.28:716-734 (its complete open book is collected herein by reference) is such as three esters, phosphite ester, phosphoramidite and H-phosphonate ester chemical synthesis.In one embodiment, use the preferred codon of expression host cell institute in the design of coding DNA.Perhaps, the DNA of encoded peptide can change over one or more variants of coding by using recombinant DNA technology, such as site-specific mutation (Kunkel et al., 1991, Methods Enzymol.204:125-139; Carter et al., 1986, Nucl.Acids Res.13:4331; Zoller et al., 1982, Nucl.Acids Res.10:6487), cassette mutagenesis (Wells et al., 1985, Gene 34:315), restricted selection mutation (Carter, 1991, at " Directed Mutagenesis:APractical Approach ", M.J.McPherson compiles, IRL Press, Oxford), or the like.
According to above-mentioned preferred aspect, the SABM that nucleic acid coding can binding target molecule.Target molecule comprises that for example the outer molecule of born of the same parents is such as various serum factors, include but not limited to plasma proteins such as serum albumin, immunoglobulin, apolipoprotein or transferrin, the perhaps protein of on erythrocyte or lymphocytic cell surface, finding, certainly, if the not words of the normal function of interference cell that combine basically of SABM and cell cortex protein.Being preferred for of the present invention is with the SABM of expectation affinity in conjunction with serum albumin, for example, and with high-affinity, or to promote and the useful tissue picked-up of the bioactive molecule that SABM merges and the affinity of diffusion.
According to another preferred aspect of the present invention, nucleic acid coding comprises the conjugate molecule of SABM sequence and TA.Aspect this, TA can comprise any polypeptide compound of useful as therapeutics or diagnostic agent, as enzyme, hormone, cytokine, antibody or antibody fragment of the present invention.The nucleic acid molecule encoding conjugate molecule of this aspect according to the present invention, and the nucleic acid of coding SABM sequence can be operatively connected (with regard to DNA sequence be successive and be in the reading frame with regard to) nucleic acid of encoding human activating agent.Optional is, the nucleotide sequence that these DNA sequence can encoded joint domain amino acid sequence connects.
According to this aspect, the present invention further comprises the expression control sequenc that the dna molecular with code book invention peptide can be operatively connected; The expression vector that comprises described dna molecular, such as plasmid, wherein control sequence is subjected to the identification with this carrier transformed host cells; Reach with this carrier transformed host cells.Generally speaking, plasmid vector comprises duplicating and control sequence derived from the species compatible with host cell.Carrier carries replication site usually and encodes can provide the proteinic sequence of Phenotypic Selection in transformant.
For in prokaryotic hosts, expressing, suitable carriers comprises pBR322 (ATCC No.37,017), phGH107 (ATCC No.40,011), any carrier of pBO475, pS0132, pRIT5, pRIT20 or pRIT30 series (Nilsson and Abrahmsen, 1990, Meth.Enzymol.185:144-161), pRIT2T, pKK233-2, pDR540 and pPL-λ.The prokaryotic host cell that comprises expression vector of the present invention comprises e. coli k12 strain 294 (ATCC No.31,446), coli strain JM101 (Messing et al., 1981, Nucl.AcidRes.9:309), coli strain B, coli strain _ 1776 (ATCC No.31537), escherichia coli c600, escherichia coli W3110 (F-, γ-, prototroph, ATCC No.27,325), coli strain 27C7 (W3110, tonA, phoA E15, (argF-lac) 169, ptr3, degP41, ompT, kan
r) (United States Patent (USP) 5,288,931, ATCC No.55,244), bacillus subtilis (Bacillus subtilis), Salmonella typhimurium (Salmonellatyphimurium), serratia marcescens (Serratia marcesans) and Rhodopseudomonas (Pseudomonas) species.
Except that prokaryote, most eukaryotes such as yeast, or can be used as host cell derived from the cell of multicellular organisms.For in yeast host cell, expressing, such as common bakery yeast or saccharomyces cerevisiae (Saccharomyces cerevisiae), suitable carriers comprises episomal replication carrier, integrating vector and yeast artificial chromosome (YAC) carrier based on 2 microns plasmids.For expressing in insect host cell, such as the Sf9 cell, suitable carriers comprises baculovirus vector.For in plant host cell, expressing, dicotyledon host particularly, such as Nicotiana tabacum L., suitable expression vector comprises the carrier derived from Agrobacterium tumefaciems (Agrobacterium tumefaciens) Ti-plasmids.
The example of useful mammalian host cell comprises the monkey kidney CV1 system (COS-7, ATCC CRL 1651) that SV40 transforms; Human embryo kidney (HEK) system (for 293 or 293 cells of growth sub-clone in suspension culture, Graham et al., 1977, J. Gen Virol. 36:59); Baby hamster kidney cell (BHK, ATCCCCL 10); Chinese hamster ovary cell/-DHFR (CHO, Urlaub and Chasin, 1980, Proc.Natl.Acad. Sci.USA, 77:4216); Mice Sai Ertuoli (Sertoli) cell (TM4, Mather, 1980, Biol.Reprod.23:243-251); Monkey-kidney cells (CV1, ATCC CCL 70); African green monkey kidney cell (VERO-76, ATCC CRL-1587); Human cervical carcinoma cell (HELA, ATCC CCL 2); Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL1442); Human pneumonocyte (W138, ATCC CCL 75); Human liver cell (Hep G2, HB 8065); Mice lacteal tumor (MMT 060562, ATCC CCL 51); TRI cell (Mather et al., 1982, Annals N.Y.Acad.Sci.383:44-68); MRC 5 cells; The FS4 cell; And Bel7402 (Hep G2).For expressing in mammalian host cell, useful carrier comprises carrier derived from SV40, comprises pRK5 and pRK7 (Suva et al., 1987, Science 237:893-896 derived from carrier such as the pRK carrier of cytomegalovirus; EP307,247 (3/15/89); EP278,776 (8/17/88)), derived from the carrier of vaccinia virus or other poxvirus, and retroviral vector such as carrier derived from Moloney (Moloney) murine leukemia virus (MoMLV).
Optional is that the DNA of coding purpose peptide can be operatively connected the secretion targeting sequencing, causes host cell that expression product is secreted in the culture medium.The example of secretion targeting sequencing comprises STII, colicine, lamB, herpes GD, lpp, alkali phosphatase, invertase and alpha factor.What also be applicable to this paper is 36 amino acid whose targeting sequencings (Abrahmsen et al., 1985, EMBO J. 4:3901) of protein A
Host cell is with above-mentioned expression of the present invention or cloning vehicle transfection and preferred the conversion, and for evoked promoter, select to cultivate in the conventional Nutrient medium of the appropriate improvement of gene of transformant or amplification coding expectation sequence.
The prokaryote host cell that is used to generate peptide of the present invention can be substantially as people such as Sambrook, cultivation described in seeing above.
The mammalian host cell that is used for generating peptide of the present invention can be cultivated in multiple culture medium.Commercially available culture medium such as HamShi F10 (Sigma), minimum essential medium (MEM, Sigma), (DMEM Sigma) is suitable for cultivating host cell for the EagleShi culture medium of RPMI-1640 (Sigma) and DulbeccoShi improvement.In addition, this area any culture medium on the books (Ham and Wallace for example, 1979, Meth.Enz.58:44; Barnes and Sato, 1980, Anal.Biochem.102:255; United States Patent (USP) 4,767,704; 4,657,866; 4,927,762; 4,560,655; WO90/03430; WO87/00195; United States Patent (USP) reexamination 30,985; Or United States Patent (USP) 5,122,469, openly book is collected herein by reference separately) all can be used as the culture medium of host cell.Any of these culture medium optionally supplementing hormone and/or other somatomedin (such as insulin, transferrin or epidermal growth factor), salt (such as sodium chloride, calcium, magnesium and phosphate), buffer agent (such as HEPES), nucleoside (such as adenosine and thymidine), antibiotic (such as Gentamycin
TMMedicine), trace element (being defined as the inorganic compound that exists with the micro-molar range final concentration usually) and the glucose or the equivalent energy.Any debita spissitudo that other must fill-in can also those skilled in the art should be known is included.Condition of culture, such as temperature, pH, or the like, be exactly that those before had been used to the host cell of expressing and selecting, be conspicuous for those of ordinary skills.
The host cell of mentioning in the disclosure book is contained cell and the interior cell of host animal in the In vitro culture.
E.
Chemosynthesis
The another kind of method that generates SABM of the present invention involves chemosynthesis.This can by use methodology well-known in the art realize (referring to Kelley and Winkler, 1990, in " GeneticEngineering Principles and Methods ", Setlow, J.K. compiles, Plenum Press, N.Y., the 12nd volume, 1-19 page or leaf; Stewart et al., 1984, J.M.Young, J.D., Solid Phase PeptideSynthesis, Pierce Chemical Co., Rockford, IL.Also can be referring to United States Patent (USP) 4,105,603; 3,972,859; 3,842,067; 3,862,925).
SABM of the present invention can use the synthetic preparation easily of solid-phase peptide.Merrifield，1964，J. Am.Chem.Soc.85：2149；Houghten，1985，Proc.Natl.Acad.Sci.USA 82：5132。Solid-phase peptide is synthetic also to can be used for preparing conjugate molecular composition of the present invention, if TA is polypeptide or comprises polypeptide.
Solid phase synthesis starts from the carboxyl terminal of nascent peptide, by shielded aminoacid is coupled to the inertia solid support.The inertia solid support can be any macromole that can serve as the anchor of initial amino acid C-end.Be typically, the macromole holder is as Stewart and Young, the 2nd and 4 page of Fig. 1-1 that sees above and the cross-linked polymeric resin (as polyamide or polystyrene resin) shown in the 1-2.In one embodiment, the C-end amino acid is coupled to polystyrene resin to form the benzyl ester.The selection of macromole holder should make and be connected peptide by the peptide anchor to be used in synthetic to be sealed under the condition of amino acid whose alpha-amido deprotection be stable.If use alkali labile α-protecting group, be desirably in so and use the unstable connection of acid between peptide and the solid support.For example, sour unsettled ether resin is synthetic for alkali labile Fmoc-amino acid peptide is that effectively as Stewart and Young, the 16th page of seeing above is described.Perhaps, can use the unsettled peptide anchor connection of acid hydrolysis difference and α-protecting group.For example, the synthetic running of aminomethyl resin such as phenyl acetamide methyl (Pam) resin-bonded Boc-amino acid peptide is fine, and as Stewart and Young, the 11-12 page or leaf that sees above is described.
After initial amino acid is coupled to the inertia solid support, the alpha-amido protecting group of removing initial amino acid with the trifluoroacetic acid (TFA) in the dichloromethane for example, and in for example triethylamine (TEA), neutralize.After the alpha-amido deprotection of initial amino acid, add following-individual alpha-amido and the shielded aminoacid of side chain in synthesizing.Then by condensation with expectation remaining alpha-amido of sequence coupling and the shielded aminoacid of side chain in case of necessity, the intermediate compound that is connected with solid support with acquisition.Perhaps, some aminoacid each other coupling subsequently fragments of peptides is added to the solid phase peptide chain in the growth to form the fragment of expectation peptide.
Can carry out the condensation reaction between two aminoacid or aminoacid and peptide or peptide and the peptide according to method of condensing commonly used, such as axide method, mixed anhydride method, DCC (N, N '-dicyclohexylcarbodiimide) or DIC (N, N '-DIC) method, active ester method, p-nitrophenyl ester method, BOP (benzotriazole-1-base-oxygen-three [dimethylamino] phosphine hexafluorophosphoric acid ester) method, N-hydroxy succinic acid polyurethane method etc., and Woodward reagent K method.
In the chemosynthesis of peptide, usually protect amino acid whose any reactive side chain group with suitable protecting group.Finally, after assembled in sequence is expected polypeptide chain well, remove these protecting groups.Equally; alpha-amido when usually the free N-terminal amino group of the solid-phase polypeptide chain in the C-of aminoacid or fragments of peptides terminal carboxyl group and growth reacts on protection aminoacid or the fragments of peptides, selectivity is removed alpha-amido and to allow next aminoacid or fragments of peptides is added to the solid-phase polypeptide chain then.Therefore, in polypeptide is synthetic, usually generate contain each amino acid residue and they in peptide chain with the intermediate compound of expectation positioned in sequence, wherein each residue still carries Side chain protective group.These protecting groups can removed after solid phase is removed basically simultaneously, to generate the polypeptide product of expectation.
α-and the epsilon-amino side chain can be with benzyloxycarbonyl group (being abbreviated as Z), different cigarette oxygen carbonyl (iNOC), adjacent benzyloxycarbonylchloride base [Z (2Cl)], to nitro benzyloxycarbonyl group [Z (NO
2)], to methoxyl group benzyloxy carbonyl [Z (OMe)], tertbutyloxycarbonyl (Boc), uncle's penta oxygen carbonyl (Aoc), isoborneol oxygen carbonyl, Buddha's warrior attendant alkoxy carbonyl group, 2-(4-the xenyl)-2-third oxygen carbonyl (Bpoc), 9-fluorenylmethyloxycarbonyl (Fmoc), methylsulfonyl carbethoxyl group (Msc), trifluoroacetyl group, phthalein (acyl) base, formyl, 2-Nitrobenzol sulfinyl (NPS), diphenylphosphothioy (Ppt) and dimethyl disulfide phosphino-(Mpt), or the like the protection.
The example that is used for the protecting group of carboxyl functional group have benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), tertiary butyl ester (Obut), 4-pyridylmethyl ester (OPic), or the like.Usually arginine, cysteine and the serine of functional group are subjected to the protection of appropriate protection base to the expectation specific amino acids in addition such as having amino and carboxyl.For example, arginic guanidine radicals can use nitro, p-toluenesulfonyl, benzyloxycarbonyl group, Buddha's warrior attendant alkoxy carbonyl group, to methoxybenzene sulfonyl, 4-methoxyl group-2,6-dimethyl benzene sulfonyl (Nds), 1 sulfonyl (Mts), or the like protection.The sulfydryl of cysteine can use to methoxybenzyl, trityl, or the like protection.
Above-mentioned many can the acquisition of being sealed in the aminoacid from commercial source, such as Novabiochem (SanDiego, CA), Bachem CA (Torrence, CA) or Peninsula Labs (Belmont, CA).
Stewart and Young, seeing above provides details about the method for preparing peptide.Alpha-amino protection is recorded in the 14-18 page or leaf, and the side chain sealing is recorded in the 18-28 page or leaf.The protecting group tabulation that is used for amine, hydroxyl and mercapto functional group is provided in the 149-151 page or leaf.
After finishing the expectation aminoacid sequence, peptide can downcut from solid support, reclaim, and purification.Peptide downcuts from solid support by the reagent that can destroy peptide-solid phase connection, and optional side chain functionalities deprotection to being sealed on the peptide.In one embodiment, peptide downcuts from solid phase by acid hydrolysis with liquid hydrogen fluoric acid (HF), and HF has also removed the Side chain protective group of any remnants.Preferably, for avoiding the residue alkanisation (for example alkanisation of methionine, cysteine and tyrosine residue) in the peptide, the acidolysis reaction mixture contains thiocresol and cresol scavenger.After the HF cutting, clean resin, and the continuous wash by acetic acid solution is from the Solid-Phase Extraction free peptide with ether.With the cleanout fluid lyophilizing that merges, with peptide purification.
F.
The chemical coupling of conjugate molecule
In certain embodiments, the conjugate molecule can comprise as the TA with diagnosis or organic compound of therapeutic use, perhaps, and between SABM and the peptide T A, the fusions that can not in a nucleic acid, encode of its structure.The example of a kind of embodiment in back comprises the fusions between the amino terminal of the amino terminal of SABM and TA, or the fusions between the carboxyl terminal of the carboxyl terminal of SABM and TA.
Chemical coupling can be used for preparing these embodiments of conjugate molecule; use multiple bifunctional protein coupling agent; such as N-succinimido-3-(2-pyridine radicals dithio)-propionic ester (SPDP); imino group sulfane (IT); imino-ester (all example hydrochloric acid adipyl imidic acid dimethyl esters); active esters (such as suberic acid two succinimido esters); aldehydes (such as glutaraldehyde); double azido compound (such as two (right-the azido benzoyl base) hexamethylene diamine); dual azepine derivatives (such as two (right-the diazobenzene formoxyl)-ethylenediamines); vulcabond is (such as toluene 2; the 6-vulcabond); with the dual-active fluorine compounds (such as 1; 5-two fluoro-2,4-dinitro benzene) dual-function derivative.The method that can be used for cytotoxic agent and polypeptide such as antibody coupling is known.
G.
The peptide that disulfide bond connects
As mentioned above, some embodiment of the present invention comprises the SABM of cyclisation.SABM can be by forming disulfide bond and cyclisation between cysteine residues.Employed any method that makes things convenient for prepared during this type of peptide can form by disulfide bond then by aforesaid chemosynthesis.For example, peptide can reclaim its sulfydryl from solid phase synthesis and be in the reduction form, be dissolved in weak solution, wherein the intramolecularly semicystinol concentration surpasses intermolecular semicystinol concentration to optimize intramolecular disulfide bond formation, peptide concentration such as 25mM-1 μ M, preferred 500 μ M-1 μ M, more preferably 25 μ M-1 μ M, then by free sulfhydryl groups being exposed to the weak oxidant that is enough to produce intramolecular disulfide bond and oxidation, as have or do not have catalyst such as metal cation, the potassium ferricyanide, sodium tetrathionate, or the like molecular oxygen.Perhaps, peptide can be as Pelton et al., and 1986, described in the J. Med.Chem.29:2370-2375 and cyclisation.
Cyclisation can be by for example forming first and second residues of disulfide bond, for example Cys, Pen, Mpr and Mpp and contain disulfide bond between the equivalent of 2-amino or the formation of lactam bond realizes.The residue that can form lactam bridges comprises for example Asp, Glu, Lys, Orn, α β-DAB, diamino-acetic acid, amino benzoic Acid and mercaptobenzoic acid.Chemical compound herein can cyclisation can utilize the covalent attachment of the side-chain radical of non-conterminous residue with formation and Cys or other amino acid whose N-terminal amino group via for example lactam bond.Alternative bridge construction also can be used for cyclisation chemical compound of the present invention, and comprising for example can be through S-S, CH
2-S, CH
2-O-CH
2, lactams ester or other connect the peptide and the peptide mimics of cyclisation.
H. Pharmaceutical composition
The Pharmaceutical composition that comprises conjugate molecule of the present invention can be used in any suitable manner, comprises parenteral, part (topical), oral or local (local) (such as aerosol or through skin), or its combination in any.
Other suitable present composition comprises any above-mentioned conjugate molecule and pharmacopedics can be accepted carrier.The character of carrier is different with insecticide-applying way.For example, for oral dispenser, preferred solid carrier; For intravenous administration, use the liquid salt solution carrier usually.
Compositions of the present invention comprises the pharmacopedics compatible with protein of the present invention with the experimenter can accept component.These generally include suspension, solution and elixir, the most especially biology buffer agent, such as phosphate buffered saline (PBS), normal saline, DulbeccoShi culture medium, or the like.Aerosol also can use, or such as starch, sugar, microcrystalline Cellulose, diluent, granulating agent, lubricant, binding agent, disintegrating agent, or the like carrier (in the situation of oral solid formulation) such as powder, capsule and tablet.
When being used for this paper, term " pharmacopedics is acceptable " means the approval that obtains federation or administrative organization of state government usually or lists in American Pharmacopeia or pharmacopeia that other is generally acknowledged, for animal, more particularly people's use.
The preparaton of selecting can use multiple aforementioned buffer agent, or even excipient comprise pharmaceutical grade for example mannitol, lactose, starch, magnesium stearate, saccharin sodium cellulose, magnesium carbonate, or the like finish." PEGization " of compositions can use technology known in the art to realize (referring to for example International Patent Publication No. WO 92/16555; The United States Patent (USP) 5,122,614 of Enzon; With International Patent Publication No. WO 92/00748).
Preferred dispenser of the present invention path is aerosol or suction form.Chemical compound of the present invention with the dispersant combination, can be used as dry powder and uses with aerosol or use with solution or suspension with diluent.
When being used for this paper, term " dispersant " refers to help chemical compound aerosolization or protein to absorb or the medicament of the two in lung tissue.Preferably, dispersant is that pharmacopedics is acceptable.Suitable dispersant is well-known in the art, includes but not limited to surfactant or the like.For example, can use this area to be generally used for reducing the accumulative surfactant that solution atomization forms chemical compound, the especially peptide compounds of the caused spatial induction of liquid aersol.The non-limitative example of this type of surfactant is such as polyoxyethylene fatty acid ester and alcohol, and the surfactant of polyoxyethylene sorbitan aliphatic ester.The consumption of surfactant can change, usually preparaton weight about 0.001% to about 4% scope.One specific aspect, surfactant is polyoxyethylene sorbitan monoleate or sorbitan trioleate.Suitable surfactant is well-known in the art, can select based on desired characteristic according to concrete dosage form, compound concentration, diluent (in liquid dosage form) or powder type (in therapy in dry powder form) or the like.
In addition, according to quality (as ill or healthy lung) and many other factorses of the selection of conjugate molecule, desired therapeutic effect, lung tissue, liquid or dry dosage form can comprise other component, as discussed further below.
The liquid aerosol dosage forms can accept to contain in the diluent conjugate molecule and dispersant the physiology usually.Dry powder aerosol dosage form of the present invention is made up of the conjugate molecule and the dispersant of the solid form of fine dispersion.No matter be liquid or dry powder aerosol dosage form, this dosage form all must aerosolization.That is to say that it must be fragmented into the liquid or solid granule, really arrive alveolar to guarantee aerosolization dosage.Generally speaking, dynamic diameter (mass median dynamic diameter) should be 5 microns or littler in the middle of the agglomerate, arrives alveolar (Wearley, 1991, Crit. Rev. in Ther.Drug CarrierSystems 8:333) to guarantee drug particles.Term " aerosol particles " is used for description in this article and is suitable for pulmonary administration, promptly should arrive the liquid or solid granule of alveolar.Other structure, other component in the preparaton and particle characteristics of considering item such as delivery apparatus is important.These aspects of medicine pulmonary administration are well-known in the art, and the structure of the operation of dosage form, aerosolization means and delivery apparatus requires those of ordinary skills to carry out routine test at the most.
Structure for delivery apparatus, any aerosolization form known in the art, include but not limited to the aerosolization (aerosolization) of spraying (nebulization), atomizing (atomization) or the pump atomizing (pumpaerosohzation) and the therapy in dry powder form of liquid dosage form, all can be used for practice of the present invention.Predicted to using the delivery apparatus of solid dosage forms unique design.Usually, the aerosolization of liquid or therapy in dry powder form needs propellant.Propellant can be the normally used any propellant in this area.The concrete non-limitative example of this type of useful propellant is CFC (chlorofluorocarbon), carbon-hydrogen fluoride (hydrofluorocarbon), hydrogen CFC (hydochlorofluorocarbon) or Hydrocarbon (hydrocarbon), comprise fluoroform, dichlorodifluoromethane, dichloro-tetrafluoro ethanol and 1,1,1,2-tetrafluoroethane, or its combination.
Of the present invention one preferred aspect, the device that is used for aerosolization is pressure metered-dose inhaler (metered dose inhaler).The pressure metered-dose inhaler provides given dose when dispenser, but not depends on the variable dose of using.This type of pressure metered-dose inhaler can use with liquid or dry powder aerosol dosage form.The pressure metered-dose inhaler is well-known in the art.
In case the conjugate molecule arrives lung, the factor of many dependence dosage forms can influence drug absorption.Will be appreciated that disposal require chemical compound cyclical level disease or when disorderly, such as aerosol particles size, aerosol particles shape, infect whether exist, the factor of lung disease or thromboembolism can influence the absorption of chemical compound.For every kind of dosage form describing herein, some lubricant, absorption enhancer, protein stabilizing agent or suspending agent may be appropriate.The selection of these additional agent will change with purpose.Will be appreciated that in hope or seek in the local situation of delivering chemical compound, will be not too important such as absorbing variable such as enhancings grade.
I.
The liquid aerosol dosage forms
Liquid aerosol dosage forms of the present invention will be used with aerosol apparatus usually.Aerosol apparatus can be compressed air-driven or hyperacoustic.Any aerosol apparatus known in the art all can be united use with the present invention, such as, but not limited to: Ultravent, Mallinckrodt, Inc. (St.Louis, MO); AcornII aerosol apparatus (Marquest Medical Products, Englewood CO).Can be recorded in the United States Patent (USP) 4,624,251 of bulletin on November 25th, 1986 with the aerosol apparatus that the present invention unites use; 3,703,173 of on November 21st, 1972 bulletin; 3,561,444 of on February 9th, 1971 bulletin; With 4,635,627 of on January 13rd, 1971 bulletin.
Preparaton can comprise carrier.Carrier is the macromole that dissolves in blood circulation, and it is that the physiology is acceptable, and wherein the acceptable those skilled in the art of meaning of physiology will approve to the described carrier of patient infusion, as the part of therapeutic scheme.Carrier is preferably metastable in blood circulation, has the acceptable elimination half-life.Such macromole includes but not limited to soybean lecithin, oleic acid and sorbitan trioleate, preferred sorbitan trioleate.
The preparaton of this embodiment also can comprise other medicament that can be used for stable protein or regulate osmotic pressure.The example of medicament includes but not limited to salt, such as sodium chloride or potassium chloride, and carbohydrate, such as glucose, galactose or mannose, or the like.
J.
The aerosol therapy in dry powder form
Also having imagined medicinal proportional preparation of the present invention will use as the SABM of the powder type that comprises fine dispersion and the Foradil Aerolizer formoterol fumarate type of dispersant.The form of chemical compound is freeze-dried powder normally.The lyophilized form of conjugate molecule can obtain by standard technique.
In another embodiment, therapy in dry powder form will comprise dry powder, dispersant and the filler of the fine dispersion that contains one or more The compounds of this invention.The filler that preparaton can associating of the present invention uses comprises the medicament such as lactose, sorbitol, sucrose or mannitol, and its amount promotes powder to scatter from device.
Complete being collected herein by reference of all publications of quoting herein (comprising patent and patent application).
This description and claims in full in, word " comprises " or changes to be interpreted as meaning such as " comprising " or " containing " and includes described integer or integer group, but does not get rid of any other integer or integer group.
The following example provides as illustration, absolutely not restriction.The disclosure of all quoted passages clearly is collected herein by reference in the description.
Embodiment
Embodiment 1-material
For these research, with p185
HER2The Fab of the bonded humanized antibody of ectodomain (HER2) by modified recombinant to comprise albumin binding peptide (AB).The variable region sequences of the antibody humAb4D5-8 that uses in this research is found in Carter etc., (1992) PNAS 89:4285-4289.Before, humanized Fab is already derived from mouse monoclonal antibody muMAb4D5 (at this 4D5), this monoclonal antibody is by in American type culture collection (Manassas, Virginia) preservation and have ATCC and go into to hide that the hybridoma of registration number CRL 10463 produces.Prepare humanized anti--method of Her-2 antibody and the evaluation of exemplary variable region sequence be provided in as United States Patent (USP) 5,821,337 and 6,054,297 and Carter etc., among (1992) PNAS 89:4285-4289.
By the coding joint sequence, the nucleotide sequence of GGGS (SEQ ID NO:422), the albumin binding peptide of will encoding (" AB "), the nucleotide sequence of QRLMEDICLPRWGCLWEDDF (SEQ ID NO:1) is connected with the nucleotide sequence of coding Fab.The nucleotide sequence of coding joint is connected on the terminal KTHT residue of heavy chain C-of Fab.In contrast, by the recombinant DNA engineering, make up the anti-tissue factor Fab that contains the D3H44 antibody variable region that merges by its light chain and AB.About the D3H44 aminoacid sequence, referring to Presta, L., etc., (2001) Thromb.Haemost.85:379-389.
Resultant construct is expressed justacrine as fusion rotein (" AB.Fab4D5-H " or " rhuABFabATFL ") from escherichia coli, separate then and purification.Then, fusion rotein is coupled to monomethylauristatin (MMAE).For tumor function research,, fusion rotein is connected with MMAE by having valine-citrulline (val-cit or vc) two peptide linker reagent of maleimide amine moiety and PAB carbamyl (PAB) sept.About the example of method that MMAE is connected with antibody referring to Klussman, K etc., (2004) Bioconjugate Chem.15:765-773.For toxicity research; through for example using succinimido acetyl thio acetas (succinimidylacetylthioacetate) (Sata) to produce free mercaptan; be coupled to valine-citrulline-MMAE (" vc-MMAE ") afterwards; the coupling of lysine by fusion rotein and the MMAE of the activated derivatives modification of using maleimide caproyl (maleimidocaproyl) is connected fusion rotein with MMAE.MMAE at the ratio on the resulting AB.Fab4D5-H usually on average at about about 1: 1, the highest 4: 1, minimum 0: 1, make 0-4 MMAE partly be randomized to either on the lysine of exposure, grand average is about 1 MMAE of each AB.Fab4D5-H.
Embodiment 2-uses the efficacy study of MMAE conjugate
To MMTV-HER2 transgenic mammary neoplasms (Fo5) the test AB.Fab-4D5-H-MMAE conjugate of having set up.Herceptin does not reply in this tumor system, but replys Herceptin -MC-vc-PAB-MMAE conjugate well.
With the intravenous 1650 μ g MMAE/m of single agent
2RhuAB.Fab-4D5-H-vc-MMAE (promptly having AB), rhuFab4D5vcMMAE (promptly not having AB) or rhuABFabATFL-vc-MMAE (negative control) are administered to the mice that has mMMTV-HER2 Fo5 tumor.The mice of every processing has 100-200mm
3Mean tumour volume.At the 0th day of research, use link coupled molecule of MMAE or phosphate buffered saline (PBS) (" media "), and carry out twice measurement of tumor weekly, continue 17 days.Use a kind of known valid MMAE conjugate Herceptin -MC-vc-PAB-MMAE at 1245 μ g/m
2Under the MMAE dosage as a comparison.Enforcement is killed (Log Cell Kill) based on the logarithm cell of tumour doubling time and is analyzed.The logarithm cell kills to analyze and has used based on compared with the control, handles the mathematical calculation of the tumor growth delay of beginning back tumor size multiplication required time.This mathematical equation is:
Fig. 1 has shown by FisherShi PSLD, between rhuAB.Fab4D5-H-vc-MMAE and Herceptin -MC-vc-PAB-MMAE group, there is not significant difference (p=0.0001), and negative MMAE contrast Fab, rhuABFabATFL-vc-MMAE and media contrast do not have significant difference (p=0.6).
Embodiment 3-IgG-MMAE, F (ab ')
2The toxicity of-MMAE conjugate and free MMAE
(Charles RiverLaboratories, Hollister CA) are used for following research with more free MMAE, Fab-MMAE and F (ab') with female Spraque-Dawley (SD) rat of weight between the 75-80 gram
2The toxicity of-MMAE conjugate.
The administration group:
| Group | Administration | mg/kg | μg MMAE/m 2 | MMAE/MAb | Connection site | Number/ |
| 1 | Media (PBS) | 0 | 0 | 0 | NA | 6/ |
| 2 | Herceptin-val-cit-MMAE | 20.2 | 2105 | 5.3 | Cysteine | 6/ |
| 3 | HerceptinF(ab’)2-val-cit-MMAE | 10.83 | 840 | 2.7 | Lysine | 6/ |
| 4 | HerceptinF(ab’)2-val-cit-MMAE | 27.14 | 2105 | 2.7 | lvsine | 6/ |
| 5 | Free MMAE | 0.516 | 2105 | NA | 6/F |
The administration group:
For Herceptin -val-cit-MMAE, use 718 molecular weight (MW) and 145167 molecular weight to calculate μ g MMAE/m as Herceptin as MMAE
2For Herceptin F (ab ')
2-val-cit-MMAE, use 718 as the molecular weight of MMAE and 100000 as Herceptin F (ab ')
2Molecular weight calculate μ g MMAE/m
2Body surface area is calculated as follows: { [(body weight gram number
0.667) x 11.8]/10000}.(industry instructs and comment.Safe initial dose is estimated in clinical trial at the therapeutic agent that is used for the adult healthy volunteer.Healthy and the Department of Public Enterprises FDA of the U.S., medicine assessment and research center (CDER), biological preparation assessment and research center (CBER).In December, 2002 (Guidance for Industry and Reviewers.Estimating the SafetyStarting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers.U.S.Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for BiologicsEvaluation and research (CBER), December 2002)).
At the 1st day that studies, with the dose volume (in PBS, diluting) of 10mL/kg, by the single intravenous push, through tail vein injection application dosage solution.Carrying out a routine clinical every day observes.Carry out twice M ﹠ M inspection (morning and afternoon) every day.Before administration in the 1st day of research, measure the body weight of animal, after this measure once every day.Whole blood collection is used for hematologic parameter (as average serum AST level, ALT level, GGT level and bilirubin level) and different cell counting (as average numeration of leukocyte and platelet count) in containing the test tube of EDTA.Whole blood collection is used for clinical chemistry parameters in serum separator tube.Before administrations in the-4 days of research, the 3rd day and the 5th day of research collect blood sample during at necropsy.When necropsy, also with whole blood collection in containing the test tube of Lithium acid heparin, and with plasma freezing in-70 ℃ of confession analyses subsequently.Collect following tissue in necropsy: liver, kidney, the heart, thymus, spleen, brain, breastbone and gastrointestinal tract (GI tract) section comprises stomach, large intestine and small intestinal.When necropsy, only weigh spleen and thymus.Body weight to the 5th day is carried out all statistical analysiss, uses One Way ANOVA, Tukey ' s check, (SigmaStat 2.03 softwares).
The 3rd day of research, the 4th and 5 administration groups (be respectively 27.14mg/kg Herceptin F (ab ')
2The free MMAE of-val-cit-MMAE and 516ug/kg) animal is dying.All animals of the 4th and 5 group are extremely lethargy all, and great majority have yellow ejection in genito-urinary area.At the 3rd day the animal in these administration groups is carried out necropsy.At the 5th day that studies, the 3rd administration group (10.83mg/kg Herceptin F (ab ')
2-animal in val-cit-MMAE) also has yellow ejection in genito-urinary area.
Only obtain the clinical chemistry of 1-3 group and the complete set (baseline, the 3rd and 5 day) of hematology's data; The the 4th and 5 group the 5th day data can't obtain because since significantly morbid state or these animals that lose weight at the 3rd day just by necropsy.When relatively the time, when the histology of these animals of interpretation changes, also considering these discoveries with observed those results in the animal of finishing in the 5th day.
At the 3rd day, the 4th group (the Herceptin F of high dose (ab ')
2) in relevant sero-enzyme ALT, AST of liver and GGT, show the highest rising with the animal of the 5th group (free MMAE), and minimum platelet and numeration of leukocyte.AST and ALT rising is similarly in these two groups, still, compares with the 5th group, and the 4th group shows that significantly higher GGT and total bilirubin raise.GGT and total bilirubin raise and can indicate excretory liver function or biliary system to go wrong.For this observed result, Histological assessment does not show morphologic relevant, and the PK property difference between unclear whether free MMAE and the immune conjugate just can be explained this discovery.At the 3rd day, low dosage Herceptin F (ab ')
2The 3rd group of rising that shows of short duration liver function test of-vc-MMAE, it returned baseline values at the 5th day.But, keep reducing in the 5th day platelet and numeration of leukocyte, there not be the sign of recovery.The animal of handling with total length immune conjugate Herceptin -vc-MMAE (dosage of MMAE is matched with the 4th and 5 group) shows liver function test enhancing and leukocyte and thrombocytopenic typical toxicology feature (profile).During 5 days, all parameters except that bilirubin have all shown and have got along with; But, at the 3rd day, to compare with 5 groups with the 4th, the variation in the 2nd group is less.
In morphology, observed toxicity pattern is identical in 2-5 group, and with before research in the coupling seen.Although have only the minority organ evaluated, the clinical pathology data do not hint at other positions has tangible organ specificity to damage.At the 3rd group (low dosage Herceptin F (ab ')
2) in the metamorphosis minimum, and at the 4th and 5 group (high dose Herceptin F (ab ')
2With free MMAE) middle maximum.Described variation comprises that medullary cell is very few, the atrophy of thymus gland that has obvious apoptosis activity, mitosis and apoptosis cell increase in the intestinal mucosa with mucosa degeneration in various degree and atrophy, and mitosis and apoptosis cell increase in hepatocyte and biliary tract epithelium.2nd, 4 and 5 groups animal has also shown the sign in hepatocyte (dropout) out of control and accidental hepatic necrosis district.
Compare the 4th and 5 group (be respectively 27.14mg/kg Herceptin F (ab ') with the 1st day body weight
2The free MMAE of-val-cit-MMAE and 516ug/kg) animal in lost 14.5 and 12 gram body weight respectively at the 3rd day.Use comparable amount MMAE (2105ug MMAE/m
2) the 2nd treated animal lose weight and not as the 4th and 5 treated animals serious.As shown in Figure 2, with Fa (b ')
2Drug regimen (not having serum albumin binding proteins) is the same, and free MMAE drug regimen produces the feature (profile) that similarly loses weight.Losing weight is toxic indication.
In a word, Herceptin F (ab ')
2-val-cit-MMAE (lysine) causes acute toxicity in the dose dependent mode.Compare as the animal of Herceptin -val-cit-MMAE with the medicine of accepting same amount, the animal among high dose Herceptin F (the ab ') 2-val-cit-MMAE shows obviously bigger losing weight.With with the Herceptin F of high dose (ab ')
2Comparable dosage (the 2150ug/m of-val-cit-MMAE
2) animal of using free MMAE has the relevant sero-enzyme level of the comparable regulating liver-QI that loses weight and the variation of leukocyte and platelet count.Described discovery and consistent (the Wood KW of medicine that uses the formation of inhibition tubulin, Cornwell WD and Jackson JR.Past and future ofthe mitotic spindle as an oncology target.Current Opinion in Pharmacology, 1:370-377.2001).
Embodiment 4-uses the toxicity research of MMAE-Fab conjugate
The toxicity that in female Sprague-Dawley rat (80-100 gram), compares Herceptin -monomethylauristatin (MMAE) immune conjugate, Fab4D5-MMAE and AB.Fab4D5-H-MMAE immune conjugate.
With the dose volume (in PBS, diluting) of 10ml/kg, use dose,equivalent (2105ug MMAE/m to female rats through tail vein injection
2).All dosage solution is used with the single bolus injection.
The administration group:
1=PBS, 6 female
2=20.2mg/kg Herceptin -val-cit-MMAE, 6 female
3＝5.7mg/kg Fab4D5-vc-MMAE
4=14.24mg/kg Fab4D5-vc-MMAE, 6 female
5＝7.85mg/kg AB.Fab4D5-H-vc-MMAE
6=19.62mg/kg AB.Fab4D5-H-vc-MMAE, 6 female
In research before, the 2105ug MMAE/m2 dosage of Herceptin -val-cit-MMAE causes sero-enzyme level and the medium serious variation of hematologic parameter that liver is relevant.In present research, also use the dosage of 5.7mg/kg rhuFab4D5-val-cit-MMAE and 7.85mg/kgrhuFab4D5-H-val-cit-MMAE, to obtain about 840ug MMAE/m
2MMAE expose.
In these are measured, generally the-3 days (before the administration) of research and the 3rd day, under isoflurane anesthesia by eye socket after hole (retro-orbital sinus) collection blood sample (about 500ul) be used for clinical chemistry and hematology.Also when the 5th day necropsy under Patients Under Ketamine Anesthesia, collect blood by postcava.Clinical observation and body weight record carry out once every day, and cage side mortality rate is checked and carried out twice (am/pm) every day.Make dying animal euthanasia.
In the 5th day necropsy process of research, the blood of collecting rat by ventral aorta is used for clinical chemistry and hematology.Collect following tissue: the heart, lung, trachea, liver, kidney, thymus, spleen, brain, axillary gland, complete gastrointestinal tract, skin, bladder and bone marrow.In addition, the organ weight of record liver, thymus, spleen and brain.In necropsy, the liver of weighing, spleen and thymus.Check comprises the check that the cell mean of the weight of animals, numeration of leukocyte, platelet count, AST level, ALT level, GGT level and abnormal level of serum total bilirubin changes.
At the 4th day of research, the animal in the 3rd and 4 administration groups (5.7 and 14.25mg/kgrhuFab4D5-val-cit-MMAE) had medium perpendicular hair, many animal lethargy.At the 4th day, two animals in 14.25mg/kg rhuFab4D5-val-cit-MMAE administration group were dying, and it is carried out necropsy.
The link coupled full length antibody of auristatin E (Herceptin -MC-val-cit-PAB-MMAE, the 2nd group) shown with research before in the identical toxic characteristic (profile) seen: at the 3rd and 5 day, liver function test raises, and has shown the sign that recovered at the 5th day except that GGT.In 5 days research, identical animal shows progressive neutrophil cell and thrombocytopenia.Animal show dose dependency toxicity with two types antibody fragments (rhuFab4D5 and rhuFab4D5-H) processing.At the 3rd and 5 day, for the 3rd and 5 group (being respectively the rhuFab4D5 and the rhuFab4D5-H of low dosage), the substantially the same animal of handling of the sero-enzyme level that liver is relevant in media.In the animal of the 5th group (rhuFab4D5-H-val-cit-MMAE), AST and ALT slight rising in ten fens, still, no matter whether this variation is statistically significant, no matter in fact whether it represented liver toxicity is unclear.At the 5th day, animal in the 3rd group (low dosage rhuFab4D5-val-cit-MMAE) has shown very slight thrombocytopenia, leukopenia 50%, and at the 5th day, animal in the 5th group shows normal numeration of leukocyte (after the decline of the 3rd day slight short time), and at the 5th day slight thrombocytosis is arranged.At the 3rd and 5 day, the animal in the 4th and 6 group (being respectively the rhuFab4D5 and the rhuFab4D5-H of high dose) showed clearly toxicity sign.In the 4th group, as if toxicity is more serious, at the 4th day, makes two animal euthanasias based on ill sign.At these two time points, compare AST, ALT and the GGT higher level of the animal in the 4th group with 6 groups (Herceptin -val-cit-MMAE and rhuFab4D5-H-val-cit-MMAE of comparable drug dose) with the 2nd.Compare with the 2nd group, the level of the animal in the 6th group is lower a little.At the 5th day, the animal in the 4th and 6 group showed very serious platelet and leukopenia.As if with comparing that those are seen in the 2nd treated animal, level is lower, and the animal in the 6th group is much better than the animal in the 4th group a little.
The result of histopathological evaluation is very relevant with clinical observation (body weight determination) and clinical pathology data.2nd, the animal in 3,4 and 6 groups shows that tangible medullary cell reduces; Regenerated sign only appears in the 3rd group the animal.In contrast, the animal in the 5th group shows the bone marrow that constitutes near normal cell.Observed similar discovery in thymus: 2nd, 4 and 6 groups animal shows tangible atrophy and apoptosis activity, and the 3rd and 5 group animal then shows slight atrophy, does not have the obvious apoptosis activity.As if variation in liver, small intestinal and the large intestine more is difficult to quantitatively, still, compares with 5 groups with the 3rd, and mitosis in these organs of the 2nd, 4 and 6 treated animals and/or apoptosis cell are bigger.The necrosis of zonule is only observed in two animals of the 4th group.What is interesting is to have only the little focus (foci) of animal (outside the animal that media is handled) reservation extramedullary hemopoiesis in liver of the 5th group, hint has the free drug of reduced levels in these animals.Compare with the animal of handling with the full length antibody conjugate, in the animal of handling with the Fab immune conjugate, clinical pathology or histopathology data do not show the sign of any different toxicity pattern.
Animal show dose dependency toxicity with two types antibody fragments processing.At the 3rd and 5 day, the animal of administered with high dose rhuFab 4D5-val-cit-MMAE and rhuFab 4D5-H-val-cit-MMAE (containing the albumin binding peptide) showed clearly toxicity sign.As if more serious in rhuFab 4D5 group toxic, at two animal euthanasias of the 4th angel, because they are dying.Histopathological evaluation result is very relevant with clinical observation result (body weight determination) and clinical pathology data.Compare with the animal of handling with the full length antibody conjugate, in the animal of handling with the Fab immune conjugate, clinical pathology or histopathology data do not show the sign of any different toxicity pattern.Described discovery and the result consistent (Wood etc., 2001) who uses the medicine that suppresses tubulin formation.
Fig. 3 shows that albumin changes the toxicity of drug conjugates in conjunction with Toplink.Compare with Fab4D5-vc-MMAE, at the 5th day of research, Ab.Fab4D5-H-vc-MMAE in rat (containing the albumin binding peptide) had obviously littler toxicity.In the animal of using 5.7mg/kg rhuFab4D5-val-cit-MMAE and 7.85mg/kg rhuFab4D5-H-val-cit-MMAE (the 3rd and 5 group), the group mean change of body weight there is no significant difference each other.2nd, 4 and 6 administration groups (being respectively 20.2mg/kgHerceptin -val-cit-MMAE, 14.24mg/kg rhuFab4D5-val-cit-MMAE and 19.62mg/kg rhuFab4D5-H-val-cit-MMAE) have all been accepted 2105ug/M
2MMAE.In the 2nd and 4 treated animals, the group decreased average of body weight is more serious than the 6th treated animal.
Embodiment 5-measures by surperficial plasmon resonance carrying out affinity
Use BIAcore 3000 (BIAcore, Inc., Piscataway, NJ) binding affinity between acquisition SA peptide and the albumin.Use amine to be coupled in the CM5 chip down in about 5000 resonance units (RU) and catch albumin.SA peptide (0,0.625,1.25,2.5,5 and 10 μ M) was injected 30 seconds with 20 μ l/min flow velocitys.Before substrate regeneration, use the 10mM glycine, pH3 allows bonded peptide dissociate 5 minutes.
The signal that will deduct fixed groove (cell) from the signal of the injection by not link coupled groove (cell) is to produce corresponding to the sensing figure as the amount of the bonded peptide of function of time.The buffer that is moved contains the PBS of 0.05%TWEEN-20T, is used to all diluted samples.BIAcore kinetics assessment software (v 3.1) is used for determining dissociation constant (K according to the combination and the speed of dissociating
d), used combination model one to one.
Assess selected peptide in conjunction with people (HAS), rabbit (BuSA), rat (RSA) and the albuminised affinity of mice (MSA) by BIAcore mensuration and SA08 peptide competitive assay.As the digital proof as shown in the following table 8 IC that in competitive assay, obtains
50Value can be successfully and the K that obtains in BIAcore measures
dValue relatively.In competitive assay, representative is used for having minimum IC in conjunction with the peptide SA15 of the albuminised total polypeptide of rabbit
50Value, but the rabbit albumin is had the highest affinity by surperficial plasmon resonance.A kind of SA06 that is same as, but two Cys residues are all changed into the linear peptides of Ala, have the IC greater than 50 μ M
50, proved the importance of disulfide bond.
The mensuration of the relative Kd of embodiment 6-
Foreword:
In the binding ability between evaluating protein matter, method selected is ELISA.Because therefore exploitation high specific and quantitative mensuration make ELISA to be used widely easily.But, directly be fixed at protein under the situation of the surface of solids, because potential pseudomorphism can appear in degeneration or cover in conjunction with epi-position.
For detect with the link coupled albumin binding peptide of Fab molecule (Fab-H) to albuminised affinity, we have developed two kinds of ELISA.First kind comprises albumin is adsorbed on hole surface, and detects bonded Fab with goat-anti--huFab-HRP.Second kind is included in the solution with albumin in conjunction with Fab-H, and measures dissociation constant (Kd).The ultimate principle of second kind of mensuration is to allow the Fab-H of constant density combine with not commensurability albumin reaching balance in solution, and measures unconjugated Fab-H at bag in by albuminised ELISA hole.Can use Scatchard analysis (Scatchard Analysis) by analytical data determine the Kd value (Munson etc., 1980, Anal.Biochem., 107:220).
Material and method:
Material:
Mice albumin-lyophilized form, catalog number (Cat.No.) A3139
Rat albumin-lyophilized form, catalog number (Cat.No.) A6414
Rabbit albumin-lyophilized form, catalog number (Cat.No.) A0639
By the 10mg albumin being dissolved in preparation 1mg/ml albumin soln among the 10ml PBS.Solution is housed in 4 ℃.
Measure buffer: PBS+0.5% egg white powder (Sigma#A5503)+0.5%Tween 20, pH7.4.
Direct bonded ELISA measures
Under 4 ℃, mice, rat or rabbit albumin (Sigma) be fixed on the NUNCMaxisorp 96 hole flat boards with 2 μ g/ml spend the night.After removing coating buffer, sealed dull and stereotyped 1 hour with binding buffer liquid (PBS, 0.5% ovalbumin and 0.05%Tween 20) down at 25 ℃.The Fab-Hx of serial dilution in binding buffer liquid is added with the 100ul/ hole, and under 25 ℃, make it in conjunction with the albumin of bag quilt 30 minutes.By removing unconjugated Fab-Hx, by descending and the anti-human Fabs of goat ' at 25 ℃ with the 0.05%PBS/Tween20 washing hole
2-HRP incubation detected bonded Fab-Hx molecule in 1 hour.Then, with tetramethyl benzidine (TMB)/H
2O
2The bonded HRP of measured in solution., react after 15 minutes at incubation by adding the cancellation of 1M phosphoric acid.Read out in the absorbance of 450nm with the reference wavelength of 650nm.
Kd (having the solution combination of precincubation) ELISA measures
At first, the albumin that is dissolved in the mensuration buffer of Fab-H of incubation fixed concentration (in above-mentioned bonded ELISA, measuring) and variable concentrations in solution.At room temperature 〉=2 behind hour incubation, the mixed liquor of 100 μ l is transferred in the ELISA flat board of albumin bag quilt.Then, measure the concentration of free F ab-Hx by aforesaid direct bonded ELISA.
During the Fab-H of fixed concentration and the albumin of initial concentration are listed in the table below.Albumin is with serial dilution in 1: 38 times.
| Molecule | Fab-H | Fab-H4 | Fab-H8 | Fab-H10 | Fab-H11 |
| [Fab-H] | 0.5nM | 200nM | 22.5nM | 3μM | 3μM |
| [rabbit albumin] | 3μM | 3μM | 3μM | 30μM | 30μM |
| [Fab-H] | 0.25nM | 0.125nM | 0.125nM | 12nM | 800nM |
| [rat albumin] | 3μM | 3μM | 3μM | 30μM | 60μM |
| [Fab-H] | 0.25nM | 6.25pM | 31.25pM | 62.5nM | 62.5nM |
| [mice albumin] | 3μM | 3μM | 3μM | 119μM | 119μM |
The result:
Friguet etc. at first disclose in 1985 and have used the affinity of ELISA to measure.Measuring Kd and selecting in the humanized antibody of HER2 ECD, we have used this method (Carter etc., Proc.Natl.Acad. Sci.89,4285,1992).
Generally speaking, binding equilibrium research requires the concentration of antibody to approach, or is lower than the dissociation constant value.Because dissociation constant is in advance unknown, therefore preferably select to provide total Fab-H concentration of enough absorbances in conjunction with ELISA what be used for measuring free F ab-Hx.Measure this concentration by titration Fab-Hx in direct bonded ELISA.
For check antibody-albumin has reached balance, with Fab-H and rabbit albumin incubation 1 hour, 2 hours and spend the night, assaying reaction mixed liquor in bonded ELISA then.Behind 2 hours incubations, arrive balance.
The Best Times that needs for the albumin of determining the bag quilt in the free F ab-H combined hole will wrap the albumin incubation 15,30,45,60 and 120 minutes of quilt in Ab-albumin mixed liquor and the hole.Measure based on this, determine 30 minutes be in conjunction with the needed minimum time of all free F ab-H.Kd (having the solution combination of the precincubation) result that ELISA measures is as shown in table 10.
Sequence table
| SEQ ID No. | |
| 1 | Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser- |
| 2 | Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys- |
| 3 | Cys-Tyr-Xaa1-Pro-Gly-Xaa- |
| 4 | Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu- |
| 5 | Trp-Cys-Asp-Xaa-Xaa-Leu-Xaa-Ala-Xaa-Asp-Leu-Cys |
| 6 | Asp-Leu-Val-Xaa-Leu-Gly-Leu-Glu-Cys-Trp |
| 7 | DLCLRDWGCLW |
| 8 | DICLPRWGCLW |
| 9 | |
| 10 | QRLMEDICLPRWGCLWEDDE |
| 11 | QGLIGDICLPRWGCLWGDSV |
| 12 | QGLIGDICLPRWGCLWGDSVK |
| 13 | EDICLPRWGCLWEDD |
| 14 | |
| 15 | MEDICLPRWGCLWEDD |
| 16 | MEDICLPRWGCLWED |
| 17 | RLMEDICLARWGCLWEDD |
| 18 | EVRSFCTRWPAEKSCKPLRG |
| 19 | |
| 20 | EMCYFPGICWM |
| 21 | CXXGPXXXXC |
| 22 | XXXXCXXGPXXXXCXXXX |
| 23 | CXXXXXXCXXXXXXCCXXXCXXXXXXC |
| 24 | |
| 25 | CCXXXXXCXXXXCXXXXCC |
| 26 | CXCXXXXXXXCXXXCXXXXXX |
| 27 | GENWCDSTLMAYDLCGQVNM |
| 28 | MDELAFYCGIWECLMHQEQK |
| 29 | DLCDVDFCWF |
| 30 | KSCSELHWLLVEECLF |
| 31 | EVRSFCTDWPAEKSCKPLRG |
| 32 | CEVALDACRGGESGCCRHICELIRQLC |
| 33 | RNEDPCVVLLEMGLECWEGV |
| 34 | DTCVDLVRLGLECWG |
| 35 | QRQMVDFCLPQWGCLWGDGF |
| 36 | CGCVDVSDWDCWSECLWSHGA |
| 37 | GEDWCDSTLLAFDLCGEGAR |
| 38 | GENWCDWVLLAYDLCGEDNT |
| 39 | MELWCDSTLMAYDLCGDFNM |
| 40 | EVRSFCTDWPAHYSCTSLQG |
| 41 | GRSFCMDWPAHKSCTPLML |
| 42 | GVRTFCQDWPAHNSCKLLRG |
| 43 | QTRSFCADWPRHESCKPLRG |
| 44 | RRTCDWPHNSCKLRG |
| 45 | RAAESSVCYWPGICFDRTEQ |
| 46 | MEPSRSVCYAEGICFDRGEQ |
| 47 | REPASLVCYFEDICFVRAEA |
| 48 | RGPDVCYWPSICFERSMP |
| 49 | LVPERIVCYFESICYERSEL |
| 50 | RMPASLPCYWETICYESSEQ |
| 51 | RTAESLVCYWPGICFAQSER |
| 52 | RAPERWVCYWEGICFDRYEQ |
| 53 | EICYFPGICWI |
| 54 | ELCYFPGICWT |
| 55 | DICYIPGICWM |
| 56 | KLCYFPGICWS |
| 57 | DLCYFPGICWM |
| 58 | GMCYFPGICWA |
| 59 | EMCYFPGICWS |
| 60 | EMCYFPGICWT |
| 61 | KTCYFPGICWM |
| 62 | KVCYFPGICWM |
| 63 | DVCYFPGICWM |
| 64 | EICYFPGICWM |
| 65 | ALCYFPGICWM |
| 66 | ELCYFPGICWP |
| 67 | ELCYFPGICWM |
| 68 | DMCYFPGICWL |
| 69 | DMCYFPGICFN |
| 70 | ETCYFPGICWL |
| 71 | EVCYFPGICWF |
| 72 | EVCYFPGICWE |
| 73 | EVCYFPGICWM |
| 74 | LAEMCYFPGICWMSA |
| 75 | GGEICYFPGICRVLP |
| 76 | EHDMCYFPGICWIAD |
| 77 | VQEVCYFPGICWMQE |
| 78 | SREVCYYPGICWNGA |
| 79 | DSEVCYFPGICWSGT |
| 80 | GTEVCYFPGICWGGG |
| 81 | SYAPCYFPGICWMGN |
| 82 | HAEICYFPGICWTER |
| 83 | NDEICYFPGVCWKSG |
| 84 | RDTVCYFPGICWMAS |
| 85 | VRDMCYFPGICWKSE |
| 86 | ASEICYFPGICWMVE |
| 87 | QTELCYFPGICWNES |
| 88 | TTEMCYFPGICWKTE |
| 89 | KTEICYFPGICWMSG |
| 90 | QCFPGWVK |
| 91 | IVEMCYYPGICWISP |
| 92 | SGAICYVPGICWTHA |
| 93 | QRHPEDICLPRWGCLWGDDD |
| 94 | NRQMEDICLPQWGCLWGDDF |
| 95 | QRLMEDICLPRWGCLWGDRF |
| 96 | QWHMEDICLPQWGCLWGDVL |
| 97 | QWQMENVCLPKWGCLWEELD |
| 98 | LWAMEDICLPKWGCLWEDDF |
| 99 | LRLMDNICLPRWGCLWDDGF |
| 100 | HSQMEDICLPRWGCLWGDEL |
| 101 | QWQVMDICLPRWGCLWADEY |
| 102 | HRLVEDICLPRWGCLWGNDF |
| 103 | QMHMMDICLPKWGCLWGDTS |
| 104 | LRIFEDICLPKWGCLWGEGF |
| 105 | QSYMEDICLPRWGCLSDDAS |
| 106 | QGDFWDICLPRWGCLSGEGY |
| 107 | RWQTEDVCLPKWGCLFGDGV |
| 108 | LIFMEDVCLPQWGCLWEDGV |
| 109 | QRDMGDICLPRWGCLWEDGV |
| 110 | QRHMMDFCLPKWGCLWGDGY |
| 111 | QRPIMDFCLPKWGCLWEDGF |
| 112 | ERQMVDFCLPKWGCLWGDGF |
| 113 | QGYMVDFCLPRWGCLWGDAN |
| 114 | KMGRVDFCLPKWGCLWGDEL |
| 115 | QSQLEDFCLPKWGCLWGDGF |
| 116 | QGGMGDFCLPQWGCLWGEDL |
| 117 | QRLMWEICLPLWGCLWGDGL |
| 118 | QRQIMDFCLPHWGCLWGDGF |
| 119 | GRQVVDFCLPKWGCLWEEGL |
| 120 | QMQMSDFCLPQWGCLWGDGY |
| 121 | KSRMGDFCLPEWGCLWGDEL |
| 122 | ERQMEDFCLPQWGCLWGDGV |
| 123 | QRQVVDFCLPQWGCLWGDGS |
| 124 | DICLPEWGCLW |
| 125 | DICLPVWGCLW |
| 126 | DLCLPEWGCLW |
| 127 | DLCLPKWGCLW |
| 128 | DLCLPVWGCLW |
| 129 | DICLPAWGCLW |
| 130 | DICLPDWGCLW |
| 131 | DICLERWGCLW |
| 132 | EWDVCLPHWGCLWDG |
| 133 | WDDICFRDWGCLWGS |
| 134 | MDDICLHHWGCLWDE |
| 135 | MDDLCLPNWGCLWGD |
| 136 | FEDFCLPNWGCLWGS |
| 137 | FEDLCVVRWGCLWGD |
| 138 | WEDLCLPDWGCLWED |
| 139 | SEDFCLPVWGCLWED |
| 140 | DFDLCLPDWGCLWDD |
| 141 | NWDLCFPDWGCLWDD |
| 142 | EEDLCLPVWGCLWGA |
| 143 | EEDVCLPVWGCLWEG |
| 144 | MFDLCLPKWGCLWGN |
| 145 | EFDLCLPTWGCLWED |
| 146 | MWDVCFPDWGCLWDV |
| 147 | EWDVCFPAWGCLWDQ |
| 148 | VWDLCLPQWGCLWDE |
| 149 | DTCADLVRLGLECWA |
| 150 | NTCADLVRLGLECWA |
| 151 | DTCDDLVQLGLECWA |
| 152 | DTCEDLVRLGLECWA |
| 153 | DSCGDLLRLGLECWA |
| 154 | DTCSDLVGLGLECWA |
| 155 | X 5DXCLPXWGCLWX 4 |
| 156 | X 4DXCLPXWGCLWX 3 |
| 157 | AAQVGDICLPRWGCLWSEYA |
| 158 | AGWAADVCLPRWGCLWEEDV |
| 159 | ASVVDDICLPVWGCLWGEDI |
| 160 | ATMEDDICLPRWGCLWGAEE |
| 161 | DEDFEDYCLPPWGCLWGSSM |
| 162 | EGTWDDFCLPRWGCLWLGER |
| 163 | ERWEGDVCLPRWGCLWGESG |
| 164 | GDWMHDICLPKWGCLWDEKA |
| 165 | GIEWGDTCLPKWGCLWRVEG |
| 166 | GQQGEDVCLPVWGCLWDTSS |
| 167 | GRYPMDLCLPRWGCLWEDSA |
| 168 | GSAGDDLCLPRWGCLWERGA |
| 169 | HASDWDVCLPGWGCLWEEDD |
| 170 | LGVTHDTCLPRWGCLWDEVG |
| 171 | LVWEEDFCLPKWGCLWGAED |
| 172 | NVGWNDICLPRWGCLWAQES |
| 173 | QGVEWDVCLPQWGCLWTREV |
| 174 | RLDAWDICLPQWGCLWEEPS |
| 175 | SEAPGDYCLPRWGCLWAQEK |
| 176 | TAMDEDVCLPRWGCLWGSGS |
| 177 | TEIGQDFCLPRWGCLWVPGT |
| 178 | TLGWPDFCLPKWGCLWRESD |
| 179 | TLSNQDICLPGWGCLWGGIN |
| 180 | TSTGGDLCLPRWGCLWDSSE |
| 181 | VSEMDDICLPLWGCLWADAP |
| 182 | VSEWEDICLPSWGCLWETQD |
| 183 | VVGDGDFCLPKWGCLWDQAR |
| 184 | VVWDDDVCLPRWGCLWEEYG |
| 185 | WSDSDDVCLPRWGCLWGNVA |
| 186 | WVEEGDICLPRWGCLWESVE |
| 187 | AQAMGDICLPRWGCLWEAEI |
| 188 | ASDRGDLCLPYWGCLWGPDG |
| 189 | ASDPGDVCLPRWGCLWGESF |
| 190 | ASNWEDVCLPRWGCLWGERN |
| 191 | ASTPRDICLPRWGCLWSEDA |
| 192 | DGEEGDLCLPRWGCLWALEH |
| 193 | EGEEVDICLPQWGCLWGYPV |
| 194 | EVGDLDLCLPRWGCLWGNDK |
| 195 | FRDGEDFCLPQWGCLWADTS |
| 196 | GDMVNDFCLPRWGCLWGSEN |
| 197 | GRMGTDLCLPRWGCLWGEVE |
| 198 | HEWERDICLPRWGCLWRDGD |
| 199 | KKVSGDICLPIWGCLWDNDY |
| 200 | LLESDDICLPRWGCLWHEDG |
| 201 | MQAESDFCLPHWGCLWDEGT |
| 202 | MQGPLDICLPRWGCLWGGVD |
| 203 | QMPLEDICLPRWGCLWEGRE |
| 204 | REEWGDLCLPTWGCLWETKK |
| 205 | RVWTEDVCLPRWGCLWSEGN |
| 206 | SIREYDVCLPKWGCLWEPSA |
| 207 | SPTEWDMCLPKWGCLWGDAL |
| 208 | SSGLEDICLPNWGCLWADGS |
| 209 | SVGWGDICLPVWGCLWGEGG |
| 210 | TEENWDLCLPRWGCLWGDDW |
| 211 | TSGSDDICLPVWGCLWGEDS |
| 212 | TWPGDLCLPRWGCLWEAES |
| 213 | WDHELDFCLPVWGCLWAEDV |
| 214 | WTESEDICLPGWGCLWGPEV |
| 215 | WVPFEDVCLPRWGCLWSSYQ |
| 216 | EEDSDICLPRWGCLWNTS |
| 217 | EGYWDLCLPRWGCLWELE |
| 218 | ELGEDLCLPRWGCLWGSE |
| 219 | ETWSDVCLPRWGCLWGAS |
| 220 | GDYVDLCLPGWGCLWEDG |
| 221 | GVLDDICLPRWGCLWGPK |
| 222 | HMMDDVCLPGWGCLWASE |
| 223 | IDYTDLCLPAWGCLWELE |
| 224 | IEHEDLCLPRWGCLWAVD |
| 225 | ISEWDLCLPRWGCLWDRS |
| 226 | ISWADVCLPKWGCLWGKD |
| 227 | ISWGDLCLPRWGCLWEGS |
| 228 | KLWDDICLPRWGCLWSPL |
| 229 | LAWPDVCLPRWGCLWGGM |
| 230 | LNESDICLPTWGCLWGVD |
| 231 | LPEQDVCLPVWGCLWDAN |
| 232 | MAWGDVCLPRWGCLWAGG |
| 233 | NEEWDVCLPRWGCLWGGV |
| 234 | QELQDFCLPRWGCLWGVG |
| 235 | QREWDVCLPRWGCLWSDV |
| 236 | QRFWDTCLPRWGCLWGGD |
| 237 | RVFTDVCLPRWGCLWDLG |
| 238 | SGWDDVCLPVWGCLWGPS |
| 239 | SSASDYCLPRWGCLWGDL |
| 240 | SWQGDICLPRWGCLWGVD |
| 241 | SYETDVCLPYWGCLWEDA |
| 242 | SYWGDVCLPRWGCLWSEA |
| 243 | TLEWDMCLPRWGCLWTEQ |
| 244 | VGEFDICLPRWGCLWDAE |
| 245 | VTSWDVCLPRWGCLWEED |
| 246 | WLWEDLCLPKWGCLWEED |
| 247 | |
| 248 | ASEWDVCLPTWGCLWMEG |
| 249 | AYSADICLPRWGCLWMSE |
| 250 | EDWEDICLPQWGCLWEGM |
| 251 | EDWTDLCLPAWGCLWDTE |
| 252 | EEWEDLCLPRWGCLWSAE |
| 253 | EFWQDICLPNWGCLWAES |
| 254 | EGFSDICLPRWGCLWSQE |
| 255 | ETWEDLCLPNWGCLWDLE |
| 256 | GEVNDFCLPRWGCLWEGD |
| 257 | GGEWDVCLPAWGCLWGEE |
| 258 | KDWYDICLPRWGCLWGGE |
| 259 | KLGQDICLPRWGCLWDFA |
| 260 | LEEWDICLPQWGCLWREG |
| 261 | LVLPDICLPKWGCLWGDT |
| 262 | MDLADICLPKWGCLWESD |
| 263 | MVLDDICLPRWGCLWSEK |
| 264 | MWSGDLCLPRWGCLWGET |
| 265 | NRMGDICLPRWGCLWDGH |
| 266 | RDWEDLCLPNWGCLWELS |
| 267 | RGDWDLCLPKWGCLWEGV |
| 268 | RQWEDICLPRWGCLWGVG |
| 269 | RVEYDLCLPRWGCLWEPP |
| 270 | SIWSDICLPRWGCLWESD |
| 271 | TDEWDICLPNWGCLWEAG |
| 272 | TEDVDFCLPLWGCLWEEP |
| 273 | VKEEDFCLPRWGCLWEAG |
| 274 | WDFEDICLPRWGCLWADM |
| 275 | WEDWDVCLPRWGCLWGGG |
| 276 | YEDIDICLPRWGCLWDLS |
| 277 | AGLDEDICLPRWGCLWGKEA |
| 278 | AGMMGDICLPRWGCLWQGEP |
| 279 | APGDWDFCLPKWGCLWDDDA |
| 280 | AQLFDDICLPRWGCLWSDGY |
| 281 | ARTMGDICLPRWGCLWGASD |
| 282 | AWQDFDVCLPRWGCLWEPES |
| 283 | DTTWGDICLPRWGCLWSEEA |
| 284 | EGFLGDICLPRWGCLWGHQA |
| 285 | EQWLHDICLPKWGCLWDDTD |
| 286 | ETGWPDICLPRWGCLWEEGE |
| 287 | FELGEDICLPRWGCLWEEHN |
| 288 | GASLGDICLPRWGCLWGPED |
| 289 | GEWWEDICLPRWGCLWGSSS |
| 290 | GSLESDICLPRWGCLWGIDE |
| 291 | GWLEEDICLPKWGCLWGADN |
| 292 | HEQWDDICLPRWGCLWGGSY |
| 293 | QRVDDDICLPRWGCLWGENS |
| 294 | SVGWGDICLPKWGCLWAESD |
| 295 | TLMSNDICLPRWGCLWDEPK |
| 296 | TLVLDDICLPRWGCLWDMTD |
| 297 | TWQGEDICLPRWGCLWDTEV |
| 298 | VGVFDDICLPRWGCLWEQPV |
| 299 | VPAMGDICLPRWGCLWEARN |
| 300 | VSLGDDICLPKWGCLWEPEA |
| 301 | VWIDRDICLPRWGCLWDTEN |
| 302 | WRWNEDICLPRWGCLWEEEA |
| 303 | AVSWADICLPRWGCLWERAD |
| 304 | AWLDEDICLPKWGCLWNTGV |
| 305 | FSLDEDICLPKWGCLWGAEK |
| 306 | GDLGDDICLPRWGCLWDEYP |
| 307 | GEGWSDICLPRWGCLWAEDE |
| 308 | GLMGEDICLPRWGCLWKGDI |
| 309 | GWHDRDICLPRWGCLWEQND |
| 310 | LLGGHDICLPRWGCLWGGDV |
| 311 | MRWSSDICLPKWGCLWGDEE |
| 312 | QFEWDDICLPRWGCLWEVEV |
| 313 | QGWWHDICLPRWGCLWEEGE |
| 314 | REGWPDICLPRWGCLWSETG |
| 315 | RELWGDICLPRWGCLWEHAT |
| 316 | RLELMDICLPRWGCLWDPQD |
| 317 | SGVLGDICLPRWGCLWEEAG |
| 318 | SLGLTDLCLPRWGCLWEEEQ |
| 319 | SSLEQDICLPRWGCLWGQDA |
| 320 | SVLSDDICLPRWGCLWWDFS |
| 321 | TSLLDDICLPRWGCLWYEEG |
| 322 | TSLADDICLPRWGCLWSEDG |
| 323 | VEMWHDICLPRWGCLWDSNA |
| 324 | WDLASDICLPRWGCLWEEEA |
| 325 | FITQDICLPRWGCLWGEN |
| 326 | FLWRDICLPRWGCLWSEG |
| 327 | FVHEDICLPRWGCLWGEG |
| 328 | GLGDDICLPRWGCLWGRD |
| 329 | GMFDDICLPKWGCLWGLG |
| 330 | GPGWDICLPRWGCLWGEE |
| 331 | GPWYDICLPRWGCLWDGV |
| 332 | GWDDDICLPRWGCLWGDG |
| 333 | LEYEDICLPKWGCLWGGE |
| 334 | LLDEDICLPRWGCLWGVR |
| 335 | LMSPDICLPKWGCLWEGD |
| 336 | LVLGDICLPRWGCLWESD |
| 337 | MLSRDICLPRWGCLWEEE |
| 338 | MPWTDICLPRWGCLWSES |
| 339 | RLGSDICLPRWGCLWGAG |
| 340 | RLGSDICLPRWGCLWDYQ |
| 341 | SPWMDICLPRWGCLWESG |
| 342 | STFTDICLPRWGCLWELE |
| 343 | SVLSDICLPRWGCLWEES |
| 344 | TWFSDICLPRWGCLWEPG |
| 345 | VHQADICLPRWGCLWGDT |
| 346 | VLLGDICLPLWGCLWGED |
| 347 | VNWGDICLPRWGCLWGES |
| 348 | VVWSDICLPRWGCLWDKE |
| 349 | VWYKDICLPRWGCLWEAE |
| 350 | WDYGDICLPRWGCLWEEG |
| 351 | WEVQDICLPRWGCLWGDD |
| 352 | YIWRDICLPRWGCLWEGE |
| 353 | YRDYDICLPRWGCLWDER |
| 354 | AFWSDICLPRWGCLWEED |
| 355 | DWGRDICLPRWGCLWDEE |
| 356 | EAWGDICLPRWGCLWELE |
| 357 | LILSDICLPRWGCLWDDT |
| 358 | LKLEDICLPRWGCLWGES |
| 359 | LLTRDICLPKWGCLWGSD |
| 360 | LRWSDICLPRWGCLWEET |
| 361 | LYLRDICLPKWGCLWEAD |
| 362 | NWYDDICLPRWGCLWDVE |
| 363 | QDWEDICLPRWGCLWGD |
| 364 | QSWPDICLPKWGCLWGEG |
| 365 | TLLQDICLPRWGCLWESD |
| 366 | VRLMDICLPRWGCLWGEE |
| 367 | VRWEDICLPRWGCLWGEE |
| 368 | WDVADICLPRWGCLWAED |
| 369 | WHMGDICLPRWGCLWSEV |
| 370 | WKDFDICLPRWGCLWDDH |
| 371 | WLSEDICLPQWGCLWEES |
| 372 | WLSEDICLPRWGCLWAAD |
| 373 | WLSDDICLPRWGCLWDDL |
| 374 | EVREWDICLPRWGCLWENWR |
| 375 | FGQEWDICLPRWGCLWGNEQ |
| 376 | TWQLEDICLPRWGCLWEDGL |
| 377 | NTPTYDICLPRWGCLWGDVP |
| 378 | QPVWSDICLPRWGCLWGEDH |
| 379 | SWYGGDICLP-WGCLWSEES |
| 380 | WGMARDWCLPMWGCLWRGGG |
| 381 | WHLTDDICLPRWGCLWGDEQ |
| 382 | NWAENDICLPRWGCLWGDEN |
| 383 | SAREWDICLPTWGCLWEKDI |
| 384 | AGEWDICLPRWGCLWDVE |
| 385 | EIRWDFCLPRWGCLWDED |
| 386 | ESLGDICLPRWGCLWGSG |
| 387 | EYWGDICLPRWGCLWDWQ |
| 388 | KMWSDICLPRWGCLWEEE |
| 389 | MGTKDICLPRWGCLWAEA |
| 390 | MHEWDICLPRWGCLWESS |
| 391 | RGLHDACLPWWGCLWAGS |
| 392 | RLFGDICLPRWGCLWQGE |
| 393 | SGEWDICLPRWGCLWGEG |
| 394 | SMFFDHCLPMWGCLWAEQ |
| 395 | VGEWDICLPNWGCLWERE |
| 396 | WWMADRCLPLWGCLWRGD |
| 397 | WWVRDLCLPTWGCLWSGK |
| 398 | YFDGDICLPRWGCLWGSD |
| 399 | TLFQDICLPRWGCLWEES |
| 400 | WFPKDRCLPVWGCLWERH |
| 401 | QRLMEDICLPRWGCLWEDDF |
| 402 | RLIEDICLPRWGCLWEDD |
| 403 | QRLMEDICLPRWGCLWE |
| 404 | GEWWEDICLPRWGCLWEEED |
| 405 | QRLIEDICLPRWGCLWEDDF |
| 406 | RLIEDICLPRWGCLWED |
| 407 | RLIEDICLPRWGCLWE |
| 408 | RLIEDICLPRWGCLW |
| 409 | RLIEDICLPRWGCL |
| 410 | RLIEDICLPRWGC |
| 411 | LIEDICLPRWGCLWED |
| 412 | IEDICLPRWGCLWED |
| 413 | EDICLPRWGCLWED |
| 414 | DICLPRWGCLWED |
| 415 | ICLPRWGCLWED |
| 416 | CLPRWGCLWED |
| 417 | IEDICLPRWGCLWE |
| 418 | EDICLPRWGCLW |
| 419 | DICLPRWGCL |
| 420 | ICLPRWGCLW |
| 421 | ICLPRWGC |
| 422 | GGGS |
| 423 | DXCLPXWGCLW |
| 424 | X 4DICLPRWGCLWX 3 |
| 425 | X 5DICLPRWGCLWX 4 |
| 426 | XXEMCYFPGICWMXX |
| 427 | XXDLCLRDWGCLWXX |
| 428 | Light chain variable sequence described in Fig. 4 |
| 429 | Weight chain variable sequence described in Fig. 4 |
Table 1
Albumin is in conjunction with the species specificity of phage display peptide
| SEQ ID NO： | The library | The bacteriophage combination | |||
| SA selects rabbit | Rabbit | The people | Rat | ||
| 27 28 29 30 | BA BB BC BD | GENWCDSTLMAYDLCGQVNM MDELAFYCGIWECLMHQEQK DLCDVDFCWF KSCSELHWLLVEECLF selects at people SA | +++ +++ +++ +++ | - - - - | - - - - |
| 31 19 20 32 | HA HB HC HE | EVRSFCTDWPAEKSCKPLRG RAPESFVCYWETICFERSEQ EMCYFPGICWM CEVALDACRGGESGCCRHICELIRQLC selects at rat SA | - - - - | +++ ++ +++ (+) | - (+) ++ - |
| 33 34 35 7 36 | RA RD RB RC RE | RNEDPCVVLLEMGLECWEGV DTCVDLVRLGLECWG QRQMVDFCLPQWGCLWGDGF DLCLRDWGCLW CGCVDVSDWDCWSECLWSHGA | - - ++ - - | - - + - - | +++ +++ +++ +++ +++ |
Table 2
| In conjunction with | |||||
| The sequence of selecting at the rabbit albumin | SEQ ID | The people | Rabbit | Rat | |
| Library BA BA-B44 BA-B37 BA-B39 | GENWCDSTLMAYDLCGQVNM GEDWCDSTLLAFDLCGEGAR GENWCDWVLLAYDLCGEDNT MELWCDSTLMAYDLCGDFNM | 27 37 38 39 | - - - | +++ +++ +++ | - - - |
| The sequence of selecting at HSA | |||||
| HB HB-H2 HB-H8 HB-H3 HB-H6 HB-H4 HB-H16 HB-H18 HB-H1 library, HA HA-H74 HA-H83 HA-H73 HA-H76 HA-H84 library, library HC HB-H12 HB-H13 HC-H6 HC-H2 HC-H3 HC-H4 HC-H7 HC-H9 HC-H10 | EVRSFCTDWPAEKSCKPLRG EVRSFCTDWPAHYSCTSLQG G-RSFCMDWPAHKSCTPLML GVRTFCQDWPAHNSCKLLRG QTRSFCADWPRHESCKPLRG R-RT-C-DWP-HNSCK-LRG RAPESFVCYWETICFERSEQ RAAESSVCYWPGICFDRTEQ MEPSRSVCYAEGICFDRGEQ REPASLVCYFEDICFVRAEA RGPD-V-CYWPSICFERSMP LVPERIVCYFESICYERSEL RMPASLPCYWETICYESSEQ RTAESLVCYWPGICFAQSER RAPERWVCYWEGICFDRYEQ EMCYFPGICWM EICYFPGICWI ELCYFPGICWT DICYIPGICWM KLCYFPGICWS DLCYFPGICWM GMCYFPGICWA EMCYFPGICWS EMCYFPGICWT KTCYFPGICWM | 31 40 41 42 43 44 19 45 46 47 48 49 50 51 52 20 53 54 55 56 57 58 59 60 61 | +++ +++ +++ +++ +++ +++ +++ + + + + + (+) ++ ++ ++ ++ ++ ++ ++ ++ ++ | - - - - - - - - - - - - - - - - - - - - - - | - - - - - - - - - - - - - - - - - - - - - - |
| The people | Rabbit | Rat | |||
| HC-H5 | KVCYFPGICWM | 62 | |||
| HC-H8 HC-H17 HC-H14 HC-H15 HC-H20 HC-H13 HC-H18 HC-H12 HC-H11 HC-H16 HC-H19 library HBC HBC-H7 HBC-H4 HBC-H6 HBC-H10 HBC-H2 HBC-H1 HBC-H3 HBC-H8 HBC-H17 HBC-H11 HBC-H18 HBC-H19 HBC-H12 HBC-H13 HBC-H14 HBC-H15 HBC-H16 HB-H10 HB-H7 | DVCYFPGICWM EICYFPGICWM ALCYFPGICWM ELCYFPGICWP ELCYFPGICWM DMCYFPGICWL DMCYFPGICFN ETCYFPGICWL EVCYFPGICWF EVCYFPGICWE EVCYFPGICWM XXEMCYFPGICWMXX LAEMCYFPGICWMSA GGEICYFPGICRVLP EHDMCYFPGICWIAD VQEVCYFPGICWMQE SREVCYYPGICWNGA DSEVCYFPGICWSGT GTEVCYFPGICWGGG SYAPCYFPGICWMGN HAEICYFPGICWTER NDEICYFPGVCWKSG RDTVCYFPGICWMAS VRDMCYFPGICWKSE ASEICYFPGICWMVE QTELCYFPGICWNES TTEMCYFPGICWKTE KTEICYFPGICWMSG Q---C-FPG--WV-K IVEMCYYPGICWISP SGAICYVPGICWTHA | 63 64 65 66 67 68 69 70 71 72 73 426 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 | ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ | - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - | - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - |
| The sequence of selecting at the rat albumin | SEQ ID NO： | ||||
| Library RB RB-H1 RB-H6 RB-B2 RB-B5 RB-B6 RB-B4 RB-B7 RB-B8 RB-B11 RB-B12 RB-B16 RB-B9 RB-B14 RB-B3 RB-B10 RB-B1 RB-R8 RB-R16 HC-R10 RB-R4 RB-R7 RB-R11 RB-R12 RB-R13 RB-R15 RB-R17 RB-R5 RB-R10 RB-R2 RB-R3 RB-R9 RB-R1 RB-R14 | QRQMVDFCLPQWGCLWGDGF QRHPEDICLPRWGCLWGDDD NRQMEDICLPQWGCLWGDDF QRLMEDICLPRWGCLWGDRF QWHMEDICLPQWGCLWGDVL QWQMENVCLPKWGCLWEELD LWAMEDICLPKWGCLWEDDF LRLMDNICLPRWGCLWDDGF HSQMEDICLPRWGCLWGDEL QWQVMDICLPRWGCLWADEY QGLIGDICLPRWGCLWGDSV HRLVEDICLPRWGCLWGNDF QMHMMDICLPKWGCLWGDTS LRIFEDICLPKWGCLWGEGF QSYMEDICLPRWGCLSDDAS QGDFWDICLPRWGCLSGEGY RWQTEDVCLPKWGCLFGDGV QGLIGDICLPRWGCLWGDSV LIFMEDVCLPQWGCLWEDGV QRDMGDICLPRWGCLWEDGV QRHMMDFCLPKWGCLWGDGY QRPIMDFCLPKWGCLWEDGF ERQMVDFCLPKWGCLWGDGF QGYMVDFCLPRWGCLWGDAN KMGRVDFCLPKWGCLWGDEL QSQLEDFCLPKWGCLWGDGF QGGMGDFCLPQWGCLWGEDL QRLMWEICLPLWGCLWGDGL QRQIMDFCLPHWGCLWGDGF GRQVVDFCLPKWGCLWEEGL QMQMSDFCLPQWGCLWGDGY KSRMGDFCLPEWGCLWGDEL ERQMEDFCLPQWGCLWGDGV QRQVVDFCLPQWGCLWGDGS | 35 93 94 95 96 97 98 99 100 101 11 102 103 104 105 106 107 11 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 | The people ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ (+) (+) (+)--++ ++ ++-------------- | Rabbit +++++++++++++++++++++++++++++++++++++++++++++++++++++++++(+) (+) (+) (+) (+) (+) (+)------- | Rat +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ |
| SEQ ID | The people | Rabbit | Rat | ||
| RC RC-R6 RC-R8 RC-R15 RC-R1 RC-R2 RC-R3 RC-R10 RC-R12 RC-R18 RC-R9 RC-R4 RC-R5 RC-R20 RC-R17 RC-R13 RC-R16 library, library RBC RBC-R16 RBC-R7 RBC-R1 RBC-R2 RBC-R4 RBC-R6 RBC-R5 RBC-R9 RBC-R10 RBC-R8 RBC-R14 RBC-R20 RBC-R12 RBC-R13 RBC-R15 RBC-R18 RBC-R11 | DLCLRDWGCLW DICLPEWGCLW DICLPEWGCLW DICLPEWGCLW DICLPVWGCLW DICLPVWGCLW DICLPVWGCLW DICLPVWGCLW DICLPVWGCLW DICLPVWGCLW DLCLPEWGCLW DLCLPKWGCLW DLCLPVWGCLW DICLPAWGCLW DICLPDWGCLW DICLPRWGCLW DICLERWGCLW XXDLCLRDWGCLWXX EWDVCLPHWGCLWDG WDDICFRDWGCLWGS MDDICLHHWGCLWDE MDDLCLPNWGCLWGD FEDFCLPNWGCLWGS FEDLCVVRWGCLWGD WEDLCLPDWGCLWED SEDFCLPVWGCLWED DFDLCLPDWGCLWDD NWDLCFPDWGCLWDD EEDLCLPVWGCLWGA EEDVCLPVWGCLWEG MFDLCLPKWGCLWGN EFDLCLPTWGCLWED MWDVCFPDWGCLWDV EWDVCFPAWGCLWDQ VWDLCLPQWGCLWDE | 7 124 124 124 125 125 125 125 125 125 126 127 128 129 130 8 131 427 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 | - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - | - - - - - - - - - - - - - - - - (+) - - - - - - - - - - - - - - - - | ++ ++ ++ ++ ++ ++ ++ ++ ++ (+) ++ (+) ++ ++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ |
| SEQ ID | The people | Rabbit | Rat | ||
| Library RD RD-R2 RD-R7 RD-R11 RD-R5 RD-R6 RD-R1 | DTCVDLVRLGLECWG DTCADLVRLGLECWA NTCADLVRLGLECWA DTCDDLVQLGLECWA DTCEDLVRLGLECWA DSCGDLLRLGLECWA DTCSDLVGLGLECWA | 34 149 150 151 152 153 154 | - - - - - - | - - - - - - | +++ +++ +++ +++ +++ +++ |
Table 3
Many species bond
| Bacteriophage | SEQ ID NO： | In conjunction with | |||
| The people | Rabbit | Rat | |||
| RB RB-H1 RB-H6 RB-B12 RB-B8 RB-B7 RB-B5 RB-B6 RB-B4 RB-B11 RB-B16 RB-B2 RB-R8 RB-R16 HC-R10 | QRQMVDFCLPQWGCLWGDGF QRHPEDICLPRWGCLWGDDD NRQMEDICLPQWGCLWGDDF QGLIGDICLPRWGCLWGDSV HSQMEDICLPRWGCLWGDEL LRLMDNICLPRWGCLWDDGF QWHMEDICLPQWGCLWGDVL QWQMENVCLPKWGCLWEELD LWAMEDICLPKWGCLWEDDF QWQVMDICLPRWGCLWADEY HRLVEDICLPRWGCLWGNDF QRLMEDICLPRWGCLWGDRF QGLIGDICLPRWGCLWGDSV LIFMEDVCLPQWGCLWEDGV QRDMGDICLPRWGCLWEDGV | 35 93 94 11 100 99 96 97 98 101 102 95 11 108 109 | + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ | ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ | +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ |
Table 4
The sequence of selecting at the rat albumin
| SEQ ID NO： | Strong randomization library (Hard Randomization Library) |
| 155 157 8 9 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 | XXXXXDXCLPXWGCLWXXXX AAQVGDICLPRWGCLWSEYA AGWAADVCLPRWGCLWEEDV ASVVDDICLPVWGCLWGEDI ATMEDDICLPRWGCLWGAEE DEDFEDYCLPPWGCLWGSSM EGTWDDFCLPRWGCLWLGER ERWEGDVCLPRWGCLWGESG GDWMHDICLPKWGCLWDEKA GIEWGDTCLPKWGCLWRVEG GQQGEDVCLPVWGCLWDTSS GRYPMDLCLPRWGCLWEDSA GSAGDDLCLPRWGCLWERGA HASDWDVCLPGWGCLWEEDD LGVTHDTCLPRWGCLWDEVG LVWEEDFCLPKWGCLWGAED NVGWNDICLPRWGCLWAQES QGVEWDVCLPQWGCLWTREV RLDAWDICLPQWGCLWEEPS SEAPGDYCLPRWGCLWAQEK TAMDEDVCLPRWGCLWGSGS TEIGQDFCLPRWGCLWVPGT TLGWPDFCLPKWGCLWRESD TLSNQDICLPGWGCLWGGIN TSTGGDLCLPRWGCLWDSSE VSEMDDICLPLWGCLWADAP VSEWEDICLPSWGCLWETQD VVGDGDFCLPKWGCLWDQAR VVWDDDVCLPRWGCLWEEYG WSDSDDVCLPRWGCLWGNVA WVEEGDICLPRWGCLWESVE AQAMGDICLPRWGCLWEAEI ASDRGDLCLPYWGCWGPDG |
Table 4 (continuing)
| SEQ ID NO： | Strong randomization library |
| 155 | XXXXXDXCLPXWGCLWXXXX |
| 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 | ASDPGDVCLPRWGCLWGESF ASNWEDVCLPRWGCLWGERN ASTPRDICLPRWGCLWSEDA DGEEGDLCLPRWGCLWALEH EGEEVDICLPQWGCLWGYPV EVGDLDLCLPRWGCLWGNDK FRDGEDFCLPQWGCLWADTS GDMVNDFCLPRWGCLWGSEN GRMGTDLCLPRWGCLWGEVE HEWERDICLPRWGCLWRDGD KKVSGDICLPIWGCLWDNDY LLESDDICLPRWGCLWHEDG MQAESDFCLPHWGCLWDEGT MQGPLDICLPRWGCLWGGVD QMPLEDICLPRWGCLWEGRE REEWGDLCLPTWGCLWETKK RVWTEDVCLPRWGCLWSEGN SIREYDVCLPKWGCLWEPSA SPTEWDMCLPKWGCLWGDAL SSGLEDICLPNWGCLWADGS SVGWGDICLPVWGCLWGEGG TEENWDLCLPRWGGLWGDDW TSGSDDICLPVWGCLWGEDS TWP-GDLCLPRWGCLWEAES WDHELDFCLPVWGCLWAEDV WTESEDICLPGWGCLWGPEV WVPFEDVCLPRWGCLWSSYQ |
Table 4 (continuing)
| SEQ ID NO： | Strong randomization library |
| 156 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 | XXXXDXCLPXWGCLWXXX EEDSDICLPRWGCLWNTS EGYWDLCLPRWGCLWELE ELGEDLCLPRWGCLWGSE ETWSDVCLPRWGCLWGAS GDYVDLCLPGWGCLWEDG GVLDDICLPRWGCLWGPK HMMDDVCLPGWGCLWASE IDYTDLCLPAWGCLWELE IEHEDLCLPRWGCLWAVD ISEWDLCLPRWGCLWDRS ISWADVCLPKWGCLWGKD ISWGDLCLPRWGCLWEGS KLWDDICLPRWGCLWSPL LAWPDVCLPRWGCLWGGM LNESDICLPTWGCLWGVD LPEQDVCLPVWGCLWDAN MAWGDVCLPRWGCLWAGG NEEWDVCLPRWGCLWGGV QELQDFCLPRWGCLWGVG QREWDVCLPRWGCLWSDV QRFWDTCLPRWGCLWGGD RVFTDVCLPRWGCLWDLG SGWDDVCLPVWGCLWGPS SSASDYCLPRWGCLWGDL SWQGDICLPRWGCLWGVD SYETDVCLPYWGCLWEDA SYWGDVCLPRWGCLWSEA TLEWDMCLPRWGCLWTEQ VGEFDICLPRWGCLWDAE VTSWDVCLPRWGCLWEED WLWEDLCLPKWGCLWEED ALFEDVCLPVWGCLWGGE ASEWDVCLPTWGCLWMEG AYSADICLPRWGCLWMSE |
Table 4 (continuing)
| SEQ ID NO： | Strong randomization library |
| 156 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 | XXXXDXCLPXWGCLWXXX EDWEDICLPQWGCLWEGM EDWTDLCLPAWGCLWDTE EEWEDLCLPRWGCLWSAE EFWQDICLPNWGCLWAES EGFSDICLPRWGCLWSQE ETWEDLCLPNWGCLWDLE GEVNDFCLPRWGCLWEGD GGEWDVCLPAWGCLWGEE KDWYDICLPRWGCLWGGE KLGQDICLPRWGCLWDFA LEEWDICLPQWGCLWREG LVLPDICLPKWGCLWGDT MDLADICLPKWGCLWESD MVLDDICLPRWGCLWSEK MWSGDLCLPRWGCLWGET NRMGDICLPRWGCLWDGH RDWEDLCLPNWGCLWELS RGDWDLCLPKWGCLWEGV RQWEDICLPRWGCLWGVG RVEYDLCLPRWGCLWEPP SIWSDICLPRWGCLWESD TDEWDICLPNWGCLWEAG TEDVDFCLPLWGCLWEEP VKEEDFCLPRWGCLWEAG WDFEDICLPRWGCLWADM WEDWDVCLPRWGCLWGGG YEDIDICLPRWGCLWDLS |
Table 5
The sequence of selecting at the rabbit albumin
| SEQ ID NO： | Strong randomization library |
| 155 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 | XXXXXDXCLPXWGCLWXXXX AGLDEDICLPRWGCLWGKEA AGMMGDICLPRWGCLWQGEP APGDWDFCLPKWGCLWDDDA AQLFDDICLPRWGCLWSDGY ARTMGDICLPRWGCLWGASD AWQDFDVCLPRWGCLWEPES DTTWGDICLPRWGCLWSEEA EGFLGDICLPRWGCLWGHQA EQWLHDICLPKWGCLWDDTD ETGWPDICLPRWGCLWEEGE FELGEDICLPRWGCLWEEHN GASLGDICLPRWGCLWGPED GEWWEDICLPRWGCLWGSSS GSLESDICLPRWGCLWGIDE GWLEEDICLPKWGCLWGADN HEQWDDICLPRWGCLWGGSY QRVDDDICLPRWGCLWGENS SVGWGDICLPKWGCLWAESD TLMSNDICLPRWGCLWDEPK TLVLDDICLPRWGCLWDMTD TWQGEDICLPRWGCLWDTEV VGVFDDICLPRWGCLWEQPV VPAMGDICLPRWGCLWEARN VSLGDDICLPKWGCLWEPEA VWIDRDICLPRWGCLWDTEN WRWNEDICLPRWGCLWEEEA AVSWADICLPRWGCLWERAD AWLDEDICLPKWGCLWNTGV FSLDEDICLPKWGCLWGAEK GDLGDDICLPRWGCLWDEYP GEGWSDICLPRWGCLWAEDE GLMGEDICLPRWGCLWKGDI |
| 155 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 | XXXXXDXCLPXWGCLWXXXX GWHDRDICLPRWGCLWEQND LLGGHDICLPRWGCLWGGDV MRWSSDICLPKWGCLWGDEE QFEWDDICLPRWGCLWEVEV QGWWHDICLPRWGCLWEEGE REGWPDICLPRWGCLWSETG RELWGDICLPRWGCLWEHAT RLELMDICLPRWGCLWDPQD SGVLGDICLPRWGCLWEEAG SLGLTDLCLPRWGCLWEEEQ SSLEQDICLPRWGCLWGQDA SVLSDDICLPRWGCLWWDFS TSLLDDICLPRWGCLWYEEG TSLADDICLPRWGCLWSEDG VEMWHDICLPRWGCLWDSNA WDLASDICLPRWGCLWEEEA FITQDICLPRWGCLWGEN FLWRDICLPRWGCLWSEG FVHEDICLPRWGCLWGEG GLGDDICLPRWGCLWGRD GMFDDICLPKWGCLWGLG GPGWDICLPRWGCLWGEE GPWYDICLPRWGCLWDGV GWDDDICLPRWGCLWGDG LEYEDICLPKWGCLWGGE LLDEDICLPRWGCLWGVR LMSPDICLPKWGCLWEGD LVLGDICLPRWGCLWESD MLSRDICLPRWGCLWEEE MPWTDICLPRWGCLWSES RLGSDICLPRWGCLWGAG RLGSDICLPRWGCLWDYQ SPWMDICLPRWGCLWESG STFTDICLPRWGCLWELE SVLSDICLPRWGCLWEES TWFSDICLPRWGCLWEPG VHQADICLPRWGCLWGDT |
| 155 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 | XXXXXDXCLPXWGCLWXXXX VLLGDICLPLWGCLWGED VNWGDICLPRWGCLWGES VVWSDICLPRWGCLWDKE VWYKDICLPRWGCLWEAE WDYGDICLPRWGCLWEEG WEVQDICLPRWGCLWGDD YIWRDICLPRWGCLWEGE YRDYDICLPRWGCLWDER AFWSDICLPRWGCLWEED DWGRDICLPRWGCLWDEE EAWGDICLPRWGCLWELE LILSDICLPRWGCLWDDT LKLEDICLPRWGCLWGES LLTRDICLPKWGCLWGSD LRWSDICLPRWGCLWEET LYLRDICLPKWGCLWEAD NWYDDICLPRWGCLWDVE QDWEDICLPRWGCLWGD- QSWPDICLPKWGCLWGEG TLLQDICLPRWGCLWESD VRLMDICLPRWGCLWGEE VRWEDICLPRWGCLWGEE WDVADICLPRWGCLWAED WHMGDICLPRWGCLWSEV WKDFDICLPRWGCLWDDH WLSEDICLPQWGCLWEES WLSEDICLPRWGCLWAAD WLSDDICLPRWGCLWDDL |
Table 6
The sequence of selecting at HSA
| SEQ ID NO： | Strong randomization library |
| 155 374 375 376 377 378 379 380 381 382 383 156 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 | XXXXXDXCLPXWGCLWXXXX EVREWDICLPRWGCLWENWR FGQEWDICLPRWGCLWGNEQ IWQLEDICLPRWGCLWEDGL NTPTYDICLPRWGCLWGDVP QPVWSDICLPRWGCLWGEDH SWYGGDICLP-WGCLWSEES WGMARDWCLPMWGCLWRGGG WHLTDDICLPRWGCLWGDEQ NWAENDICLPRWGCLWGDEN SAREWDICLPTWGCLWEKDI XXXXDXCLPXWGCLWXXX AGEWDICLPRWGCLWDVE EIRWDFCLPRWGCLWDED ESLGDICLPRWGCLWGSG EYWGDICLPRWGCLWDWQ KMWSDICLPRWGCLWEEE MGTKDICLPRWGCLWAEA MHEWDICLPRWGCLWESS RGLHDACLPWWGCLWAGS RLFGDICLPRWGCLWQGE SGEWDICLPRWGCLWGEG SMFFDHCLPMWGCLWAEQ VGEWDICLPNWGCLWERE WWMADRCLPLWGCLWRGD WWVRDLCLPTWGCLWSGK YFDGDICLPRWGCLWGSD TLFQDICLPRWGCLWEES WFPKDRCLPVWGCLWERH |
Table 7
In conjunction with the albuminised peptide of many species
| Peptide | SEO ID NO： | IC 50(nM) | |||
| Rabbit | Rat | Mouse | |||
| SA02 SA04 SA05 SA06 SA07 SA08 SA09 SA10 SA11 SA12 SA13 D3H44- L D3H44- Ls | 7 8 16 401 11 12 13 14 15 16 17 401 401 | DLCLRDWGCLW-n DICLPRWGCLW-n MEDICLPRWGCLWED-n QRLMEDICLPRWGCLWEDDF-n QGLIGDICLPRWGCLWGDSV-n AcQGLIGDICLPRWGCLWGDSVK-n AcEDICLPRWGCLWEDD-n AcRLMEDICLPRWGCLWEDD-n AcMEDICLPRWGCLWEDD-n AcMEDICLPRWGCLWED-n AcRLMEDICLARWGCLWEDD-n QRLMEDICLPRWGCLWEDDF-n QRLMEDICLPRWGCLWEDDF-n | 8543 804 128 30 63 1687 86 1213 1765 3200 241 75 | 787 161 68 35 68 258 77 232 205 2480 | 40 6 8 6 10 6 4 17 13 188 |
Table 8
The competition of surface plasmon resonance peptide
Kd(nM) IC
50(nM)
| HuSA | BuSA | RSA | SA | ID | Sequence | BuSA | MuSA |
| 467±47 | 320±22 | 266±6 | 21 | 402 | Ac-RLIEDICLPRWGCLWEDD -NH2 | 270±110 | 7±2 |
| 803±82 | 143±5 | 229+9 | 06 | 403 | QRLMEDICLPRWGCLWE | 130±50 | 6±2 |
| 858±59 | 108± | 158±3 | 08 | 11 | Ac-QGLIGDICLPRWGCLWGDSVK -NH2 | 51±11 | 12±2 |
| 878±58 | 65±3 | 150±5 | 15 | 404 | GEWWEDICLPRWGCLWEEED -NH2 | 13±2 | 5±1 |
Table 9
| Peptide | SEQ ID NO： | Sequence | RSA IC 50(nM) |
| SA20 | 405 | AcQRLIEDICLPRWGCLWEDDF NH2 | 260 |
| SA21 | 402 | AcRLIEDICLPRWGCLWEDD NH2 | 270±110 |
| SA22 | 406 | AcRLIEDICLPRWGCLWED NH2 | 430±70 |
| SA29 | 407 | AcRLIEDICLPRWGCLWE NH2 | 400±90 |
| SA31 | 408 | AcRLIEDICLPRWGCLW NH2 | 200 |
| SA33 | 409 | AcRLIEDICLPRWGCL NH2 | 4310±2770 |
| SA35 | 410 | AcRLIEDICLPRWGC NH2 | ＞250000 |
| SA23 | 411 | AcLIEDICLPRWGCLWED NH2 | 360±140 |
| SA24 | 412 | AcIEDICLPRWGCLWED NH2 | 1380±410 |
| SA25 | 413 | AcEDICLPRWGCLWED NH2 | 2730±1300 |
| SA26 | 414 | AcDICLPRWGCLWED NH2 | 3120±660 |
| SA27 | 415 | AcICLPRWGCLWED NH2 | 86700±21800 |
| SA28 | 416 | AcCLPRWGCLWED NH2 | ＞400000 |
| SA30 | 417 | AcIEDICLPRWGCLWE NH2 | 1800±590 |
| SA32 | 418 | AcEDICLPRWGCLW NH2 | 2170±520 |
| SA04 | 8 | DICLPRWGCLW NH2 | 8540±4620 |
| SA34 | 419 | AcDICLPRWGCL NH2 | 28210±6500 |
| SA19 | 419 | DICLPRWGCL NH2 | 24510±2100 |
| SA18 | 420 | ICLPRWGCLW NH2 | 124900 |
| SA36 | 421 | AcICLPRWGC NH2 | ＞250000 |
Table 10
Kd (with the solution combination of precincubation) ELISA measures
| Molecule | EC50 is directly in conjunction with ELISA | The combination of Kd solution phase | Kd passes through BIAcore |
| Rabbit SA | |||
| 4D5Fab-H | 25nM | 36nM | 150nM |
| 4D5Fab-H4 | ～500nM | 444nM | 500nM |
| 4D5Fab-H8 | ～500nM | 247nM | 710nM |
| 4D5Fab-H10 | ＞2uM | 1065nM | |
| 4D5Fab-H11 | ＞2uM | 1110nM | |
| Rat SA | |||
| 4D5Fab-H | 65pM | 92nM | 20nM |
| 4D5Fab-H4 | 75pM | 149nM | 40nM |
| 4D5Fab-H8 | 45pM | 145nM | 40nM |
| 4D5Fab-H10 | 8,000pM | 493nM | |
| 4D5Fab-H11 | ＞1μM | ＞2μM | |
| Mouse SA | |||
| 4D5Fab-H | 70pM | 44nM | 20nM |
| 4D5Fab-H4 | 77pM | 52nM | 30nM |
| 4D5Fab-H8 | 43pM | 41nM | 30nM |
| 4D5Fab-H10 | 14,520pM | 2,500nM | |
| 4D5Fab-H11 | ＞1μM | 1,250nM | |
Claims (27)
1. a conjugate molecule comprises at least one serum albumin bound fraction (SABM), at least one targeting agent (TA) and at least one cytotoxic agent (CA).
2. according to the conjugate molecule of claim 1, wherein SABM comprises at least 50% samely as the aminoacid sequence of DICLPRWGCLW (SEQ ID NO:8) sequence, and wherein aminoacid sequence has two Cys residues, has 5 amino acid residues between the Cys residue.
3. according to the conjugate molecule of claim 1, wherein SABM comprises the variant of the aminoacid sequence of DICLPRWGCLW (SEQ ID NO:8), and wherein except that the Cys residue, the 1-5 of SEQ ID NO:8 any residue replaced by different amino acid residues.
4. according to the conjugate molecule of claim 1, wherein SABM comprises linearity or cyclic amino acid sequence, is selected from:
Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa
Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys[SEQ ID NO：1]
Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe[SEQ ID NO：2]
Cys-Tyr-Xaal-Pro-Gly-Xaa-Cys[SEQ ID NO：3]
Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp[SEQ ID NO：4]
Trp-Cys-Asp-Xaa-Xaa-Leu-Xaa-Ala-Xaa-Asp-Leu-Cys[SEQ ID NO：5]；
Asp-Leu-Val-Xaa-Leu-Gly-Leu-Glu-Cys-Trp[SEQ ID NO：6]；
CXXGPXXXXC[SEQ ID NO：21]
XXXXCXXGPXXXXCXXXX[SEQ ID NO：22]
CXXXXXXCXXXXXXCCXXXCXXXXXXC[SEQ ID NO：23]
CCXXXCXXXXXXC[SEQ ID NO：24]
CCXXXXXCXXXXCXXXXCC[SEQ ID NO：25]
CXCXXXXXXXCXXXCXXXXXX[SEQ ID NO：26]
XXXXXDXCLPXWGCLWXXXX[SEQ ID NO：155]
XXXXDXCLPXWGCLWXXX[SEQ ID NO：156]
D X C L P X W G C L W[SEQ ID NO：423]
X X X X D I C L P R W G C L W X X X[SEQ ID NO：424]，
X X X X X D I C L P R W G C L W X X X X[SEQ ID NO：425]
X X E M C Y F P G I C W M X X[SEQ ID NO：426]
X X D L C L R D W G C L W X X[SEQ ID NO：427]
Wherein X is an arbitrary amino acid residue.
5. according to the conjugate molecule of claim 1, wherein SABM comprises the aminoacid sequence of the arbitrary SEQ of being selected from IDNOs:7-20,27-154 and 157-421.
6. according to the conjugate molecule of claim 1, wherein SABM comprises and is selected from SEQ ID NOs:7,8,9,10,11,12,13,14,15,16,17,18,19 and 20 aminoacid sequence.
7. according to the conjugate molecule of claim 1, wherein SABM comprises the arbitrary peptide sequence described in the table 1-9.
8. according to the conjugate molecule of claim 1, wherein SABM combines with serum albumin, has about 100 μ M or littler K
d
9. according to the conjugate molecule of claim 1, wherein TA is an antibody.
10. according to the conjugate molecule of claim 9, wherein antibody is Fab, F (ab)
2, scFv or double antibody.
11. according to the conjugate molecule of claim 1, wherein TA combines with the cell surface protein that raises in cancer.
12. according to the conjugate molecule of claim 11, wherein cell surface protein is HER2, PSMA, PCMA, KDR and Flt-1.
13. according to the conjugate molecule of claim 11, wherein cell surface protein is the B cell surface marker.
14. according to the conjugate molecule of claim 11, wherein cell surface protein is the B cell surface marker, is CD20 or BR3.
15. according to the conjugate molecule of claim 1, wherein TA is anti--HER2 antibody.
16. according to the conjugate molecule of claim 15, wherein TA has the VH of SEQ ID NO:428 and 429 and the antibody of VL sequence.
17. according to the conjugate molecule of claim 15, wherein TA comprises the variant sequence of anti--HER2 antibody, described resisting-HER2 antibody comprises SEQ ID NO:428 and SEQ ID NO:429.
18. according to the conjugate molecule of claim 1, wherein cytotoxic agent is monomethylauristatin (MMAE).
19. according to the conjugate molecule of claim 1, wherein the blank area between described SABM and targeting agent or cytotoxic agent is GGGS.
20. according to the conjugate molecule of claim 1, wherein SABM is in conjunction with people's albumin.
21. a compositions that is used for the treatment of purposes comprises and the blended conjugate molecule according to claim 1 of pharmaceutical carrier.
22. a method that is used to reduce therapeutic agent toxicity comprises with coupling having the serum albumin bound fraction (SABM) of therapeutic agent to produce the step of therapeutic agent.
23. a method that is used for reducing the mammal therapeutic agent toxicity comprises the conjugate molecule according to claim 1 to administration treatment effective dose.
24., further comprise and measure the interior therapeutic agent: the toxic step of SABM conjugate according to the method for claim 22 or 23.
25. a method for the treatment of mammalian cancer comprises the mammiferous step of suffering from cancer with the conjugate molecular therapy according to claim 1 of treatment effective dose.
26. method for the treatment of mammal autoimmune sexual disorder, comprise with the conjugate molecular therapy according to claim 1 of treatment effective dose and suffer from the mammiferous step of autoimmune sexual disorder, described conjugate molecule with help or cause that the B-cell of autoimmune sexual disorder combines.
27. a product comprises the compositions that comprises in container, the container according to the conjugate molecule of claim 1, the package insert that contains the operation instruction of administering therapeutic effective dose.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US61650704P | 2004-10-05 | 2004-10-05 | |
| US60/616,507 | 2004-10-05 | ||
| US60/641,534 | 2005-01-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101072581A true CN101072581A (en) | 2007-11-14 |
Family
ID=38899430
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2005800417589A Pending CN101072581A (en) | 2004-10-05 | 2005-09-22 | Therapeutic agents with decreased toxicity |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN101072581A (en) |
| ZA (1) | ZA200703478B (en) |
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- 2005-09-22 ZA ZA200703478A patent/ZA200703478B/en unknown
- 2005-09-22 CN CNA2005800417589A patent/CN101072581A/en active Pending
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