CN101605906A - In conjunction with human antibodies of CD70 and uses thereof - Google Patents

In conjunction with human antibodies of CD70 and uses thereof Download PDF

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Publication number
CN101605906A
CN101605906A CNA2007800512313A CN200780051231A CN101605906A CN 101605906 A CN101605906 A CN 101605906A CN A2007800512313 A CNA2007800512313 A CN A2007800512313A CN 200780051231 A CN200780051231 A CN 200780051231A CN 101605906 A CN101605906 A CN 101605906A
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antibody
variable region
seq
heavy chain
light chain
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Chinese (zh)
Inventor
M·A·科恰
J·A·科雷特
D·J·金
C·帕恩
J·卡尔达雷利
M·亚马纳卡
K·A·亨宁
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ER Squibb and Sons LLC
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Medarex LLC
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Abstract

The invention provides with high-affinity specifically in conjunction with the isolating monoclonal antibody, particularly human monoclonal antibody of CD70.Preferably, the human CD70 of these antibodies.In certain embodiments, these antibody capables maybe can be mediated the antigen dependent cellular cytotoxicity by internalization to the cell of expressing CD70.The method that antibody of the present invention is carried out nucleic acid molecules encoding, expression vector, host cell and is used to express antibody of the present invention also is provided.The pharmaceutical composition that antibody-mating partner molecule conjugate, bispecific molecule also is provided and has comprised antibody of the present invention.The present invention also provide use the present invention anti--CD70 antibody detects the method for CD70 and the method for treatment cancer (as kidney and lymphoma).

Description

In conjunction with human antibodies of CD70 and uses thereof
Related application
Present patent application is advocated in the U.S. Provisional Application series number 60/870 of submission on December 14th, 2006,091, the U.S. Provisional Application series number of submitting on May 1st, 2,007 60/915,314 and the U.S. Provisional Application series number 60/991 submitted on November 30th, 2007,702 right of priority, its content merges as a reference at this.
Background of invention
Cytokine receptor CD27 is the member of Tumor Necrosis Factor Receptors (TFNR) superfamily, and it and is played a role in apoptosis or programmed death in cell growth and differentiation.The part of CD27 is CD70, and it belongs to the tnf family cytokines of part.CD70 is 193 amino acid whose polypeptide, has the hydrophilic N-terminal structural domain of 20 amino acid and comprises C-terminal structural domain (Goodwin, people such as R.G. (1993) Cell that 2 potential N-connect glycosylation sites 73: 447-56; People such as Bowman (1994) Immunol 152: 1756-61).Based on these features, CD70 is confirmed as having the II type transmembrane protein of extracellular C-terminal part.
Having found that CD70 is instantaneous comes across activated but not T and the bone-marrow-derived lymphocyte and dendritic cell (people (1994) J.Immunol. such as Hintzen of resting state 152: 1762-1773; People such as Oshima (1998) Int.Immunol. 10: 517-26; People such as Tesselaar (2003) J.Immunol. 170: 33-40).Except that on normal cell, expressing, reported that CD70 expresses in dissimilar cancer cells, comprise renal cell carcinoma, transfevent breast cancer, cerebral tumor, leukemia, lymphoma and nasopharyngeal carcinoma (people (2005) J Urol. such as Junker 173: 2150-3; People such as Sloan (2004) Am J Pathol. 164: 315-23; Held-Feindt and Mentlein (2002) Int J Cancer 98: 352-6; People such as Hishima (2000) Am J Surg Pathol. 24: 742-6; People such as Lens (1999) Br JHaematol. 106: 491-503).In addition, find CD70 overexpression on the T cell of handling with dnmt rna inhibitor or ERK approach restrainer, may cause drug-induced or primary lupus (people (2004) Arthritis Rheum. such as Oelke 50: 1850-60).Propose, the interaction of CD70 and CD27 is at cell-mediated autoimmune disease and suppress to work in the generation of TNF-α (people (2000) J.Neuroimmunol. such as Nakajima 109: 188-96).
Therefore, CD70 has represented and has been used for the treatment of the valuable target that is expressed as cancer, autoimmune disorder and many other diseases of feature with CD70.
General introduction
The invention provides isolating monoclonal antibody, particularly human monoclonal antibody, these antibodies specifiies are in conjunction with CD70 and have the multiple desirable characteristic.These characteristics comprise high-affinity in conjunction with human CD70, by the ability of the cell internalizing of expressing CD70, mediate antibody dependent cellular cytotoxicity, in conjunction with the ability of renal cell carcinoma tumor cell line and/or in conjunction with the ability of lymphoma cell line (for example B cell tumour clone).The growth of the cell that antibody of the present invention can be used for for example detecting CD70 albumen or be used to suppress to express CD70 (as expressing the tumour cell of CD70).
The method of the disease of using isolating monoclonal antibody of the present invention and the multiple CD70 mediation of combination treatment thereof also is provided.
On the one hand, the present invention relates to the antigen-binding portion thereof of isolating monoclonal antibody or this antibody, this antibodies CD70 wherein, and show at least a in the following characteristic:
(a) with 1 * 10 -7M or littler K DCombine with human CD70;
(b) in conjunction with the renal cell carcinoma tumor cell line;
(c) in conjunction with lymphoma cell line, B cell tumour clone for example;
(d) expressed the cell internalizing of CD70;
(e) show antibody dependent cellular cytotoxicity (ADCC) at the cell of expressing CD70; And
(f) when suppressing to express the growth of the cell of CD70 mutually during coupling in vivo with cytotoxin.
Preferably, this antibody show characteristic (a) and (b), (c), (d), (e) and (f) at least two kinds.More preferably, this antibody show characteristic (a) and (b), (c), (d), (e) and (f) at least three kinds.More preferably, this antibody show characteristic (a) and (b), (c), (d), (e) and (f) in four kinds.Even more preferably, this antibody show characteristic (a) and (b), (c), (d), (e) and (f) in five kinds.Even more preferably, this antibody show characteristic (a) and (b), (c), (d), (e) and (f) in whole six kinds.In another preferred embodiment, when this antibody and cytotoxin mutually during coupling this antibody suppress to express the growth of the tumour cell of CD70 in vivo.
Preferably, this antibodies renal cell carcinoma tumor cell line, this clone is selected from: 786-O (ATCC accession number CRL-1932), A-498 (ATCC accession number HTB-44), ACHN (ATCC accession number CRL-1611), Caki-1 (ATCC accession number HTB-46) and Caki-2 (ATCC accession number HTB-47).
Preferably, this antibodies B cell carcinoma tumor cell line, this clone is selected from Daudi (ATCC accession number CCL-213), HuT 78 (ATCC accession number TIB-161), Raji (ATCC accession number CCL-86) or Granta-519 (DSMZ accession number 342) cell.
Preferably, this antibody is human antibodies, though in embodiment alternately, this antibody can be rodent antibody, chimeric antibody or humanized antibody.
In a more preferred embodiment, this antibody is with 5.5 * 10 -9M or littler K DCombine with human CD70, or with 3 * 10 -9M or littler K DCombine with human CD70, or with 2 * 10 -9M or littler K DCombine with human CD70, or with 1.5 * 10 -9M or littler K DCombine with human CD70.
In another embodiment, the CD70 that expresses on this antibody and the 786-O renal cell carcinoma tumour cell combines the back by those cell internalizings.
In another embodiment, the invention provides the antigen-binding portion thereof of isolating monoclonal antibody or this antibody, wherein this antibody cross competition is gone up the epi-position of being discerned by reference antibody, wherein this reference antibody in conjunction with CD70: (a) with 1 * 10 -7M or littler K DIn conjunction with human CD70; And (b) in conjunction with the renal cell carcinoma tumor cell line.
In different embodiments, this comprises with reference to antibody:
(a) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:1; And (b) variable region of light chain, comprise aminoacid sequence SEQ ID NO:7;
Or should comprise (a) variable region of heavy chain with reference to antibody, comprise aminoacid sequence SEQ ID NO:2; And (b) variable region of light chain, comprise aminoacid sequence SEQ ID NO:8;
Or should comprise (a) variable region of heavy chain with reference to antibody, comprise aminoacid sequence SEQ ID NO:3; And (b) variable region of light chain, comprise aminoacid sequence SEQ ID NO:9;
Or should comprise (a) variable region of heavy chain with reference to antibody, comprise aminoacid sequence SEQ ID NO:4; And (b) variable region of light chain, comprise aminoacid sequence SEQ ID NO:10;
Or should comprise (a) variable region of heavy chain with reference to antibody, comprise aminoacid sequence SEQ ID NO:5 or 73; And (b) variable region of light chain, comprise aminoacid sequence SEQ ID NO:11;
Or should comprise (a) variable region of heavy chain with reference to antibody, comprise aminoacid sequence SEQ ID NO:6; And (b) variable region of light chain, comprise aminoacid sequence SEQ ID NO:12.
In another embodiment, reference antibody of the present invention is antibody 69A7Y.69A7Y is identical with antibody 69A7, but at the V of aminoacid sequence SEQ ID NO:5 HIn contain conservative the modification, cause sporting Y (tyrosine) at amino acid position 100 C of place (halfcystine).The V of 69A7Y HAminoacid sequence is provided by SEQ ID NO:73.Sudden change from C to Y is by the V at 69A7 HThe single base pair that nucleotide position 323 places of nucleotide sequence (SEQ ID NO:53) take place from G to A replaces formed.The V of 69A7Y HNucleotide sequence is provided by SEQ ID NO:74.69A7Y has variable region of heavy chain CDR3, comprises the given aminoacid sequence as SEQ ID NO:75.
On the other hand, the present invention relates to the isolating monoclonal antibody that is connected with therapeutical agent or its antigen-binding portion thereof, this monoclonal antibody or its antigen-binding portion thereof comprise as human V H3-30.3 the product of gene or derived from the variable region of heavy chain of this gene, wherein this antibodies specific is in conjunction with CD70.The present invention also provides isolating monoclonal antibody, comprises the monoclonal antibody that is connected on the therapeutical agent or its antigen-binding portion thereof, and wherein this antibody comprises as human V HThe product of 3-33 gene or derived from the variable region of heavy chain of this gene, wherein this antibody combines with the CD70 specificity.The present invention also provides isolating monoclonal antibody, comprises the monoclonal antibody that is connected on the therapeutical agent or its antigen-binding portion thereof, and wherein this antibody comprises as human V HThe product of 4-61 gene or derived from the variable region of heavy chain of this gene, wherein this antibody combines specifically with CD70.The present invention also provides isolating monoclonal antibody, comprises the monoclonal antibody that is connected on the therapeutical agent or its antigen-binding portion thereof, and wherein this antibody comprises as human V HThe product of 3-23 gene or derived from the variable region of heavy chain of this gene, wherein this antibodies specific ground is in conjunction with CD70.
The present invention also further provides isolating monoclonal antibody, comprises the monoclonal antibody that is connected on the therapeutical agent or its antigen-binding portion thereof, and wherein this antibody comprises as human V KThe product of L6 gene or derived from the variable region of light chain of this gene, wherein this antibody is specifically in conjunction with CD70.The present invention also further provides isolating monoclonal antibody, comprises the monoclonal antibody that is connected on the therapeutical agent or its antigen-binding portion thereof, and wherein this antibody comprises as human V KThe product of L18 gene or derived from the variable region of light chain of this gene, wherein this antibody is specifically in conjunction with CD70.The present invention further provides isolating monoclonal antibody, comprised the monoclonal antibody that is connected on the therapeutical agent or its antigen-binding portion thereof, wherein this antibody comprises as human V KThe product of L15 gene or derived from the variable region of light chain of this gene, wherein this antibody is specifically in conjunction with CD70.The present invention further provides isolating monoclonal antibody, comprised the monoclonal antibody that is connected on the therapeutical agent or its antigen-binding portion thereof, wherein this antibody comprises as human V KThe product of A27 gene or derived from the variable region of light chain of this gene, wherein this antibody is specifically in conjunction with CD70.
Particularly preferred antibody or its antigen-binding portion thereof comprise:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:13;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:19;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:25;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:31;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:37; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:43.
Another kind of preferred combination comprises:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:14;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:20;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:26;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:32;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:38; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:44.
Another kind of preferred combination comprises:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:15;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:21;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:27;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:33;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:39; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:45.
Another kind of preferred combination comprises:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:16;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:22;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:28;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:34;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:40; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:46.
Another kind of preferred combination comprises:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:17;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:23;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:29 or 75;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:35;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:41; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:47.
Another kind of preferred combination comprises:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:18;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:24;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:30;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:36;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:42; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:48.
Other preferred antibody of the present invention have antibody or its antigen-binding portion thereof, and it comprises: the variable region of heavy chain that (a) comprises aminoacid sequence SEQ ID NO:1; And the variable region of light chain that (b) comprises aminoacid sequence SEQID NO:7.
Another kind of preferred combination comprises that (a) comprises the variable region of heavy chain of aminoacid sequence SEQ ID NO:2; And the variable region of light chain that (b) comprises aminoacid sequence SEQ ID NO:8.
Another kind of preferred combination comprises that (a) comprises the variable region of heavy chain of aminoacid sequence SEQ ID NO:3; And the variable region of light chain that (b) comprises aminoacid sequence SEQ ID NO:9.
Another kind of preferred combination comprises that (a) comprises the variable region of heavy chain of aminoacid sequence SEQ ID NO:4; And the variable region of light chain that (b) comprises aminoacid sequence SEQ ID NO:10.
Another kind of preferred combination comprises that (a) comprises the variable region of heavy chain of aminoacid sequence SEQ ID NO:5 or 73; And the variable region of light chain that (b) comprises aminoacid sequence SEQ ID NO:11.
Another kind of preferred combination comprises that (a) comprises the variable region of heavy chain of aminoacid sequence SEQ ID NO:6; And the variable region of light chain that (b) comprises aminoacid sequence SEQ ID NO:12.
In another embodiment, a kind of antibody of the present invention is antibody 69A7Y.69A7Y is identical with antibody 69A7, but at the V of SEQ ID NO:5 HComprise conservative the modification in the aminoacid sequence, cause sporting Y (tyrosine) at amino acid position 100 C of place (halfcystine).The V of 69A7Y HAminoacid sequence is provided by SEQ ID NO:73.Sudden change from C to Y is the V at 69A7 HThe single base pair of nucleotide position 323 places generation from G to A of nucleotide sequence (SEQ ID NO:53) replaces formation.The V of 69A7Y HNucleotide sequence is provided by SEQ ID NO:74.69A7Y has variable region of heavy chain CDR3, comprises the given aminoacid sequence as SEQ ID NO:75.
For example, antibody of the present invention can be full length antibody, for example IgG1 or IgG4 isotype.Alternately, this antibody can be such as Fab or Fab ' 2The antibody fragment of fragment or single-chain antibody.
The present invention also provides immune conjugate, comprises the antibody of the present invention that is connected to therapeutical agent (for example cytotoxin or radio isotope) or its antigen-binding portion thereof.In an especially preferred embodiment, the invention provides immune conjugate, this conjugate comprises and (for example is connected to cytotoxin, Shuo Ming cytotoxin or be illustrated in the U.S. Patent application 60/882 of on December 28th, 2006 application herein, the Application No. 60/991 of application on November 30th, 461 or 2007, in 300, it merges as a reference at this in full) antibody of the present invention or its antigen-binding portion thereof of (for example connecting) by the thiol alcohol radical.In certain embodiments, the cytotoxin of this immune conjugate and connector have N1 or N2 structure.
For example, in different embodiments, the invention provides following preferred immune conjugate:
(i) immune conjugate comprises antibody or its antigen-binding portion thereof, comprising:
(a) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:1 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:7;
(b) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:2 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:8;
(c) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:3 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:9;
(d) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:4 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:10;
(e) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:5 or 73 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:11, and
(f) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:6 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:12, wherein this antibody or its antigen-binding portion thereof are connected on the cytotoxin;
(ii) a kind of immune conjugate comprises antibody or its antigen-binding portion thereof, comprising:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:13;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:19;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:25;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:31;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:37; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:43;
Perhaps antibody or its antigen-binding portion thereof comprise:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:14;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:20;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:26;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:32;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:38; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:44;
Perhaps antibody or its antigen-binding portion thereof comprise:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:15;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:21;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:27;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:33;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:39; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:45;
Perhaps antibody or its antigen-binding portion thereof comprise:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:16;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:22;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:28;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:34;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:40; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:46;
Perhaps plant antibody or its antigen-binding portion thereof, comprising:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:17;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:23;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:29 or 75;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:35;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:41; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:47;
Perhaps antibody or its antigen-binding portion thereof comprise:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:18;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:24;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:30;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:36;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:42; And
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:48,
Wherein this antibody or its antigen-binding portion thereof are connected on the cytotoxin; And
(iii) a kind of immune conjugate comprises the antibody or its antigen-binding portion thereof that are connected on the cytotoxin, and it is in conjunction with the identical epi-position of being discerned by following antibody (as combining human CD70 with this antibody cross competition), and this antibody comprises:
(a) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:1 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:7;
(b) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:2 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:8;
(c) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:3 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:9;
(d) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:4 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:10;
(e) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:5 or 73 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:11; And
(f) comprise the variable region of heavy chain of aminoacid sequence SEQ ID NO:6 and comprise the variable region of light chain of aminoacid sequence SEQ ID NO:12.
The present invention also provides bispecific molecule, this bispecific molecule comprises antibody of the present invention or its antigen-binding portion thereof, this antibody or its antigen-binding site are connected with second funtion part, and this second funtion part has with described antibody or its antigen-binding portion thereof compares different binding specificities.
Composition also is provided, has comprised antibody of the present invention or its antigen-binding portion thereof or immune conjugate or bispecific molecule, and pharmaceutically acceptable carrier.
The present invention also comprises the nucleic acid molecule of code book invention antibody or their antigen-binding portion thereof and comprises this type of expression of nucleic acids carrier and the host cell that comprises this type of expression vector.Also provide to use to comprise that the host cell of this type of expression vector prepares the method for anti-CD70 antibody, and the step that these methods can comprise has: (i) in host cell, express this antibody and (ii) from host cell, separate this antibody.
Again on the other hand in, the present invention relates to prepare the method for anti-CD70 antibody.This method comprises:
(a) provide: (i) variable fragments of heavy chain sequence comprises the CDR1 sequence that is selected from SEQ ID NO:13 to 18; Be selected from the CDR2 sequence of SEQ ID NO:19 to 24; And/or be selected from the CDR3 sequence of SEQID NO:25 to 30 and 75; And/or (ii) variable region of light chain antibody sequence, comprise the CDR1 sequence that is selected from SEQ ID NO:31 to 36; Be selected from the CDR2 sequence of SEQ ID NO:37 to 42, and/or be selected from the CDR3 sequence of SEQ ID NO:43 to 48;
(b) in variable fragments of heavy chain sequence and/or variable region of light chain antibody sequence, change at least one amino-acid residue, to produce at least a reformed antibody sequence; And
(c) reformed antibody sequence is expressed as protein.
The present invention also provides with high-affinity and the isolating anti-CD70 antibody of CD70 specificity bonded-mating partner molecule conjugate, and particularly those comprise anti-CD70 antibody-mating partner molecule conjugate of human monoclonal antibody.There is some can be to the cell of expressing CD70 in this antibody-like-mating partner molecule conjugate by internalization, and can the effect of mediate antibody dependent cellular cytotoxicity.The present invention also provides and has used anti-CD70 antibody disclosed here-mating partner molecule conjugate treatment method for cancer, and these cancers are renal cell carcinoma or lymphoma for example.
Also provide and comprised and the mating partner molecule of the present invention composition of link coupled antibody or its antigen-binding portion thereof mutually.Can be advantageously with antibody mating partner molecule conjugate disclosed here in antibody mutually link coupled mating partner molecule include but not limited to: as molecule, cytotoxin, tagged molecule (as radio isotope), protein and the therapeutical agent of medicine.Also disclosed the composition that comprises antibody-mating partner molecule conjugate and pharmaceutically acceptable carrier at this.
On the one hand, this antibody-like-mating partner molecule conjugate is by the coupling of chemical linkers phase.In some embodiments, this connector is the peptidyl connector, and is represented as (L at this 4) p-F-(L 1) mOther connectors comprise hydrazine and disulfide linkers, and are expressed as (L respectively at this 4) p-H-(L 1) mOr (L 4) p-J-(L 1) mExcept with connector that mating partner combines, the present invention also provides and has been applicable to and is bonded to the connecting arm that cuts of any molecular species basically.
On the other hand, the present invention relates to suppress express the method for the growth of tumour cell of CD70.This method comprises that the tumour cell that will express CD70 contacts with antibody of the present invention-mating partner molecule conjugate, and the feasible growth of expressing the tumour cell of CD70 is suppressed.In a preferred embodiment, this mating partner molecule is a therapeutical agent, as cytotoxin.The tumour cell of particularly preferred expression CD70 is kidney cancer cell and lymphoma cell.
On the other hand, the present invention relates to treat experimenter's method for cancer.This method comprises and gives this experimenter antibody of the present invention-mating partner molecule conjugate, makes this experimenter's cancer obtain medical treatment.In a preferred embodiment, this mating partner molecule is a therapeutical agent, as cytotoxin.The particularly preferred cancer that can treat is kidney and lymphoma.
On the other hand, the present invention relates to treat the method for experimenter's autoimmune disease, inflammation or virus infection.This method comprises and gives this experimenter antibody of the present invention-mating partner molecule conjugate, makes this experimenter's autoimmune disorder obtain medical treatment.
Below detailed specification sheets and embodiment other features and advantages of the present invention will clearly be described, these specification sheetss and embodiment must not be construed as limitation of the present invention.All reference that the application quotes in full, Genbank accession number, patent and disclosed patent application are all integrated with this paper as a reference clearly.
Brief Description Of Drawings
Figure 1A shows the nucleotide sequence (SEQID NO:49) and the aminoacid sequence (SEQ ID NO:1) of 2H5 human monoclonal antibody's variable region of heavy chain.Describe CDR1 (SEQ ID NO:13), CDR2 (SEQ ID NO:19) and CDR3 (SEQ ID NO:25) district, and indicated the kind system source (germline derivations) of V and J.
Figure 1B shows the nucleotide sequence (SEQID NO:55) and the aminoacid sequence (SEQ ID NO:7) of 2H5 human monoclonal antibody's variable region of light chain.Describe CDR1 (SEQ ID NO:31), CDR2 (SEQ ID NO:37) and CDR3 (SEQ ID NO:43) district, and indicated the kind system source of V and J.
Fig. 2 A shows the nucleotide sequence (SEQID NO:50) and the aminoacid sequence (SEQ ID NO:2) of 10B4 human monoclonal antibody's variable region of heavy chain.Describe out CDR1 (SEQ IDNO:14), CDR2 (SEQ ID NO:20) and CDR3 (SEQ ID NO:26) district, and indicated the kind system source of V, D and J.
Fig. 2 B shows the nucleotide sequence (SEQID NO:56) and the aminoacid sequence (SEQ ID NO:8) of 10B4 human monoclonal antibody's variable region of light chain.Describe CDR1 (SEQ ID NO:32), CDR2 (SEQ ID NO:38) and CDR3 (SEQ ID NO:44) district, and indicated the kind system source of V and J.
Fig. 3 A shows the nucleotide sequence (SEQID NO:51) and the aminoacid sequence (SEQ ID NO:3) of 8B5 human monoclonal antibody's variable region of heavy chain.Describe CDR1 (SEQ ID NO:15), CDR2 (SEQ ID NO:21) and CDR3 (SEQ ID NO:27) district, and indicated the kind system source of V, D and J.
Fig. 3 B shows the nucleotide sequence (SEQ IDNO:57) and the aminoacid sequence (SEQ ID NO:9) of 8B5 human monoclonal antibody's variable region of light chain.Describe CDR1 (SEQ ID NO:33), CDR2 (SEQ ID NO:39) and CDR3 (SEQ ID NO:45) district, and indicated the kind system source of V and J.
Fig. 4 A shows the nucleotide sequence (SEQID NO:52) and the aminoacid sequence (SEQ ID NO:4) of 18E7 human monoclonal antibody's variable region of heavy chain.Describe CDR1 (SEQ ID NO:16), CDR2 (SEQ ID NO:22) and CDR3 (SEQ ID NO:28) district, and indicated the kind system source of V, D and J.
Fig. 4 B shows the nucleotide sequence (SEQID NO:58) and the aminoacid sequence (SEQ ID NO:10) of 18E7 human monoclonal antibody's variable region of light chain.Describe CDR1 (SEQ ID NO:34), CDR2 (SEQ ID NO:40) and CDR3 (SEQ ID NO:46) district, and indicated the kind system source of V and J.
Fig. 5 A shows the nucleotide sequence (SEQID NO:53) and the aminoacid sequence (SEQ ID NO:5) of 69A7 human monoclonal antibody's variable region of heavy chain.Describe CDR1 (SEQ ID NO:17), CDR2 (SEQ ID NO:23) and CDR3 (SEQ ID NO:29) district, and indicated the kind system source of V, D and J.
Fig. 5 B shows the nucleotide sequence (SEQID NO:59) and the aminoacid sequence (SEQ ID NO:11) of 69A7 human monoclonal antibody's variable region of light chain.Describe CDR1 (SEQ IDNO:35), CDR2 (SEQ ID NO:41) and CDR3 (SEQ ID NO:47) district, and indicated the kind system source of V and J.
Fig. 6 A shows the nucleotide sequence (SEQ IDNO:54) and the aminoacid sequence (SEQ ID NO:6) of 1F4 human monoclonal antibody's variable region of heavy chain.Describe CDR1 (SEQ ID NO:18), CDR2 (SEQ ID NO:24) and CDR3 (SEQ ID NO:30) district, and indicated the kind system source of V, D and J.
Fig. 6 B shows the nucleotide sequence (SEQ IDNO:60) and the aminoacid sequence (SEQ ID NO:12) of 1F4 human monoclonal antibody's variable region of light chain.Describe CDR1 (SEQ ID NO:36), CDR2 (SEQ ID NO:42) and CDR3 (SEQ ID NO:48) district, and indicated the kind system source of V and J.
The aminoacid sequence that Fig. 7 shows the variable region of heavy chain of 2H5 and 10B4 is V with human the kind H3-30.3 the comparison of aminoacid sequence (SEQ ID NO:61).
The aminoacid sequence that Fig. 8 shows the variable region of heavy chain of 8B5 and 18E7 is V with human the kind HThe comparison of 3-33 aminoacid sequence (SEQ ID NO:62).
The aminoacid sequence that Fig. 9 shows the variable region of heavy chain of 69A7 is V with human the kind HThe comparison of 4-61 aminoacid sequence (SEQ ID NO:63).
The aminoacid sequence that Figure 10 shows the variable region of heavy chain of 1F4 is V with human the kind HThe comparison of 3-23 aminoacid sequence (SEQ ID NO:64).
The aminoacid sequence that Figure 11 shows the variable region of light chain of 2H5 is V with human the kind KThe comparison of L6 aminoacid sequence (SEQ ID NO:65).
The aminoacid sequence that Figure 12 shows the variable region of light chain of 10B4 is V with human the kind KThe comparison of L18 aminoacid sequence (SEQ ID NO:66).
The aminoacid sequence that Figure 13 shows the variable region of light chain of 8B5 and 18E7 is V with human the kind KThe comparison of L15 aminoacid sequence (SEQ ID NO:67).
The aminoacid sequence that Figure 14 shows the variable region of light chain of 69A7 is V with human the kind KThe comparison of L6 aminoacid sequence (SEQ ID NO:65).
The aminoacid sequence that Figure 15 shows the variable region of light chain of 1F4 is V with human the kind KThe comparison of A27 aminoacid sequence (SEQ ID NO:68).
Figure 16 shows the ELSIA result of experiment, proves that human monoclonal antibody's specificity at human CD70 is in conjunction with CD70.
Figure 17 shows the flow cytometry result of experiment, proves that anti--CD70 human monoclonal antibody 2H5 combines with renal carcinoma cell line.
Figure 18 A and B show the flow cytometry experimental result, prove at the human monoclonal antibody of human CD70 to combine with renal cell carcinoma (RCC) clone in the mode of concentration dependent.(A) 786-ORCC clone (B) A498RCC clone.
Figure 18 C shows the flow cytometry result of experiment, proves that the human monoclonal antibody at human CD70 combines with the 786-O renal carcinoma cell line.
Figure 18 D shows the flow cytometry result of experiment, proves at the HuMAb69A7 antibody of the human CD70 mode with concentration dependent to combine with renal cell carcinoma (RCC) clone 786-O.
Figure 19 shows the flow cytometry result of experiment, proves that anti-CD70 human monoclonal antibody 2H5 combines with human lymphoma clone.
Figure 20 A and B show the flow cytometry result of experiment, prove that anti--CD70 human monoclonal antibody 2H5 combines with human lymphoma clone in the mode of concentration dependent.(A) Raji lymphoma cell line (B) Granta-519 lymphoma cell line.
Figure 20 C shows the flow cytometry result of experiment, proves that human monoclonal antibody at human CD70 is in conjunction with the Raji lymphoma cell line.
Figure 20 D shows the result that competitive flow cytometry is measured, and proves the shared epi-position that similarly combines of HuMAb 2H5 and 69A7.
Figure 20 E shows the flow cytometry result of experiment, proves that the human monoclonal antibody at human CD70 combines with Daudi lymphoma cell line and 786-O renal carcinoma cell line.
Figure 21 shows Hum-Zap internalization result of experiment, proves that the human monoclonal antibody at human CD70 can internalization enter in the CD70 positive cell.
Figure 22 A to C shows the result of cell proliferating determining, prove the cytotoxic human monoclonal of coupling anti--CD70 antibody kills renal cell carcinoma cell (RCC) and is.(A)Caki-2RCC(B)786-ORCC(C)ACHN?RCC。
Figure 23 A to D shows the result that ADCC measures, and proves that human monoclonal anti-CD70 antibody kills human leukemia and lymphoma cell line in the mode that ADCC relies on.(A) ARH-77 leukemia cell's system's (B) HuT 78 lymphoma cell lines (C) Raji lymphoma cell line and (D) L-540 clone, it does not express CD70.
Figure 24 shows the result of cell proliferating determining, prove the cytotoxic human monoclonal of coupling anti--CD70 antibody kills human lymphoma clone.
Figure 25 A to B shows the result of cell proliferating determining, prove the cytotoxic human monoclonal of coupling anti--CD70 antibody demonstrate (A) washing three hours and (B) when continuing washing to the cytotoxicity of Raji cell.
Figure 26 A to B shows the result of mouse tumor model research in vivo, prove with coupling is cytotoxic to resist-CD70 antibody 2H5 treats has restraining effect in the direct body to renal cell carcinoma (RCC) tumour.(A) A-498RCC tumour (B) ACHN RCC tumour.
Figure 27 A to F shows the result that ADCC measures, prove non-fucosylated human monoclonal anti--CD70 antibody has the cytotoxicity that relies on the increase of mode with ADCC to the human leukaemia cell.(A) ARH-77 cell; (B) MEC-1 cell; (C) with the MEC-1 cell of anti--CD16 antibody treatment; (D) SU-DHL-6 cell; (E) IM-9 cell; (F) HuT 78 cells.
Figure 28 shows the result that ADCC measures, and proves that human monoclonal anti-CD70 antibody kills the human leukaemia cell in ADCC concentration dependent mode.
Figure 29 shows the result that antibody dependent cellular cytotoxicity (ADCC) is measured, and prove that human monoclonal anti-CD70 antibody kills the human leukaemia cell in ADCC dependency mode, but cytotoxicity depends on CD16.
Figure 30 shows the result that ADCC measures, and proves that human monoclonal anti-CD70 antibody kills mankind's activated T cells, and this effect can obtain with the adding of anti--CD16 antibody reversing.
Figure 31 shows the result that blocking-up is measured, and proves that some human monoclonal antis-CD70 antibody blocking CD70 combines with CD27's, and other human monoclonal anti-CD70 antibody are not blocked combining of CD70 and CD27.
Figure 32 A to B shows the result of mouse tumor model research in vivo, proves that the treatment of exposed anti--CD70 antibody 2H5 has restraining effect in the direct body to lymphoma.(A) Raji tumour (B) ARH-77 tumour.
Figure 33 A to C shows the result of the research of mouse tumor model in vivo, prove with coupling is cytotoxic to resist-treatment of CD70 antibody 2H5 has restraining effect in the direct body to lymphoma.(A) ARH-77 tumour; (B) Granta 519 tumours; (C) Raji tumour.
Figure 34 shows a result of study, shows that anti--CD70 antibody 69A7 and the CD70 that fastens expression at the positive B lymphoma cell of rhesus monkey CD70 carry out cross reaction.
Figure 35 shows the result that blocking-up is measured, and proves the combination of the human resisting-known mouse anti of CD70 antibody blocking-human CD70 antibody.
Figure 36 A and B show the result who handles with the non-fucosylated form of anti--CD70 antibody or this antibody.(A) anti--CD70 antibody suppresses the CD70 propagation of stimulated cells altogether in dose-dependent mode.(B) anti--CD70 antibody suppresses the IFN-γ secretion that CD70 stimulates altogether in dose-dependent mode.
Figure 37 A to C shows the result who handles with the non-fucosylated form of anti--CD70 antibody or this antibody on the peptide stimulated cells.(A) propagation (expansion) of anti--specific CD8 positive T cell of CD70 antibody inhibiting peptide.(B) do not observe the obvious decline of total cell survival.(C) do not observe the obvious decline of total CD8 positive cell number.
Anti--CD16 antibody blocking that Figure 38 shows anti--CD70 antibody is added the effect of the CD8 positive T cell propagation of peptide specific.
Figure 39 A to B shows the result of the research of mouse tumor model in vivo, prove with the cytotoxin link coupled resist-treatment of CD70 antibody 2H5 has restraining effect in the direct body to the kidney tumour.(A) 786-O tumour (B) Caki-1 tumour.
Figure 40 shows immune conjugate, and anti--CD70-N1 resists the effect that tumour forms with anti--CD70-N2 in the body in 786-O renal cell carcinoma xenotransplantation NOD-SCID mouse model.
The immune conjugate that Figure 41 shows single dose resists-the CD70-N2 effect that the opposing tumour forms in the body in 786-O renal cell carcinoma xenotransplantation SCID mouse model.
The immune conjugate that Figure 42 shows various dose resists-the CD70-N2 effect that the opposing tumour forms in the body in 786-O renal cell carcinoma xenotransplantation SCID mouse model.
The immune conjugate that Figure 43 shows various dose resists-the CD70-N2 effect that the opposing tumour forms in the body in Caki-1 renal cell carcinoma xenotransplantation SCID mouse model.
Anti--CD70-N2 effect that the opposing tumour forms in the body in Raji cell lymphoma SCID mouse model that Figure 44 shows immune conjugate.
Anti--CD70-N2 security in vivo that Figure 45 shows in BALB/c mouse immune conjugate.
Figure 46 A to D illustrates with free medicine in dog and compares, and immune conjugate resists-CD70-N2 security in vivo.
Figure 47 shows the result that ADCC measures.HIgG1nf Neg Ctrl=IgG 1NF negative control antibody.HIgG1Neg Ctrl=IgG 1 negative control antibody.MIgG1NegCtrl=mouse IgG1 negative control antibody.(A) 2H5 is bonded to the facs analysis on the activated B cell.(B) 2H5NF on the human B cell of activated and the ADCC of 2H5 measure.(C) ADCC that adds anti-CD16 antibody measures.
Figure 48 described anti--CD70 antibody by ADCC utilize in the human PBMC culture of irriate naturally occurring effector cell's mediation through Ag activate, the ability of the positive human T lysis of CD70.
Figure 49 has described the binding characteristic of anti--CD70 antibody to natural expression CD70 male human cancer cell line 786-0 cell.
Figure 50 has described fucosylated and non-fucosylated anti--CD70 antibody and mediated the ability of ADCC on CD70 positive lymphomas clone ARH77.
Figure 51 shows that the anti-CD70-cytotoxin E of single dose resists effect in the body that tumour forms in 786-O renal cell carcinoma xenotransplantation SCID mouse model.
Figure 52 shows that the anti-CD70-cytotoxin E of single dose resists effect in the body that tumour forms in A498 renal cell carcinoma xenotransplantation SCID mouse model.
Figure 53 shows that the anti-CD70-cytotoxin E of single dose resists effect in the body that tumour forms in Caki-1 renal cell carcinoma xenotransplantation SCID mouse model.
Figure 54 shows that the anti-CD70-cytotoxin E of single dose resists effect in the body that tumour forms in Raji cell lymphoma SCID mouse model.
Figure 55 shows that the anti-CD70-cytotoxin E of single dose resists effect in the body that tumour forms in Dauli cell lymphoma SCID mouse model.
Figure 56 shows that anti-CD70-cytotoxin E resists effect in the body that tumour forms in Caki-1 renal cell carcinoma xenotransplantation rat model.
Figure 57 shows security in the body of anti-CD70-cytotoxin E in BALB/c mouse.
Figure 58 shows security in the body of anti-CD70-cytotoxin E in dog.
Figure 59 shows security in the body of anti-CD70-cytotoxin E in monkey.
Figure 60 shows that the anti-CD70-cytotoxin F of single dose resists effect in the body that tumour forms in 786-O renal cell carcinoma xenotransplantation SCID mouse model.
Figure 61 shows that the anti-CD70-cytotoxin F of single dose resists effect in the body that tumour forms in Caki-1 renal cell carcinoma xenotransplantation SCID mouse model.
Figure 62 shows effect in the body of the antitumor formation of anti-CD70-cytotoxin F in Raji cell lymphoma SCID mouse model of single dose.
Figure 63 shows that the anti-CD70-cytotoxin G of single dose resists effect in the body that tumour forms in 786-O renal cell carcinoma xenotransplantation SCID mouse model.
Figure 64 shows that the anti-CD70-cytotoxin G of single dose resists effect in the body that tumour forms in Caki-1 renal cell carcinoma xenotransplantation SCID mouse model.
Figure 65 shows that the anti-CD70-cytotoxin H of single dose resists effect in the body that tumour forms in A498 renal cell carcinoma xenotransplantation SCID mouse model.
Figure 66 shows that the anti-CD70-cytotoxin H of single dose resists effect in the body that tumour forms in Caki-1 renal cell carcinoma xenotransplantation SCID mouse model.
Figure 67 shows that the anti-CD70-cytotoxin I of single dose resists effect in the body that tumour forms in 786-O renal cell carcinoma xenotransplantation SCID mouse model.
Figure 68 shows that the anti-CD70-cytotoxin I of single dose resists effect in the body that tumour forms in Caki-1 renal cell carcinoma xenotransplantation rat model.
Figure 69 shows that the anti-CD70-cytotoxin J of single dose resists effect in the body that tumour forms in 786-O renal cell carcinoma xenotransplantation SCID mouse model.
Figure 70 shows, and anti--CD70 antibody 2H5 functionally blocks the human T cell proliferation that CD70 stimulates.
Figure 71 is the structure of cytotoxin B.
Figure 72 is the structure of cytotoxin C.
Figure 73 is the structure of cytotoxin D.
Figure 74 is the structure of cytotoxin E.
Figure 75 is the structure of cytotoxin F.
Figure 76 is the structure of cytotoxin G.
Figure 77 is the structure of cytotoxin H.
Figure 78 is the structure of cytotoxin I.
Figure 79 is the structure of cytotoxin J.
Describe in detail
The monoclonal antibody that the present invention relates to separate, particularly human monoclonal antibody, these antibody are in conjunction with mankind CD70 and have desirable functional characteristic. In certain embodiments, antibody of the present invention is derived from specific heavy chain and light chain germline sequence and/or comprise specific architectural feature, for example comprises the CDR district of specific amino acid sequence. The invention provides the antibody of separation, the method for preparing this antibody-like, antibody-gametophyte molecule conjugate and comprise the bispecific molecule of this antibody-like, and the pharmaceutical composition that comprises antibody of the present invention, antibody-gametophyte molecule conjugate or bispecific molecule. The invention still further relates to this antibody and for example detect the CD70 method of protein, and the method for growth that suppresses to express the cell (for example tumour cell) of CD70 with anti-CD70 antibody of the present invention. Therefore, the present invention also provides the method for using anti-CD70 antibody of the present invention and antibody-polytype cancer of gametophyte molecule conjugate treatment, for example, and treatment clear-cell carcinoma or lymthoma.
, in order more easily to understand the present invention, at first defined some term. Other are defined in whole detailed description and provide.
Comprise variant, isotype, homologue, straight homologues and plant interior homologue as term used herein " CD70 ". For example mankind CD70 albumen is had specific antibody and can carry out cross reaction with the CD70 albumen of species from except the mankind in some cases. In another embodiment, it may be fully special for mankind CD70 albumen that mankind CD70 albumen is had specific antibody, and may not show the cross reactivity of species or other types, perhaps may with from some other species but the CD70 of not all other species carries out cross reaction (as carrying out cross reaction with primate CD70 but with mouse CD70, not carrying out cross reaction). Term " mankind CD70 " refers to human sequence CD70, as has the complete amino acid sequence (SEQ ID NO:76) of the mankind CD70 of Genbank accession number P32970. Term " mouse CD70 " refers to mouse sequence C D70, as has the complete amino acid sequence of the mouse CD70 of Genbank accession number NP_035747. By for example having conservative sudden change or in the sudden change in non-conservative district, mankind CD70 sequence may be different from the mankind CD70 of Genbank accession number P32970, and this CD70 has basically identical with the mankind CD70 of Genbank accession number P32970 biological function. For example, people CD70 biological function is in conjunction with cytokine receptor CD27.
The mankind CD70 of specific mankind CD70 sequence and Genbank accession number P32970 has at least 90% homogeneity usually on amino acid sequence; and contain such amino acid residue; when the CD70 amino acid sequence with other species (as muroid) was compared, it was the people source that these amino acid residues are accredited as this amino acid sequence. In some cases, mankind CD70 and Genbank accession number are that the CD70 of P32970 can have at least 95% on amino acid sequence, perhaps at least 96%, 97%, 98% or 99% homogeneity even. In certain embodiments, with the CD70 sequence of Genbank accession number P32970, compare, mankind CD70 sequence can show and be no more than 10 amino acid whose differences. In certain embodiments, with the CD70 sequence of Genbank accession number P32970, compare, this mankind CD70 can show and be no more than 5, does not perhaps even surpass 4,3,2 or 1 amino acid whose differences. Percentage homogeneity so place explanation is determined.
Term " immune response " refers to lymphocyte for example, antigen presenting cell, phagocyte, granulocyte and the effect of the soluble large molecule (comprising antibody, cell factor and complement) that produced by above cell or liver, and this effect causes to the invasive pathogen, by selective infringement, the destruction of the cell or tissue of pathogenic infection, cancer cell or the normal human subject cell or tissue in autoimmunity inflammation or Inflammation situation or they are removed from human body.
" signal transduction pathway " refers to the biochemistry relation between the multi-signal transduction molecule, and these signal transducers are passed to from the part of cell at signal the transmission of another part of cell and play a role. Phrase used herein " cell surface receptor " comprises, for example, and can acknowledge(ment) signal and the sort signal transmission is passed the molecule of cell membrane of cell and the compound of molecule. An example of " cell surface receptor " of the present invention is the CD70 acceptor.
The term of herein mentioning " antibody " comprises complete antibody and their any Fab (i.e. " antigen-binding portion thereof ") or their strand. " antibody " refers to comprise glycoprotein or its antigen-binding portion thereof of at least two weights (H) chain and two light (L) chains, wherein interconnects by disulfide bond between two heavy chains and two light chains. Each heavy chain includes variable region of heavy chain and (at this, is abbreviated as VH) and CH. CH is by three domain CsH1、C H2And CH3Form. Each light chain includes variable region of light chain and (at this, is abbreviated as VLOr Vk) and constant region of light chain. Constant region of light chain comprises a domain, CL。V HAnd VLDistrict can further be divided into hypervariable region (being called complementary determining region (CDR)), is scattered with the conservative district that is called framework region (FR) therebetween. Each VHAnd VLForm by three CDR and four FR, arrange from aminoterminal to c-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of heavy chain and light chain comprises the binding structural domain with AI. The constant region of antibody can the mediated immunity globulin and the combination of host tissue or the factor, and these host tissues or the factor comprise first composition (Clq) of immune various kinds of cell (for example effector cell) and classical complement system.
" antigen-binding portion thereof " (or referred to as " antibody moiety ") of term used herein " fragment of antibody " and antibody refers to the fragment (as CD70) of the antibody of one or more abilities that kept specific binding antigen. The antigen binding function that has been found that antibody can be realized by the fragment of full length antibody. The example of the binding fragment that " antigen-binding portion thereof " of term antibody contains comprises: (i) Fab fragment, and by VL、V H、C LAnd CH1The unit price fragment that domain forms; (ii) F (ab ')2Fragment, comprise the divalence fragment by two Fab fragments of the connection of the disulfide bridge bond at hinge area; (iii) Fab ' fragment, it is in fact that Fab with hinge area part is (referring to, FUNDAMENTAL IMMUNOLOGY (Paul writes, the third edition, 1993); (iv) by VHAnd CH1The Fd fragment that domain forms; (v) by the V of antibody single armedLAnd VHThe Fv fragment that domain forms; (vi) dAb fragment (people such as Ward, (1989) Nature 341:544-546), it is by VHDomain forms; (vii) complementary determining region (CDR) that separates; And (viii) nano antibody (nanobody), contain the variable region of heavy chain of single variable domains and two constant domain. In addition, although two domain V of Fv fragmentLWith VHBe by independent gene code, but can use recombination method by synthetic linker, they to be coupled together, make them form single protein chain, wherein VLWith VHDistrict's pairing forms monovalent molecule and (is called scFv (scFv); Referring to, as the people such as Bird (1988) Science242: 423-426; And people (1988) the Proc.Natl.Acad.Sci.USA 85:5879-5883 such as Huston). " antigen-binding portion thereof " of term antibody also is intended to contain this type of single-chain antibody. These antibody fragments are to use conventional method well known by persons skilled in the art to obtain, and to these fragments, adopt the mode identical with complete antibody to carry out the serviceability screening.
" antibody of separation " as used herein is intended to refer to there is no the antibody (for example, the antibody of the separation of specific binding CD70 is the antibody that there is no the antigen of specific binding except CD70) of other antibody that possess different antigentic specificities. Yet, may have cross reactivity in conjunction with the antibody of the separation of CD70 to other antigens CD70 molecule of other species (for example from) specifically. In certain embodiments, the antibody specific binding mankind CD70 of separation and not with other non-human CD70 antigenic cross-reaction. In addition, the antibody of separation may be substantially free of other cell materials and/or chemicals.
Term used herein " monoclonal antibody " or " monoclonal antibody combination " refer to have the goods of single molecular antibody molecule. Monoclonal antibody combination has been showed single binding specificity and the affinity for defined epitope.
Term used herein " human antibodies " is intended to comprise the antibody with such variable region, and in these variable regions, framework region and CDR district are all derived from mankind's racial immunity protein sequence. In addition, if this antibody comprises constant region, this constant region is also derived from mankind's racial immunity globulin sequence. Human antibodies can comprise modification subsequently, comprises natural or synthetic modification. Human antibodies of the present invention can comprise the amino acid residue of by mankind's racial immunity globulin sequence, not encoding (for example, external by random or fixed point specificity mutagenesis or the sudden change introduced by somatic mutation in vivo). Yet term used herein " human antibodies " not is intended to comprise such antibody, wherein from the CDR sequence of other mammalian species (as mouse) germline, has been transplanted on mankind's Frame sequence.
Term " human monoclonal antibody " refers to show the antibody of single binding specificity, and these antibody have framework region wherein and CDR district all derived from the variable region of mankind's racial immunity globulin sequence. In one embodiment, the human monoclonal antibody is produced by hybridoma, this hybridoma comprises the B cell that obtains from transgenic nonhuman animal (for example transgenic mice) that merges to immortalized cell, and it has the human heavy chain transgene of comprising and the genetically modified genome of light chain.
Term used herein " recombinant human antibody " comprises by recombinant means preparation, express, produce or separate everyone antibody-like, for example (a) from animal (for example mouse) or by the antibody that separates its hybridoma of preparing (following further illustrating), this animal for human immunoglobulin gene be transgene or transfection chromosome; (b) from through with the host cell of express human antibody (for example transforming, from transfectoma) the antibody that separates, (c) antibody that separates from the human antibodies library of recombinating, make up, and (d) by the antibody of additive method preparation, expression, generation or separation, these methods relate to the montage of human immunoglobulin gene sequence to other DNA sequence dnas. This type of recombinant human antibody has wherein framework region and CDR district all derived from the variable region of mankind's racial immunity globulin sequence. Yet in certain embodiments, the human antibodies of this type of restructuring can carry out mutagenesis in vitro (perhaps, when the transgenic animals of using for mankind Ig sequence, carrying out the mutagenesis of body endosome), and therefore recombinant antibodies VHAnd VLThe amino acid sequence in district is perhaps not naturally to be present in the sequence in human antibodies germline express spectra in body, although they are derived from the V of mankind's germlineHAnd VLSequence is also relevant to it.
" isotype " used herein refers to the antibody type (for example IgM or IgG1) by the weight chain constant area gene coding.
The phrase antibody of antigen " identification " and " to the antibody of antigen-specific " herein can with term " antibody of specific binding antigen " Alternate.
Term " human antibodies derivative " refers to any modified form of human antibodies, for example, and the conjugate of this antibody and another kind of reagent or antibody.
Term " humanized antibody " is intended to expression and wherein derived from the CDR sequence of another mammalian species (as mouse) germline, has been transplanted to antibody on mankind's Frame sequence. Can carry out extra framework region in mankind's Frame sequence modifies.
Term " chimeric antibody " is intended to represent that wherein variable region sequences is derived from species and the constant region sequence is derived from the antibody of another species, as variable region sequences wherein, is derived from mouse antibodies and the constant region sequence is derived from the antibody of human antibodies.
Term " antibody analog " is intended to represent the molecule of conjugated antigen ability that can analog antibody, but they are not limited to the natural antibody structure. The example of this type of antibody analog includes but not limited to affine body (Affibody), the ankyrin repetitive proteins (DARPin) through design, anti-transporter (Anticalin), Avimers (Avimer) and reverse antibody (Versabody), the traditional antibody of simulation in conjunction with the time, all these antibody analogs adopt the integrated structure that produces and play a role by different mechanism from different mechanism.
Term used herein " gametophyte molecule " refers to be coupled to the entity of antibody in antibody-gametophyte molecule conjugate. The example of gametophyte molecule comprises medicine, toxin, labelled molecule (including but not limited to peptide and little molecular marked compound, as the fluorochrome label thing, and monatomic label, as radio isotope), protein and therapeutic agent.
The antibody of " specific binding mankind CD70 " used herein is intended to refer to 5 * 10-8M or less KDIn conjunction with mankind CD70, more preferably with 1 * 10-8M or less, more preferably 6 * 10-9M or less, more preferably 3 * 10-9M or less, even more preferably 2 * 10-9M or less KDAntibody in conjunction with mankind CD70.
Term " K used hereinassoc" or " Ka" be intended to refer to the association rate of specific antibody-AI, and term " K used hereindis" or " Kd, " be intended to refer to the dissociation rate of specific antibody-AI. Term " K used hereinD" be intended to refer to dissociation constant, it is by KdWith KaRatio (be Kd/K a) obtain, and be expressed as molar concentration (M). The K of antibodyDThe method that value can use this area fully to determine is determined. Determine the K of antibodyDMethod for optimizing be by using surface plasma resonance, preferred use such as
Figure A20078005123100381
The bio-sensor system of system.
" high-affinity " of term IgG antibody used herein refers to the K that has for target antigen antibodyDBe 1 * 10-7M or less, more preferably 1 * 10-8M or less, more preferably 1 * 10-9M or less, and even more preferably 1 * 10-10M or less. Yet for other antibody isotypes, " high-affinity " is in conjunction with changing. For example, for the IgM isotype, " high-affinity " is in conjunction with referring to the K that antibody hasDValue is 1 * 10-7M or less, more preferably 1 * 10-8M or less, even more preferably 1 * 10-9M or less.
Term used herein " basically not in conjunction with " protein or cell refer to not in conjunction with or not with high-affinity conjugated protein or cell, that is, and with 1 * 10-6M or larger, more preferably 1 * 10-5M or larger, more preferably 1 * 10-4M or larger, more preferably 1 * 10-3M or larger, even more preferably 1 * 10-2M or larger KDConjugated protein or cell.
Term used herein " experimenter " comprises any mankind or non-human animal. Term " non-human animal " comprises all vertebrates, for example mammal and nonmammalian, for example non-human primate, sheep, dog, cat, horse, ox, chicken, amphibian, fish, reptile etc.
Symbol "-", represent all that as key or while being shown as perpendicular to key such point, the part of in this place, showing are connected on the remainder of molecule, solid phase support part etc. no matter be.
Unless otherwise mentioned, term " alkyl " refers to straight or branched or cyclic hydrocarbon radical or their combination with regard to itself or with regard to another substituent part, it may be fully saturated, monounsaturated or polyunsaturated, and can comprise divalence and polyad, having specified carbon atom number (is C1-C 10Refer to 1 to 10 carbon atom). The example of saturated hydrocarbyl includes but not limited to following group, for example methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, the tert-butyl group, isobutyl group, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropyl methyl and for example homologue or the isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl is the group with one or more pairs of keys or triple bond. The example of unsaturated alkyl includes but not limited to vinyl, 2-acrylic, cyclobutenyl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(1, the 4-pentadienyl), acetenyl, 1-propinyl and 3-propinyl, 3-butynyl, and more higher homologue and isomers. Except as otherwise noted, term " alkyl " also is intended to comprise those following alkyl derivatives that describes in detail, as " assorted alkyl ". The alkyl group that is limited to alkyl is called as " homotype alkyl ".
Term " alkylidene " is with regard to itself or as the divalent group that refers to regard to another substituent part derived from alkane, such as, but be not limited to-CH2CH 2CH 2CH 2-, and further comprise the group of those following being illustrated as " assorted alkylidene ". Generally, alkyl (or alkylidene) should have 1 to 24 carbon atom, and those groups that wherein have 10 or carbon atom still less are preferred in the present invention. " low alkyl group " or " low-grade alkylidene " is shorter alkyl group or alkylidene, generally has eight or carbon atom still less.
Unless otherwise mentioned, term " assorted alkyl " makes up with regard to itself or with another term, refer to stable straight or branched or cyclic hydrocarbon radical or their combination, carbon atom and at least one hetero atom by mentioned number form, this hetero atom is selected from: O, N, Si and S, and wherein nitrogen, carbon and sulphur atom optionally are subject to oxidation, and nitrogen heteroatom optionally is subject to quaternized. One or more hetero atom O, N, S and Si can be positioned at any interior location of assorted alkyl, maybe can be positioned on the position that alkyl is connected in this molecule remainder. Example includes but not limited to :-CH2-CH 2-O-CH 3、 -CH 2-CH 2-NH-CH 3、-CH 2-CH 2-N(CH 3)-CH 3、-CH 2-S-CH 2-CH 3、 -CH 2-CH 2，-S(O)-CH 3、-CH 2-CH 2-S(O) 2-CH 3、-CH＝CH-O-CH 3、-Si(CH 3) 3、 -CH 2-CH＝N-OCH 3And-CH=CH-N (CH3)-CH 3. Two hetero atoms can be continuous at the most, for example-CH2-NH-OCH 3And-CH2-O-Si(CH 3) 3. Similarly, term " assorted alkylidene " is with regard to itself or as the bilvalent radical that refers to regard to another substituent part derived from assorted alkyl, such as but not limited to-CH2-CH 2-S-CH 2-CH 2-and-CH2-S-CH 2-CH 2-NH-CH 2-. For assorted alkylidene, hetero atom also can occupy an one or both ends (for example, alkylidene oxygen base, alkylenedioxy group, alkylidene amino, alkylidene diaminourea and analog) of chain end. Term " assorted alkyl " and " assorted alkylidene " comprise PEG and derivative thereof (referring to for example, Shearwater Polymers Catalog, 2001). In addition, for alkylidene and assorted alkylidene linking group, the direction of writing the formula of linking group does not represent the direction of linking group. For example, formula-C (O)2R '-representative-C (O)2R '-and-R ' C (O)2-。
Refer to have the part of 1 to 6 carbon atom with term " alkyl " or " assorted alkyl " term " rudimentary " that is used in combination.
Term " alkoxyl ", " alkylamino ", " alkyl sulphonyl " and " alkylthio group " (or thio alkoxy) are all the conventional meaning uses with them, and refer to respectively by oxygen atom, amino group, SO2Group or sulphur atom are connected in those alkyl groups of this molecule remainder. Term " aryl sulfonyl " refers to pass through SO2Group is connected in the aryl of molecule remainder, and term " sulfydryl " refers to the SH group.
Generally speaking, " acyl substituent " also is selected from above given group. Term " acyl substituent " refers to be connected in carbonyl carbon and meets its valent group as used herein, and this carbonyl carbon is connected on many nucleolus of the compounds of this invention directly or indirectly.
Unless otherwise mentioned, term " cycloalkyl " and " Heterocyclylalkyl ",, with regard to they itself or with regard to other terms combinations, represent respectively ring-like replacement or unsubstituted " alkyl " and replacement or unsubstituted " assorted alkyl ". In addition, for Heterocyclylalkyl, hetero atom can occupy heterocycle and be connected in the position of the remainder of molecule. The example of cycloalkyl includes but not limited to cyclopenta, cyclohexyl, 1-cyclohexenyl group, 3-cyclohexenyl group, suberyl and analog. The example of Heterocyclylalkyl includes but not limited to 1-(1,2,5,6-tetrahydro pyridyl), 1-piperidyl, 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, oxolane-2-base, oxolane-3-base, thiophane-2-base, thiophane-3-base, 1-piperazinyl, 2-piperazinyl and analog. The hetero atom of ring structure and carbon atom optionally are subject to oxidation.
Unless otherwise mentioned, term " halogen " or " halogen ", with regard to they self or refer to fluorine, chlorine, bromine or iodine atom with regard to another substituent part. In addition, term refers to comprise single alkylhalide group and poly-alkylhalide group as " alkylhalide group ". For example, term " halogen (C1-C 4) alkyl " be intended to include but not limited to trifluoromethyl, 2,2,2-trifluoroethyl, 4-chloro butyl, 3-bromopropyl and analog.
unless otherwise mentioned, term " aryl " refers to replace or unsubstituted polyunsaturated aromatic hydrocarbon substituting group, and it can be monocycle or many rings (preferably from 1 to 3 ring), and these rings condense together or with covalent bond and are connected. term " heteroaryl " refers to contain from one to four and is selected from N, O and the heteroatomic aromatic yl group of S (or ring), and wherein nitrogen, carbon and sulphur atom are optionally oxidized, and nitrogen-atoms is optionally quaternized. heteroaryl can be connected in by hetero atom the remainder of molecule. the limiting examples of aryl and heteroaryl comprises phenyl, the 1-naphthyl, the 2-naphthyl, the 4-xenyl, the 1-pyrrole radicals, the 2-pyrrole radicals, the 3-pyrrole radicals, the 3-pyrazolyl, the 2-imidazole radicals, the 4-imidazole radicals, pyrazinyl, the 2-oxazolyl, the 4-oxazolyl, 2-phenyl-4-oxazolyl, the 5-oxazolyl, the 3-isoxazolyl, the 4-isoxazolyl, the 5-isoxazolyl, the 2-thiazolyl, the 4-thiazolyl, the 5-thiazolyl, the 2-furyl, the 3-furyl, the 2-thienyl, the 3-thienyl, the 2-pyridine radicals, the 3-pyridine radicals, the 4-pyridine radicals, the 2-pyrimidine radicals, the 4-pyrimidine radicals, the 5-benzothiazolyl, purine radicals, the 2-benzimidazolyl, the 5-indyl, the 1-isoquinolyl, the 5-isoquinolyl, the 2-quinoxalinyl, the 5-quinoxalinyl, 3-quinolyl and 6-quinolyl. the substituting group of above-mentioned each aryl and heteroaryl ring system is selected from the acceptable substituting group of following explanation. " aryl " and " heteroaryl " also comprises member ring systems, and wherein one or more non-aromatic member ring systems are condensed or otherwise are bonded on aryl or heteroaryl system.
In brief, term " aryl " (as aryloxy group, aryl sulphur oxygen base (arylthioxy), aralkyl) when with other terms, being used in combination comprises aryl rings and the heteroaryl ring of above definition. Therefore, term " aryl alkyl " is intended to comprise those groups, and wherein aryl is connected in alkyl (for example benzyl, phenethyl, picolyl and analog); This alkyl comprises that wherein carbon atom (for example, methylene group) is by for example those alkyl groups (as Phenoxymethyl, 2-pyridine oxygen methyl, 3-(1-naphthoxy) propyl group and analog) of oxygen atom replacement.
Above term (as " alkyl ", " assorted alkyl ", " aryl " and " heteroaryl ") includes separately the replacement of specifying group and does not replace this two kinds of forms. The preferred substituents of each type group below is provided.
Be called " alkyl substituent " and " assorted alkyl substituent " the substituting group of alkyl and assorted alkyl (comprise those often mention as alkylidene, alkenyl, assorted alkylidene, assorted thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl), and they can be the one or more of following multiple group, and these groups are selected from but are not limited to :-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R ' " ,-OC (O) R ' ,-C (O) R ' ,-CO2R’、 -CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R’”、 -NR”C(O) 2R’、-NR-C(NR’R”R’”)＝NR””、-NR-C(NR’R”)＝NR’”、-S(O)R’、 -S(O) 2R’、-S(O) 2NR’R”、-NRSO 2R ' ,-CN and-NO2, quantity from 0 in the scope of (2m '+1), wherein m ' is the sum of the carbon atom of this group. R ', R ", R ' " and R " " all preferably refer to independently separately hydrogen, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, 1 to 3 aryl, replacement or unsubstituted alkyl, alkoxyl or thio alkoxy or aryl alkyl that halogen replaces for example. When compound of the present invention comprised more than a R group, for example, when existing more than a group in these groups, each R group was selected independently, as to each R ', R ", R ' " and R " " group selects. As R ' and R " while being connected in identical nitrogen-atoms, they can be combined into 5,6 or 7 rings with nitrogen-atoms. For example ,-NR ' R " be intended to including, but not limited to, 1-pyrrolidinyl and 4-morpholinyl. From above substituent discussion, those of skill in the art can understand that term " alkyl " is intended to comprise the group that is attached to the carbon atom on the group that is not the hydrogen base, and for example alkylhalide group is (as-CF3And-CH2CF 3) and acyl group (as-C (O) CH3、-C(O)CF 3、 -C(O)CH 2OCH 3And analog).
Be similar to the substituting group illustrated to alkyl, aryl substituent and heteroaryl substituting group are called " aryl substituent " and " heteroaryl substituting group " usually, and be changeable and be selected from, for example: halogen ,-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R ' " ,-OC (O) R ' ,-C (O) R ' ,-CO2R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、 -NR’-C(O)NR”R’”、-NR”C(O) 2R’、-NR-C(NR’R”)＝NR’”、-S(O)R’、 -S(O) 2R’、-S(O) 2NR’R”、-NRSO 2R ' ,-CN and-NO2、-R’、-N 3、-CH(Ph) 2, fluorine (C1-C 4) alkoxyl and fluorine (C1-C 4) alkyl, and its number is opened in the scope of (open) valent sum from 0 to the aromatic rings system; And wherein R ', R ", R ' " and R " " preferably be independently selected from hydrogen, (C1-C 8) alkyl and assorted alkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C 4) alkyl and (unsubstituted aryl) oxygen-(C1-C 4) alkyl. When compound of the present invention comprised more than a R group, for example, when existing more than a group in these groups, each R group was selected independently, as to each R ', R ", R ' " and R " " group selects.
Optionally, two aryl substituents on the adjacent atom of aryl or heteroaryl ring can be-T-C (O)-(CRR ') by having formulaqThe substituting group of-U-is replaced, and wherein, T and U be independently-NR-,-O-,-CRR '-or singly-bound; And q is from 0 to 3 integer. Alternately, two substituting groups on the adjacent atom of aryl or heteroaryl ring can be chosen wantonly by having formula and be-A-(CH2) rThe substituting group of-B-replaces, and wherein, A and B be independently-CRR '-,-O-,-NR-,-S-,-S (O)-,-S (O)2-、-S(O) 2NR '-or singly-bound; And r is from 1 to 4 integer. One of singly-bound of the new ring that so forms can be chosen wantonly by two keys and replace. Alternately, two substituting groups on the adjacent atom of aryl or heteroaryl ring optionally by have formula for-(CRR ')s-X-(CR”R’”) d-substituting group replace, wherein s and d are from 0 to 3 integer independently, and X be-O-,-NR '-,-S-,-S (O) ,-S (O)2-or-S (O)2NR '-. Preferably, substituent R, R ', R " and R ' " be independently selected from hydrogen or replacement or unsubstituted (C1-C 6) alkyl.
Term used herein " bisphosphate " is including, but not limited to the ester of the phosphoric acid that comprises two bound phosphate groups. Term " triguaiacyl phosphate " is including, but not limited to the ester of the phosphoric acid that comprises three bound phosphate groups. For example, the specific medicine that has bisphosphate or a triguaiacyl phosphate comprises:
Term used herein " hetero atom " comprises oxygen (O), nitrogen (N), sulphur (S) and silicon (Si).
Symbol " R " is a general abbreviation, it represents substituting group, and this substituting group is selected from: replace or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted heterocyclic radical group.
In following each branch, different aspect of the present invention has been described in further detail.
Have resisting-CD70 antibody of specific functional features
The specific functional features that is characterized as antibody or the characteristic of antibody of the present invention. For example, this antibody is specifically in conjunction with mankind CD70, the mankind CD70 that for example expresses at cell surface. Preferably, antibody of the present invention with high-affinity in conjunction with CD70, for example with 1 * 10-7M or less KD, more preferably with 5 * 10-8M or less KD, even more preferably with 1 * 10-8M or less KD are in conjunction with CD70. Assessment antibody is as known in the art to the standard test of the binding affinity of CD70, comprises for example ELISA, Western blotting and RIA. Understand in detail in an embodiment suitable mensuration. Also can, with the binding kinetics (as binding affinity) of standard test assessment antibody as known in the art, for example by ELISA, Scatchard and Biacore, analyze. As another example, antibody of the present invention can be in conjunction with the kidney tumor cell line, for example 786-O, A-498, ACHN, Caki-1 or Caki-2 clone. As another example again, antibody of the present invention can be in conjunction with B cell tumour clone, for example Daudi, HuT 78, Raji or Granta-519 clone.
Of the present invention anti--CD70 antibody is in conjunction with mankind CD70, and preferably shows one or more in following characteristic:
(a) with 1 * 10-7M or less KDIn conjunction with mankind CD70; And
(b) in conjunction with the clear-cell carcinoma tumor cell line;
(c) in conjunction with lymphoma cell line, as B cell tumour clone;
(d) be expressed the cell internalizing of CD70;
_ _ (e) demonstrates ADCC (ADCC) to the cell of expressing CD70; And
(f) suppress in vivo to express the growth of the cell of CD70 when with the coupling of cytotoxin phase.
Preferably, this antibody show characteristic (a), (b), (c), (d), (e) and (f) at least two kinds. More preferably, this antibody show characteristic (a), (b), (c), (d), (e) and (f) at least three kinds. More preferably, this antibody show characteristic (a), (b), (c), (d), (e) and (f) in four kinds. Even more preferably, this antibody show characteristic (a), (b), (c), (d), (e) and (f) in five kinds. Even more preferably, this antibody shows whole six specific characters (a), (b), (c), (d), (e) and (f).
In another preferred embodiment, this antibody affinity of with CD70, being combined is 5 * 10-9M or less. In another preferred embodiment again, when this antibody during with the coupling of cytotoxin phase in vivo antibody suppression express the growth of the tumour cell of CD70.
The technology of having set up in conjunction with available one or more this areas of antibody of the present invention and CD70 is assessed. For example, in preferred embodiments, antibody can be tested with flow cytometry, wherein this antibody reacts with the clone of expressing mankind CD70, for example obtained transfection and can be at the Chinese hamster ovary celI of its surface expression CD70, or express the clone of CD70, as 786-O, A498, ACHN, Caki-1 and/or Caki-2 (referring to the suitable mensuration as in embodiment 4 and 5, and further illustrating clone). Additionally or selectively, the combination of antibody, comprise that the available BIAcore of binding kinetics (as the KD value) tests in conjunction with mensuration. What other were suitable comprises that in conjunction with measuring ELISA measures, and for example utilizes the CD70 albumen (referring to the suitable mensuration as in embodiment 1) of restructuring.
Preferably, the protein bound K of antibody of the present invention and CD70DBe 5 * 10-8M or less, with the protein bound K of CD70DBe 3 * 10-8M or less, with the protein bound K of CD70DBe 1 * 10-8M or less, with the protein bound K of CD70DBe 7 * 10-9M or less, with the protein bound K of CD70DBe 6 * 10-9M or less or with the protein bound K of CD70DBe 5 * 10-9M or less. This antibody can be analyzed to assess by for example standard BIACORE to the binding affinity of CD70.
In the standard test (referring to Hum-ZAP and immunofluorescence assay as in embodiment 7 and 21 explanation) of the cell by expression CD70 known in the art for assessment of the internalization of anti--CD70 antibody. Also become known in the art assessing the combination of CD70 and CD27 and pass through the standard test (referring to mensuration as among embodiments 17 explanation) of anti--CD70 antibody to its inhibition. For assessment of the standard test of the ADCC of the cell for expressing CD70 be also in the art known (referring to as the ADCC of explanation in embodiment 9 measure). By anti--CD70 antibody with and the cytotoxin conjugate for assessment of the standard test of inhibition tumor cell growth in vivo be also in the art known (referring to as the tumour heterograft mouse model of explanation in embodiment 18,19,24 to 31 and 36 to 41).
The preferred antibody of the present invention is the human monoclonal antibody. Additionally or alternately, this antibody can be, for example chimeric or humanized monoclonal antibody.
Monoclonal antibody 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4
Exemplary antibodies of the present invention comprises human monoclonal antibody 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4, and they are as separating illustrated with 2 at embodiment 1 and carrying out structural sign. The V of 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4HAmino acid sequence shows respectively in SEQ ID NO:1,2,3,4,5,73 and 6. The V of 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4LAmino acid sequence shows in SEQ ID NO:7,8,9,10,11,11 and 12 that respectively (69A7 and 69A7Y all have the V of amino acid sequence SEQ ID NO:11L). Consider in these antibody that each all can be in conjunction with CD70, can be with VHAnd VLSequence is carried out " mix and mate ", to produce other anti--CD70 binding molecules of the present invention. Can use combination above and explanation in an embodiment to measure (as FACS or ELISA) and test this type of antibody that " mixes and mate " and the combination of CD70. Preferably, work as VHAnd VLWhen chain is mixed and is mated, from specific VH/V LRight VHSequence is by the V of structural similarityHSequence replaces. Similarly, preferably from specific VH/V LRight VLSequence is by the V of structural similarityLSequence replaces.
Therefore, on the one hand, the invention provides monoclonal antibody or its antigen-binding portion thereof of separation, comprising:
(a) variable region of heavy chain, comprise and be selected from SEQ ID NO:1,2,3,4,5,6 and 73 amino acid sequence; And
(b) variable region of light chain comprises and being selected from: SEQ ID NO:7,8,9,10,11 and 12 amino acid sequence;
Wherein this antibody is specifically in conjunction with CD70.
Preferred heavy chain and light chain combination comprise:
(a) variable region of heavy chain, comprise amino acid sequence SEQ ID NO:1; And (b) variable region of light chain, comprise amino acid sequence SEQ ID NO:7; Or
(a) variable region of heavy chain, comprise amino acid sequence SEQ ID NO:2; And (b) variable region of light chain, comprise amino acid sequence SEQ ID NO:8; Or
(a) variable region of heavy chain, comprise amino acid sequence SEQ ID NO:3; And (b) variable region of light chain, comprise amino acid sequence SEQ ID NO:9; Or
(a) variable region of heavy chain, comprise amino acid sequence SEQ ID NO:4; And (b) variable region of light chain, comprise the amino acid sequence of SEQ ID NO:10; Or
(a) variable region of heavy chain, comprise amino acid sequence SEQ ID NO:5 or 73; And (b) variable region of light chain, comprise amino acid sequence SEQ ID NO:11; Or
(a) variable region of heavy chain, comprise amino acid sequence SEQ ID NO:6; And (b) variable region of light chain, comprise amino acid sequence SEQ ID NO:12.
On the other hand, the invention provides the heavy chain that comprises 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4 and the antibody of light chain CDR1, CDR2 and CDR3 or their combination. The V of 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4HThe CDR1 amino acid sequence shows in SEQ ID NO:13,14,15,16,17,17 and 18 that respectively (69A7 and 69A7Y all have the V of SEQ ID NO:17HThe CDR1 sequence). The V of 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4HThe amino acid sequence of CDR2 shows in SEQ ID NO:19,20,21,22,23,23 and 24 that respectively (69A7 and 69A7Y all have the V that shows in SEQ ID NO:23HThe CDR2 sequence). The V of 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4HThe amino acid sequence of CDR3 shows respectively in SEQ ID NO:25,26,27,28,29,75 and 30.
The V of 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4KThe amino acid sequence of CDR1 shows in SEQ ID NO:31,32,33,34,35,35 and 36 that respectively (69A7 and 69A7Y all have the V that shows in SEQ ID NO:35KThe CDR1 sequence). The V of 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4KThe amino acid sequence of CDR2 shows in SEQ ID NO:37,38,39,40,41,41 and 42 that respectively (69A7 and 69A7Y all have the V that shows in SEQ ID NO:41KThe CDR2 sequence). The V of 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4KThe amino acid sequence of CDR3 shows in SEQ ID NO:43,44,45,46,47,47 and 48 that respectively (69A7 and 69A7Y all have the V that shows in SEQ ID NO:47KThe CDR3 sequence). These CDR districts draw (Kabat with the Kabat system, E.A., Deng people (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).
In view of each in these antibody all can be in conjunction with CD70, and mainly by CDR1, CDR2 and CDR3 district, provide antigen-binding specificity, VHCDR1, CDR2, and CDR3 sequence and VkCDR1, CDR2, and the CDR3 sequence can obtain " mixes and mate ", and (that is, from the CDR of different antibodies, can be mixed and mate, still every kind of antibody all must comprise VHCDR1, CDR2, and CDR3, and VkCDR1, CDR2, and CDR3), to produce other anti--CD70 binding molecules of the present invention. Use combination above and explanation in an embodiment to measure (as FACS, ELISA, Biacore, analyzing) and can test the CD70 combination of this type of antibody of " mixing and mate ". Preferably, work as VHWhen the CDR sequence is mixed and is mated, from specific VHThe CDR1 of sequence, CDR2 and/or CDR3 sequence are replaced by the CDR sequence of structural similarity. Similarly, work as VkWhen CDR is mixed and is mated, from specific VkThe CDR1 of sequence, CDR2 and/or CDR3 sequence are replaced by the CDR sequence of structural similarity. It will be clear that for those of ordinary skills, by replacing one or more V from the structure similar sequence of the CDR sequence of monoclonal antibody 2H5 disclosed here, 10B4,8B5,18E7,69A7,69A7Y and 1F4HAnd/or VLThe CDR region sequence, can produce new VHAnd VLSequence.
Therefore, on the other hand, the invention provides monoclonal antibody or its antigen-binding portion thereof of separation, comprising:
(a) variable region of heavy chain CDR1 comprises and being selected from: SEQ ID NO:13,14,15,16,17 and 18 amino acid sequence;
(b) variable region of heavy chain CDR2 comprises and being selected from: SEQ ID NO:19,20,21,22,23 and 24 amino acid sequence;
(c) variable region of heavy chain CDR3 comprises and being selected from: SEQ ID NO:25,26,27,28,29,30 and 75 amino acid sequence;
(d) variable region of light chain CDR1 comprises and being selected from: SEQ ID NO:31,32,33,34,35 and 36 amino acid sequence;
(e) variable region of light chain CDR2 comprises and being selected from: SEQ ID NO:37,38,39,40,41 and 42 amino acid sequence; And
(f) variable region of light chain CDR3 comprises and being selected from: SEQ ID NO:43,44,45,46,47 and 48 amino acid sequence,
Wherein this antibody is specifically in conjunction with CD70, preferred combination mankind CD70.
In preferred embodiments, this antibody comprises:
(a) variable region of heavy chain CDR1, comprise SEQ ID NO:13;
(b) variable region of heavy chain CDR2, comprise SEQ ID NO:19;
(c) variable region of heavy chain CDR3, comprise SEQ ID NO:25;
(d) variable region of light chain CDR1, comprise SEQ ID NO:31;
(e) variable region of light chain CDR2, comprise SEQ ID NO:37; And
(f) variable region of light chain CDR3, comprise SEQ ID NO:43.
In another preferred embodiment, this antibody comprises:
(a) variable region of heavy chain CDR1, comprise SEQ ID NO:14;
(b) variable region of heavy chain CDR2, comprise SEQ ID NO:20;
(c) variable region of heavy chain CDR3, comprise SEQ ID NO:26;
(d) variable region of light chain CDR1, comprise SEQ ID NO:32;
(e) variable region of light chain CDR2, comprise SEQ ID NO:38; And
(f) variable region of light chain CDR3, comprise SEQ ID NO:44.
In another preferred embodiment, this antibody comprises:
(a) variable region of heavy chain CDR1, comprise SEQ ID NO:15;
(b) variable region of heavy chain CDR2, comprise SEQ ID NO:21;
(c) variable region of heavy chain CDR3, comprise SEQ ID NO:27;
(d) variable region of light chain CDR1, comprise SEQ ID NO:33;
(e) variable region of light chain CDR2, comprise SEQ ID NO:39; And
(f) variable region of light chain CDR3, comprise SEQ ID NO:45.
In another preferred embodiment, this antibody comprises:
(a) variable region of heavy chain CDR1, comprise SEQ ID NO:16;
(b) variable region of heavy chain CDR2, comprise SEQ ID NO:22;
(c) variable region of heavy chain CDR3, comprise SEQ ID NO:28;
(d) variable region of light chain CDR1, comprise SEQ ID NO:34;
(e) variable region of light chain CDR2, comprise SEQ ID NO:40; And
(f) variable region of light chain CDR3, comprise SEQ ID NO:46.
In another preferred embodiment, this antibody comprises:
(a) variable region of heavy chain CDR1, comprise SEQ ID NO:17;
(b) variable region of heavy chain CDR2, comprise SEQ ID NO:23;
(c) variable region of heavy chain CDR3, comprise SEQ ID NO:29 or 75;
(d) variable region of light chain CDR1, comprise SEQ ID NO:35;
(e) variable region of light chain CDR2, comprise SEQ ID NO:41; And
(f) variable region of light chain CDR3, comprise SEQ ID NO:47.
In another preferred embodiment, this antibody comprises:
(a) variable region of heavy chain CDR1, comprise SEQ ID NO:18;
(b) variable region of heavy chain CDR2, comprise SEQ ID NO:24;
(c) variable region of heavy chain CDR3, comprise SEQ ID NO:30;
(d) variable region of light chain CDR1, comprise SEQ ID NO:36;
(e) variable region of light chain CDR2, comprise SEQ ID NO:42; And
(f) variable region of light chain CDR3, comprise SEQ ID NO:48.
Well known CDR3 domain does not rely on CDR1 and/or CDR2 domain, can determine alone the binding specificity of antibody to isoantigen, and predictably produce Multiple Antibodies, these antibody have the identical combination specificity based on common CDR3 sequence. Referring to for example, the people such as Klimka, British J.of Cancer83(2): 252-260 (2000) (illustrated and only with the weight chain variable domain C DR3 of the anti-CD30 antibody of muroid Ki-4, produced Humanized CD 3-resisting 0 antibody); The people such as Beiboer, J.Mol.Biol.296: 833-849 (2000) (restructuring Glycoproteins in Epithelial-2 (EGP-2) antibody of the heavy chain CDR3 sequence of only using the anti-EGP-2 antibody of parental generation muroid MOC-31 has been described); The people such as Rader, Proc.Natl.Acad.Sci.U.S.A.95: 8910-8915 (1998) (has illustrated use muroid anti-alpha 2 integrin αvβ 3A series of humanization anti-alpha 2 integrin α of the heavy chain of antibody LM609 and light chain variable CDR3 domainvβ 3Antibody, wherein each member's antibody all comprises different sequences outside the CDR3 domain, and all can be the same as with the parental generation rodent antibody identical epi-position, and its binding affinity is the same with the parental generation rodent antibody high or higher than parental generation rodent antibody); The people such as Barbas, J.Am. Chem.Soc.116: 2161-2162 (1994) (disclosing the CDR3 domain maximum to the effect of antigen combination); The people such as Barbas, Proc.Natl.Acad.Sci.U.S.A.92: 2529-2533 (1995) (has illustrated the heavy chain CDR3 sequence of three Fab for the mankind placenta DNA (SI-1, SI-40 and SI-32) has been transplanted on the heavy chain of anti-tetanus toxoid Fab, the heavy chain CDR3 that replace to exist by this, and show that the CDR3 domain given alone binding specificity); And the people such as Ditzel, J. Immunol.157: 739-749 (1996) (has illustrated and has transplanted research, wherein only the heavy chain CDR3 of parental generation polyspecific Fab LNA3 is transplanted on the monospecific IgG heavy chain in conjunction with tetanus toxoid Fab p313 antibody, just is enough to keep the binding specificity of parental generation Fab); The people such as Berezov, BIAjournal 8:Scientific Review 8 (2001) (peptide mimics based on the CDR3 of anti-HER 2 monoclonal antibody has been described); The people such as Igarashi, J.Biochem (Tokyo) 117:452-7 (1995) (12 amino acid whose synthetic polypeptide corresponding to the CDR3 domain of anti-phosphatidylserine antibody have been described); The people such as Bourgeois, J.Virol 72:807-10 (1998) (shown derived from single peptide of the heavy chain CDR3 domain of anti respiratory syncytial virus (RSV) antibody can in this virus of external neutralization); The people such as Levi, Proc.Natl.Acad.Sci.U.S.A.90:4374-8 (1993) (peptide based on the heavy chain CDR3 domain of the anti-HIV antibody of muroid has been described); Polymenis and Stoller, J. Immunol.152:5218-5329 (1994) (illustrated by the heavy chain CDR3 district of transplanting the Z-DNA binding antibody make scFv can in conjunction with); And Xu and Davis, Immunity 13:37-45 (2000) (diversity that heavy chain CDR3 has been described is enough to make the identical IgM molecule of other parts to distinguish multiple haptens and proteantigen). Also be illustrated in U.S. Patent number 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905; And 5,760,185, the patented antibody by single CDR domain definition has been described. In these lists of references, the full text of each piece all merges as a reference thus.
Therefore, the invention provides the monoclonal antibody that comprises from derived from the one or more heavy of the mankind or non-human animal's antibody and/or light chain CDR3 domain, wherein this monoclonal antibody can specific binding CD70. In some aspects, the invention provides the monoclonal antibody that comprises from non-human antibody's's (for example mouse or rat antibody) one or more heavy and/or light chain CDR3 domain, wherein this monoclonal antibody can specific binding CD70. In some embodiments, this type of creationary antibody comprises from the one or more heavy of non-human antibody and/or light chain CDR3 domain, with the non-human antibody of corresponding parental generation, compares, and (a) can carry out combination with its competition; (b) reservation function characteristic; (c) be attached to identical epi-position; And/or (d) has a similar binding affinity.
In other respects, monoclonal antibody provided by the invention comprises that wherein this human antibodies can specific binding CD70 from the one or more heavy and/or light chain CDR3 domain of human antibodies (for example, available from non-human animal's human antibodies). In other respects, monoclonal antibody provided by the invention comprises the one or more heavy and/or light chain CDR3 domain from the first antibody-like (for example available from non-human animal human antibodies), wherein this first antibody-like can specific binding CD70, and wherein from the CDR3 domain of this first antibody-like, replace the CDR3 domain of the human antibodies that lacks the CD70 binding specificity, with produce can be specifically in conjunction with the second human antibodies of CD70. In some embodiments, this type of antibody of the present invention comprises compares with the first antibody-like of corresponding parental generation from the one or more heavy of the first antibody-like and/or light chain CDR3 domain, (a) can carry out combination with its competition; (b) reservation function characteristic; (c) be attached to identical epi-position; And/or (d) has a similar binding affinity.
Antibody with specific germline sequence
In certain embodiments, antibody of the present invention comprises from the variable region of heavy chain of specific germline heavy chain immunoglobulin gene and/or from the variable region of light chain of specific germline light chain immunoglobulin gene.
For example, in preferred embodiments, the invention provides the monoclonal antibody of separation or its antigen-binding portion thereof, comprise the V as the mankindH3-30.3 the product of gene or derived from the variable region of heavy chain of this gene, wherein this antibody is specifically in conjunction with CD70. In another preferred embodiment, the invention provides the monoclonal antibody of separation or its antigen-binding portion thereof, comprise the V as the mankindHThe product of 3-33 gene or derived from the variable region of heavy chain of this gene, wherein this antibody is specifically in conjunction with CD70. In another preferred embodiment, the invention provides the monoclonal antibody of separation or its antigen-binding portion thereof, comprise the V as the mankindHThe product of 4-61 gene or derived from the variable region of heavy chain of this gene, wherein this antibody is specifically in conjunction with CD70. In another preferred embodiment, the invention provides the monoclonal antibody of separation or its antigen-binding portion thereof, comprise the V as the mankindHThe product of 3-23 gene or derived from the variable region of heavy chain of this gene, wherein this antibody is specifically in conjunction with CD70.
In another preferred embodiment, the invention provides the monoclonal antibody of separation or its antigen-binding portion thereof, comprise the V as the mankindKThe product of L6 gene or derived from the variable region of light chain of this gene, wherein this antibody specific binding CD70. In another preferred embodiment, the invention provides the monoclonal antibody of separation or its antigen-binding portion thereof, comprise the V as the mankindKThe product of L18 gene or derived from the variable region of light chain of this gene, wherein this antibody specific binding CD70. In another preferred embodiment, the invention provides the monoclonal antibody of separation or its antigen-binding portion thereof, comprise the V as the mankindKThe product of L15 gene or derived from the variable region of light chain of this gene, wherein this antibody specific binding CD70. In another preferred embodiment, the invention provides the monoclonal antibody of separation or its antigen-binding portion thereof, comprise the V as the mankindKThe product of A27 gene or derived from the variable region of light chain of this gene, wherein this antibody specific binding CD70.
In other another preferred embodiment, the invention provides the monoclonal antibody of separation or its antigen-binding portion thereof, wherein this antibody:
(a) comprise variable region of heavy chain, this variable region of heavy chain is mankind VH3-30.3, the product of 3-33,4-61 or 3-23 gene or derived from this gene (amino acid sequence of these coded by said gene provides respectively in SEQ ID NO:61,62,63 and 64);
(b) comprise variable region of light chain, this variable region of light chain is human gene VKThe product of L6, L18, L15 or A27 gene or derived from this gene (amino acid sequence of these coded by said gene provides respectively in SEQ ID NO:65,66,67 and 68); And
(c) specifically in conjunction with CD70.
This antibody-like also may have one or more functional characteristics of above detailed description, for example in conjunction with the high-affinity of mankind CD70, by the cell internalizing of expressing CD70, mediation for the ability of the ADCC of the cell of expressing CD70 and/or suppress in vivo to express the ability of tumor growth of the tumour cell of CD70 when with the coupling of cytotoxin phase.
Has respectively VH3-30.3 and VkThe V of L6HAnd VkThe example of antibody be 2H5. Has respectively VH3-30.3 and VKThe V of L18HAnd VKThe example of antibody be 10B4. Has respectively VH3-33 and VKThe V of L15HAnd VKThe example of antibody be 8B5 and 18E7. Has respectively VH4-61 and VKThe V of L6HAnd VKThe example of antibody be 69A7 and 69A7Y. Has respectively VH3-23 and VKThe V of A27HAnd VKThe example of antibody be 1F4.
This antibody-like also may have one or more functional characteristics of above detailed description, for example in conjunction with the high-affinity of mankind CD70, by the cell internalizing of expressing CD70, in conjunction with the clear-cell carcinoma tumor cell line, in conjunction with lymphoma cell line, mediation for the ability of the ADCC of the cell of expressing CD70 and/or suppress in vivo to express the ability of tumor growth of the tumour cell of CD70 when with the coupling of cytotoxin phase.
As used in this, if the variable region of human antibodies obtains from the system of using mankind's racial immunity globulin gene, this human antibodies comprises heavy chain or variable region of light chain, and it is specific germline sequence " product " or " being derived from " this specific germline sequence. This type systematic comprises with purpose antigen the transgenic mice that carries human immunoglobulin gene is carried out immunity, perhaps with purpose antigen, the human immunoglobulin gene library that is illustrated on bacteriophage is screened. The human antibodies that is " product " or " being derived from " this sequence of mankind's racial immunity globulin sequence can compare by the amino acid sequence of the amino acid sequence to this human antibodies and mankind's racial immunity globulin, and is chosen on sequence near mankind's racial immunity globulin sequence of the sequence of (being maximum % homogeneity) human antibodies and identifies like this. Be the human antibodies of " product " or " being derived from " this sequence of specific mankind's racial immunity globulin sequence, from this germline sequence, compare and can contain different amino acid, this is due to the somatic mutation of for example natural generation or the intentional rite-directed mutagenesis of introducing. Yet, human antibodies through selecting is general has at least 90% homogeneity with the amino acid sequence by mankind's racial immunity globulin gene coding on amino acid sequence, and when with other species (for example, when racial immunity globulin amino acid sequence muroid germline sequence) compares, comprise the amino acid residue that this human antibodies is accredited as human antibodies. In some cases, human antibodies can have at least 95% or even at least 96%, 97%, 98% with the amino acid sequence by racial immunity globulin gene coding on amino acid sequence, or 99% homogeneity. Generally, and by the coded amino acid sequence of mankind's racial immunity globulin gene, compared, the human antibodies that is derived from this specific mankind's germline sequence is no more than displaying the amino acid difference of 10. In some cases, with the amino acid sequence of by the racial immunity globulin gene, being encoded, compare, this human antibodies can show and be no more than 5, or even surpasses 4,3,2 or 1 's amino acid difference.
Homologous antibody
In another embodiment, antibody of the present invention comprises heavy chain and variable region of light chain, these variable regions comprise the amino acid sequence with the amino acid sequence homologous of preferred antibody in this explanation, and wherein this antibody has kept the desirable functional characteristic of the anti-CD70 antibody of the present invention.
For example, the invention provides monoclonal antibody or its antigen-binding portion thereof of separation, comprise variable region of heavy chain and variable region of light chain, wherein:
(a) this variable region of heavy chain comprises and the amino acid sequence that is selected from SEQ ID NO:1,2,3,4,5,6 and 73 amino acid sequence and has 80% homology at least;
(b) this variable region of light chain comprises and is selected from SEQ ID NO:7,8,9,10,11, and amino acid sequence with homology of at least 80% of 12 amino acid sequence;
(c) this antibody specificity combines with CD70.
Additionally or alternately, this antibody can have one or more in following functions characteristic discussed above, for example in conjunction with the high-affinity of mankind CD70, by the cell internalizing of expressing CD70, in conjunction with the clear-cell carcinoma tumor cell line, in conjunction with lymphoma cell line, mediation for the ability of the ADCC of the cell of expressing CD70 and/or suppress in vivo to express the ability of tumor growth of the tumour cell of CD70 when with the coupling of cytotoxin phase.
In different embodiments, this antibody can be for example human antibodies, humanized antibody or chimeric antibody.
In other embodiments, V HAnd/or V LAminoacid sequence can have 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the above sequence that provides.Have with more than the V of the sequence that provides HAnd V KThere is the V of high homology (that is, 80% or higher) in the district HWith V LThe antibody in district can obtain in the following manner: the nucleic acid molecule to coding SEQ ID NO:1-12 and 73 (for example carries out mutagenesis, site-directed mutagenesis or PCR mediated mutagenesis), detect the function (given function promptly) of the reservation of coded, reformed antibody then with the functional examination method of explanation herein.
As used herein, the per-cent homology between two aminoacid sequences is equal to two per-cent identity between the sequence.In the per-cent identity between these two sequences is to consider under the situation of room number and each room length, the function of the number of the same position that these sequences are total (promptly, the overall number of the number/position of % homology=same position * 100), need to introduce these rooms to be used for the best comparison of two sequences.As what in following non-limiting example, illustrate, use mathematical algorithm can finish determining of sequence comparison between the two sequences and per-cent identity.
Can use the E.Meyers and the W.Miller (Comput.Appl.Biosci. that have been incorporated into (2.0 version) in the ALIGN program, 4:11-17 (1988)) algorithm, use PAM120 weight residue table, room length point penalty be 12 and gap penalty be 4, determine the per-cent identity of two aminoacid sequences.In addition, the per-cent identity of two aminoacid sequences can be used the Needleman of GAP program (can the obtain) lining that has been incorporated in the GCG software package and the algorithm of Wunsch (J.Mol.Biol.48:444-453 (1970)) on www.gcg.com, use Blossum 62 matrixes or PAM250 matrix, and room weight 16,14,12,10,8,6 or 4 and length weight 1,2,3,4,5 or 6 determine.
Additionally or alternately, protein sequence of the present invention can be further used as " search sequence " and use, and public database is retrieved, and for example identifies correlated series.Use Altschul, wait people (1990) J.Mol.Biol. 215: the XBLAST program of 403-10 (version 2 .0) can be carried out this type of retrieval.Use the XBLAST program, keep the score=50, the retrieval of BLAST protein is carried out in word length=3, to obtain and antibody molecule homologous aminoacid sequence of the present invention.For obtaining to be used for the room comparison of comparison purpose, as at people such as Altschul (1997) Nucleic Acids Res.25 (17): the used Gapped BLAST that illustrates among the 3389-3402.When using BLAST and Gapped blast program, the default parameters of corresponding program (as XBLAST and NBLAST) is useful.Referring to www.ncbi.nlm.nih.gov.
Has the antibody that conservative property is modified
In certain embodiments, antibody of the present invention comprises the variable region of heavy chain that contains CDR1, CDR2 and CDR3 sequence and contains CDR1, CDR2 and the variable region of light chain of CDR3 sequence, wherein the one or more sequences in these CDR sequences comprise the specific amino acids sequence of modifying based on known anti-CD70 antibody or their conservative propertys, and wherein these antibody have kept the desirable functional performance of the anti-CD70 antibody of the present invention.This area is understood that can carry out some conservative property sequence modification, and these conservative property sequence modifications are not removed the antigen combination.Referring to, for example, people such as Brummell (1993) Biochem 32:1180-8 (explanation) at mutation analysis to the special antibody CDR3 heavy chain structural domain of Salmonellas; People (1997) Prot.Eng.10:835-41 (mutation research of anti-UA1 antibody has been described) such as de Wildt; People such as Komissarov (1997) J.Biol.Chem.272:26864-26870 (having shown the elimination or the attenuating that cause avidity in the sudden change of HCDR3 intermediary); People such as Hall (1992) J.Immunol.149:1605-12 (illustrate single amino acid in the CDR3 district changes eliminated) in conjunction with active; Kelley and O ' Connell (1993) Biochem.32:6862-35 (effect of tyrosine residues in the antigen combination has been described); People (2000) Clin.Can.Res.6:2835-43 (HCDR3 amino acid mutation body has been described) such as people such as Adib-Conquy (1998) Int.Immunol.10:341-6 (effect of hydrophobicity in combination has been described) and Beers.Therefore, the invention provides isolating monoclonal antibody or its antigen-binding portion thereof, comprise the variable region of heavy chain that contains CDR1, CDR2 and CDR3 sequence and contain CDR1, CDR2 and the variable region of light chain of CDR3 sequence, wherein:
(a) variable region of heavy chain CDR3 sequence comprises and is selected from aminoacid sequence SEQ ID NO:25,26,27,28,29,30 and 75, and the aminoacid sequence modified of their conservative property;
(b) variable region of light chain CDR3 sequence comprises and is selected from aminoacid sequence SEQ ID NO:43,44,45,46,47 and 48, and the aminoacid sequence modified of their conservative property;
(c) antibody combines with the CD70 specificity.
Additionally or alternately, this antibody can have one or more in the following functional performance of above explanation, for example in conjunction with the high-affinity of human CD70, expressed CD70 cell internalizing, in conjunction with the renal cell carcinoma tumor cell line, in conjunction with lymphoma cell line, mediation at the ability of the ADCC of the cell of expressing CD70 and/or when suppressing to express the ability of tumor growth of the tumour cell of CD70 mutually during coupling in vivo with cytotoxin.
In preferred embodiments, variable region of heavy chain CDR2 sequence comprises and is selected from aminoacid sequence SEQ ID NO:19,20,21,22,23 and 24, and the aminoacid sequence modified of their conservative property; And variable region of light chain CDR2 sequence comprises and is selected from aminoacid sequence SEQ ID NO:37,38,39,40,41 and 42, and the aminoacid sequence modified of their conservative property.In another preferred embodiment, variable region of heavy chain CDR1 sequence comprises and is selected from aminoacid sequence SEQ ID NO:13,14,15,16,17 and 18, and the aminoacid sequence modified of their conservative property; And variable region of light chain CDR1 sequence comprises and is selected from aminoacid sequence SEQ ID NO:31,32,33,34,35 and 36, and the aminoacid sequence modified of their conservative property.
In different embodiments, this antibody can be for example human antibodies, humanized antibody or chimeric antibody.
As used herein, term " conservative property sequence modification " is intended to represent that not remarkably influenced or change comprise binding characteristic amino acid modified of the antibody of this aminoacid sequence.This type of conservative property is modified and is comprised amino acid whose displacement, insertion and disappearance.By standard technique known in the art, for example site-directed mutagenesis and PCR mediated mutagenesis can be modified these and be introduced in the antibody of the present invention.The conservative amino acid displacement is following these displacements, and wherein amino-acid residue is replaced by the amino-acid residue with similar side chain.Defined amino-acid residue family in this area with similar side chain.These families comprise that the amino acid with basic side chain is (as Methionin, arginine, Histidine), amino acid with acid side-chain is (as aspartic acid, L-glutamic acid), amino acid with uncharged polar side chain is (as glycine, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine, tryptophane), amino acid with non-polar sidechain is (as L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met)), amino acid with β-branched building block is (as Threonine, Xie Ansuan, Isoleucine), and the amino acid with aromatic side chain (as tyrosine, phenylalanine, tryptophane, Histidine).Therefore, one or more amino-acid residues in the CDR district of antibody of the present invention can be by being replaced from other amino-acid residues of identical side chain family, and use at the functional examination of this explanation and can test reformed antibody at the function (that is, in the above function that provides) that keeps.
Antibody with of the present invention resisting-identical epi-position of CD70 antibodies
In another embodiment, the invention provides antibody, epi-position on its human CD70 that is discerned in conjunction with any CD70 monoclonal antibody of the present invention (that is, have the ability with monoclonal antibody of the present invention in any antibody carry out cross competition and combine the antibody of CD70).What in preferred embodiments, be used for cross competition research can be that monoclonal antibody 2H5 (has the V that is shown in SEQ ID NO:1 and 7 respectively with reference to antibody HAnd V LSequence) or monoclonal antibody 10B4 (have the V that is shown in SEQ ID NO:2 and 8 respectively HAnd V LSequence) or monoclonal antibody 8B5 (have the V that is shown in SEQ ID NO:3 and 9 respectively HAnd V LSequence) or monoclonal antibody 18E7 (have the V that is shown in SEQ ID NO:4 and 10 respectively HAnd V LSequence) or monoclonal antibody 69A7 (have the V that is shown in SEQ ID NO:5 and 11 respectively HAnd V LSequence) or monoclonal antibody 69A7Y (have the V that is shown in SEQ ID NO:73 and 11 respectively HAnd V LSequence) or monoclonal antibody 1F4 (have the V that is shown in SEQ ID NO:6 and 12 respectively HAnd V LSequence).
This type of cross competition antibody can be at the CD70 of standard in conjunction with carrying out the ability of cross competition based on them and 2H5,10B4,8B5,18E7,69A7,69A7Y or 1F4 in measuring and being differentiated.Can use standard ELISA to measure, the human CD70 proteopexy that wherein will recombinate onboard, a kind of in these antibody carries out mark with fluorescence, and estimated the be at war with bonded ability of the antibody of sloughing mark of unmarked antibody.Additionally or alternately, BIAcore analyzes the ability of the cross competition that also can be used to estimate antibody.For example, use the epi-position of BIAcore in conjunction with having experimental results show that 2H5,10B4,8B5,18E7,69A7,69A7Y or 1F4 antibodies CD70 go up different epi-positions.Test antibody suppress for example 2H5,10B4,8B5,18E7,69A7,69A7Y or 1F4 in conjunction with the ability of human CD70 prove this test antibody can with the combining of 2H5,10B4,8B5,18E7,69A7,69A7Y or 1F4 competition and human CD70, and so be bonded to by 2H5 and (have the V that is shown in SEQ ID NO:1 and 7 respectively HAnd V LSequence) or 10B4 (have the V that is shown in SEQ ID NO:2 and 8 respectively HAnd V LSequence) or 8B5 (have the V that is shown in SEQ ID NO:3 and 9 respectively HAnd V LSequence) or 18E7 (have the V that is shown in SEQ ID NO:4 and 10 respectively HAnd V LSequence) or 69A7 (have the V that is shown in SEQ ID NO:5 and 11 respectively HAnd V LSequence) or 69A7Y (have the V that is shown in SEQ ID NO:73 and 11 respectively HAnd V LSequence) or 1F4 (have the V that is shown in SEQ ID NO:6 and 12 respectively HAnd V LSequence) mankind that discerned CD70'sOn the identical epi-position.
In a preferred embodiment, the antibody that is bonded to the identical epi-position on the human CD70 that is discerned by 2H5,10B4,8B5,18E7,69A7,69A7Y or 1F4 is the human monoclonal antibody.As illustrated in an embodiment, can be prepared and separate this type of human monoclonal antibody.
Through that transform and modified antibody
Can further prepare antibody of the present invention in the following manner: can use to have one or more V disclosed herein HAnd/or V LThe antibody of sequence is as parent material, and to transform modified antibody as, this modified antibody is compared the characteristic that may have through changing with initial antibody.By (being V in one or two variable region HAnd/or V L) in, for example the one or more residues of middle modification in one or more CDR district and/or in one or more framework regions can be transformed by antagonist.Additionally or alternately,, for example change the effector function of this antibody, can transform by antagonist by modifying the residue in the constant region.
In certain embodiments, can use CDR to transplant transforms with the variable region of antagonist.Antibody and target antigen mainly interact by the amino-acid residue that is arranged in six heavy chains and light chain complementary determining region (CDR).Owing to this reason, the aminoacid sequence of the CDR between each antibody has more diversity than the sequence outside CDR.Because the CDR sequence is responsible for most of antibody-AI, so comprise expression vector from the CDR sequence of transplanted special natural generation antibody to framework sequence (from different antibodies) with different qualities by structure, the recombinant antibodies of expressing the characteristic of the special natural generation antibody of simulation be possible (referring to, Riechmann for example, people such as L. (1998) Nature 332: 323-327; Jones, people such as P. (1986) Nature 321: 522-525; Queen, people such as C. (1989) Proc.Natl.Acad.See.U.S.A. 86: 10029-10033; The U.S. Patent number 5,225,539 of Winter, and people's such as Queen U.S. Patent number 5,530,101; 5,585,089; 5,693,762 and 6,180,370).
Therefore, another embodiment of the invention relates to isolating monoclonal antibody or its antigen-binding portion thereof, this monoclonal antibody or its antigen-binding portion thereof comprise the variable region of heavy chain that contains CDR1, CDR2 and CDR3 sequence, this CDR1, CDR2 and CDR3 sequence comprise respectively and being selected from: SEQID NO:13,14,15,16,17 and 18, SEQ ID NO:19,20,21,22,23 and 24, and SEQ ID NO:25,26,27,28,29,75 and 30 aminoacid sequence; And the variable region of light chain that contains CDR1, CDR2 and CDR3 sequence, this CDR1, CDR2 and CDR3 sequence comprise respectively and being selected from: SEQ ID NO:31,32,33,34,35 and 36, SEQ ID NO:37,38,39,40,41 and 42, and SEQ ID NO:43,44,45,46,47 and 48 aminoacid sequence.Therefore, this antibody-like contains the V of monoclonal antibody 2H5,10B4,8B5,18E7,69A7,69A7Y or 1F4 HAnd V LThe CDR sequence, but the framework sequence different can be comprised with these antibody.
This class framework sequence can obtain from comprising kind of public DNA database that is the antibody gene sequence or disclosed document.For example, kind at human heavy chain and chain variable region gene is that dna sequence dna can be to find (obtaining on http://www.mrc-cpe.cam.ac.uk/vbase in the internet) in the sequence library " Vbase " the human kind, together with in following offering, finding: Kabat, E.A., Deng people (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S. Department of Health and Human Service, NIH Publication No.91-3242; Tomlinson, I.M. waits people (1992) " The Repertoire of Human Germline V HSequencesReveals about Fifty Groups of V HSegments with Different HypervariableLoops " J.Mol.Biol. 227: 776-798; And Cox, people such as J.P.L. (1994) " ADirectory of Human Germ-line V HSegments Reveals a Strong Bias in theirUsage " Eur.J.Immunol. 24: 827-836; The content of each piece is all as a reference incorporated herein by reference clearly in these documents.As another example, be that dna sequence dna can find in the Genbank database at the kind of human heavy chain and chain variable region gene.For example, the following heavy chain kind of finding in the HCo7HuMAb mouse is that sequence can obtain in appended Genbank accession number: 1-69 (NG_0010109, NT_024637 and BC070333), 3-33 (NG_0010109 and NT_024637) and 3-7 (NG_0010109 and NT_024637).As another example, the following heavy chain kind of finding in the HCo12HuMAb mouse is that sequence can obtain in appended Genbank accession number: 1-69 (NG_0010109, NT_024637 and BC070333), 5-51 (NG_0010109 and NT_024637), 4-34 (NG_0010109 and NT_024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).Yet human heavy chain and light chain kind are another source of sequence is the human immunoglobulin gene database, and this database can be available from IMGT (http://imgt.cines.fr).
Use Gapped BLAST method people (1997) Nucleic Acids Research 25:3389-3402 such as () Altschul that is called of one of sequence similarity search method that antibody protein sequence and the protein sequence database worked out are compared, this knows to those skilled in the art.BLAST is a kind of heuritic approach because between antibody sequence and the database sequence significance on statistics comparison may comprise compare character high score fragment to (HSP).Can not improve fragment by expansion or correction the fractional fragment is hit (hit) to being called as.In brief, translate the nucleotide sequence (http://vbase.mrc-cpe.cam.ac.uk/vbase1/list2.php) in VBASE source, and be retained in the district's (comprising FR1 to FR3 framework region) between FR1 to the FR3 framework region.The mean length that these database sequences have is 98 residues.Remove tumor-necrosis factor glycoproteins (they are the accurate couplings on the protein total length).Be used for proteinic BLAST and retrieve service routine blastp, its use is given tacit consent to, the canonical parameter except low-complexity filters (it is closed), and permutation matrix BLOSUM62 are at preceding 5 filtrations of hitting item that realize that sequence matches.By whole six reading frames translation nucleotide sequences, and think that the reading frame that does not have terminator codon in the coupling fragment of database sequence is that potential hits item.And then use blast program tblastx right This conclusionConfirm that this program is with whole six reading frames translation antibody sequences, and the VBASE nucleotide sequences of these being translated and dynamically translating with whole six reading frames compares.The method that can use as described above be similar to VBASE is that sequence library (for example database that can obtain from IMGT (http://imgt.cines.fr)) is retrieved to other human kinds.
Identity is the accurate amino acid coupling on the total length of sequence between antibody sequence and the Protein Data Bank.These positive (identity+replacements mated) is not identical, but the aminoacid replacement that is guided by the BLOSUM62 substitution matrix.Hit the sequence that item will be judged as coupling and hit item if antibody sequence, then has maximum male with the two sequences in the identical identity matching database sequence
The preferred framework sequence that can be used for antibody of the present invention is and structurally similar those framework sequences of the used framework sequence of selected antibody of the present invention that it is employed with underframe sequence: V for example to be similar to the preferred monoclonal antibody of the present invention H3-30.3 framework sequence (SEQ ID NO:61) and/or V H3-33 framework sequence (SEQ ID NO:62) and/or V H4-61 framework sequence (SEQ ID NO:63) and/or V H3-23 framework sequence (SEQ ID NO:64) and/or V KL6 framework sequence (SEQ IDNO:65) and/or V KL18 framework sequence (SEQ ID NO:66) and/or V KL15 framework sequence (SEQ ID NO:67) and/or V KA27 framework sequence (SEQ ID NO:68).
V HCDR1, CDR2 and CDR3 sequence, and V KCDR1, CDR2 and CDR3 sequence can be transplanted to such skeleton construction district, its have with the racial immunity globulin gene that therefrom derives this framework sequence in the identical sequence of sequence found, perhaps these CDR sequences can be transplanted to being that sequence is compared in the skeleton construction district of containing one or more sudden changes with kind.For example, it may be useful having found in framework region residue to be suddenlyd change in some cases, with the antigen binding capacity that keeps or strengthen this antibody (referring to, people's such as Queen U.S. Patent number 5,530,101 for example; 5,585,089; 5,693,762 and 6,180,370).
It is at V that the variable region of another kind of type is modified HAnd/or V KIn CDR1, CDR2 and/or the CDR3 district amino-acid residue is suddenlyd change, improve one or more binding characteristics (for example avidity) of purpose antibody thus.Can carry out site-directed mutagenesis or PCR mediated mutagenesis introduce one or more the sudden change, and the influence of the combination of antagonist or other purpose functional performances can by in body this explanation and that provide in an embodiment or external test assess.The preferred conservative property of introducing is modified (as discussed above).These sudden changes can be amino-acid substitution, insertion or disappearance, but preferably displacement.In addition, generally in the CDR district, change and be no more than one, two, three, four or five residues.
Therefore, in another embodiment, the invention provides isolating anti-CD70 monoclonal antibody or its antigen-binding portion thereof, comprise variable region of heavy chain, this variable region of heavy chain comprises: (a) VH CDR1 district, comprise being selected from SEQ ID NO:13,14,15,16,17 and 18 aminoacid sequence, perhaps compare with 18 with SEQ ID NO:13,14,15,16,17 have one, two, three, four or five amino acid displacement, disappearance or the aminoacid sequence that adds.(b) VH CDR2 district, comprise being selected from SEQ ID NO:19,20,21,22,23 and 24 aminoacid sequence, perhaps compare with 24 with SEQ IDNO:19,20,21,22,23 have one, two, three, four or five amino acid displacement, disappearance or the aminoacid sequence that adds; (c) VH CDR3 district, comprise being selected from SEQ IDNO:25,26,27,28,29,75 and 30 aminoacid sequence, perhaps compare with 30 with SEQ ID NO:25,26,27,28,29,75 have one, two, three, four or five amino acid displacement, disappearance or the aminoacid sequence that adds; (d) VK CDR1 district, comprise being selected from SEQ IDNO:31,32,33,34,35 and 36 aminoacid sequence, perhaps compare with 36 with SEQ ID NO:31,32,33,34,35 have one, two, three, four or five amino acid displacement, disappearance or the aminoacid sequence that adds; (e) VK CDR2 district, comprise being selected from SEQ ID NO:37,38,39,40,41 and 42 aminoacid sequence, perhaps compare with 42 with SEQ ID NO:37,38,39,40,41 have one, two, three, four or five amino acid displacement, disappearance or the aminoacid sequence that adds; And (f) VK CDR3 district, comprise being selected from SEQ ID NO:43,44,45,46,47 and 48 aminoacid sequence, perhaps compare with 48 with SEQ ID NO:43,44,45,46,47 have one, two, three, four or five amino acid displacement, disappearance or the aminoacid sequence that adds.
The antibody that the present invention transforms comprises wherein at V HAnd/or V KIn the framework residue has been carried out the antibody modified, as improved the characteristic of antibody.Generally this class framework is modified to reduce the immunogenicity of antibody.For example, a method is that one or more framework residues " reverse mutation " are become the residue that corresponding kind is a sequence.Or rather, may to comprise with the kind that therefrom derives this antibody be the different framework residue of sequence to the antibody that has experienced somatic mutation.This type of residue can be by being that sequence compares and identified with this antibody framework sequence with the kind that therefrom derives this antibody.The present invention also is intended to contain the antibody of this type of " reverse mutation ".For example, for 10B4, the #2 amino acid of VH (being arranged in FR1) is Isoleucine and should be that this residue of sequence is a Xie Ansuan in corresponding VH 3-30.3 kind.For the kind that the framework region sequence is back to they is a sequence, by for example site-directed mutagenesis or PCR mediated mutagenesis, can be sequence (can be Xie Ansuan from the Isoleucine reverse mutation for example) for planting with somatic mutation " reverse mutation " with No. 2 residues of FR1 among the VH of 10B4.
As another example, for 10B4, V HAmino-acid residue #30 (in FR1) be glycine, and at V H3-30.3 plant is that corresponding this residue is a Serine in the sequence.For the kind that makes this framework region sequence turn back to them is a configuration, for example, the V of 10B4 HThe residue 30 of FR1 can be from glycine " reverse mutation " to Serine.
As another example, for 8B5, V HAmino-acid residue #24 (in FR1) be Threonine, and at V HThe 3-33 kind is that corresponding this residue is a L-Ala in the sequence.For the kind that makes this framework region sequence turn back to them is a configuration, for example, the V of 8B5 HThe residue 24 of FR1 can be from Threonine " reverse mutation " to L-Ala.
As another example, for 8B5, V HAmino-acid residue #77 (in FR3) be Methionin, and at V HThe 3-33 kind is that corresponding this residue is a l-asparagine in the sequence.For the kind that makes this framework region sequence turn back to them is a configuration, for example, the V of 8B5 HThe residue 11 of FR3 can be from Methionin " reverse mutation " to l-asparagine.
As another example, for 8B5, V HAmino-acid residue #80 (in FR3) be Serine, and at corresponding V HThe 3-33 kind is that this residue is a tyrosine in the sequence.For the kind that makes this framework region sequence turn back to them is a configuration, for example, the V of 8B5 HThe residue 14 of FR3 can be from Serine " reverse mutation " to tyrosine.
As another example, for 69A7, V HAmino-acid residue #50 (FR2 in) be leucine, and at V HThe 4-61 kind is that corresponding this residue is an Isoleucine in the sequence.For the kind that makes this framework region sequence turn back to them is a configuration, for example, the V of 69A7 HThe residue 13 of FR2 can be from leucine " reverse mutation " to Isoleucine.
As another example, for 69A7, V HAmino-acid residue #85 (in FR3) be arginine, and at V HThe 4-61 kind is that corresponding this residue is a Serine in the sequence.For the kind that makes this skeleton construction region sequence turn back to them is a configuration, for example, the V of 69A7 HThe residue 18 of FR3 can be from arginine " reverse mutation " to Serine.
As another example, for 69A7, V HAmino-acid residue #89 (in FR3) be Threonine, and at V HThe 4-61 kind is that corresponding this residue is a L-Ala in the sequence.For the kind that makes this framework region sequence turn back to them is a configuration, for example, the V of 69A7 HThe residue 22 of FR3 can be from Threonine " reverse mutation " to L-Ala.
As another example, for 10B4, V LAmino-acid residue #46 (in FR2) be phenylalanine, and at V LThe L18 kind is that corresponding this residue is a leucine in the sequence.For the kind that makes this framework region sequence turn back to them is a configuration, for example, the V of 10B4 LThe residue 12 of FR2 can be from phenylalanine " reverse mutation " to leucine.
As another example, for 69A7, V LAmino-acid residue #49 (in FR2) be phenylalanine, and at V LThe L6 kind is that corresponding this residue is a tyrosine in the sequence.For the kind that makes this framework region sequence turn back to them is a configuration, for example, the V of 69A7 LThe residue 15 of FR2 can be from phenylalanine " reverse mutation " to tyrosine.
The framework of another kind of type modify relate in framework region or even one or more residues are suddenlyd change in one or more CDR district, to remove t cell epitope, reduce the potential immunogenicity of antibody thus.This method is also referred to as " going immunization ", and in people's such as Carr U.S. Patent Publication No. 20030153043 this method has been described in further detail.
Antibody through transforming of the present invention also comprises those antibody: wherein amino-acid residue is modified, with by change on antibody with t cell epitope interactional amino acid modified increase or reduce immunogenic response (referring to, as U.S. Patent number 6,835,550; 6,897,049 and 6,936249).
Except the modification of in framework region or CDR district, carrying out, or alternately, can also transform antibody of the present invention, in the Fc district, to comprise some modifications, general one or more functional performances that change this antibody, for example cytotoxic effect of the cell of serum half-life, complement combination, Fc receptors bind and/or antigen dependence.In addition, antibody of the present invention can or be modified to change its glycosylation, so that change one or more functional performances of this antibody once more by chemically modified (for example, one or more chemical partly can be connected in this antibody).Below in these embodiments each all is described in detail.The numbering of residue is those numberings of the EU call number of Kabat in the Fc district.
In one embodiment, the hinge area of CH1 is modified, made the number of the cysteine residues in the hinge area change, as, increase or reduce.In people's such as Bodmer the US patent No. 5,677,425, further illustrate this method.The number of cysteine residues changes in the hinge area of CH1, the stability of for example being convenient to assemble light chain and heavy chain or increase or reducing this antibody.
In another embodiment, the Fc hinge area of antagonist suddenlys change to reduce the biological half-life of this antibody.Or rather, with the interface region that one or more amino acid mutations are introduced the segmental CH2-CH3 structural domain of Fc hinges, make that this antibody has weakened the combination of SpA for the combination of the SP (SpA) in natural Fc hinge arrangement territory.People's such as Ward the US patent No. 6,165,745 is understood this method in more detail.
In another embodiment, this antibody is modified to increase its biological half-life. MultipleDiverse ways is possible.For example, as explanation in the U.S. Patent number 6,277,375 of Ward, can introduce one or more following sudden changes: T252L, T254S and T256F.Alternately, for increasing biological half-life, this antibody can be at CH1 or C LChange in the district to comprise from what two rings of the CH2 structural domain in the Fc district of IgG obtained and remedy antibody (salvage receptor) in conjunction with epi-position, as people's such as Presta U.S. Patent number 5,869,046 and 6,121,022 is illustrated.
In other other embodiment, change the Fc district by replacing at least one amino-acid residue, and change one or more effector functions of this antibody with different amino-acid residues.For example, the one or more amino acid that are selected from amino-acid residue 234,235,236,237,297,318,320 and 322 can be replaced by different amino-acid residues, make this antibody for the effector part, have the avidity that has changed, but still keep the antigen binding capacity of this parental generation antibody.For example, the reformed effector part of the avidity C1 composition that can be Fc acceptor or complement.This method has more detailed description in No. 5,624,821, people's such as Winter U.S. Patent number and 5,648,260.
In another example, the one or more amino-acid residues that are selected from amino-acid residue 329,331 and 322 can be replaced by different amino-acid residues, the C1q that makes this antibody have to have changed in conjunction with and/or reduce or cytotoxic effect (CDC) that the complement eliminated relies on.This method has more detailed description in people's such as Idusogie U.S. Patent number 6,194,551.
In another example, change the one or more amino acid in the amino acid position 231 and 239, change the ability of the complement-fixing of antibody thus.This method has more detailed description in the open WO 94/29351 of people's such as Bodmer PCT.
In another example again, the Fc district is modified to improve the ability of antibody-mediated ADCC, and/or increase the avidity of antibody at Fc γ acceptor, realize by in upper/lower positions, modifying one or more amino acid: 238,239,248,249,252,254,255,256,258,265,267,268,269,270,272,276,278,280,283,285,286,289,290,292,293,294,295,296,298,301,303,305,307,309,312,315,320,322,324,326,327,329,330,331,333,334,335,337,338,340,360,373,376,378,382,388,389,398,414,416,419,430,434,435,437,438 or 439.This method has further instruction in the PCT of Presta open file WO 00/42072.Drawn out at Fc γ R1, Fc γ RII, Fc γ RIII and the binding site of FcRn on IgG 1, and illustrated and have the bonded variant that improved (referring to Shields, people such as R.L. (2001) J.Biol.Chem. 276: 6591-6604).Shown that sudden change special on position 256,290,298,333,334 and 339 improves the combination to Fc γ RIII.In addition, shown that following combination mutant improves Fc γ RIII combination: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.
In another embodiment again, illustrated as U.S. Provisional Application series number 60/957,271 (it merges as a reference at this in full), modify the C-terminal of antibody of the present invention by introducing cysteine residues.This type of modification includes but not limited at the C of total length sequence of heavy chain end place or replaces existing amino-acid residue in its vicinity, and the prolongation sequence (extension) that will comprise halfcystine is introduced into the C end of total length sequence of heavy chain.In preferred embodiments, the prolongation sequence that comprises halfcystine comprises sequence Ala-Ala-halfcystine (holding the end to C from N).
In preferred embodiments, the coupling that exists for the mating partner molecule of this type of C-terminal cysteine modified provides the site, and this mating partner molecule for example is therapeutical agent or marker molecules.Particularly, the reactive behavior sulfydryl that occurs owing to the C-terminal cysteine modified can adopt the disulfide linkers and the coupling mutually of mating partner molecule of following detailed description.Antibody and the feasible control that can strengthen of mating partner molecule coupling in this way to the specific site that is connected.In addition,, more optimized conjugate, made its reduce or eliminate interference, and allowed the simplification analysis and the quality control of coupling prepared product this antibody function characteristic by at C-terminal or introduce connection site in its vicinity.
In another embodiment again, the glycosylation of antagonist is modified.For example, can manufacture the antibody (that is this antibody deficiency glycosylation) of de-glycosylation (aglycoslated).Can change glycosylation for example to increase antibody to antigenic avidity.For example, can realize this type of carbohydrate modification by in antibody sequence, changing one or more glycosylated sites.For example, can carry out one or more amino acid whose displacements, this causes removing the glycosylation site of one or more variable regions framework, eliminates the glycosylation in this site thus.This de-glycosylation effect can improve antibody to antigenic avidity.This method is at people's such as Co U.S. Patent number 5,714,350 and 6,350, more detailed description arranged in 861.Following document understands in more detail that also other changes the method for glycosylation: people's such as Hanai United States Patent (USP) 7,214,775, the U.S. Patent number 6 of Presta, 737,056, people's such as the U.S. Patent Publication No. 20070020260 of Presta, Dickey PCT discloses people's such as WO/2007/084926, Zh u the open WO/2006/089294 of PCT, and the open WO/2007/055916 of people's such as Ravetch PCT, each piece of writing of above document its in full all by reference as a reference at this.
Additionally or alternately, can prepare the antibody of glycosylation, for example have the antibody of low fucosylated (hypofucosylated) of fucosyl residues of reduction or antibody with bisection GlcNac structure of increase with the type of being changed.The verified type of glycosylation that this type of has changed can improve the ADCC ability of antibody.For example, can realize this type of carbohydrate modification by expressing antibodies in the host cell that obtains changing in glycosylation mechanism.Cell with the glycosylation mechanism that has changed is illustrated in the art, and can produce the antibody with the glycosylation that has changed thus as the host cell of expressing recombinant antibodies of the present invention.For example, clone Ms704, Ms705 and Ms709 lack fucosyl transferase gene, FUT8 (α (1,6) fucosyl transferase) makes Fucose on the carbohydrate of antibody deficiency at them of expressing in Ms704, Ms705 and Ms709 clone.Ms704, Ms705 and Ms709FUT8 -/-Clone is by using two kinds to replace carriers and the target of the FUT8 gene in the CHO/DG44 cell is destroyed produce (referring to people (2004) Biotechnol Bioeng 87:614-22 such as people's such as Yamane U.S. Patent Publication 20040110704 and Yamane-Ohnuki).As another example, people's such as Hanai EP 1,176,195 have illustrated the clone that has ruined FUT8 gene on function (this genes encoding fucosyltransferase), make by reducing or eliminating α 1,6 key involved enzyme, the antibody of expressing in this clone show low fucosylated effect.People such as Hanai have also illustrated some clones, they have the low enzymic activity (N-acetyl-glucosamine is bonded to the Fc district of antibody) that is used for Fucose is added into N-acetyl-glucosamine, perhaps do not have this enzymic activity, for example rat myeloma cell is YB2/0 (ATCC CRL 1662).The PCT open file WO 03/035835 of Presta has illustrated that a kind of Chinese hamster ovary celI is mutation, the Lec13 cell, the ability that Fucose is connected in the carbohydrate that links to each other with Asn (297) with reduction, this also causes the low fucosylated effect (also can be referring to Shields, people such as R.L. (2002) J.Biol.Chem.277:26733-26740) of the antibody of expressing in this host cell.Illustrated among people's such as Umana the PCT open file WO 99/54342 through transform and (for example express glycosyltransferase that glycoprotein modifies, β (1,4)-N-acetylglucosaminyltransferase III (GnT III)) clone, make the antibody of expressing in the clone through transforming have the bisection GlcNac structure that increases, this causes active raising of ADCC (also can referring to people such as Umana (1999) Nat.Biotech.17:176-180) of antibody.Alternately, can use fucosidase to cut the fucosyl residues of this antibody.For example, the fucosidase alpha-L-fucosidase is removed fucosyl residues (Tarentino, people such as A.L. (1975) Biochem.14:5516-23) from antibody.
Additionally or alternately, can prepare the antibody of the type of glycosylation with change, wherein this change relates to sialic acid (sialyation) the change level of antibody.This type of change has explanation in people's such as people's such as Dickey PCT publication number WO/2007/084926 and Ravetch PCT publication number WO/2007/055916, its script of these two pieces of documents all by reference as a reference.For example, can adopt the enzyme reaction of sialidase, for example produce urea Arthrobacter sialidase (Arthrobacterureafacens sialidase).The condition of this reaction mainly is illustrated in United States Patent (USP) 5,831, and in 077, its full content merges as a reference at this.Other limiting examples of suitable enzyme are neuraminidase and N-Glycosylase F, respectively as at people J.Virology such as Schloemer, and 15 (4), people Biochem J. such as 882-893 (1975) and Leibiger, 338, explanation among the 529-538 (1999).The antibody of asialoglycoproteinization can be further purified by using affinity chromatography.Alternately, can adopt several different methods to improve sialylated level, for example adopt sialytransferase.As long as the condition of this reaction is illustrated in people such as Basset, Scandinavian Journal of Immunology, 51 (3), among the 307-311 (2000).
Being modified at the another kind of this antibody of considering by the present invention is Pegylation effect (pegylation).But antagonist carries out the biological half-life (for example serum half-life) of Pegylation for example to increase this antibody.For antagonist carries out Pegylation, generally be connected under antibody or its segmental condition at one or more polyoxyethylene glycol (PEG) group, antibody or its antibody fragment and PEG (for example reactive ester of PEG or aldehydes derivative) are reacted.Preferably, use reactive PEG molecule (or similar reaction water-soluble polymkeric substance), carry out Pegylation by acylation reaction or alkylated reaction.Term " polyoxyethylene glycol " is intended to contain and has been used to derive any form of PEG of other protein (for example single (C1-C10) alkoxyl group-or aryloxy-polyoxyethylene glycol or polyoxyethylene glycol-maleimide) as used herein.In certain embodiments, the antibody for the treatment of Pegylation is deglycosylated antibody.The method of protein being carried out Pegylation is well known in the art, and can be applied to antibody of the present invention.Referring to, people's such as people's such as Nishimura EP 0,154 316 and Ishikawa EP 0 401 384 for example.
Antibody fragment and antibody analog
The invention is not restricted to traditional antibody, and may be implemented by the use of antibody fragment and antibody analog.As following detailed description, multiple antibody fragment and antibody analog technology are developed now and are widely known by the people in this area.Though being arranged in these technology, many technology (as domain antibodies, nano antibody and UniBody) used the fragment of traditional antibody structure or other modifications of these traditional antibody structures, but also exist the technology of plurality of replaceable, as affine body, ankyrin repetitive proteins, anti-transporter, high-affinity polymer and reverse antibody through design, they have adopted the bonded structure, and these structures (although the traditional antibodies of they simulations) result from different mechanism also by different mechanism performance functions.
Domain antibodies (dAb) is the minimum function bonding unit of antibody, corresponding to the heavy chain (V of human antibodies H) or light chain (V L) the variable region.The molecular weight of domain antibodies is approximately 13kDa.Domantis has developed whole human V HWith V LDAb a series of big and high functionality library (10,000,000,000 different sequences of surpassing are arranged in each library), and use these libraries to select to the special dAb of treatment target.Different with many traditional antibody, domain antibodies is expressed in bacterium, yeast and mammiferous cell system well.The further details of domain antibodies and production method thereof may be by obtaining referring to following document, and these documents are: United States Patent (USP) 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; U.S. serial 2004/0110941; European Patent Application No. 1433846 and European patent 0368684 and 0616640; WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 and WO03/002609, wherein the full text of each all merges as a reference at this.
Nano antibody (Nanobody) is the therapeutic protein that is derived from antibody, and it comprises the unique texture and the functional performance of the heavy chain antibody of natural generation.These heavy chain antibodies comprise single variable region (VHH) and two constant regions (CH2 and CH3).Importantly, be the completely stable polypeptide that carries whole antigen binding capacities of original heavy chain antibody through clone and isolating VHH structural domain.The V of nano antibody and human antibodies HStructural domain has high homology, and can further can not lost any activity by humanization.Importantly, nano antibody has the potentiality of reduced immunogenicity, and this is confirmed in the primate study of the compound due to using nano antibody.
Nano antibody combines the advantage of conventional antibody and the key character of small-molecule drug.The same with conventional antibody, nano antibody demonstrates high targeting specific, to their high-affinity and the low intrinsic toxicity of target spot.Yet as small-molecule drug, they can suppress enzyme and arrive acceptor crack (receptor cleft) easily.In addition, nano antibody is highly stable, can carry out administration (for example, referring to WO 04/041867, it merges as a reference at this in full) by the method outside the injection, and be easy to make.Other advantages of nano antibody comprise: identify uncommon or hiding epi-position owing to its size is little; Because the handiness of three-dimensional, the medicament forms of its uniqueness enters the cavity or the avtive spot of protein targets with high-affinity and selective binding; Transformation period is repaiied system, and find medicine easily and fast.
Nano antibody is encoded by term single gene, and effectively produces in most prokaryotic hosts and eucaryon host, these hosts for example intestinal bacteria (E.coli) (referring to, as, US 6,765, and 087, it merges as a reference at this in full), mould (for example Aspergillus or Trichoderma) and yeast (for example Saccharomycodes, genus kluyveromyces, Hansenula or Pichia) (referring to, as, US6,838,254, it merges as a reference at this in full).But this production method is a ratio amplifies, and has produced the nano antibody of several kilogram quantities.Demonstrate superior stability because compare nano antibody with conventional antibody, so they can be mixed with long quality-guarantee period, available solution immediately.
The nanometer cloning process (referring to, for example WO 06/079372, it merges as a reference at this in full) be to be used to create antagonism the patented method of nano antibody of desired target, it is selected based on the automatic high-throughput of B cell, and can use in situation of the present invention.
UniBody is another kind of antibody fragment technology, yet this technology is based on the hinge area of removing IgG4 antibody.The deletion hinge area is the molecule of conventional I gG4 antibody one half-size scale basically, and this molecule has the divalence land of unit price land rather than IgG4 antibody.Therefore as everyone knows, IgG4 antibody is inert, and does not react with immunity system, and it is favourable that this may not expect to produce the disease of immunne response to treatment, and this advantage has passed to Unibody.For example, Unibody can bring into play suppress or reticent they in conjunction with the effect of cell, but do not kill these cells.In addition, the UniBody that is bonded to cancer cell does not stimulate their propagation.In addition, because Unibody is half of size of conventional I gG4 antibody, they can demonstrate better distribution on bigger solid tumor, have the favourable effect of potential.Unibody is to be eliminated from human body with the similar speed of complete IgG4 antibody and can be to combine their antigen with the similar avidity of complete antibody The further details of Unibody can obtain from patent application WO2007/059782, and it merges as a reference at this in full.
Affine body molecule is represented the rabphilin Rab matter of novel type, and it is based on the protein structure domain of 58 amino-acid residues, derived from one of IgG binding domains of SP.This triple helical bundle structural domain has been used to make up the phagemid library (combinatorial phagemidlibrary) of combination as support, use display technique of bacteriophage can from this library, select affine body varient (the Nord K of the desired molecule of target, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, NygrenPA, Binding proteins selected from combinatorial libraries of an α-helicalbacterial receptor domain, Nat Biotechnol 1997; 15:772-7.Ronmark J, Gronlund H, Uhlen M, Nygren PA, Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A, Eur JBiochem 2002; 269:2647-55).Affine body molecule simple in structure firm, and their molecular weight low (6kDa), this makes them be suitable for various application widely, for example, as detection reagent (Ronmark J, Hansson M, people such as Nguyen T, Construction andcharacterization of affibody-Fc chimeras produced in Escherichia coli, JImmunol Methods 2002; 261:199-211), and interaction (the Sandstorm K that suppresses acceptor, Xu Z, Forsberg G, Nygren PA, In hibition of theCD28-CD80co-stimulation signal by a CD28-binding Affibody liganddeveloped by combinatorial protein engineering, Protein Eng2003; 16:691-7).The further details of affine body and their production method can be by referring to United States Patent (USP) 5,831, and 012 and obtain, it merges as a reference at this in full.
The affine body of mark also can be used for determining in the imaging applications of isotype abundance.
DARPin (through the ankyrin repetitive proteins of design) is an example of antibody analog D RP (through the repetitive proteins of design) technology, this technology is developed to utilize the binding ability of non-antibody polypeptide.Repetitive proteins, ankyrin or to be rich in leucic repetitive proteins be ubiquitous binding molecule for example, different with antibody, they appear in the cell and the extracellular.The modular structure of their uniquenesses is a feature with repeated structural unit (duplicon), the prolongation repeating structure territory that these duplicons are stacked and show target mating surface variable and module to form.Based on this modularity, can generate polypeptides in combination library with highly multifarious binding specificity.This strategy comprises the consistence design of the repeat body (self-compatible repeat) (showing variable surface residue) of ego syntonia, and their random groups are fed in the repeating structure territory.
Ankyrin repetitive proteins through design can be produced with quite high productive rate in bacterial expression system, and they belong to the most stable known protein.Selected the ankyrin repetitive proteins through design at high special, the high-affinity of a large amount of target proteins, these target proteins comprise human receptor, cytokine, kinases, human protein enzyme, virus and membranin.Can obtain avidity at the ankyrin repetitive proteins through design of units nmole to the units picomole scope.
Ankyrin repetitive proteins through design has been used for using widely, comprises ELISA, sandwich ELISA, flow cytometry (FACS), immunohistochemical analysis (IHC), chip application, affinity purification or Western blotting.Proved that also the ankyrin repetitive proteins through design has high reactivity in the intracellular region chamber, for example merged as born of the same parents' internal labeling thing albumen and green fluorescent protein (GFP).Ankyrin repetitive proteins through designing further is used to suppress virus and enters its IC 50In the pM scope.Ankyrin repetitive proteins through designing also suppresses enzyme not only aspect blocks protein-protein-interacting being ideal.Proteolytic enzyme, kinases and translocator have successfully been suppressed, normally other structure suppression mode.Enrichment specifically and with the ratio of very favorable tumour and blood very fast and on tumour makes ankyrin repetitive proteins through design suit very much in-vivo diagnostic or methods of treatment.
The relevant ankyrin repetitive proteins and the other information of other DRP technology through design can find in U.S. Patent Application Publication 2004/0132028 and the open WO 02/20565 of international patent application, and their full text all merges as a reference at this.
Anti-transporter is another kind of antibody simulation technique, yet in this case, the bonded specificity is derived from lipocalin protein, and lipocalin protein is a low molecular weight protein (LMWP) family, great expression natively in tissue and body fluid.Lipocalin protein has obtained evolving, and carries out the relevant multiple function of storage with physiology transportation and chemosensitive or insoluble compound in vivo.Lipocalin protein has firm internal structure, is included in β-bucket that this proteic end is supported the high conservative of four rings (loop).These rings have formed the inlet of binding pocket, and have explained variation at the binding specificity between each lipocalin protein at this a part of conformational difference of molecule.
Although the one-piece construction by the super variable loop of conservative β-lamella framework support allows the people recall immunoglobulin (Ig), but lipocalin protein and antibody is significant difference in size, it is made of 160 to 180 amino acid whose single polypeptide chains, and this single polypeptide chain is slightly greater than single immunoglobulin domains.
Lipocalin protein is cloned, and make their ring stand to transform so that produce anti-transporter.Generated the library of structurally various anti-transporter, and the displaying of anti-transporter allows combined function is selected and screened, in protokaryon or eucaryon system, be used for further analysis subsequently by expressing and generate soluble proteins.Multinomial research has successfully proved can develop all specific anti-transporter of any human target protein almost, they can be separated, and can obtain binding affinity in nmole or higher scope.
Anti-transporter can also form two proteic forms of target, is called Duocalin.The production method of use standard, Duocalin in conjunction with two kinds of different therapeutic targets, keeps targeting specific and avidity with a monomeric protein form that is easy to produce simultaneously, regardless of its structural approach of two binding domainss.
By individual molecule a plurality of targets are adjusted in the known disease that relates to more than a kind of virulence factor and have superiority especially.In addition, for example divalence such as Duocalin or multivalence combining form bunch mediate the agonist effect of signal transduction pathway or induce at the cell surface molecule of target disease, combination by cell surface receptor and collection and has significant potentiality aspect the enhanced internalization effect.In addition, the high inherent stability of Duocalin is similar to the anti-transporter of monomer, and this makes Duocalin have to prepare flexibly and send and pass potentiality.
Other information of relevant anti-transporter can be at United States Patent (USP) 7,250,297 and International Patent Application Publication No. WO 99/16873 in find, their full text all merges as a reference at this.
Useful another kind of antibody simulation technique is high-affinity polymer (Avimer) in content of the present invention.The high-affinity polymer is to come by evolution the extended familys of external exon rearrangement and phage display receptor domain outside the human cell, produces the Multidomain albumen with combination and rejection characteristic.Shown that connecting multiple independently binding domains produces avidity, and with traditional conjugated protein comparing of single epi-position, this binding domains has produced avidity and the specificity improved.Other potential advantages are included in the intestinal bacteria simple and produce the molecule of many target-specifics effectively, the thermostability of improvement and to the resistibility of proteolytic enzyme.Obtained high-affinity polymer at the inferior nmole avidity of having of multiple target.
Can find in U.S. Patent Application Publication No. 2006/0286603,2006/0234299,2006/0223114,2006/0177831,2006/0008844,2005/0221384,2005/0164301,2005/0089932,2005/0053973,2005/0048512,2004/0175756 about polymeric other information of high-affinity, its full content all merges as a reference at this.
Oppositely antibody is the antibody simulation technique that another kind can be used for content of the present invention.Oppositely antibody is 3 to 5kDa small protein matter, has the halfcystine greater than 15%, and it forms high disulphide density support, replaces the hydrophobic core that general albumen has.The a spot of disulphide of this usefulness is replaced a large amount of hydrophobic amino acids (comprising hydrophobic core) makes protein littler, more hydrophilic (gathering and non-specific binding are littler), has bigger resistibility for proteolytic enzyme and heat, and having more low-density t cell epitope, is hydrophobic because MHC is presented the maximum residue of contribution.Whole four kinds in these characteristics all is that the meeting of knowing influences immunogenicity, and expects that they can reduce immunogenicity together significantly.
Oppositely the enlightenment of antibody comes from by leech, snake, spider, scorpion, snail, and the natural injectable biological agent of sea anemone (anemone) generation, and known these biological agents show unforeseeable reduced immunogenicity.From natural protein family,, size, hydrophobicity, the processing of proteolysis antigen and epi-position density all can be minimized to well below the level of the proteinic mean value of natural injectable by design and screening through selecting.
In view of the structure of reverse antibody, these antibody analogs provide various form, comprise the vacancy in multivalence, polyspecific, diversified transformation period mechanism, tissue target module and antibody Fc district.In addition, can in intestinal bacteria, produce reverse antibody, and because their wetting ability and size are little, oppositely the antibody height is solvable, and can be formulated into high density with high yield.Oppositely antibody has special thermostability (they can be boiled), and has the long quality guaranteed period.
Other information about reverse antibody can find in U.S. Patent Application Publication No. 2007/0191272, and it merges as a reference at this in full.
The detailed description of antibody fragment that more than provides and antibody simulation technique is not the comprehensive inventory that is intended for all technology that can use in the context of the present specification.For example, but be not construed as limiting multiple other technology, comprise alternative technology based on polypeptide, as people such as Qui, NatureBiotechnology, the fusion of the complementary determining region of general introduction among 25 (8) 921-929 (2007) (it merges as a reference at this in full), and based on the technology of nucleic acid is as at United States Patent (USP) 5,789,157,5,864,026,5,712,375,5,763,566,6,013,443,6,376,474,6,613,526,6,114,120,6,261,774 and 6, the fit technology of RNA of explanation all can be used in the content of the present invention in 387,620, and these documents all merge as a reference at this.
The physical property of antibody
Further being characterized as of antibody of the present invention be anti--the different physical propertys of CD70 antibody.Based on these physical propertys, can use different mensuration to detect and/or distinguish dissimilar antibody.
In some embodiments, antibody of the present invention may contain one or more glycosylation sites at light chain or variable region of heavy chain.Have one or more glycosylation sites in the variable region, can cause the immunogenicity of antibody to improve or the pK value of antibody changes, this is (people (1972) Annu Rev Biochem such as Marshall because the antigen combination changes 41: 673-702; Gala FA and Morrison SL (2004) J Immunol 172: 5489-94; People such as Wallick (1988) J Exp Med 168: 1099-109; Spiro RG (2002) Glycobiology 12: 43R-56R; People such as Parekh (1985) Nature 316: 452-7; People such as Mimura (2000) Mol Immunol 37: 697-706).Known glycosylation occurs in the motif that comprises the N-X-S/T sequence.Use Glycoblot mensuration can detect the glycosylation of variable region, this mensuration is sheared this antibody and is produced Fab, and then uses the mensuration of measuring periodate oxidation and Schiff's base formation to detect glycosylation.Alternately, use the photochromic spectrometry of Dionex (Dionex-LC) can test the glycosylation of variable region, in this mensuration the carbohydrate on the Fab is cut into monose, and analyze the content of every kind of sugar.In some cases, preferred use does not comprise the glycosylated antibody in variable region.No matter this can be by being chosen in the antibody that do not contain the glycosylation motif in the variable region or realizing by using standard technique well known in the art that residue in the glycosylation motif is suddenlyd change.
In a preferred embodiment, antibody of the present invention does not contain l-asparagine isometry site.Desamidation or different aspartic acid effect may take place respectively on N-G sequence or D-G sequence.Desamidation or different aspartic acid effect cause the generation of different asparagicacid residue, and it by generating winding arrangement, reduces the stability of antibody at the side chain carboxyl group end rather than on main chain.Use equal yield line to measure the generation that (iso-quant assay) can test different aspartic acid, this mensuration uses reversed-phase HPLC to detect different aspartic acid.
Every kind of antibody all has unique iso-electric point (pI), but the iso-electric point of antibody all drops in the scope between the pH6 to 9.5 usually.The pI of IgG1 antibody generally drops in the scope of pH7 to 9.5, and the pI of IgG4 antibody generally drops in the scope of pH6 to 8.Antibody may have the pI outside this scope.Though these effects also imperfectly understand, infer that pI may have some under the condition in vivo at the antibody outside the normal range and separate folding (unfolding) and unstable.Use capillary isoelectric focusing to measure and can test iso-electric point, this is measured and produces the pH gradient, and can use laser focusing to improve accuracy (people (2002) Electrophoresis such as Janini 23: 1605-11; People such as Ma (2001) Chromatographia 53: S75-89; People such as Hunt (1998) JChromatogr A 800: 355-67).In some cases, preferably have and comprise the pI value and drop on anti--CD70 antibody in the normal range.This can by select the pI value in normal range antibody or realize by using standard technique well known in the art that charged surface residue is suddenlyd change.
Every kind of antibody all has melting temperature (Tm), and it is index (the Krishnamurthy R and Manning MC (2002) the Curr Pharm Biotechnol of thermostability 3: 361-71).High more thermostability shows that antibody general stability in vivo is good more.Can use melting temperature (Tm) (people (2003) the Pharm Res such as Chen that measures antibody such as the technology of differential scanning calorimetry 20: 1952-60; People such as Ghirlando (1999) Immunol Lett 68: 47-52).T M1The folding temperature of expression antibody starting solution.T M2Expression antibody is separated folding temperature fully.Normally, the T of preferred antibody of the present invention M1Be higher than 60 ℃, preferably be higher than 65 ℃, even more preferably be higher than 70 ℃.Alternately, use the circular dichroism spectrometry can measure the thermostability of antibody (people (2002) J.Chromatogr Sci such as Murray 40: 343-9).
In preferred embodiments, select the antibody can not degrade fast.Fragmentation (the Alexander AJ and Hughes DE (1995) the Anal Chem of the anti--CD70 antibody that uses capillary electrophoresis known in the art (CE) and MALDI-MS to measure 67: 3626-32).
In another preferred embodiment, select antibody with the congregation of minimizing.Congregation may cause causing pharmacokinetic properties unwanted immune response and/or change or bad.Generally, the gathering of acceptable antibody is 25% or lower, preferred 20% or lower, even be more preferably 15% or lower, even be more preferably 10% or lower, and even more preferably be 5% or lower.Can measure to differentiate monomer, dimer, tripolymer or polymer gathering by several technology well known in the art, these technology comprise size exclusion post (SEC), high performance liquid chromatography (HPLC) and light scattering method.
The method of engineered antibody
As discussed above, by modifying V HAnd/or V kSequence, or one or more constant regions of its connection can be used to have V disclosed here HAnd V kAnti--CD70 the antibody of sequence is to produce new anti--CD70 antibody.Therefore, in another aspect of this invention, use of the present invention resisting-CD70 antibody, constitutional features as 2H5,10B4,8B5,18E7,69A7,69A7Y or 1F4, be used to produce structurally relevant anti--CD70 antibody, this antibody has kept at least a functional performance of antibody of the present invention, for example in conjunction with human CD70.For example, as discussed above, can be with one or more CDR district or their sudden change and known framework region and/or other CDR reorganization ground combination of 2H5,10B4,8B5,18E7,69A7,69A7Y or 1F4, to produce the of the present invention anti--CD70 antibody of other modified recombinant.The modification of other types comprises those modifications of explanation in the joint.The parent material that is used for remodeling method is the one or more V that provide at this HAnd/or V KSequence or their one or more CDR district.For producing the antibody of transforming, needn't make antibody (that is, being expressed as protein) practically, the V that provides at this is provided this antibody HAnd/or V kOne or more sequences in sequence or their the one or more CDR district.On the contrary, use the information that contains in this sequence, generate " s-generation " sequence of original series since then of deriving, and then prepare this " s-generation " sequence, and it is expressed as protein as parent material.
Therefore, in another embodiment, the invention provides a kind of method for preparing anti--CD70 antibody, comprising:
(a) provide: (i) variable fragments of heavy chain sequence, comprise and be selected from SEQ ID NO:13,14,15,16,17 and 18 CDR1 sequence, be selected from SEQ ID NO:19,20,21,22,23 and 24 CDR2 sequence, and/or be selected from SEQ ID NO:25,26,27,28,29,75 and 30 CDR3 sequence; And/or (ii) variable region of light chain antibody sequence, comprise and be selected from SEQID NO:31,32,33,34,35 and 36 CDR1 sequence, be selected from SEQ ID NO:37,38,39,40,41 and 42 CDR2 sequence, and/or be selected from SEQ ID NO:43,44,45,46,47 and 48 CDR3 sequence;
(b) change at least one amino-acid residue in variable fragments of heavy chain sequence and/or variable region of light chain antibody sequence is to produce at least a antibody sequence through changing; And
(c) antibody sequence through changing is expressed as a kind of protein.
Can use the Protocols in Molecular Biology of standard to prepare and express antibody sequence through changing.
Preferably, be the antibody that has kept a kind of, several or repertoire characteristic in anti--CD70 antibody of this explanation by the antibody of the coding of the antibody sequence through changing, these functional performances include but not limited to:
(a) with 1x10 -7M or littler K DIn conjunction with human CD70; And
(b) in conjunction with the renal cell carcinoma tumor cell line;
(c) in conjunction with lymphoma cell line, as B cell tumour clone;
(d) by the cell internalizing of expressing CD70;
(e) cell of expressing CD70 is shown the cytotoxicity (ADCC) that antibody relies on; And
(f) when suppressing to express the growth of the cell of CD70 mutually during coupling in vivo with cytotoxin.
Use this area those mensuration existing and/or that for example provide among the embodiment in the standard test of this explanation, (for example flow cytometry, in conjunction with measuring) can be assessed the functional performance through the antibody of change.
In some embodiment of the method through engineered antibody of the present invention, can introduce sudden change randomly or optionally along the total length or the part of anti--CD70 antibody coding sequence, and in conjunction with active and/or at other functional performances of this explanation the modified anti--CD70 antibody that obtains is screened.The method of sudden change has been described in the art.For example, the PCT open file WO02/092780 of Short has illustrated use saturation mutagenesis, synthetic being linked and packed, or their combination generates and screen the method for antibody mutation.Alternately, people's such as Lazar PCT open file WO 03/074679 has illustrated that the screening method that uses a computer is to optimize the physics-chem characteristic of antibody.
The nucleic acid molecule of code book invention antibody
Another aspect of the present invention relates to carries out nucleic acid molecules encoding to antibody of the present invention.Nucleic acid may reside in the intact cell, exist in the cell pyrolysis liquid or with partially purified form or pure basically form.Method with standard, comprise with alkalescence/SDS processing, CsCl and become band (CsClbanding), column chromatography, agarose gel electrophoresis and other methods well known in the art, when with nucleic acid from other cellular constituent or other pollutents (as other nucleus or protein) purifying when coming out, this nucleic acid obtains " separation " or " abundant pureization ".See F.Ausubel, wait the people to write (1987) Current Protocols in Molecular Biology, Greene Publishing and WileyInterscience, New York.Nucleic acid of the present invention can be, for example DNA or RNA, and can comprise or not comprise intron sequences.In preferred embodiments, nucleic acid is the cDNA molecule.
The Protocols in Molecular Biology of use standard can obtain nucleic acid of the present invention.For (for example passing through hybridoma, hybridoma from the following transgenic mice preparation of carrying human immunoglobulin gene that further specifies) antibody of expressing, pcr amplification by standard or cDNA clone technology can obtain the light chain of the antagonist made by hybridoma and the cDNA that heavy chain is encoded.For the antibody that from the immunoglobulin gene library (for example, the use display technique of bacteriophage) obtains, from this gene library, can reclaim the nucleic acid of this antibody-like of coding.
The preferred nucleic acid molecule of the present invention is those codings 2H5,10B4,8B5,18E7,69A7,69A7Y or the VH of 1F4 monoclonal antibody and the nucleic acid molecule of VL sequence.To 2H5,10B4,8B5,18E7,69A7,69A7Y, and the VH sequence of 1F4 is carried out the coded DNA sequence respectively shown in SEQ ID NO:49,50,51,52,53,74 and 54.The VL sequence of 2H5,10B4,8B5,18E7,69A7,69A7Y and 1F4 is carried out coded DNA sequence shown in SEQ ID NO:55,56,57,58,59,59 and 60 (69A7 and 69A7Y have the same DNA sequence of the coding VL sequence shown in SEQ ID NO:59) respectively.
In case obtained the dna fragmentation of coding VH and VL section, then can further operate these dna fragmentations by the recombinant DNA technology of standard, for example variable region gene is transformed into full length antibody chain gene, Fab fragment gene or scFv gene.In these operations, the dna fragmentation of coding VL or VH is connected to another dna fragmentation of the another kind of protein of coding (for example antibody constant region or flexibly connect thing) effectively.The term of Shi Yonging " connection effectively " is intended to illustrate so that connect this two dna fragmentations by two dna fragmentation amino acid sequence coded to meet the mode of reading the box form reservation in this article.
DNA by the VH that will encode is connected on the dna molecular of another encoding heavy chain constant region (CH1, CH2 and CH3) effectively, can be with coding V HThe separated DNA in district changes into the total length heavy chain gene.The sequence of human heavy chain constant region gene be well known in the art (referring to, as people (1991) Sequences of Proteins of Immunological Interest such as Kabat E.A., the 5th edition, U.S.Department of Health and Human Services, and contain these regional dna fragmentations and can obtain by the pcr amplification by standard NIH PublicationNo.91-3242).This CH can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is IgG1, IgG2, IgG3 or IgG4 constant region.For Fab fragment heavy chain gene, the DNA of coding VH can be connected to another effectively and only heavy chain CH1 constant region be carried out on the coded DNA molecule.
DNA by the VL that will encode is connected on the dna molecular of another coding constant region of light chain CL effectively, the separated DNA in coding VL district can be changed into full-length light chains gene (and Fab light chain gene).The sequence of human constant region of light chain gene known in the art (referring to, as people (1991) Sequences of Proteins of Immunological Interest such as Kabat E.A., the 5th edition, U.S., Department of Health and Human Services, and contain these regional dna fragmentations and can obtain by the pcr amplification by standard NIH PublicationNo.91-3242).In preferred embodiments, constant region of light chain can be κ or λ constant region.
Be to produce the scFv gene, the dna fragmentation of coding VH and VL is connected to the fragment that another coding flexibly connects thing effectively, as encoding amino acid sequence (Gly 4-Ser) 3Fragment, make VH and VL sequence can be expressed as adjacent single chain protein matter, wherein VH is connected by flexibly connecting thing with the VL district (for example, referring to people such as Bird (1988) Science 242: 423-426; People such as Huston (1988) Proc.Natl.Acad.Sci.USA 85: 5879-5883; And people such as McCafferty. (1990) Nature 348: 552-554).
The production of monoclonal antibody of the present invention
Can produce monoclonal antibody of the present invention (mAb) by multiple technologies, these technology comprise conventional monoclonal anti body method, for example Kohler and Milstein (1975) Nature 256: 495 standard body hybridoma technique.Though preferred body cell hybridization method can adopt other technology that are used for the manufacture order clonal antibody in principle, for example the viral conversion of bone-marrow-derived lymphocyte or carinogenicity transform.
The preferred animal system that is used to prepare hybridoma is the muroid system.Producing hybridoma in mouse is the method for fully establishing.The splenocyte that is used to separate through immunity is well known in the art with immunization protocol and the technology that merges.Fusion partner (as murine myeloma cell) and fusion program also are known.
Sequence based on the non-human monoclonal antibody of preparation as described above can prepare chimeric antibody of the present invention or humanized antibody.The Protocols in Molecular Biology of use standard can obtain heavy chain and light chain immunoglobulin (Ig) are carried out coded DNA from purpose non-human hybridoma, and transforms, to comprise non-mouse (for example human) immunoglobulin sequences.For example, be to generate chimeric antibody, use method as known in the art the muroid variable region can be connected with human constant region (referring to, as people's such as Cabilly United States Patent (USP) 4,816,567).In order to generate humanized antibody, use methods known in the art can with in the muroid CDR district insertion people class framework (referring to, as the United States Patent (USP) 5,225,539 of Winter, and people's such as Queen U.S. Patent number 5,530,101; 5,585,089; 5,693,762 and 6,180,370).
In a preferred embodiment, antibody of the present invention is the human monoclonal antibody.The transgenosis or the transchromosomic mice of groups of people's para-immunity system rather than mouse system carried in use, can produce direct this type of human monoclonal antibody at CD70.These transgenic mices or transchromosomic mice are included in this and are called HuMAb
Figure A20078005123100831
And KM
Figure A20078005123100832
Mouse, and they all are referred to as " human Ig mouse " at this.
HuMAb The human immunoglobulin gene minigene seat that human heavy chain (μ and γ) to non-rearrangement and κ light chain immunoglobulin sequences are encoded is contained in (Medarex company), and contain the target sudden change that makes endogenous μ and κ chain gene seat inactivation (referring to, as Lonberg, wait people (1994) Nature 368(6474): 856-859).Therefore, these mouse show mouse IgM or κ expression decreased; And in response to immunity, the human heavy and light chain transgenosis of institute inductive has experienced classification conversion and somatic mutation to produce IgG κ mono-clonal (Lonberg, people such as N. (1994), the supra of high-affinity; At Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113: summarize among the 49-101; Lonberg, N. and Huszar, D. (1995) Intern.Rev.Immunol. 13: 65-93 and Harding, F. and Lonberg, N. (1995) Ann.N.Y.Acad.Sci. 764: 536-546).HuMAb
Figure A20078005123100842
Preparation and use, and by the genomic modification that this class mouse is carried, be discussed in Taylor, people such as L. (1992) NucleicAcids Research 20: 6287-6295; Chen, people such as J. (1993) InternationalImmunology 5: 647-656; People such as Tuaillon (1993) Proc.Natl.Acad.Sci.USA 90: 3720-3724; People such as Choi (1993) Nature Genetics 4: 117-123; Chen, people such as J. (1993) EMBO J. 12: 821-830; People such as Tuaillon (1994) J.Immunol. 152: 2912-2920; Taylor, people such as L. (1994) International Immunology 6: 579-591; And Fishwild, people such as D. (1996) Nature Biotechnology 14: 845-851, this especially with they full text by reference as a reference.Further see the United States Patent (USP) 5,545,806 of Lonberg and Kay; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; And 5,770,429; People's such as Surani United States Patent (USP) 5,545,807; The PCT of Lonberg and Kay open WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962; And the open WO 01/14424 of people's such as Korman PCT.Can also use the transgenic mice that carries human lambda light chain gene, as those transgenic mices of explanation among the PCT publication number WO 00/26373 of Bruggemann.For example, carry human lambda light chain transgenic mice can with carry human heavy chain transgene (as HCo7) and optional mouse of also carrying human κ light chain transgenosis (as KCo5) hybridize with produce carry simultaneously human heavy chain transgene and the genetically modified mouse of light chain (referring to, as embodiment 1).
In another embodiment, use the mouse (for example, carrying the mouse of human heavy chain transgene and human light chain transfection chromosome) of the human immunoglobulin sequence that carries transgenosis and transfection chromosome can produce human antibodies of the present invention.This mouse is called " KM at this
Figure A20078005123100851
And in the open WO 02/43478 of people's such as Ishida PCT, detailed explanation is arranged.
Moreover the alternative transgenic animal system of expressing human immunoglobulin gene can obtain in this area, and can be used to produce of the present invention resisting-CD70 antibody.For example, can use the alternative transgenosis system that is called as Xenomouse (Abgenix company); This type of mouse is illustrated in, for example people's such as Kucherlapati U.S. Patent number 5,939,598; 6,075,181; 6,114,598; In 6,150,584 and 6,162,963.
In addition, the alternative trans-chromosome animal system of expressing human immunoglobulin gene can obtain in this area, and can be used to produce of the present invention resisting-CD70 antibody.For example, can use the mouse of carrying human heavy chain transfection chromosome and human light chain transfection chromosome when being called " TC mouse "; This mouse is illustrated in people Proc.Natl.Acad.Sci.USA such as Tomizuka 97: 722-727, in (2000).In addition, the ox of carrying human heavy and light chain transfection chromosome (people Nature Biotechnology such as Kuroiwa has been described in this area 20: 889-894 (2002) and PCT application WO 2002/092812), and can be used to produce of the present invention resisting-CD70 antibody.
The phage display method that use is used to screen the human immunoglobulin gene library also can prepare human monoclonal antibody of the present invention.This type of display technique of bacteriophage that is used for the separation of human antibody-like is to determine in this area.Referring to, people's such as Ladner United States Patent (USP) 5,223,409 for example; 5,403,484; And 5,571,698; People's such as Dower United States Patent (USP) 5,427,908 and 5,580,717; People's such as McCafferty United States Patent (USP) 5,969,108 and 6,172,197; People's such as Griffiths United States Patent (USP) 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.
Use the SCID mouse also can prepare human monoclonal antibody of the present invention, the human immunity cell is gone into the SCID mouse by reconstruct, can produce the human antibodies reaction when making immunity.This type of mouse is for example at people's such as Wilson United States Patent (USP) 5,476,996 and 5,698, explanation arranged in 767.
In another embodiment, use as the human Ig mouse of explanation in people's such as Buechler the United States Patent (USP) 6,794,132 and human the resisting-CD70 antibody of combined preparation of display technique of bacteriophage.Or rather, this method at first comprises by with one or more CD70 antigens human Ig mouse (for example the HuMab mouse or the KM mouse of above explanation) being carried out immunity, in these mouse, produce anti--CD70 antibody response, the nucleic acid that from the lymphocyte of these mouse, separates coding human antibodies chain subsequently, and these nucleic acid are imported in display carriers (for example phage) so that demonstration package body (displaypackage) library to be provided.Thus, each library member comprises the nucleic acid of coding human antibodies chain, and every kind of antibody chain is displayed by the demonstration package body.Then with this library of CD70 protein screening to isolate and CD70 specificity bonded library member.Then selected library member's nucleic acid is inserted fragment and separate by the method for standard and check order, to determine the light chain and the weight chain variable sequence of selected CD70 zygote.Recombinant DNA technology by standard can change into the variable region full length antibody chain, and these technology for example enter the expression vector that carries human heavy chain and constant region of light chain with variable region clone, makes V HThe district is connected to C effectively HDistrict, and V LThe district is connected to C effectively LThe district.
The immunization of human Ig mouse
When human Ig mouse is used to produce human antibodies of the present invention, goods or CD70 fusion rotein with the purified or enrichment of the clone, CD70 antigen and/or the recombinant C D70 that express CD70 can carry out immunity to this type of mouse, as Lonberg, people such as N. (1994) Nature 368(6474): 856-859; Fishwild, people such as D. (1996) Nature Biotechnology 1 4: 845-851; And PCT publication number WO 98/24884 and WO 01/14424 are illustrated.Preferably, these mouse were 6 to 16 ages in week when first infusion.For example, available CD70 antigenic purified or the reorganization prepared product (5 to 50 μ g) through intraperitoneal and/or the human Ig mouse of subcutaneous immunity.
The detailed method that is used to produce in conjunction with the total length human monoclonal antibody of CD70 has been described in following examples 1.Experience to the accumulation of multiple antigen shows that transgenic mice can be replied under the following conditions: the antigen that is used in the complete Freund's adjuvant carries out the initial immunity of intraperitoneal (IP), and the antigen in the full freund's adjuvant that toos many or too much for use carries out IP immunity (at the most totally 6 times) week about afterwards.Yet other adjuvants except that freund's adjuvant also find it is effective (for example RIBI adjuvant).In addition, find that full cell has the immunogenicity of height under the situation that does not have adjuvant.Get the plasma sample monitoring immunne response that blood obtains after can be in whole immunologic process with socket of the eye.Can screen blood plasma by ELISA (as described below), and the mouse with anti--CD70 human immunoglobulin of enough titres can be used for merging.For example, can put to death and remove spleen before 3 days, by intravenous injection antigen to the mouse booster immunization.Expection may need to carry out 2 to 3 times to each immunization and merge.Every kind of antigen generally carries out immunity to 6 to 24 mouse.Often use HCo7 and two strains of HCo12.The generation of HCo7 and HCo12 mouse species is illustrated in United States Patent (USP) 5,770 respectively, and 429 and the embodiment 2 of the open WO01/09187 of PCT.In addition, two transgenosiss of HCo7 and HCo12 can be cultivated into the single two kinds of genetically modified mouse of different human heavy chains (HCo7/HCo12) that have jointly.Alternative or other, as explanation in PCT open file WO 02/43478, can use KM
Figure A20078005123100871
Strain.
Produce the generation of human monoclonal antibody's of the present invention hybridoma
In order to generate the hybridoma that produces human monoclonal antibody of the present invention, can separate the splenocyte and/or the lymph-node cell of the mice immunized of hanging oneself, and it is merged to the immortalized cell line that is fit to, for example mouse myeloma cell line.Generation at antigen-specific antibodies can be screened the hybridoma that obtains.For example, merge mutually with can hang oneself the in the future single cell suspension of splenic lymphocyte of immune mouse and the P3X63-Ag8.653 nonsecreting type murine myeloma cell (ATCC, CRL 1580) of sixth quantity of 50%PEG.Alternately, use is based on the electric fusion method of electric field, use CytoPulse big chamber cytogamy electroporation apparatus (large chamber cell fusionelectroporator) (CytoPulse Sciences company, Glen Burnie Maryland), can merge the single cell suspension of the splenic lymphocyte of the mice immunized of hanging oneself.With about 2 * 10 5Cell cultures on flat-bottom microtiter plates, hatch in selective medium a week afterwards: this substratum contains 2 mercapto ethanol, 50 units/ml penicillin, 50mg/ml Streptomycin sulphate, 50mg/ml gentamicin and 1 * xanthoglobulin-aminopterin-induced syndrome-thymidine (HAT) substratum (Sigma of 20% embryo cloning serum (Clone Serum), 18% " 653 " conditioned medium, 5%origen (IGEN), 4mM L-glutaminate, 1mM Sodium.alpha.-ketopropionate, 5mM HEPES, 0.055mM; Merge and add HAT after 24 hours).After about 2 weeks, can be with cell cultures in substituting in the substratum of HAT with HT.These single holes are screened at human monoclonal IgM and IgG antibody with ELISA then.In case the expansion growth of hybridoma occurs, can after 10 to 14 days, observe substratum usually.Can the hybridoma of secretory antibody be inoculated, screening again, and if it IgG is still the positive, then can be by limiting dilution with this monoclonal antibody subclone at least 2 times.Can be used for characterizing so that in tissue culture medium (TCM), produce a small amount of antibody at the stable subclone of vitro culture then.
For the purifying human monoclonal antibody, can allow the hybridoma selected grow in two liters revolve and be used for the monoclonal antibody purifying in the bottle.Can filter and concentrate supernatant liquor, (Pharmacia, Piscataway N.J.) carry out affinity chromatography to use albumin A-agarose afterwards.Can check that by gel electrophoresis and high performance liquid chromatography eluted IgG is to guarantee purity.Damping fluid can be changed to PBS, and pass through OD 280The optical extinction coefficient of use 1.43 is determined its concentration.Monoclonal antibody can be divided into equal portions, and be stored under-80 ℃.
Produce the generation of the transfectoma of monoclonal antibody of the present invention
Use the dna technique of example reorganization as known in the art and the combination of gene transfection method, in the host cell transfectoma, also can produce antibody of the present invention (for example, Morrison, S. (1985) Science 229: 1202).
For example, for expressing antibodies or their antibody fragment, Protocols in Molecular Biology by standard (for example, the pcr amplification of the hybridoma that use is expressed purpose antibody or cDNA clone) can obtain the DNA of encoding part or full-length light chains and heavy chain, and DNA can be inserted in the expression vector, make gene be connected to effectively on the control sequence of transcribing and translating.Herein, term " connection effectively " is intended to explanation the antibody gene connection is entered carrier, so that the control sequence of transcribing in the carrier and translating plays the expectation function of transcribing and translating that it regulates antibody gene.Expression vector and expression control sequenc are selected so that make it compatible with employed expression host cell.Light chain of antibody gene and heavy chain of antibody gene can be inserted in the independent carrier, perhaps more typically, two genes all be inserted in the identical expression vector.Method by standard is inserted into antibody gene in the expression vector (for example, connect the complementary restriction site on antibody gene fragment and the carrier, perhaps if there is no carry out flush end during restriction site and connect).Can be by the following method with at the light chain of the antibody of this explanation and the full length antibody gene that variable region of heavy chain is used for producing any antibody isotype: light chain and variable region of heavy chain are inserted into the CH of the isotype of hope and the expression vector that constant region of light chain is encoded, make V HFragment is connected to effectively carries intravital C HFragment, and with V KFragment is connected to effectively carries intravital C LFragment.Additionally or alternately, the expression vector of reorganization can the coded signal peptide, and this signal peptide helps secretory antibody chain from host cell.The antibody chain gene clone can be entered carrier, make signal peptide to meet the N-terminal that the mode of reading frame is connected to the antibody chain gene.This signal peptide can be immunoglobulin (Ig) signal peptide or allogenic the signal peptide signal peptide of NIg (that is, from).
Recombinant expression vector of the present invention also is carried at the adjusting sequence of these antibody chain genetic expressions of control in the host cell except carrying the antibody chain gene.Term " adjusting sequence " is intended to comprise other expression controlling elementss (for example, polyadenylation signal) of promotor, enhanser and control antibody chain genetic transcription or translation.This type of regulating and controlling sequence has been described, for example, explanation in Goeddel (GeneExpression Technology.Methods in Enzymology 185, Academic Press, SanDiego, CA (1990)).Those of skill in the art will be understood that: the design of expression vector, comprise the selection of regulating and controlling sequence, and may depend on this type of factor, the selection of the transformed host cells of being ready to use in, desirable protein expression level or the like are for example arranged.Preferably be used for the regulating and controlling sequence that mammalian host cell expresses and be included in the viral element that mammalian cell instructs high-caliber protein expression, for example derived from promotor and/or the enhanser of cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (as adenovirus major late promoter (AdMLP)) and polyomavirus.Alternately, can use non-virus to regulate sequence, for example ubiquitin promoter or beta-globin promotor.Moreover regulatory element is made up of the sequence from different sources, for example comprises SR α promoter systems (Takebe, people such as Y. (1988) Mol.Cell.Biol. from the terminal repetition sequence of SV40 early promoter and I type human T-leukemia virus's length 8: 466-472).
Recombinant expression vector of the present invention also may carry extra sequence except carrying these antibody chain genes and regulating the sequence, for example regulates sequence (for example, replication origin) and the selectable marker gene that carrier duplicates in host cell.Optionally marker gene promotes wherein to have imported the selection (for example, referring to the United States Patent (USP) 4,399,216,4,634,665 and 5,179,017 that all belongs to people such as Axel) of the host cell of carrier.For example, general selectable marker gene is given the host cell that imports carrier, to the resistance such as the medicine of G418, Totomycin or methotrexate.Preferred selectable marker gene comprises Tetrahydrofolate dehydrogenase (DHFR) gene (being used for methotrexate selection/amplification in the dhfr-host cell) and neo gene (being used for G418 selects).
For the expression of light chain and heavy chain, enter in the host cell by will the encode expression vector transfection of these heavy chains and light chain of standard technique.The multi-form multiple technologies that are intended to the covering scope broadness of term " transfection ", these technology generally are used for foreign DNA is imported host cell protokaryon or eucaryon, for example electroporation, calcium phosphate precipitation, the transfection of DEAE-dextran and similar techniques.Though can all express antibody of the present invention at prokaryotic host cell or in eukaryotic host cell in theory, but expressing antibodies in eukaryotic cell, and it is most preferred most preferably expressing in mammalian host cell, because this eukaryotic cell, and mammalian cell particularly, it is suitably folding and have immunocompetent antibody more likely to assemble justacrine than prokaryotic cell prokaryocyte.It is reported that the prokaryotic expression of antibody gene is invalid (Boss, M.A. and Wood, C.R. (1985) Immunology Today for the generation of the high yield of active antibody 6: 12-13).
The mammalian host cell that preferably is used to express recombinant antibodies of the present invention comprises that Chinese hamster ovary cell (Chinese hamster ovary celI) (comprises dhfr -Chinese hamster ovary celI is illustrated in Urlaub and Chasin, (1980) Proc.Natl.Acad.Sci.USA 77: 4216-4220, use the selectable marker of DHFR, for example illustrated in R.J.Kaufman and P.A.Sharp (1982) J.Mol.Biol.159:601-621), NSO myeloma cell, COS cell and SP2 cell.Especially, for using NSO myeloma cell, another preferred expression system is the GS gene expression system, and this GS gene expression system is disclosed among WO 87/04462, WO 89/01036 and the EP 338,841.When the recombinant expression vector of encoding antibody gene imports in the mammalian host cell, can produce antibody by host cell being cultivated for some time, this time is enough to make antibody to obtain expressing in this host cell, perhaps more preferably, this antibody-secreting is entered in the substratum of host cell growth.The method of purifying protein of use standard can reclaim antibody from substratum.
The antigenic sign of antibodies
Can test combining of antibody of the present invention and CD70 by for example flow cytometry.In brief, the cell of CD70 is expressed in fresh collection from tissue culture flasks, and the preparation single cell suspension.With first antibody the cell suspension of expressing CD70 is carried out substantive dyeing, dye again after perhaps deciding with the PBS that contains 1% paraformaldehyde is liquid-solid.About 1,000,000 cells are resuspended among the PBS of the first antibody that contains 0.5%BSA and 50 to 200 μ g/ml, and hatched 30 minutes on ice.With cell with containing 0.1%BSA, 0.01%NaN 3The PBS washed twice, be resuspended in 100 μ l the FITC link coupled goat-Anti-Human's class IgG antibody with dilution in 1: 100 (Jackson ImmunoResearch, WestGrove, PA) in, and hatched again 30 minutes on ice.With cell washing twice, be resuspended in the lavation buffer solution of 0.5ml again, and (Becton-Dickinson, SanJose CA) go up analysis of fluorescence dyeing at the FACSCalibur cell counter.
Alternately, can test combining of antibody of the present invention and CD70 by standard ELISA.In brief, microtiter plate is wrapped quilt with the purified CD70 albumen of 0.25 μ g/ml among the PBS, seal with 5% bovine serum albumin among the PBS then.In each hole, add the antibody blood plasma of the dilution of CD70 immune mouse (for example from) of dilution, and under 37 ℃, hatched 1 to 2 hour.Wash this plate with the PBS/ tween, and then hatched 1 hour at 37 ℃ with second reagent that is coupled to alkaline phosphatase (for example, for human antibodies, goat-anti-IgG Fc specific polyclonal reagent).After the washing, (1mg/ml) develops to culture plate with the pNPP substrate, and analyzes for 405 to 650 times at OD.Preferably, the mouse of high titre is used for merging with forming.
ELISA as described above measures and also can be used for screening the hybridoma that the CD70 immunogen is shown positive reaction.To carrying out subclone with high affinity in conjunction with the hybridoma of CD70 and further characterizing.Can select a clone (its keeps the reactivity (passing through ELISA) of parental cell) to be used to make the cell bank of 5 to 10 bottles, it is stored in-140 ℃ and descend and be used for antibody purification from each hybridoma.
For purifying CD70 antibody, can allow the hybridoma selected grow in two liters revolve and be used for the monoclonal antibody purifying in the bottle.Supernatant liquor can be filtered and concentrate, (Pharmacia, Piscataway N.J.) carry out affinity chromatography to use albumin A-agarose afterwards.Can check that by gel electrophoresis and high performance liquid chromatography IgG through wash-out is to guarantee purity.Damping fluid can be changed to PBS, and determine its concentration by the optical extinction coefficient of OD280 use 1.43.Monoclonal antibody can be divided into equal portions, and be stored under-80 ℃.
For whether anti--CD70 monoclonal antibody of determining to have selected is bonded to unique epi-position, (Pierce, Rockford IL) can carry out biotinylation to every kind of antibody to use commercially available reagent.Use the competitiveness research of unlabelled monoclonal antibody and biotinylated monoclonal antibody, can use above explanation through the elisa plate of CD70 bag quilt and carry out.Use the combination of the mAb that streptavidin-alkaline phosphatase probe can the detection of biological elementization.Alternately,, can use, and analyze with Scatchard and to detect unlabelled competitive antibody through the radiolabeled antibody Journal of Sex Research that is at war with as further specifying in following examples.
In order to determine the isotype of purified antibody, can use the special reagent of the antibody of specific isotype is carried out isotype ELISA.For example, in order to measure human monoclonal antibody's isotype, can under 4 ℃, be spent the night by the hole of microtiter plate by the anti-human immunoglobulin bag with 1 μ g/ml.After with 1% BSA sealing, these culture plates can with 1 μ g/ml or still less tried monoclonal antibody or purified isotype contrast reacted one to two hour at ambient temperature.Subsequently these holes and IgG 1 or the specific alkaline phosphatase link coupled of human IgM-probe are reacted.As described above plate is developed and analyzes.
Can further test anti--CD70 IgG and the antigenic reactivity of CD70 by Western blotting.In brief, can prepare CD70 and it is carried out SDS-PAGE.Behind the electrophoresis antigen that separates is transferred on the nitrocellulose filter, the foetal calf serum with 10% seals, and surveys with monoclonal antibody to be tested.The combination of IgG can detect and (Sigma Chem. company, St.Louis Mo.) develops with BCIP/NBT substrate sheet with Anti-Human's class IgG alkaline phosphatase.
Also can determine the binding specificity of antibody of the present invention by monitoring antibody and combine (for example the passing through flow cytometer) of expressing the proteic cell of CD70.Can use proteic cell of natural expression CD70 or clone, for example 786-O, A498, ACHN, Caki-1 and/or Caki-2 cell (in embodiment 4 and 5, further specifying), perhaps can use the expression vector transfection of coding CD70 with clone (for example Chinese hamster ovary celI system), make CD70 on the surface of this cell, express.Protein through transfection can comprise label, and for example myc-label or his-label are preferably placed at the N-end, and it uses the antibody at label can obtain detecting.Antibody of the present invention and CD70 be proteic, and combine can be by hatching through cells transfected and antibody, detect institute's bonded antibody and determine.Can combining with antibody and label on transfection albumen as positive control.
Bispecific molecule
On the other hand, the invention is characterized in comprise of the present invention anti--CD70 antibody or its segmental bispecific molecule.Antibody of the present invention or its antigen-binding portion thereof can be connected by derivatize or with another functional molecular, for example with another peptide or protein (for example, the part of another kind of antibody or acceptor) is connected, to generate bispecific molecule in conjunction with at least two kinds of different binding sites or target molecule.In fact, antibody of the present invention can or be connected to other functional moleculars more than one by derivatize, is attached to more than two kinds the different binding sites and/or the polyspecific molecule of target molecule with generation; Also be intended to contain this type of polyspecific molecule at this used term " bispecific molecule ".In order to produce bispecific molecule of the present invention, antibody of the present invention (for example functionally can be connected with one or more other binding molecules (for example another kind of antibody, antibody fragment, peptide or in conjunction with stand-in), by chemical coupling, heredity fusion, non-covalent connection or other modes), obtain bispecific molecule thus.
Therefore, the present invention also comprises bispecific molecule, and this bispecific molecule comprises at least a first binding specificity of CD70 and at second binding specificity of the second target epi-position.In a specific embodiment of the present invention, the second target epi-position is the Fc acceptor, for example human Fc gamma RI (CD64) or human Fc α acceptor (CD89).Therefore, the bispecific molecule that the present invention includes can either be bonded to the effector cell's (for example monocyte, scavenger cell or polymorphonuclear cell (PMN)) who expresses Fc γ R or Fc α R, again can be in conjunction with the target cell of expressing CD70.These bispecific molecules will be expressed the cell-targeting of CD70 to the effector cell, and the receptor-mediated effector cell's of triggering Fc activity, for example, express the phagolysis of the cell of CD70, cytotoxicity (ADCC), the release of cytokine or the generation of super-oxide negative ion of antibody dependent cellular mediation.
In one embodiment of the invention (wherein this bispecific molecule is a polyspecific), this molecule is anti-except comprising-Fc binding specificity and anti--CD70 binding specificity, can further comprise the 3rd binding specificity.In one embodiment, the 3rd binding specificity is anti-enhancement factor (EF) part, for example, be bonded on the surface protein that relates to cytotoxic activity molecule also, strengthen immunne response thus at target cell." anti-enhancement factor part " can be antibody, function antibody fragment or be bonded to part on the given molecule (as antigen or acceptor), and cause thus for Fc acceptor or the antigenic enhanced effect in conjunction with determinant of target cell." anti-enhancement factor part " can be in conjunction with Fc acceptor or target cell antigen.Alternately, anti-enhancement factor part can binding entity, and this entity is different from first and second binding specificity bonded entity.For example, anti-enhancement factor part can in conjunction with cytotoxic T cell (as, through CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immunocytes, cause enhancing at the immunne response of target cell).
In one embodiment, because its binding specificity, bispecific molecule of the present invention comprises at least a antibody or its antibody fragment, comprises, for example Fab, Fab ', F (ab ') 2, Fv, Fd, dAb or strand Fv.Antibody can also be light chain or heavy chain dipolymer, or its any minimal segment, and as Fv or strand construction, as illustrated in people's such as Ladner United States Patent (USP) 4,946,778, its content is by specific reference and as a reference.
In one embodiment, provided by monoclonal antibody at the binding specificity of Fc γ acceptor, it is in conjunction with not blocked by human immunoglobulin G (IgG).Term " IgG acceptor " is meant any in eight γ-chain genes on karyomit(e) 1 as used herein.These genes encodings altogether 12 stride film or soluble acceptor isotype, these isotypes are divided into three kinds of Fc γ acceptor types: Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16).In a preferred embodiment, Fc γ acceptor is human high affinity Fc γ RI.This human Fc gamma RI is the molecule of 72kDa, and it has high affinity (10 to monomer I gG 8To 10 9M -1).
Some preferably anti--Fc γ MONOCLONAL ANTIBODIES SPECIFIC FOR and sign has been described in people's such as Fanger PCT open file WO 88/00052 and United States Patent (USP) 4,954,617, and the content of wherein teaching all is combined in this by reference.Therefore the site of the epi-position of these antibodies Fc γ I, Fc γ II or Fc γ III is different from the Fc γ binding site of acceptor, and their combination can not blocked by the IgG of physiological level basically.Useful in the present invention specificity is anti--and Fc γ RI antibody is mAb 22, mAb32, mAb44, mAb62 and mAb197.The hybridoma that produces mAb32 can be preserved the center available from U.S.'s typical case's culture, and the ATCC accession number is HB9469.In other embodiments, the anti-Fc γ receptor antibody humanization form (H22) that is monoclonal antibody 22.At people (1995) J.Immunol such as Graziano R.F. 155(10): production and sign that H22 antibody has been described among 4996-5002 and the PCT open file WO 94/10332.The clone that produces H22 antibody is named as HA022CL1, be preserved in American type culture collection, and accession number is CRL 11177.
In other other preferred embodiments, binding specificity to the Fc acceptor is to be provided by the antibody that is bonded to the human IgA acceptor, this human IgA acceptor for example is Fc-α acceptor (Fc α RI (CD89)), and this combination preferably can not blocked by human immunoglobulin A (IgA).Term " IgA acceptor " is intended to comprise the gene product (Fc α RI) that is positioned at a α gene on the karyomit(e) 19.The film isotype is striden in the variable shearing of known this genes encoding several 55 to 110kDa.Fc α RI (CD89) constitutive expression in monocyte/macrophage, eosinophilic granulocyte and neutrophilic granulocyte, but in non-effector cell group, do not express.Fc α RI all has medium avidity for IgA1 and IgA2, and (≈ 5 * 10 7M -1), be exposed to cytokine for example when G-CSF or GM-CSF this avidity can increase (Morton, people such as H.C. (1996) Critical Reviews inImmunology 16: 423-440).Four kinds of specific monoclonal antibodies of Fc α RI that are accredited as A3, A59, A62 and A77 are disclosed, they outside the IgA ligand binding domains in conjunction with Fc α RI (Monteiro, people such as R.C. (1992) J.Immunol. 148: 1764).
Fc α RI and Fc γ RI are the triggering acceptors that preferably is used for bispecific molecule of the present invention, because they: (1) mainly is expressed on the immune effector cell (for example monocyte, PMN, scavenger cell and dendritic cell); (2) high level expression (for example 5,000 to 100,000/ cells); (3) mediators of cellular cytoxicity activity (for example ADCC, phagolysis); And (4) mediated targeted their enhanced antigen presentation of antigen (comprising self antigen).
Though the human monoclonal antibody is preferred, other antibody that can adopt in bispecific molecule of the present invention are muroid, chimeric and Humanized monoclonal antibodies.
Use method as known in the art can prepare bispecific molecule of the present invention by coupling composition binding specificity (for example, the binding specificity of anti--FcR and anti--CD70).For example, every kind of binding specificity of bispecific molecule can generate separately, then with coupling each other.When binding specificity is protein or peptide, can use multiple coupling reagent or cross-linking reagent to carry out covalent coupling.The example of cross-linking reagent comprises albumin A, carbodiimide, N-succinimido-S-ethanoyl-thioacetate (SATA), 5; 5 '-two sulphur two (2-nitrobenzoic acids) (DTNB), neighbour-phenylene dimaleimide (oPDM), N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP) and 4-(N-maleimide ylmethyl) hexanaphthene-1-carboxylic acid sulfosuccinimide ester (sulfo group-SMCC) (referring to; for example, people (1984) J.Exp.Med. such as Karpovsky 160: 1686; Liu, people such as MA (1985) Proc.Natl.Acad.Sci.USA 82: 8648).Other method comprises that those are illustrated in Paulus (1985) Behring Ins.Mitt.No.78,118-132; People such as Brennan (1985) Science 229: 81-83); And people (1987) J.Immunol. such as Glennie 139: the method for 2367-2375.Preferred coupling reagent is SATA and sulfo group-SMCC, and the two all can (Rockford IL) buys from Pierce Chemical company.
When these binding specificities were antibody, they can pass through the sulfydryl key phase coupling of the C-terminal hinge area of two heavy chains.In particularly preferred embodiments, before coupling, hinge area is modified, made it contain the odd number sulfhydryl residue, preferably contain a sulfhydryl residue.
Alternately, two kinds of binding specificities all can obtain coding and obtain expression and assembling in same host cell in same vehicle.When bispecific molecule is mAb * mAb, mAb * Fab, Fab * F (ab ') 2Or during part * Fab fusion rotein, this method is particularly useful.Bispecific molecule of the present invention can be to comprise a kind of single-chain antibody and in conjunction with the single chain molecule of determinant, or comprises two strand bispecific molecules in conjunction with determinant.Bispecific molecule can contain at least two kinds of single chain molecules.The method for preparing bispecific molecule is disclosed in for example U.S. Patent number 5,260,203; U.S. Patent number 5,455,030; U.S. Patent number 4,881,175; U.S. Patent number 5,132,405; U.S. Patent number 5,091,513; U.S. Patent number 5,476,786; U.S. Patent number 5,013,653; U.S. Patent number 5,258,498; And U.S. Patent number 5,482,858, all these contents are combined in this by reference clearly.
Bispecific molecule is bonded to their specific target can be confirmed by following method, for example enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), facs analysis, biological assay (for example growth-inhibiting) or western blot analysis.During these are measured each all be usually by adopt to the purpose mixture special through labelled reagent (for example, antibody), detect the existence of specific purpose protein-antibody complex.For example, the enzyme len antibody or the antibody fragment that can use for example identification and specificity to be bonded to antibody-FcR mixture detects the FcR-antibody complex.Alternately, use any in multiple other immunoassay can detect these mixtures.For example, antibody can be by radio-labeling, and be used for radioimmunoassay (RIA) (referring to for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course onRadioligand Assay Techniques, The Endocrine Society, March, 1986, it is combined in this by reference).But by such as gamma counter or scintillometer or by autoradiographic means detection of radioactive isotropic substance.
Connector
The invention provides antibody-mating partner conjugate, wherein antibody is connected on the mating partner by chemical linkers.In certain embodiments, connector is the peptidyl connector, and is described as (L at this 4) p-F-(L 1) mOther connector comprises hydrazine connector and disulfide linkers, and is described as (L respectively at this 4) p-H-(L 1) mOr (L 4) p-J-(L 1) mExcept the connector that is connected in mating partner, the present invention also provides and has been applicable to and is connected in the connecting arm of shearing of any molecular species basically.Connecting arm of the present invention aspect at this by partly being illustrated with reference to the treatment that they connected.Yet clearly, these connectors can be connected in a plurality of kinds concerning those skilled in the art, including, but not limited to, diagnostic reagent, analytical reagent, biomolecules, targeting agent, detectable mark and analogue.
The operation instruction of peptidyl and other connectors is in U.S. Provisional Patent Application series number 60/295,196 in antibody-mating partner conjugate; 60/295,259; 60/295342; 60/304,908; 60/572,667; 60/661,174; 60/669,871; 60/720,499; 60/730,804; And 60/735,657, and U.S. Patent Application Serial 10/160,972; 10/161,234; 11/134,685; 11/134,826; And 11/398,854; And U.S. Patent number 6,989,452 and PCT number of patent application PCT/US2006/37793, its all the elements all are combined in this by reference.
Other connector is illustrated in U.S. Patent number 6,214,345 (Bristol-Myers Squibb), U.S. Patent application 2003/0096743 and U.S. Patent application 2003/0130189 (these two all belongs to Seattle Genetics), people such as de Groot, J.Med.Chem.42,5277 (1999); People J.Org.Chem.43 such as deGroot, 3093 (2000); People such as de Groot, J.Med.Chem.66,8815, (2001); WO 02/083180 (Syntarga); People such as Carl, J.Med.Chem.Lett.24,479, (1981); People such as Dubowchik, Bioorg ﹠amp; Med.Chem.Lett.8,3347 (1998); And 60/891,028 (submitting to) on February 21st, 2007.
On the one hand, the present invention relates to be used for the target group is connected in the connector of therapeutical agent and marker.On the other hand, the invention provides connector, these connectors are given compound stability, are reduced their toxicity in vivo or other pharmacokinetics, bioavailability and/or the pharmacodynamics that advantageously influences them.Common following situation is preferred: in this type of embodiment, in case with the site of action of drug conveying to it, connector is promptly sheared, and discharges this active medicine.Therefore, in one embodiment of the invention, connector of the present invention is traceless, in case make when connector is removed from therapeutical agent or marker (for example between pot-life), does not stay the vestige that connector exists.
In another embodiment of the invention, connector is characterised in that they are in the target cell site or close on the ability that target cell place (for example in the therapeutic action site or marker avtive spot) obtains shearing.This shearing can be enzymatic in essence.This characteristic helps to reduce system's activation, reduction toxicity and system's side effect of therapeutical agent or marker.The group of preferably can shearing that is used for the enzymatic shearing comprises peptide bond, ester connector and disulfide linkers.In other embodiment, these connectors are to the pH sensitivity, and obtain shearing by changing pH.
An important aspect of the invention is the ability of control linkage thing velocity of shear.The connector of THE ADIABATIC SHEAR IN often is desirable.Yet, in some embodiments, may preferably shear slower connector.For example, at sustained release preparation or have the snap-out release composition simultaneously and discharge at a slow speed in the preparation of composition, it may be useful that the connector of cutting than slow shear is provided.WO 02/096910 provides several special part-medicinal compositions with hydrazine connector.Yet, have no idea to come " adjusting " connector to form, and illustrated specific compound is sheared this part to be slower than many medicines-preferred speed of connector conjugate institute from this medicine according to needed cyclisation speed.By contrast, hydrazine connector of the present invention provides from very near very slow cyclisation velocity range, thereby allows to select specific hydrazine connector based on desirable cyclisation speed.
For example, use the hydrazine connector that when shearing, produces single 5 yuan of rings can realize very fast cyclic action.Be used for cytotoxin reagent targeted delivery to the preferred cyclisation speed of cell, can use the hydrazine connector of two 5 yuan rings when shearing, producing the comfortable connector that has two methyl together with the position or 6 yuan of rings and realize.Show, and compare at the single 6 yuan of rings that do not have these two methyl together with the position, should be together with-dimethyl (gem-dimetyl)Effect has been accelerated the speed of cyclization.This is because stress is released in ring.Yet may slow down speed of response rather than it is accelerated of substituting group sometimes.Usually the reason that postpones to take place can trace back to steric hindrance.For example, be CH together with carbon 2In time, compared, together with-dimethyl replaces makes cyclization take place sooner.
Yet, be important to note that in some embodiments, it can be preferred shearing slower connector.For example, at sustained release preparation or have the snap-out release composition simultaneously and discharge at a slow speed in the preparation of composition, it is useful that the slower connector of shearing is provided. In certain embodiments, use hydrazine Connector is realized cyclisation at a slow speed, in case cutting hydrazine connector, this gem-dimethyl not replaces with producing Single 6 yuan of rings or single 7 yuan of rings
These connectors also are used for stablizing therapeutical agent or marker in circulation, prevent its degraded.This characteristic provides important favorable factor, because this stabilization causes the circulating half-life of the reagent that connected or marker to prolong.Connector also can be used for weakening the reagent that connected or the activity of marker, make conjugate at circulation time relatively for optimum, and after desirable action site activates, have needed effect, for example have cytotoxicity.For the therapeutical agent conjugate, these characteristics of connector are used to improve the therapeutic index of reagent.
The preferred stabilization group of selecting, with Restriction is by possibilityBe present in enzyme in blood or the nonstandard target tissue to the removing and the metabolism of therapeutical agent or marker; And further select this stabilization group, enter in the cell with restriction reagent or marker transhipment.These stabilization groups are used to stop the degraded of reagent or marker, and also may play a role in other physical propertys of this reagent or marker are provided.The stabilization group also can improve to be configured or without the reagent of collocation form storage or the stability of marker.
Ideally; the stabilization group is useful for stablizing therapeutical agent or marker; condition is that this stabilization group role is by therapeutical agent or marker having been protected this therapeutical agent or marker avoid in human blood when 37 ℃ of storages were tested in 2 hours to degrade and having been caused under given condition determination by being present in enzyme in the human blood this therapeutical agent or marker being less than 20%; preferably be less than 10%, more preferably less than 5% and even more preferably less than 2% shearing.
The invention still further relates to the conjugate that contains these connectors.More specifically, the present invention relates to the use of prodrug, it can be used for treatment of diseases, especially for the chemotherapy of cancer.Exactly, use the connector in this explanation that multiple prodrug is provided, for the prodrug with analog structure, it shows the toxicity of high effect specificity, reduction and improved stability in blood.
Can be positioned at the different positions of mating partner molecule at the connector of the present invention of this explanation.
Therefore, provide connector, it can comprise any as the multiple group of its chain part, and with respect to the construct that lacks this type of group, these groups (for example, in blood flow) speed of shearing in vivo can strengthen.Also provide Connecting arm and therapeutical agent and diagnostic reagentConjugate.These connectors are useful for the prodrug analogue that forms therapeutical agent, and are useful to reversibly therapeutical agent or diagnostic reagent being connected to target medicament, detectable mark or solid phase carrier.Connector can be integrated into and comprise in the cytotoxic complex body.
Prodrug and the security advantages that may bring the other antibody coupling matter that surpasses conventional cytotoxic drugs being connected of antibody.At tumour cell and in several normal tissues (comprising blood plasma), all available esterase is finished the activation of prodrug.Though the activity level that has shown corresponding esterase among the mankind is than observed little in mouse, to observed very similar in rat and inhuman primate.The activation of prodrug also can be sheared by glucuronidase and be finished.
Except peptide, hydrazine or the disulphide group that can shear, one or more from disappearing (self-immolative) connector group L 1Can randomly be incorporated between cytotoxin and the targeting agent.These connector groups also can be illustrated as spacer groups, and comprise at least two reactive functional groups.Generally, on the chemical functional group of chemical functional group's bonding therapeutical agent (for example cytotoxin) of spacer groups, and other chemical functional groups of this spacer groups are used for the bonding targeting agent or the chemical functional group of the connector that can shear.The chemical functional group's of spacer groups example comprises hydroxyl, sulfydryl, carbonyl, carboxyl, amino, ketone group and mercapto groups.
From the connector that disappears, by L 1Representative generally is replacement or unsubstituted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl or replacement or unsubstituted assorted alkyl.In one embodiment, this alkyl or aryl group can comprise 1 to 20 carbon atom.They also may comprise polyalkylene glycol moiety.
Exemplary spacer groups comprises, for example 6-amino-hexanol, 6-sulfydryl hexanol, 10-hydroxydecanoic acid, glycine and other amino acid, 1,6-hexylene glycol, Beta-alanine, 2-monoethanolamine, 2-benzofuranone, carbonyl group, animal ester, nucleic acid, peptide and the analogue of cysteamine (2-aminoothyl mercaptan), 5-aminovaleric acid, 6-aminocaprolc acid, 3-maleimide yl benzoic acid, 2-benzofuranone, alpha-substitution.
Spacer can be used to guide other molecular mass and chemical functional group to enter this cytotoxin-targeting agent mixture.Usually, other quality and functional group serum half-life and other characteristics that will influence this mixture.Therefore, by careful selection spacer groups, can produce serum half-life cytotoxin mixture within the specific limits.
The spacer that is positioned at the position of drug moiety direct neighbor also is expressed as (L 1) m, wherein m is selected from 0,1,2,3,4,5 and 6 integer.As a plurality of L 1When spacer exists, can use same or different spacer.L 1May be any from disappearing group.
L 4Be the connector part, utilize the connector that comprises this part or change the hydrolysis rate of conjugate, it preferably gives the solvability of conjugate increase or the aggregation properties that reduces.L 4Connector is also nonessential from disappearing.In one embodiment, L 4Part is the assorted alkyl or the unsubstituted assorted alkyl of the alkyl that replaces, unsubstituted alkyl, the aryl of replacement, unsubstituted aryl, replacement, and any of these groups may be straight chain, side chain or cyclic.Substituting group may be for example rudimentary (C 1-C 6) alkyl, alkoxyl group, alkylthio, alkylamino or dialkyl amido.In certain embodiments, L 4Comprise the non-annularity part.In another embodiment, L 4The aminoacid polymers that comprises any positively charged or negative electricity, for example polylysine or poly arginine.L 4Can comprise polymkeric substance such as polyalkylene glycol moiety.In addition, L 4Connector for example can comprise polymer composition and small molecules chemical part both.
In a preferred embodiment, L 4Comprise polyoxyethylene glycol (PEG) part.L 4Peg moiety length can be 1 to 50 unit.Preferably, PEG has 1 to 12 repeating unit, more preferably 3 to 12 repeating units, more preferably 2 to 6 repeating units, or even more preferably 3 to 5 repeating units, and 4 repeating units most preferably.L 4Can only be made up of peg moiety, perhaps it also can comprise other replacements or unsubstituted alkyl or assorted alkyl.With PEG as L 4The part of part makes up the solubleness that can be used to improve mixture.In addition, peg moiety has reduced contingent gathering when medicine and antibody coupling.
In some embodiments, L 4Comprise and be directly connected in (AA 1) cN-terminal
Figure A20078005123101011
R 20Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group.R 25, R 25', R 26And R 26' all be independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl separately; And s and t are from 1 to 6 integer independently.Preferably, R 20, R 25, R 25', R 26And R 26' be hydrophobic.In some embodiments, R 20Be H or alkyl (preferred unsubstituted low alkyl group).In some embodiments, R 25, R 25', R 26And R 26' be H or alkyl (preferred unsubstituted C independently 1-C 4Alkyl).In some embodiments, R 25, R 25', R 26And R 26' be H.In some embodiments, t be 1 and s be 1 or 2.
Peptide connector (F)
As discussed above, peptidyl connector of the present invention can be used general formula (L 4) p-F-(L 1) mExpression, wherein F represents to comprise the connector part of peptide base section.In one embodiment, F partly comprises the optional other connector that disappears certainly, L 2And carbonyl group.In another embodiment, F partly comprises amino and the L of optional spacer group 3
Therefore, in one embodiment, the conjugate that comprises the peptidyl connector contains the structure of following formula (a):
Figure A20078005123101021
In this embodiment, L 1Be the connector that disappears certainly as described above, and L 4Be the aggregation properties of preferably giving enhanced solubleness or minimizing as described above or the part that changes hydrolysis rate.L 2Expression is from disappearing connector.In addition, m is 0,1,2,3,4,5 or 6; And o and p are 0 or 1 independently.AA 1Represent one or more natural amino acids and/or non-natural a-amino acid; C is from 1 to 20 integer.In some embodiments, c in 2 to 5 scope or c be 2 or 3.
In the peptide connector with following formula (a) of the present invention, AA 1Direct and the L at its N-terminal 4Connect or, work as L 4Direct and X when not existing 4Group (being targeting agent, detectable mark, shielded reactive functionality or not protected reactive functionality) connects.In some embodiments, work as L 4When existing, L 4Do not contain and be directly connected in (AA 1) cThe carboxyl carboxyl groups of N-terminal.Therefore, in these embodiments, needn't exist directly at L 4Or X 4With AA 1Between carboxyl acyl group unit, and, then be essential in 345 the peptide connector at U.S. Patent number 6,214.
In another embodiment, the conjugate that contains the peptidyl connector comprises the structure of following formula (b):
Figure A20078005123101031
In this embodiment, L 4Be the solubleness of preferably giving increase or the aggregation properties of minimizing or the part of change hydrolysis rate as described above; L 3Be spacer groups, comprise primary amine, secondary amine or carboxyl functional group, and L 3Amine and the side carboxyl functional group of D form amido linkage or L 3Carboxyl and the pendant amine groups functional group of D form amido linkage; And o and p are 0 or 1 independently.AA 1Represent one or more natural amino acid, and/or the non-natural a-amino acid; C is from 1 to 20 integer.In this embodiment, there is not L 1(being that m is 0 in this general formula).
In the peptide connector with following formula (b) of the present invention, AA 1Direct and the L at its N-terminal 4Connect or, work as L 4When not existing, direct and X 4Group (being targeting agent, detectable mark, shielded reactive functionality or not protected reactive functionality) connects.In some embodiments, work as L 4When existing, L 4Do not contain and be directly connected in to (AA 1) cThe carboxyl carboxyl groups of N-terminal.Therefore, in these embodiments, needn't exist directly at L 4Or X 4With AA 1Between carboxyl acyl group unit, and it is at United States Patent (USP) 6,214, is essential in 345 the peptide connector.
From the connector L that disappears 2
From the connector L that disappears 2Be the difunctionality chemical part, it can be with the normally stable three joint molecules (tripartate molecule) of the covalently bound one-tenth of the chemical part at two intervals, shear from this three joints molecule by enzyme to discharge the chemical part at described interval one; And after described enzyme is sheared, spontaneous shearing from the rest part of this molecule and discharge in the chemical part at described interval another.According to the present invention, covalently bound from the spacer that disappears at one end and peptide moiety, and it is covalently bound in the chemical reaction site of its other end and drug moiety, the derivatize of this drug moiety has suppressed pharmacological activity, so that at interval with peptide moiety and the covalently bound one-tenth three joint molecules of drug moiety, this molecule is stable and parmacodynamics-less activity under the non-existent situation of target enzyme, but this molecule can obtain enzymatic by this type of target enzyme on the key of covalently bound spacer part and peptide moiety shears, and peptide moiety is discharged from this three joints molecule.Conversely, this enzymatic is sheared the self-eliminating matter that will activate the spacer part, and causes the spontaneous fracture of covalently bound spacer part and the key of drug moiety, and influencing thus on the pharmacology is the release of the medicine of activity form.
From the connector L that disappears 2Any from disappearing group.Preferred L 2Be the alkyl that replaces, unsubstituted alkyl, the assorted alkyl of replacement, unsubstituted assorted alkyl, unsubstituted Heterocyclylalkyl, replacement Heterocyclylalkyl, replacement and unsubstituted aryl, and that replace and unsubstituted heteroaryl.
One particularly preferred from disappearing spacer L 2Can represent by chemical formula (c):
Figure A20078005123101041
The aromatic ring of aminobenzyl group can be replaced by one or more " K " group." K " group is the replacement on this aromatic ring Base, it has replaced the hydrogen on one of four unsubstituted carbon atoms being connected originally to a ring structure part." K " group is single atom, halogen atom for example, or polyatom group, for example alkyl, assorted alkyl, amino, nitro, hydroxyl, alkoxyl group, alkylhalide group and cyano group.Each K all is independently selected from: the assorted alkyl of the alkyl of replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, the Heterocyclylalkyl of replacement, unsubstituted Heterocyclylalkyl, halogen, NO 2, NR 21R 22, NR 21COR 22, OCONR 21R 22, OCOR 21And OR 21, R wherein 21And R 22Be independently selected from: the Heterocyclylalkyl and the unsubstituted Heterocyclylalkyl of the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, replacement.Exemplary K substituting group includes but not limited to F, Cl, Br, I, NO 2, OH, OCH 3, NHCOCH 3, N (CH 3) 2, NHCOCF 3And methyl.For " K i", i is an integer in 0,1,2,3 or 4.In a preferred embodiment, i is 0.
The ether oxygen atom of above display structure links to each other with carbonyl.From NR 24Functional group enters the line of aromatic ring and represents that amine function group can be bonded to any one of 5 carbon, and these 5 carbon all form ring and not by-CH 2-O-group replaces.Preferably, the NR of X 24Functional group is with respect to-CH 2The contraposition of-O-group and aromatic ring covalent attachment.R 24Be selected from: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement and unsubstituted assorted alkyl.In a specific embodiment, R 24Be H.
In one embodiment, the invention provides the peptide connector of following formula (a), wherein F comprises following structure:
Figure A20078005123101051
Wherein, R 24Be selected from: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement and unsubstituted assorted alkyl.Each K all is the members that are independently selected from down group: the assorted alkyl of the alkyl of replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, the Heterocyclylalkyl of replacement, unsubstituted Heterocyclylalkyl, halogen, NO 2, NR 21R 22, NR 21COR 22, OCONR 21R 22, OCOR 21And OR 21, R wherein 21 And R 22 Be independently selected from: the alkyl of H, replacement, unsubstituted alkyl, replacement assorted The heteroaryl of alkyl, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, replacement, not The heteroaryl that replaces, the Heterocyclylalkyl and the unsubstituted Heterocyclylalkyl of replacement, and i is an integer 0,1,2,3 or 4
In another embodiment, the peptide connector with following formula (a) comprises-F-(L 1) m-, it contains following structure:
Figure A20078005123101052
Wherein, each R 24All are the members that are selected from down group: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement and unsubstituted assorted alkyl.
In some embodiments, the at interval L that disappears certainly 1Or L 2Comprise:
Figure A20078005123101061
R wherein 17, R 18, and R 19All be independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl separately, and w is from 0 to 4 integer.In some embodiments, R 17And R 18Be H or alkyl (preferred unsubstituted C1-4 alkyl) independently.Preferably, R 17And R 18Be C 1-C 4Alkyl is as methyl or ethyl.In some embodiments, w is 0.Although do not wish by any specific theory, there has been experiment to find, this is specific disappears certainly that to be partitioned into ring speed very fast relatively.
In some embodiments, L 1Or L 2Comprise:
Figure A20078005123101062
Spacer groups L 3
Spacer groups L 3Be characterised in that it comprises primary amine or secondary amine or carboxyl functional group, and L 3The side carboxyl functional group of the amine of group and D forms amido linkage, perhaps L 3Carboxyl and the side amine functional group of D form amido linkage.L 3Can be selected from: replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl or replacement or unsubstituted Heterocyclylalkyl.In a preferred embodiment, L 3Comprise aromatic group.More preferably, L 3Comprise phenylformic acid group, aniline group or indolyl radical.As-L 3The limiting examples of-NH-structure at interval comprises following structure:
Wherein Z is selected from O, S and NR 23The member, and R wherein 23Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group.
Cutting the present invention comprises L 3Connector the time, L 3Part still is connected on the medicine D.Therefore, select L 3Part makes existence that it is connected to D can not influence the activity of D significantly.In another embodiment, the part of medicine D itself plays L 3Function at interval.For example, in one embodiment, medicine D is the derivative of many card Mi Xing (duocarmycin), and the part of its Chinese traditional medicine plays L 3Effect at interval.The limiting examples of this type of embodiment comprises those examples, wherein NH 2-(L 3)-D has and is selected from following structure:
Figure A20078005123101081
And
Figure A20078005123101082
Wherein, Z is selected from O, S and NR 23The member, R wherein 23Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group; And each structural NH wherein 2Group and (AA 1) cReact formation-(AA 1) c-NH-.
Peptide sequence AA 1
Group AA 1Expression single amino acids or a plurality of amino acid that connect together by amido linkage.These amino acid may be natural amino acid and/or non-natural a-amino acid.
Peptide sequence (AA 1) cOn function single amino acids (during c=1) or a plurality of amino acid whose amidation residue that links together by amido linkage.Select peptide of the present invention to be used for instructing at destination locations the enzyme catalysis cutting of the peptide that enzyme carries out in biosystem.For example, for use targeting agent by target to cell but not by the conjugate of this cell internalizing, select peptide, by one or more proteolytic enzyme (for example because the intracellular matter that near amort cell discharges) that may reside in the extracellular matrix this peptide is sheared, made that this peptide obtains in the extracellular shearing.Amino acid whose number can change in from 1 to 20 scope in peptide; But more preferably (AA 1) cComprise 1 to 8 amino acid, 1 to 6 amino acid or 1,2,3 or 4 amino acid.Known in this area by the peptide sequence of specific enzyme or enzyme cutting easily.
Many sequences by the peptide of cutting such as the enzyme in serum, liver, the intestines etc. are known in this area.Exemplary peptides sequence of the present invention comprises the peptide sequence that is cut by proteolytic enzyme.Below be clarity about the center of the discussion of the purposes of the responsive sequence of proteolytic enzyme in order to set forth, be not limited to scope of the present invention.
When the enzyme of cutting peptide was proteolytic enzyme, connector generally comprised the peptide that comprises proteolytic enzyme cutting recognition sequence.The cutting recognition sequence of proteolytic enzyme is the specific aminoacid sequence of being discerned by proteolytic enzyme in the proteolysis cutting process.Many proteolytic enzyme cuttings site is well known in the art, and these and other cleavage site can be included in the connector part.See, for example people Science 247:954 (1990) such as Matayoshi; People Meth.Enzymol.241:254 (1994) such as Dunn; People Meth.Enzymol.244:175 (1994) such as Seidah; Thornberry, Meth.Enzymol.244:615 (1994); People Meth.Enzymol.244:595 (1994) such as Weber; People Meth.Enzymol.244:412 (1994) such as Smith; People Meth.Enzymol.248:614 (1995) such as Bouvier, people such as Hardy, in Amyloid Protein Precursor in Development, Aging, and Alzheimer ' s Disease, people pp.190-198 (1994) such as ed.Masters.
Based on peptide sequence (AA 1) cAmino acid for the suitability that carries out selective enzyme restriction by specific molecular (for example tumor correlated albumen enzyme), to peptide sequence (AA1) cAmino acid select.Used amino acid can be natural or alpha-non-natural amino acid.They may be L or D configuration.In one embodiment, at least three kinds of different amino acid have been used.In another embodiment, two seed amino acids have only been used.
In a preferred embodiment, select peptide sequence (AA 1) cBe based on the ability that it is sheared by the lysosomal protein enzyme, the limiting examples of these proteolytic enzyme comprises cathepsin B, C, D, H, L and S.Preferably, this peptide sequence (AA 1) cCan be organized proteolytic enzyme B cutting external, this can use external proteolytic enzyme cutting assay method known in the art to test.
In another embodiment, select peptide sequence (AA 1) cBe based on the ability that it is sheared by the tumor correlated albumen enzyme, near this proteolytic enzyme proteolytic enzyme that for example extracellular is found tumour cell, its limiting examples comprises phorate oligopeptidase (TOP) and CD10.Toplink can be tested with external proteolytic enzyme cutting assay method known in the art by the ability of TOP or CD10 cutting.
Suit in conjugate of the present invention, to use suitable but nonrestrictive peptide sequence example comprises Val-Cit, Cit-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9-tosyl group-Arg, Phe-N 9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO:77), β-Ala-Leu-Ala-Leu (SEQ ID NO:78), Gly-Phe-Leu-Gly (SEQ.ID NO:79), Val-Ala, Leu-Leu-Gly-Leu (SEQ ID NO:91), Leu-Asn-Ala and Lys-Leu-Val.Preferred peptide sequence is Val-Cit and Val-Lys.
In another embodiment, the amino acid that is positioned at the drug moiety proximal most position is selected from: Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.In another embodiment, the amino acid that is positioned at the drug moiety proximal most position is selected from: Ala, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.
Proteolytic enzyme is relevant with cancer metastasis.It is relevant that proteolytic enzyme urokinase synthetic is increased in many cancers the increase with transfer ability.Urokinase activates Profibrinolysin and is plasmin, and Profibrinolysin extensively exists in ECS, and its activation causes the proteolytic degradation in the extracellular matrix, metastatic cancer cell intrusion thus.Plasmin also can activate collagenase, therefore promotes to allow tumour cell to invade (Dano waits people Adv.Cancer.Res., 44:139 (1985)) in the target tissue thus in the degraded of collagen in kapillary and lymphoid basilar membrane.Therefore, use the peptide sequence sheared by urokinase also within the scope of the invention as connector.
The present invention also provides the purposes to the responsive peptide sequence of tryptase cutting.Human mast cell-expressed at least four kinds of different tryptases are known as α, β I, β II and β III.These enzymes are not controlled by the plasma proteins enzyme inhibitors, and only at some physiology substrates of external cutting.The tryptase family of serine protease is relevant with the multiple anaphylactic disease and the inflammatory diseases that relate to mastocyte, raises because found the tryptase level in the patient's who suffers from these illnesss biological liquid.Yet, still need tryptase the definite effect in the physiopathology of disease to describe.The scope of the biological function of tryptase and corresponding physiology result are limited by their substrate specificity basically.
Tryptase is effective activator of preceding-urokinase plasminogen activator (uPA), and uPA is and the metastases and the zymogen forms of invading the proteins associated enzyme.The activation of Profibrinolysin cascade reaction (cause the destruction of extracellular matrix and produce cell exosmose and move) can be before the P4-P1 of Pro-Arg-Phe-Lys sequence (SEQ ID NO:80) goes up tryptase and activates-function of urokinase plasminogen activator (people Journal of BiologicalChemistry 269 (13) such as Stack: 9416-9419 (1994)).Vasoactive intestinal peptide (a kind of neuropeptide, relevant with the regulation and control of the saturating property of blood vessel) also cut by tryptase, mainly in Thr-Arg-Leu-Arg (SEQ.ID NO:81) sequence place cutting (Tam waits the people, Am.J.Respir.Cell Mol.Biol.3:27-32 (1990)).G-protein linked receptor PAR-2 can be sheared by tryptase on Ser-Lys-Gly-Arg (SEQ.IDNO:82) sequence and be activated, to promote fibroblasts proliferation, and thrombin activation acceptor PRA-1 on Pro-Asn-Asp-Lys (SEQ.ID NO:83) sequence by tryptase inactivation (people such as Molino, Journal of Biological Chemistry 272 (7): 4043-4049 (1997)).Combine, this evidence prompting proteinoid kinases plays the role of a nucleus in the tissue remodeling that disease causes.This is consistent with observed deep variation in the illness of several mast cell mediated.The characteristics of chronic asthma and other long-term respiratory tract diseases are fibrosiss of covering weave (underlying tissue) down and thicken that this may be the result that tryptase activates its physiology target.Similarly, a series of reports shown mastocyte density in vasculogenesis and the multiple cancer, tryptase activity and bad prognosis relevant (people such as Coussens, Genesand Development 13 (11): 1382-97 (1999)); People such as Takanami, Cancer 88 (12): 2686-92 (2000); People such as Toth-Jakatics, Human Pathology 31 (8): 955-960 (2000); People such as Ribatti, International Journal of Cancer 85 (2): 171-5 (2000)).
The method whether specific proteolytic enzyme of evaluation known in the art cuts the peptide sequence of having selected.For example, using 7-amino-4-methylcoumarin (AMC) fluorescence peptide substrates is the method (Zimmerman, people such as M., (1977) Analytical Biochemistry78:47-51) of determining the specific fine establishment of proteolytic enzyme.The specificity cutting of N-anilide key (anilide bond) discharges fluorescence AMC leavings group, allows the cutting speed of each substrate is simply measured.Recently, take a sample by substrate in single experiment, can adopt array people (1999) Bioorganic and Medicinal Chemistry Letters 9:1667-72 such as () Lee D. in AMC peptide substrates library and scanning (positional-scanning) library, position people (1997) Chemistryand Biology 4:149-55 such as () Rano T.A. to describe the specificity of the N-terminal of proteolytic enzyme fast wide region.Therefore, those of skill in the art can easily evaluate the array of peptide sequence, with definite their effectiveness in the present invention, and need not take the over-drastic experiment.
Antibody of the present invention-mating partner conjugate can randomly comprise two or more connectors.These connectors can be identical or different.For example, the peptidyl connector can be used for medicine is connected on the part, and the second peptidyl connector can be connected to diagnostic reagent on the complex body.Other purposes of other connector comprise analytical reagent, biomolecules, targeting agent and detectable label are connected to antibody-mating partner complex body.
Same is compound of the present invention within the scope of the invention, and they are polyvalent kinds, comprise, for example, such as dimer, tripolymer, the tetramer or the higher homologue of The compounds of this invention or its reactive analogue.The polyvalent kind can be assembled by single kind of the present invention or more than a kind.For example, the dimer construct can be " all dimers " or " heterodimer ".In addition, the polyvalent construct, compound wherein of the present invention or its reaction analogue is connected to that (for example polylysine, dextran, hydroxyethylamyle and analogue) also is within the scope of the invention on oligomerization or the poly framework, this framework preferably polyfunctional (that is, having a collection of reactive site that is used to connect The compounds of this invention).In addition, this framework can be derived with single kind of the present invention or kind more than one of the present invention.
In addition, the present invention includes multiple compound, these compounds functionalised the compound that has the water solubility that has strengthened with generation for the similar compound that does not functionalised similarly.Therefore, any substituting group in the substituting group given herein can similarly be had the water miscible group that has strengthened and replaced.For example, replace oh group with glycol or the amine that has quaternary ammonium, oxyamine or similarly have more water-soluble portion, this also within the scope of the invention.In a preferred embodiment, by replacing, given additional water solubility with active optional site of the part that strengthens the parent compound water solubility to the ionic channel of compound given herein.The method that strengthens the organic compound water solubility is well known in the art.These class methods include, but are not limited to, with permanent charged part, for example quaternary ammonium or under relevant pH condition on the physiology charged group (for example carboxylic acid, amine) make organic core functionalized.Additive method comprises to organic core and adds the group comprise hydroxyl or amine, for example alcohol, polyvalent alcohol, polyethers, or the like.Representational example is including, but not limited to, polylysine, polymine, poly-(ethylene glycol) and poly-(propylene glycol).The suitable functionalized chemistry and the scheme of these compounds is well known in the art.See that for example, Dunn, people such as R.L. write Polymeric Drugs and Drug Delivery Systems, ACS Symposium Series Vol.469, American Chemical Society, Washington, D.C.1991.
Hydrazine connector (H)
In second embodiment, conjugate of the present invention comprises hydrazine from disappearing connector, and wherein this conjugate has following structure:
X 4——(L 4) p-H-(L 1) m-D
Wherein, D, L 1, L 4, and X 4As above limits, and further specify herein, and H is the connector that comprises following structure:
Figure A20078005123101131
N wherein 1It is from 1 to 10 integer; n 2Be 0,1 or 2; Each R 24All are the members that are independently selected from down group: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement and unsubstituted assorted alkyl; And I or key (that is, the carbon atom on the main chain and close on key between the nitrogen-atoms) or following structure:
Figure A20078005123101141
Wherein, n 3Be 0 or 1, condition is to work as n 3Be 0 o'clock, n 2Not 0; And n 4Be 1,2 or 3, wherein working as I is a key, n 1Be 3, and n 2Be 1 o'clock, D can not be:
Figure A20078005123101142
Wherein R is Me or CH 2-CH 2-NMe 2
In one embodiment, the replacement on the phenyl ring is a para-orientation.In preferred embodiments, n 1Be 2,3 or 4 or n 1Be 3.In preferred embodiments, n 2Be 1.In preferred embodiments, I is a key (that is, the carbon atom on the main chain and close on key between the nitrogen-atoms).On the one hand, hydrazine connector H can form 6 yuan the connector that disappears certainly when shearing, for example work as n 3Be 0 and n 4It is 2 o'clock.On the other hand, hydrazine connector H can form two 5 yuan the connector that disappears certainly when shearing.In other respects, during shearing, H forms the connector that disappears certainly that disappear certainly connector or H that 5 yuan the connector that disappears certainly, H form 7 yuan form 5 yuan disappear certainly connector and 6 yuan.The influence of the ring size that forms when shearing rate is sheared.Therefore, according to desirable shearing rate, the suitable big circlet that forms in the time of can selecting to shear.
Five yuan hydrazine connector
In one embodiment, this hydrazine connector comprises 5 yuan hydrazine connector, and wherein H comprises following structure:
Figure A20078005123101151
In a preferred embodiment, n 1Be 2,3 or 4.In another preferred embodiment, n 1Be 3.
In above structure, each R 24Be independently to be selected from: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement and the member of unsubstituted assorted alkyl.In one embodiment, each R 24All be H or C independently 1-C 6Alkyl.In another embodiment, each R 24All be H or C independently 1-C 3Alkyl, more preferably H or CH 3In another embodiment, at least one R 24Be methyl group.In another embodiment, each R 24All are H.To each R 24Select with the steric effect of adjusting compound and be used to change solubleness.
5 yuan hydrazine connector can experience once or more cyclizations, medicine and connector is separated, and can describe by for example following formula:
Figure A20078005123101152
The exemplary synthetic route of 5 yuan of connectors of preparation the present invention is:
Figure A20078005123101161
In thionyl chloride solution, be subjected to the DMDA b and 2 of Cbz protection, 2-dimethyl malonic acid a reaction forms Cbz-DMDA-2,2-dimethyl malonic acid c.In the presence of EDC, compound c and Boc-N-methyl hydrazine d reaction form DMDA-2,2-dimethyl malonic acid-Boc-N-methyl hydrazine e.
Hexavalent hydrazine connector
In another embodiment, this hydrazine connector comprises one 6 yuan hydrazine connector, and wherein H comprises following structure:
Figure A20078005123101162
In a preferred embodiment, n 1Be 3.In above structure, each R 24Be the assorted alkyl and the unsubstituted assorted alkyl of the alkyl that is independently selected from following member: H, replacement, unsubstituted alkyl, replacement.In one embodiment, each R 24Be H or C independently 1-C 6Alkyl.In another embodiment, each R 24All be H or C independently 1-C 3Alkyl, more preferably H or CH 3In another embodiment, at least one R 24Be methyl group.In another embodiment, each R 24All are H.Each R 24Be selected as the steric effect of this compound is adjusted and is used to change solubleness.In a preferred embodiment, H comprises following structure:
Figure A20078005123101171
In one embodiment, H comprises that gem-dimethyl replaces.In an embodiment of above structure, each R 24All be H or replacement or unsubstituted alkyl independently.
6 yuan of hydrazine connectors can experience cyclization, and this reaction separates this medicine and this connector, and can be described to:
Figure A20078005123101172
The exemplary synthetic route for preparing 6 yuan of connectors of the present invention is:
Figure A20078005123101173
In containing the solution of methylene dichloride, reacted by the dimethyl propylene propylhomoserin a of Cbz protection and HOAt and CPI, form the dimethyl propylene aminoacylhydrazine b that protected by Cbz.This hydrazine b by deprotection, forms compound c by the methyl alcohol effect.
Other hydrazine connectors
Expection the present invention includes the heptatomic connector.This connector is can not be as five yuan or hexa-atomic connector fast carries out cyclisation, but this may be preferred for some antibody-mating partner conjugate.Similarly, this hydrazine connector can comprise two six-rings or have the hydrazine connector of a hexa-atomic and five-ring product.Five yuan and seven yuan of connectors and hexa-atomic and heptatomic connector have also been considered.
Another kind of hydrazine structure H has following formula:
Figure A20078005123101181
Wherein q is 0,1,2,3,4,5 or 6; And
Each R 24All are the members that are independently selected from down group: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement and unsubstituted assorted alkyl.This hydrazine structure also can form five, six or seven-membered ring, and can add supplementary component to form a plurality of rings.
Disulfide linkers (J)
In another embodiment, this connector comprises can carry out the disulphide group that enzymatic is sheared.In one embodiment, the invention provides a kind of cytotoxic antibody-mating partner compound with formula (d) structure:
Wherein, D, L 1, L 4, and X 4As defined above, and further specify, and J is the disulfide linkers that comprises the group with following structure at this:
Figure A20078005123101183
Wherein, each R 24All are the members that are independently selected from down group: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement and unsubstituted assorted alkyl; Each K all is the members that are independently selected from down group: the assorted alkyl of the alkyl of replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, the Heterocyclylalkyl of replacement, unsubstituted Heterocyclylalkyl, halogen, NO 2, NR 21R 22, NR 21COR 22, OCONR 21R 22, OCOR 21, and OR 21, wherein, R 21And R 22Be the member who is independently selected from down group: the Heterocyclylalkyl and the unsubstituted Heterocyclylalkyl of the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, the aryl of replacement, unsubstituted aryl, the heteroaryl of replacement, unsubstituted heteroaryl, replacement; I is 0,1,2,3 or 4 integer; And d is 0,1,2,3,4,5 or 6 integer.
The aromatic nucleus of this disulfide linkers can replace with one or more " K " group." K " group is the substituting group on the aromatic nucleus, and its replacement is connected in the hydrogen of one of four unsubstituted carbon into the part ring structure originally." K " group can be single atom, as halogen, maybe can be polyatomic group, as alkyl, assorted alkyl, amino, nitro, hydroxyl, alkoxyl group, alkylhalide group and cyano group.Exemplary K substituting group includes but not limited to F, Cl, Br, I, NO independently 2, OH, OCH 3, NHCOCH 3, N (CH 3) 2, NHCOCF 3, and methyl.For " K i", i is 0,1,2,3 or 4 integer.In a particular, i is 0.
In a preferred embodiment, but this connector comprises the disulphide group that enzymatic with following formula is sheared:
Figure A20078005123101191
In this embodiment, L 4, X 4, p and R 24Identity as described above, and d is 0,1,2,3,4,5 or 6.In a particular, d is 1 or 2.
Disulfide linkers is shown below more specifically
Figure A20078005123101192
A particular instance of this embodiment is as follows:
Figure A20078005123101193
Preferably, d is 1 or 2.
Another kind of disulfide linkers is shown below:
Figure A20078005123101201
A particular instance of this embodiment is as follows:
Figure A20078005123101202
Preferably, d is 1 or 2.
In different embodiments, disulphide is at the ortho position of amine.In another specific embodiment, a is 0.In a preferred embodiment, R 24Be independently selected from H and CH 3
The exemplary synthetic route for preparing disulfide linkers of the present invention is as follows:
Figure A20078005123101203
The solution of 3-thiohydracrylic acid a and bipyridyl two sulphur (aldrithiol-2) react, and form iodate 3-methylbenzothiazole (3-methyl benzothiazolium iodide) b.Iodate 3-methylbenzothiazole c and sodium hydroxide react, and form compound d.The methanol solution and the compound b of compound d are further reacted, and form Verbindung.By the effect of Acetyl Chloride 98Min. and methyl alcohol, Verbindung is formed compound f by deprotection.
For the type of cytotoxin, connector and the further discussion that therapeutical agent is coupled to the method for antibody, also referring to people's such as Gangwar PCT open file WO 2007/05940, and title is " Cytotoxic Compounds And Conjugates; " Saito, people such as G. (2003) Adv.Drug Deliv.Rev.55:199-215; Trail, people such as P.A. (2003) Cancer Immunol.Immunother.52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, T.M. (2002) Nat.Rev.Cancer 2:750-763; Pastan, I.and Kreitman, R.J. (2002) Curr.Opin.Investig.Drugs 3:1089-1091; Senter, P.D. and Springer, C.J. (2001) Adv.Drug Deliv.Rev.53:247-264, the full text of each in them is all incorporated this paper into as a reference.
The mating partner molecule
Feature of the present invention is the antibody of coupling mating partner molecule (for example cytotoxin, medicine (as immunosuppressor) or radiotoxin).This type of conjugate is also referred to as " immune conjugate " at this.Comprise that one or more cytotoxic immune conjugates are called " immunotoxin ".Cytotoxin or cytotoxic agent comprise the reagent of any pair cell harmful (for example cell killing).
The example of mating partner molecule of the present invention comprises taxol, cytochalasin B, Gramicidin D, ethidium bromide, ipecamine, mitomycin, Etoposide, teniposide, vincristine(VCR), vinealeucoblastine(VLB), colchicine, Zorubicin, daunorubicin, dihydroxyl anthracin diketone (dihydroxyanthracin dione), mitoxantrone, Plicamycin, dactinomycin, the 1-boldenone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Proprasylyte, and tetracycline, with and analogue or homologue.The example of mating partner molecule also comprises, for example metabolic antagonist is (as methotrexate, Ismipur, the 6-Tioguanine, cytosine arabinoside, 5 FU 5 fluorouracil, dacarbazine), alkylating reagent is (as mustargen, thio-tepa, Chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), endoxan (cyclothosphamide), busulfan, mitobronitol, U-9889, ametycin, and cis-platinum (II) (DDP cis-platinum), anthracycline antibiotics (as Daunorubicin (claiming daunorubicin in the past) and Zorubicin), microbiotic is (as gengshengmeisu (claiming actinomycin in the past), bleomycin, Plicamycin, reach Antramycin (AMC)), and antimitotic agent (as vincristine(VCR) and vinealeucoblastine(VLB)).
Other can comprise with the mating partner molecule preferred embodiment of antibody coupling of the present invention all Ka-7038, calicheamicin, maytenin and Ali Si Dating (auristatin), with and derivative.A kind of example of calicheamicin antibody coupling matter be commercially available (
Figure A20078005123101221
American HomeProducts).
The preferred example of mating partner molecule is CC-1065 and Ka-7038s all.CC-1065 separates (people such as Hanka, J.Antibiot.31:1211 (1978) by Upjohn company from Ze Er streptomycete (streptomyces zelensis) first; People such as Martin, J.Antibiot.33:902 (1980); And be found in external and in laboratory animal, all have effective antitumour and antimicrobial acivity (people such as Li, Cancer Res.42:999 (1982)) people such as Martin, J.Antibiot.34:1119 (1981)).CC-1065 be attached to double-stranded b form dna have priority sequence be 5 '-ditch of d (A/GNTTA)-3 ' and 5 '-d (AAAAA)-3 ' in (people such as Swenson, CancerRes.42:2821 (1982)), and by the CPI left-hand unit that it exists in molecule make 3 '-the N3 position alkylation (people such as Hurley, Science 226:843 (1984)) of VITAMIN B4.Although CC-1065 has effectively and anti-tumor activity widely, because CC-1065 has caused the slow death of laboratory animal, so it can not be used to the mankind.
CC-1065 and all the many analogues and the derivative of Ka-7038 are known in this area.The structure of chemical compound lot, synthetic and The Characteristic Study are summarized.Referring to, for example, people such as Boger, Angew.Chem.Int.Ed.Engl.35:1438 (1996); And people such as Boger, Chem.Rev.97:787 (1997).
Kyowa Hakko Kogya Co., the team of Ltd. has prepared many CC-1065 derivatives.Referring to, for example, U.S. Patent number 5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,703,080; 5,070,092; 5,641,780; 5,101,038; And 5,084,468; And disclosed PCT application WO 96/10405 and disclosed European application 0537 575A1.
Upjohn Company (Pharmacia Upjohn) is at the derivative that actively prepares CC-1065.Referring to, for example, U.S. Patent number 5,739,350,4,978,757,5,332,837 and 4,912,227.
A particularly preferred aspect of the present invention provides cytotoxin compounds, and this compound has the structure according to following formula (e):
Figure A20078005123101231
Wherein, member ring systems A is the member who is selected from replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or the unsubstituted Heterocyclylalkyl.Exemplary member ring systems comprises phenyl and pyrroles.
Symbol E and G are independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, heteroatoms, singly-bound, perhaps E and G can randomly carry out combination and form a ring-type system, and this system is selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl.
Symbol X represents to be selected from O, S and NR 23In the member.R 23Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group.
Symbol R 3Expression be selected from (=O), SR 11, NHR 11And OR 11In the member, R wherein 11Be H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, phosplate, bisphosphate, triguaiacyl phosphate, sulphonate, acyl group, C (O) R 12R 13, C (O) OR 12, C (O) NR 12R 13, P (O) (OR 12) 2, C (O) CHR 12R 13, SR 12Or SiR 12R 13R 14Symbol R 12, R 13, and R 14Represent H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl, wherein R independently 12And R 13Can randomly be connected to form replacement or unsubstituted Heterocyclylalkyl ring-type system with nitrogen-atoms or carbon atom that they connected, this ring bodies cording has 4 to 6 yuan, can randomly comprise 2 or a plurality of heteroatoms.R 12, R 13Or R 14In one or morely in its structure, can comprise the group that can shear.
R 4, R 4', R 5And R 5' be to be independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl, halogen, NO 2, NR 15R 16, NC (O) R 15, OC (O) NR 15R 16, OC (O) OR 15, C (O) R 15, SR 15, OR 15, CR 15=NR 16, and O (CH 2) nN (CH 3) 2The member, wherein, n is from 1 to 20 integer, perhaps R 4, R 4', R 5And R 5' any adjacent a pair of carbon atom that is connected with them be joined together to form replacement or a unsubstituted cycloalkyl or a Heterocyclylalkyl ring-type system that has 4 to 6 yuan.R 15And R 16Represent H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted peptidyl, wherein R independently 15And R 16Can randomly be connected to form replacement or unsubstituted Heterocyclylalkyl ring-type system with the nitrogen-atoms that they connected, this ring bodies cording has 4 to 6 yuan, can randomly comprise two or more heteroatomss.An exemplary structure is an aniline.
R 4, R 4', R 5, R 5', R 11, R 12, R 13, R 15And R 16In their structure, can randomly comprise one or more groups of shearing, for example the substrate that maybe can shear of the connector that can shear.The exemplary group of shearing includes, but are not limited to peptide, amino acid, hydrazine, disulphide and cephalosporins derivatives.
In some embodiments, R 4, R 4', R 5, R 5', R 11, R 12, R 13, R 15And R 16In at least one be used for the substrate that medicine and connector of the present invention or enzyme can be sheared is combined, as described here, for example be connected to L 1Go up (if existence), or be connected to F, H, J or X 2Or on the J.
In another exemplary, R 4, R 4', R 5, R 5', R 11, R 12, R 13, R 15And R 16In at least one have the reactive group that is suitable for this compound of coupling.In another exemplary, R 4, R 4', R 5, R 5', R 11, R 12, R 13, R 15And R 16Be independently selected from the alkyl of H, replacement and the assorted alkyl of replacement, and have reactive functionality at the free-end of alkyl or assorted moieties.R 4, R 4', R 5, R 5', R 11, R 12, R 13, R 15And R 16In one or more can being coupled on another kind, for example, targeting agent, detectable mark, solid support etc.
R 6Be singly-bound, it exists or does not exist.Work as R 6When existing, R 6And R 7In conjunction with forming cyclopropyl rings.R 7Be CH 2-X 1Or-CH 2-.Work as R 7Be-CH 2In-time, it is the integral part of cyclopropane ring.Symbol X 1Expression leavings group such as halogen, for example Cl, Br or F.Explain R in the mode that can not violate the chemical valence principle 6And R 7Combination.
X 1It can be any leavings group.Useful leavings group includes but not limited to that halogen, trinitride, sulfo group ester (as alkyl sulphonyl, aryl sulfonyl), oxonium ion, perchloric acid alkyl ester, amino alkane sulphonate, alkyl fluoride RF are for sulphonate and fluorinated compound (as fluoroform sulphonate, perfluoro butyl mesylate (nonaflates), trifluoro esilate (tresylates)) or the like.Concrete halogen as leavings group is F, Cl and Br.For the setting of special reaction condition within these and other leavings group appropriate selection one skilled in the relevant art's the ability (referring to, for example, March J, Advanced Organic Chemistry, second edition, John Wiley and Sons, 1992; Sandler SR, Karo W, Organic Functional Group Preparations, second edition, Academic Press, Inc., 1983; And Wade LG, Compendium of OrganicSynthetic Methods, John Wiley and Sons, 1980).
Curve in the six-ring shows that this ring can have one or more degrees of unsaturation, and it can be an aromatics.Therefore, ring structure (as following given) and dependency structure are in the scope of formula (f):
Figure A20078005123101251
In some embodiments, R 4, R 4', R 5, and R 5' at least one described medicine is connected to L 1In (if existence), or be connected to F, H, J or X 2On, and comprise
Figure A20078005123101261
Wherein v is from 1 to 6 integer; And R 27, R 27', R 28And R 28' all be independently selected from separately H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl.In some embodiments, R 27, R 27', R 28And R 28' all be H.In some embodiments, v is from 1 to 3 integer (preferably 1).This unit can be used for aryl substituent and medicine are separated, and therefore hinders or avoid producing the compound of multiple resistant substrate.
In one embodiment, R 11Comprise a part X 5, it can self-cyclisation, and medicine is connected to L 1In (if existence), or be connected to F, H, J or X 2On.Part X 5The preferred enzyme that uses can be sheared, and active medicine is provided when being sheared.As an example, R 11Can have the following structure rest part coupling mutually of medicine (its right side with):
In an exemplary, the ring-type system A of formula (e) replaces or unsubstituted phenyl ring.Ring-type system A can be replaced by the one or more aryl substituent that provides in this paper definitional part.In some embodiments, this phenyl ring is replaced by CN or methoxyl group part.
In some embodiments, R 4, R 4', R 5, and R 5' at least one with described medicine and L 1If (existence) is connected, or with F, H, J or X 2Be connected, and R 3Be selected from SR 11, NHR 11And OR 11R 11Be selected from-SO (OH) 2,-PO (OH) 2,-AA n,-Si (CH 3) 2C (CH 3) 3,-C (O) OPhNH (AA) m,
Or any other sugared or sugared combination,
Figure A20078005123101272
And pharmacy acceptable salt, wherein n is the arbitrary integer in 1 to 10 scope, and m is the arbitrary integer in 1 to 4 scope, and p is the arbitrary integer in 1 to 6 scope, and AA is any natural or alpha-non-natural amino acid.In some embodiments, AA nOr AA mBe selected from above explanation peptide connector (F) same acid sequence and randomly with at R 4, R 4', R 5Or R 5' the used aminoacid sequence of connector part identical.In at least some embodiments, R 3Be in vivo can shear so that active pharmaceutical compounds to be provided.In at least some embodiments, R 3Improve compound solubleness in vivo.In some embodiments, the speed that active medicine concentration reduces in the blood is in fact faster than the R that active medicine is provided 3Shearing rate.When the toxicity of active medicine was higher than the toxicity of prodrug form in fact, this may be useful especially.In other embodiment,, active medicine shears R for being provided 3Speed faster than the concentration changing down of active medicine in the blood.
In another exemplary, the invention provides the compound that has according to the structure of formula (g):
Figure A20078005123101281
In this embodiment, substituent R 3, R 4, R 4', R 5, R 5', R 6, R 7And the identity of X, and it is for the preference of specific embodiment, described as the above explanation of being done for formula (a) basically.Symbols Z is independently to be selected from O, S and NR 23The member.Symbol R 23Expression is selected from the member of H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group.To each R 23Select independently.Symbol R 1Represent H, replacement or unsubstituted low alkyl group, or C (O) R 8Or CO 2R 8R 8Be alkyl, unsubstituted alkyl, the NR that is selected from replacement 9R 10, NR 9NHR 10And OR 9The member.R 9And R 10Be independently selected from H, replacement or unsubstituted alkyl and replacement or unsubstituted assorted alkyl.R 2Be H or replacement or unsubstituted low alkyl group.Usually preferably work as R 2When being the alkyl that replaces, it is different from perfluoroalkyl, for example CF 3In one embodiment, R 2Be the alkyl that replaces, wherein substituting group is not a halogen.In another embodiment, R 2It is unsubstituted alkyl.
In some embodiments, R 1Be ester moiety, CO for example 2CH 3In some embodiments, R 2Be low alkyl group, it can be replacement or unsubstituted.At present preferred low alkyl group is CH 3In some preferred embodiments, R 1Be CO 2CH 3And R 2Be CH 3
In some embodiments, R 4, R 4', R 5, and R 5' be to be independently selected from H, halogen, NH 2, OMe, O (CH 2) 2N (R 29) 2, and NO 2The member.Each R 29All be H or low alkyl group (for example methyl) independently.
In some embodiments, medicine is selected, made leavings group X 1Be the member who is selected from down group: halogen, alkyl sulphonyl, aryl sulfonyl and trinitride.In some embodiments, X 1Be F, Cl or Br.
In some embodiments, Z is O or NH.In some embodiments, X is O.
In another exemplary, the invention provides the compound that has according to formula (h) or structure (i):
Figure A20078005123101291
Another preferred construction of all Ka-7038s analogue of formula (e) is that wherein ring-type system A is unsubstituted or the structure of the phenyl ring of replacement.When ring-type system A is the pyrroles, preferred substituted on the drug molecule of the structure with formula 7 of above explanation also be when ring-type system A be unsubstituted or preferred substituted during the phenyl ring that replaces.
For example, in a preferred embodiment, medicine (D) comprises structure (j):
Figure A20078005123101301
In this structure, R 3, R 6, R 7, X is as with explanation in the following formula (e).In addition, Z is selected from O, S and NR 23The member, R wherein 23Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group;
R 1Be H, replacement or unsubstituted low alkyl group, C (O) R 8Or CO 2R 8, R wherein 8Be to be selected from NR 9R 10And OR 9The member, R wherein 9And R 10Be the member who is independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl;
R 1' be H, replacement or unsubstituted low alkyl group or C (O) R 8, R wherein 8Be to be selected from NR 9R 10And OR 9The member, R wherein 9And R 10Be the member who is independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl;
R 2Be H or replacement or unsubstituted low alkyl group or unsubstituted assorted alkyl or cyano group or alkoxyl group; R 2' be H or replacement or unsubstituted low alkyl group or unsubstituted assorted alkyl.
R 4, R 4', R 5, R 5', R 11, R 12, R 13, R 15Or R 16In at least one medicine is connected to L 1In (if existence), or be connected to F, H, J or X 2On.
Another embodiment of medicine (D) comprises structure (k), wherein R 4And R 4' formation Heterocyclylalkyl has combined:
Figure A20078005123101302
In this structure, R 3, R 5, R 5', R 6, R 7, X is as illustrated with following formula (e).In addition, Z is selected from O, S and NR 23The member, R wherein 23Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group;
R 32Be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl, halogen, NO 2, NR 15R 16, NC (O) R 15, OC (O) NR 15R 16, OC (O) OR 15, C (O) R 15, SR 15, OR 15, CR 15=NR 16, and O (CH 2) nN (CH 3) 2, wherein, n is from 1 to 20 integer.R 15And R 16Represent H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted peptidyl, wherein R by oneself 15And R 16Can randomly be connected to form replacement or unsubstituted Heterocyclylalkyl ring-type system with the nitrogen-atoms that they connected, this ring bodies cording has 4 to 6 yuan, randomly comprises two or more heteroatomss.R 32In its structure, randomly comprise one or more groups of shearing, for example the substrate that maybe can shear of the connector that can shear.The exemplary group of shearing includes, but are not limited to peptide, amino acid, hydrazine, disulphide and cephalosporins derivatives.And, Shuo Ming R herein 4, R 4', R 5, R 5', R 15, and R 16Substituent any selection also can be applicable to R 32
R 5, R 5', R 11, R 12, R 13, R 15, R 16Or R 32In at least one this medicine is connected to L 1In (if existence), or be connected to F, H, J or X 2On.In at least some embodiments, R 32Medicine is connected to L 1In (if existence), or be connected to F, H, J or X 2On.
An embodiment preferred of this compound is:
Figure A20078005123101311
R 1Be H, replacement or unsubstituted low alkyl group, C (O) R 8Or CO 2R 8, R wherein 8Be to be selected from NR 9R 10And OR 9The member, R wherein 9And R 10Be the member who is independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl;
R 1' be H, replacement or unsubstituted low alkyl group or C (O) R 8, R wherein 8Be to be selected from NR 9R 10And OR 9The member, R wherein 9And R 10Be the member who is independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl;
R 2Be H or replacement or unsubstituted low alkyl group or unsubstituted assorted alkyl or cyano group or alkoxyl group; R 2' be H or replacement or unsubstituted low alkyl group or unsubstituted assorted alkyl.
Another embodiment has following formula:
Figure A20078005123101321
In this structure, A, R 6, R 7, X, R 4, R 4', R 5And R 5' as illustrated with following formula (e).In addition, Z is selected from O, S and NR 23The member, R wherein 23Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group;
R 33Be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl, halogen, NO 2, NR 15R 16, NC (O) R 15, OC (O) NR 15R 16, OC (O) OR 15, C (O) R 15, SR 15, OR 15, CR 15=NR 16, and O (CH 2) nN (CH 3) 2, wherein, n is from 1 to 20 integer.R 15And R 16Represent H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted peptidyl, wherein R by oneself 15And R 16Can randomly be connected to form replacement or unsubstituted Heterocyclylalkyl ring-type system with the nitrogen-atoms that they connected, this ring bodies cording has 4 to 6 yuan, randomly comprises two or more heteroatomss.R 33Medicine is connected to L 1In (if existence), or be connected to F, H, J or X 2On.
Preferably, A replaces or unsubstituted phenyl or replacement or unsubstituted pyrroles.In addition, explanation herein is used for R 11Substituent any selection also can be applicable to R 33
Part
X 4Expression is selected from: the part of shielded reactive functionality, not protected reactive functionality, detectable mark and targeting agent.Preferred part is a targeting agent, as antibody and fragment thereof.
In some embodiments, radicals X 4Can be illustrated as and be selected from R 29, COOR 29, C (O) NR 29, and C (O) NNR 29The member, R wherein 29Be the member who is selected from replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted heteroaryl.In another exemplary embodiment, R 29Be the thiol reactant member.In another exemplary, R 29Be the thiol reactant member, be selected from halo ethanoyl and alkylogen derivative, maleimide, aziridine and acryloyl derivative.Above thiol reactant member can serve as the reaction blocking group, and these reaction blocking groups can react with the amino acid side chain of for example targeting agent (as antibody), and this targeting agent is connected on this connector-drug moiety.
Detectable mark
But specific markers that is used in combination with The compounds of this invention or detection moiety and the inventive method are not critical aspects of the present invention usually, as long as it does not significantly disturb the activity or the effectiveness of The compounds of this invention.Detectable group can be any material with detectable physics or chemical property.This type of is detectable to be marked in the field of immunoassay and well to be developed, and usually, any mark useful in these class methods can both be applied among the present invention.Therefore, mark is by the detectable any component of spectroscopy, photochemistry, biological chemistry, immunochemistry, electricity, optics or chemical means.Useful mark comprises magnetic bead (DYNABEADS for example among the present invention TM), fluorescence dye (for example, fluorescein isothiocyanate, texas Red, rhodamine etc.), radio-labeling (for example 3H, 125I, 35S, 14C or 32P), enzyme (for example, horseradish peroxidase, alkaline phosphatase and other enzyme commonly used in ELISA) and colorimetric mark, as Radioactive colloidal gold or tinted shade or plastic bead (for example, polystyrene, polypropylene, latex etc.).
According to method well known in the art, mark can directly or indirectly be coupled on the compound of the present invention.As above indication, can use multiple mark, the selection of mark depend on required sensitivity, with the easy degree of compound link coupled, durability requirements, available instrument and governable supplies.
When compound of the present invention and detectable mark mutually during coupling, this mark preferably is selected from down the member of group: radio isotope, fluorescent reagent, fluorescent reagent precursor, chromophore, enzyme and their combination.The method that various groups is coupled to antibody is known in this area.For example, the detectable mark that often is coupled to antibody is an enzyme, as horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes and glucose oxidase.
The nonradioactive labeling usually connects by indirect means.Usually, ligand molecular (biological example element) covalently is attached on the composition of this conjugate.This part combines with another molecule (as streptavidin) then, this molecule be detectable inherently or with signalling system (as detectable enzyme, fluorescent chemicals or chemiluminescence compound) covalent attachment.
The composition of conjugate of the present invention can also directly be coupled on the compound that produces signal, for example by with enzyme or fluorophore coupling mutually.The purpose enzyme that serves as a mark mainly is lytic enzyme, particularly Phosphoric acid esterase, esterase and Glycosylase or oxydase (oxidotase), particularly peroxidase.Fluorescent chemicals comprises fluorescein and derivative, rhodamine and derivative thereof, dansyl, Umbelliferone etc.Chemiluminescence compound comprises luciferin and 2,3-dihydro phthalazine diketone, for example luminol,3-aminophthalic acid cyclic hydrazide.For the commentary of operable various marks or signal generation system, referring to U.S. Patent number 4,391,904.
The means of certification mark are that those of ordinary skills know.Therefore, for example, when mark was radio-labeling, detection means comprised scintillometer or as the photographic film in radioautography.When mark is fluorescent mark, can it be detected by the fluorescence that produces with suitable optical wavelength fluorescence excitation dyestuff and detection.Can be by photographic film, by using electronic detector (for example charge-coupled device (CCD) or photomultiplier or the like) fluorescence is carried out visual detection.Similarly, can come enzyme labelling is detected by suitable substrate that enzyme is provided and the reaction product that detects generation.At last, can detect simply simple colorimetric mark by observing the color relevant with mark.Therefore, measure in (dipstick assay) in various test strip, institute's link coupled gold often is shown as pink, and various link coupled pearl demonstrates the color of pearl.
Fluorescent mark is preferred at present, and the preventive measures that need in the operation are few because they have, the advantage of also suitable high-throughput imaging technique (optical analysis is included in the digitizing of the image that is used to analyze in the integrated system that comprises computer).Preferred mark typically has following one or multinomial feature: the high specific of highly sensitive, high stability, low background, low environment susceptibility and mark.Many fluorescent marks can be commercially available from following company: SIGMA chemical company (Saint Louis, MO), Molecular Probes (Eugene, OR), R﹠amp; D systems (Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECHLaboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich ChemicalCompany (Milwaukee, WI), Glen Research, Inc., GIBCO BRL LifeTechnologies, Inc. (Gaithersburg, MD), Fluka Chemica-BiochemikaAnalytika (Fluka Chemie AG, Buchs, Switzerland), and AppliedBiosystems (Foster City, and many other commercial source known to the skilled CA).In addition, those of ordinary skill in the art will recognize how to select suitable fluorophore to be used for specific application, if and it be not easy commercially available, then can de novo synthesis essential fluorophore or commercially available fluorescent chemicals carried out synthetic modification to obtain desirable fluorescent mark.
Except the small molecules fluorophore, the fluorescin of natural generation and also useful in the present invention to the analogue of this type of proteinic transformation.This proteinoid comprises green fluorescent protein (people such as Ward, the Photochem.Photobiol.35:803-808 (1982) of (for example) cnidarian; People such as Levine, Comp.Biochem.Physiol., 72B:77-85 (1982)), come the yellow fluorescence protein (people such as Baldwin of Zi Feishi vibrios bacterial strain, Biochemistry 29:5509-15 (1990)), perdinin-chlorophyll (the people such as Morris who belongs to dinoflagellate (dinoflagellate Symbiodinium sp.) from the symbiosis dinoflagellate, Plant Molecular Biology 24:673:77 (1994)), from the ocean blue bacterium phycobiliprotein of (belonging to (Synechococcus)) as synechococcus, phycoerythrin and Phycocyanins, C-(people such as Wilbanks for example, J.Biol.Chem.268:1226-35 (1993)), reach analogue.
Usually, before, at least one among these chemical functional groups can be activated to form and be connected (and optional be spacer groups) between cytotoxin and target (or other) reagent.Those of ordinary skill in the art will understand, and can use many kinds of standard methods and condition to come activation functional group, comprise hydroxyl, amino and carboxylic group.For example, can be by forming corresponding chlorocarbonate with the light gas disposal or handling the hydroxyl that the corresponding carbonate of formation comes activating cells toxin or targeting agent with the p-nitrophenyl chlorocarbonate.
In an exemplary, utilization of the present invention comprises carboxyl functionality's targeting agent.Carboxyl can pass through, and for example, changes corresponding carboxylic acid halides or active ester into and is activated.Carry out under the multiple condition that this reaction can be set forth in March (the same) 388-89 page or leaf.In an exemplary, react by carboxylic group and oxalyl chloride and to prepare carboxylic acid halides.Reagent that is activated and cytotoxin or cytotoxin-connector arm combination is reacted, to form conjugate of the present invention.Those of ordinary skill in the art will understand that to use carboxylic targeting agent only be illustrative, and the reagent with many other functional groups can be coupled on the connector of the present invention.
Reactive functionality
For the clarity of setting forth, the discussion of back concentrates on the coupling of cytotoxin and targeting agent.The focus illustration one embodiment of the invention, from this embodiment, those skilled in the art is easy to infer other embodiment.The single embodiment of concentrated discussion does not also mean that limitation of the present invention.
Exemplary compounds of the present invention has reactive functionality, and this functional group is usually located on replacement or unsubstituted alkyl or the assorted alkyl chain, and they easily are connected on another kind.The vantage point of this reactive group is the terminal position of this chain.
Implement reactive group useful when of the present invention and reactive species normally those in known reactive group of biological coupling chemical field and reactive species.Reactive functionality can be shielded or not protected, and the shielded person's character of this functional group can change by the known method in organic synthesis field.With the obtainable preferred reactive species of reactive cytotoxin analogue is those reactive species of carrying out under gentle relatively condition.These reactive species include but not limited to that nucleophilic substitution (for example, the reaction of amine and alcohol and carboxylic acid halides, active ester), electrophilic substitution (for example, enamine reaction) and the addition of carbon-to-carbon and a plurality of keys of carbon-heteroatoms (for example, michael reaction, Diels-Alder reaction).These and other useful reaction is discussed at for example March, Advanced OrganicChemistry, the third edition, John Wiley ﹠amp; Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; And people such as Feeney, Modification of Proteins; Advances in Chemistry Series, Vol.198, American Chemical Society, Washington, D.C., 1982.
The exemplary reaction type comprises the reaction of carboxyl and different derivative thereof, and carboxy derivatives includes but not limited to N-hydroxy-succinamide ester, N-hydroxybenzotriazole ester, carboxylic acid halides, acylimidazole, thioesters, p-nitrophenyl ester, alkyl, thiazolinyl, alkynyl and aromatic ester.Oh group can be changed into ester, ether, aldehyde etc.By reacting with for example amine, carboxylate anion, mercaptan negatively charged ion, carbanion or alkoxide ion, the alkylhalide group group can be changed into new kind.Dienophile (for example maleimide) group participates in Diels-Alder reaction.Aldehyde radical or ketone group group can be changed into imines, hydrazone, semicarbazone or oxime, or finish these transformations by for example Grignard addition reaction or the such mechanism of lithium alkylide addition reaction.Sulfonic acid halide easily and amine react, for example form sulphonamide.Amine or mercapto groups are by for example acidylate, alkylation or oxidation.Use cycloaddition, acylation, Michael addition etc., alkene can be changed into a series of new kinds.Epoxide easy and amine and oxy-compound reaction.
Those of ordinary skill in the art will readily appreciate that, has during these connect manyly can and use multiple condition to produce by several different methods.For the preparation of ester, for example see 1157 pages of March supra; For thioesters, see March, (the same) 362-363 page or leaf, 491 pages, 720-722 page or leaf, 829 pages, 941 pages and 1172 pages; For carbonic ether, see March, (the same) 346-347 page or leaf; For carbamate, see March, (the same) 1156-57 page or leaf; For acid amides, see March, (the same) the 1152nd page; For urea and thiocarbamide, see March, (the same) the 1174th page; For acetal and ketal, see people such as Greene, (the same) 178-210 and March, (the same) the 1146th page; For the acyloxy alkyl derivative, see Prodrugs:Topical and Ocular DrugDelivery, K.B.Sloan writes, Marcel Dekker, Inc., New York, 1992; For enol ester, see March, (the same) the 1160th page; For the inferior acid amides (N-sulfonylimidates) of the inferior sulphur of N-sulfo group, for example see people such as Bundgaard, J.Med.Chem., 31:2066 (1988); For acid anhydrides, see March, (the same) 355-56 page or leaf, 636-37 page or leaf, 990-91 page or leaf and 1154 pages; For the N-acid amides, see March, (the same) the 379th page; For the N-Mannich base, see March, (the same) 800-02 page or leaf and 828 pages; For the methylol ketone ester, see people Annals NY Acad.Sci. such as Petracek, 507:353-54 (1987); For disulphide, see the 1160th page of March (the same); And for the preparation of phosphoric acid ester and phosphonic amide ester.
Reactive functionality can be not protected and selected, makes them not participate in or does not interfere reaction.Alternatively, the reactive functional group group can be protected by the existence of blocking group and not participate in reacting.Those of ordinary skill in the art will understand the reaction conditions setting of how protecting the particular functional group not disturb to have selected.For the example of useful protecting group, referring to people such as Greene, Protective Groups inOrganic Synthesis, John Wiley ﹠amp; Sons, New York, 1991.
Typically, use the chemical technology of standard target reagent is covalently bound to cytotoxin by its chemical functional group separately.Randomly, connector or reagent are by one or more spacer groups and this reagent coupling.When being used in combination, spacer groups can be equivalence or different.
Usually, between cytotoxin and reactive functional groups (and randomly being spacer groups), form is connected before, at least one among the chemical functional group will obtain activation.Those of ordinary skill in the art will understand, and use many kinds of standard methods and condition can activate number of chemical functional group, comprise hydroxyl, amino and carboxylic group.In an exemplary, the present invention comprises the carboxyl functional group as reactive functionality.Carboxyl can as described abovely obtain activating.
The substrate that can shear
The substrate that the present invention can be sheared is illustrated as: " X 2".Preferably, the substrate that can shear is the enzyme substrates of shearing that can be sheared by enzyme.Preferably, preferred direct or indirect and pending tumour or other target cells of this kind of enzyme is associated.This enzyme can be produced by pending tumour or other target cells.For example, this substrate that can shear can be a peptide, this peptide preferably can by around tumour or other target cells or the enzyme of finding therein shear.Additionally or alternatively, this enzyme can be connected in the targeting agent that carries out specific combination with tumour cell, for example special to tumour antigen antibody.
As the substrate example that the enzyme of the medicine that is suitable for being coupled to above explanation can be sheared, PCT patent application open file WO 00/33888, WO 01/95943, WO 01/95945, WO 02/00263 and WO 02/100353 (they all incorporate this paper into as a reference) disclose being connected of the peptide that can shear and medicine.This peptide can be sheared with the tumour involved enzyme, for example trouase (as the phorate oligopeptidase), CD10 (neutral lyase), matrix metalloproteinase (as MMP2 or MMP9), II type transmembrane serine protease (as Hepsin, testis albumen, TMPRSS4 or matriptase/MT-SP1) or kethepsin.In this embodiment, prodrug comprises the medicine, peptide, stabilization group of above explanation and the linking group between medicine and peptide randomly.The stabilization group is connected in the peptide end, is not degraded before arriving tumour or other target cells with the protection prodrug.The example of suitable stabilization group comprises non-amino acid, for example succsinic acid, diglycollic acid, toxilic acid, polyoxyethylene glycol, Pyrrolidonecarboxylic acid, acetic acid, naphthalene monocarboxylic acid, terephthalic acid and glutaric acid derivatives; And the amino acid of non-genetic coding or aspartic acid or L-glutamic acid, it is connected in the N-terminal of peptide at the β-carboxyl of aspartic acid or the γ of L-glutamic acid-carboxyl place.
Peptide generally comprises the individual amino acid of 3 to 12 (or more).The near small part of the selection of specific amino acids depends on and will be used to shear the enzyme of this peptide, and this peptide stability in vivo.An example of the suitable peptide sheared is β-AlaLeuAlaLeu (SEQ ID NO:92).This can with the stabilization moiety combinations, form succinyl-β-AlaLeuAlaLeu (SEQ ID NO:92).The example of the peptide sheared that other are suitable is provided in the document cited above.
As an illustrative example, CD10 is also referred to as neutral lyase, neutral endopeptidase (NEP) and common acute lymphocytoblast leukemia antigen (CALLA), is a kind of II type cell-surperficial zinc dependent form metalloprotease.The substrate of shearing that is suitable for CD10 comprises LeuAlaLeu and IleAlaLeu.The substrate of other known CD10 of being used for comprises length 50 amino acid whose peptides at the most, reduces greatly although catalytic efficiency often becomes with substrate.
Another illustrative example is based on matrix metalloproteinase (MMP).As perhaps being the proteolytic ferment that the best relevant with tumour characterizes, there is tangible dependency in the activation of MMP in tumor microenvironment.Particularly, dissolvable matrix enzyme MMP2 (gelatin enzyme A) and MMP9 (gelatinase B) are furtherd investigate, and they demonstrate in comprising the tissue remodeling of tumor growth and are optionally activated.The peptide sequence that to be sheared by MMP2 and MMP9 and the conjugate of having tested following material have been designed: dextran and Rheumatrex people such as (, Bioconjugate Chem.15:931-941 (2004)) Chau; PEG (polyoxyethylene glycol) and Zorubicin (people such as Bae, Drugs Exp.Clin.Res.29:15-23 (2004)); And albumin and Zorubicin (people such as Kratz, Bioorg.Med.Chem.Lett.11:2001-2006 (2001)).The example of the suitable sequence of using with MMP includes but not limited to: ProValGlyLeuIleGly (SEQ IDNO:84), GlyProLeuGlyVal (SEQ ID NO:85), GlyProLeuGlyIleAlaGlyGln (SEQ ID NO:86), ProLeuGlyLeu (SEQ ID NO:87), GlyProLeuGlyMetLeuSerGln (SEQ ID NO:88) and GlyProLeuGlyLeuTrpAlaGln (SEQ ID NO:89).(referring to for example, the reference that preamble is quoted is together with people such as Kline, people such as Mol.Pharmaceut.1:9-22 (2004) and Liu, Cancer Res.60:6061-6067 (2000)).The also substrate that can use other to shear.
Another example is an II type transmembrane serine protease.This group enzyme comprises for example hepsin, testis albumen and TMPRSS4.GlnAlaArg is a kind of substrate sequence, this sequence is used with protein lyase (matriptase)/MT-SP1 (crossed in mammary cancer and ovarian cancer and express), and LeuSerArg uses with hepsin (crossed in prostate gland and some other tumor type and express).(referring to for example, Lee et.al., J.Biol.Chem.275:36720-36725 and Kurachi and Yamamoto, Handbook of Proeolytic Enzymes second volume, second edition (BarrettAJ, Rawlings ND ﹠amp; Woessner JF, eds) 1699-1702 page or leaf (2004)).The also substrate that can use other to shear.
The arrangement of the substrate that another type can be sheared comprises that preparation can shear the independent enzyme that this can shear substrate, and it becomes and is associated with tumour or cell.For example, endonuclease capable is coupled on the TS antibody (or other entities, this entity preferably are attracted on the tumour or on other target cells, for example receptors ligand), and then enzyme-antibody coupling matter can offer patient.Enzyme-antibody coupling matter is orientable to tumour relevant antigen and combination with it.Subsequently, the substrate conjugate with this medicine-can shear offers patient as prodrug.When the substrate conjugate of medicine-can shear when becoming with enzyme interacting that tumour combines, medicine only discharges near tumour, the substrate that can shear obtains shearing and medicine obtains discharging like this.For example, U.S. Patent number 4,975,278; 5,587,161; 5,660,829; 5,773,435; And 6,132,722 disclose this arrangement, and all documents are all incorporated this paper into as a reference.The suitable enzyme and the example of substrate include but not limited to the folate derivative of β-Nei Xiananmei and cephalosporins derivatives, carboxypeptidase G 2 and L-glutamic acid and aspartic acid.
In one embodiment, enzyme-antibody coupling matter comprises antibody or antibody fragment, and this antibody or antibody fragment are based on it and select for the specificity of the related antigen of expressing on purpose target cell or the target site.In the discussion that antibody above is provided.An example of the substrate of suitable cynnematin-can shear is
Figure A20078005123101411
The example of conjugate
Connector of the present invention and the substrate that can shear can be used in the conjugate that contains multiple mating partner molecule.The example of conjugate of the present invention is in the further detailed explanation of following do.Except as otherwise noted, as above given in the part of cells involved toxin, connector and the substrate that can shear, substituting group is limited.
A. connector conjugate
An example of suitable conjugate is the compound with following formula:
Figure A20078005123101412
L wherein 1Be from disappearing connector; M is an integer 0,1,2,3,4,5 or 6; F is the connector that comprises following structure:
Figure A20078005123101413
AA wherein 1Be the one or more members that are independently selected from down group: natural amino acid and non-natural a-amino acid; C is from 1 to 20 integer; L 2Be from disappearing connector and comprising
Figure A20078005123101414
R wherein 17, R 18And R 19Be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl independently of one another, and w is from 0 to 4 integer; O is 1; L 4Be the connector member; P is 0 or 1; X 4Be the member who is selected from down group: shielded reactive functionality, not protected reactive functionality, detectable mark and targeting agent; And D comprises following structure:
Figure A20078005123101421
Wherein ring-type system A is the member who is selected from replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl; E and G are independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, heteroatoms, single bonded member, perhaps E and G combine, form a ring-type system, this ring-type system is selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl; X is selected from O, S and NR 23The member; R 23Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group; R 3Be OR 11, R wherein 11Be the member who is selected from down group: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, phosplate, bisphosphate, triguaiacyl phosphate, sulphonate, acyl group, C (O) R 12R 13, C (O) OR 12, C (O) NR 12R 13, P (O) (OR 12) 2, C (O) CHR 12R 13, SR 12, and SiR 12R 13R 14, R 4, R 4', R 5And R 5' be the member who independently is selected from down group: the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO 2, NR 15R 16, NC (O) R 15, OC (O) NR 15R 16, OC (O) OR 15, C (O) R 15, SR 15, OR 15, CR 15=NR 16, and O (CH 2) nN (CH 3) 2, or R 4, R 4', R 5And R 5' the carbon atom that is connected with them of any phase adjacency pair be joined together to form have 4 to 6 yuan, replacement or unsubstituted cycloalkyl or Heterocyclylalkyl ring-type system; Wherein n is from 1 to 20 integer; R 15And R 16Be to be independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted peptidyl, wherein R 15And R 16With the nitrogen-atoms that they connected can randomly be connected with formation have 4 to 6 yuan, randomly comprise two or more heteroatomic replacements or unsubstituted Heterocyclylalkyl ring-type system; R 6Be singly-bound, it exists or does not exist, and works as R 6When existing, R 6And R 7Combine, form cyclopropyl rings; And R 7Be in described cyclopropyl rings with R 6Bonded CH 2-X 1Or-CH 2-, X wherein 1Be leavings group, R wherein 11Described medicine is connected to L 1If in (existence), or be connected on the F.
In some embodiments, medicine has above structure (c) or (f).A concrete example of the suitable compound that uses as conjugate is
Figure A20078005123101431
Another example of one class conjugate is the compound with following formula
Figure A20078005123101432
L wherein 1Be from disappearing connector; M is an integer 0,1,2,3,4,5 or 6; F is the connector that comprises following structure:
AA wherein 1Be the one or more members that are independently selected from down group: natural amino acid and non-natural a-amino acid; C is from 1 to 20 integer; L 2Be from disappearing connector; O is 0 or 1; L 4Be the connector member; P is 0 or 1; X 4Be the member who is selected from shielded reactive functionality, not protected reactive functionality, detectable mark and targeting agent; And D comprises following structure:
Figure A20078005123101434
Wherein ring-type system A is the member who is selected from replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted heterocycloalkyl; E and G independently are selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, heteroatoms, single bonded member, perhaps E and G combine and form the ring-type system, and this ring-type system is selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl; X is selected from O, S and NR 23The member; R 23Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group; R 3Be the member who is selected from down group: (=O), SR 11, NHR 11And OR 11, R wherein 11Be selected from down the member of group: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, phosplate, bisphosphate, triguaiacyl phosphate, sulphonate, acyl group, C (O) R 12R 13, C (O) OR 12, C (O) NR 12R 13, P (O) (OR 12) 2, C (O) CHR 12R 13, SR 12And SiR 12R 13R 14, R wherein 12, R 13, and R 14Be the member who independently is selected from down group: H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl, wherein R 12And R 13Can randomly connect to form replacement or unsubstituted Heterocyclylalkyl ring-type system with nitrogen-atoms or carbon atom that they connected, this ring bodies cording has 4 to 6 yuan, randomly comprises two or more heteroatomss; R 4, R 4', R 5And R 5' be the member who independently is selected from down group: the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO 2, NR 15R 16, NC (O) R 15, OC (O) NR 15R 16, OC (O) OR 15, C (O) R 15, SR 15, OR 15, CR 15=NR 16, and O (CH 2) nN (CH 3) 2, perhaps R 4, R 4', R 5And R 5' the carbon atom that is connected with them of any phase adjacency pair be joined together to form have 4 to 6 yuan, replacement or unsubstituted cycloalkyl or Heterocyclylalkyl ring-type system, wherein n is from 1 to 20 integer; R 15And R 16Be independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted peptidyl, wherein R 15And R 16Can randomly be connected to form replacement or unsubstituted Heterocyclylalkyl ring-type system with the nitrogen-atoms that they connected, this ring bodies cording has 4 to 6 yuan, randomly comprises two or more heteroatomss; R wherein 4, R 4', R 5And R 5' at least one connect described medicine to L 1If in (existence), or be connected on the F, and comprise
Figure A20078005123101451
Wherein v is from 1 to 6 integer; And R 27, R 27, R 28And R 28' be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl independently of one another; R 6Be singly-bound, it exists or does not exist, and works as R 6When existing, R 6And R 7Combine, form cyclopropyl rings; And R 7Be in described cyclopropyl rings with R 6Bonded CH 2-X 1Or-CH 2-, X wherein 1It is leavings group.
In some embodiments, medicine has above structure (c) or (f).A concrete example of the suitable compound that uses as conjugate is
Figure A20078005123101452
Wherein r is the integer in from 0 to 24 scope.
Another example of suitable conjugate is the compound with following formula:
Figure A20078005123101453
L wherein 1Be from disappearing connector; M is an integer 0,1,2,3,4,5 or 6; F is the connector that comprises following structure:
Figure A20078005123101454
AA wherein 1Be the one or more members that are independently selected from down group: natural amino acid and non-natural a-amino acid; C is from 1 to 20 integer; L 3It is the spacer groups that contains primary amine or secondary amine or carboxyl functional group; If L wherein 3Exist, then m is 0, and L 3Amine and the side carboxyl functional group of D form amido linkage or L 3Carboxyl and the side amine functional group of D form amido linkage; O is 0 or 1; L 4Be connector member, wherein L 4Comprise
Figure A20078005123101461
It is directly connected to (AA 1) cThe N end on, R wherein 20Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group, R 25, R 25', R 26, and R 26' be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl independently of one another; And s and t are from 1 to 6 integer independently; P is 1; X 4Be the member who is selected from down group: shielded reactive functionality, not protected reactive functionality, detectable mark and targeting agent; And D contains following structure:
Figure A20078005123101462
Wherein ring-type system A is the member who is selected from replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted heterocycloalkyl; E and G are independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, heteroatoms, single bonded member, perhaps E and G combine and form the ring-type system, and this ring-type system is selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl; X is selected from O, S and NR 23The member; R 23Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group; R 3Be the member who is selected from down group: (=O), SR 11, NHR 11And OR 11, R wherein 11Be the member who is selected from down group: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, phosplate, bisphosphate, triguaiacyl phosphate, sulphonate, acyl group, C (O) R 12R 13, C (O) OR 12, C (O) NR 12R 13, P (O) (OR 12) 2, C (O) CHR 12R 13, SR 12And SiR 12R 13R 14, R wherein 12, R 13And R 14Be the member who is independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl, wherein R 12And R 13Can randomly be connected to form replacement or unsubstituted Heterocyclylalkyl ring-type system with nitrogen-atoms or carbon atom that they connected, this ring bodies cording has 4 to 6 yuan, randomly comprises two or more heteroatomss; R 4, R 4', R 5And R 5' be the member who independently is selected from down group: the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO 2, NR 15R 16, NC (O) R 15, OC (O) NR 15R 16, OC (O) OR 15, C (O) R 15, SR 15, OR 15, CR 15=NR 16, and O (CH 2) nN (CH 3) 2, or R 4, R 4', R 5And R 5' the carbon atom that is connected with them of any phase adjacency pair be joined together to form have 4 to 6 yuan, replacement or unsubstituted cycloalkyl or Heterocyclylalkyl ring-type system, wherein n is from 1 to 20 integer; R 15And R 16Be to be independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted peptidyl, wherein R 15And R 16Can randomly be connected to form a replacement or unsubstituted Heterocyclylalkyl ring-type system with the nitrogen-atoms that they connected, this ring bodies cording has 4 to 6 yuan, randomly comprises two or more heteroatomss; R 6Be singly-bound, exist or do not exist, and work as R 6When existing, R 6And R 7Combine, form cyclopropyl rings; And R 7Be in described cyclopropyl rings with R 6Bonded CH 2-X 1Or-CH 2-, X wherein 1Be leavings group, R wherein 4, R 4', R 5, R 5', R 15Or R 16In at least one described medicine is connected to L 1If in (existence), or be connected on the F.
In some embodiments, this medicine has above structure (c) or (f).A concrete example of the suitable compound that uses as conjugate is
Figure A20078005123101471
Wherein r is the integer of scope from 0 to 24.
Other examples of the suitable compounds of using as conjugate comprise:
Figure A20078005123101481
And
Figure A20078005123101491
Figure A20078005123101501
Wherein r is
Figure A20078005123101511
And r is the integer in from 0 to 24 scope.
Can also form conjugate with medicine, for example following compound with structure (g):
Figure A20078005123101512
Figure A20078005123101521
(wherein r is the integer from 0 to 24 scope).
Can also use medicine to form conjugate with following structure:
Figure A20078005123101522
Figure A20078005123101531
Figure A20078005123101541
And
Figure A20078005123101542
This type of is cytotoxic synthetic, and is disclosed in the U.S. Patent Application Serial of submitting on November 30th, 2,007 60/991,300 about the details that they are connected to antibody.
In certain embodiments, the anti-CD70 therapeutical agent that is coupled to connector and has structure N1:
In certain embodiments, the anti-CD70 therapeutical agent that is coupled to connector and has structure N2:
B. the connector conjugate that can shear
An example of suitable conjugate is the compound with following structure:
Figure A20078005123101553
L wherein 1Be from disappearing at interval; M is 0,1,2,3,4,5 or 6 integer; X 2It is the substrate that to shear; And D contains following structure:
Figure A20078005123101554
Wherein ring-type system A is the member who is selected from replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted heterocycloalkyl; E and G are independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, heteroatoms, single bonded member, perhaps E and G are in conjunction with forming a ring-type system, and this ring-type system is selected from and replaces or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl; X is selected from O, S and NR 23The member; R 23Be the member who is selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and acyl group; R 3Be the member who is selected from down group: (=O), SR 11, NHR 11And OR 11, R wherein 11Be the member who is selected from down group: the assorted alkyl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted assorted alkyl, phosplate, bisphosphate, triguaiacyl phosphate, sulphonate, acyl group, C (O) R 12R 13, C (O) OR 12, C (O) NR 12R 13, P (O) (OR 12) 2, C (O) CHR 12R 13, SR 12And SiR 12R 13R 14, R wherein 12, R 13And R 14Be the member who independently is selected from down group: H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl and replacement or unsubstituted aryl, wherein R 12And R 13Can randomly be connected to form replacement or unsubstituted Heterocyclylalkyl ring-type system with nitrogen-atoms or carbon atom that they connected, this ring bodies cording has 4 to 6 yuan, randomly comprises two or more heteroatomss; R 6Be singly-bound, it exists or does not exist, and works as R 6When existing, R 6And R 7In conjunction with forming cyclopropyl rings; And R 7Be in described cyclopropyl rings with R 6Bonded CH 2-X 1Or-CH 2-, X wherein 1Be leavings group, R 4, R 4', R 5And R 5' be the member who independently is selected from down group: the Heterocyclylalkyl of the heteroaryl of the aryl of the alkyl of H, replacement, unsubstituted alkyl, replacement, unsubstituted aryl, replacement, unsubstituted heteroaryl, replacement, unsubstituted Heterocyclylalkyl, halogen, NO 2, NR 15R 16, NC (O) R 15, OC (O) NR 15R 16, OC (O) OR 15, C (O) R 15, SR 15, OR 15, CR 15=NR 16, and O (CH 2) nN (CH 3) 2, or R 4, R 4', R 5And R 5' any adjacent carbon atom to being connected with them be joined together to form have 4 to 6 yuan, replacement or unsubstituted cycloalkyl or Heterocyclylalkyl ring-type system, wherein n is from 1 to 20 integer; R 15And R 16Be independently selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl, replacement or unsubstituted Heterocyclylalkyl and replacement or unsubstituted peptidyl, wherein R 15And R 16Can randomly be connected to form replacement or unsubstituted Heterocyclylalkyl ring-type system with the nitrogen-atoms that they connected, this ring bodies cording has 4 to 6 yuan, can randomly comprise two or more heteroatomss; R wherein 4, R 4', R 5And R 5' at least one described medicine is connected to L 1In (if existence), or be connected to X 2On, and be selected from:
Figure A20078005123101571
R wherein 30, R 30', R 31And R 31' be selected from H, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted heteroaryl and replacement or unsubstituted Heterocyclylalkyl independently of one another; And v is from 1 to 6 integer.
The example of the suitable connector sheared comprise β-AlaLeuAlaLeu (SEQ ID NO:92) and
Figure A20078005123101572
Pharmaceutical composition
On the other hand, the disclosure provides composition, pharmaceutical composition for example, and said composition comprises a kind of monoclonal antibody of the present invention or its antigen-binding portion thereof, or it is the combination of multiple monoclonal antibody or their antigen-binding portion thereof, and formulated jointly with pharmaceutically acceptable carrier.This composition can comprise or its combination (for example, two or more are different) of antibody of the present invention or immunoconjugates or bispecific molecule.For example, pharmaceutical composition of the present invention can comprise the combination of antibody (or immunoconjugates or bispecific molecule), the different epi-positions on these antibody (or immunoconjugates or bispecific molecule) and the target antigen in conjunction with or have a complementary activity.
Can also in combination therapy, use pharmaceutical composition of the present invention (that is, with other agent combination).For example, this conjoint therapy can comprise of the present invention resisting-CD70 antibody, this anti--CD70 antibody and at least a other carcinostatic agents, antiphlogistic or immunosuppressor combination.Below the example of the therapeutical agent that can be used for combination therapy has been carried out more detailed explanation of the present invention in about the chapters and sections of antibody purposes.
Comprise solvent compatible on any and whole physiology, dispersion medium, dressing, antibacterial agent and anti-mycotic agent, isotonic agent and absorb delayer etc. as " the pharmaceutically acceptable carrier " that uses herein.Preferably, this carrier is suitable for intravenously, intramuscular, subcutaneous, parenteral, spinal cord or epidermis administration (for example by injection or infusion).According to route of administration, active compound (being antibody, immunoconjugates or bispecific molecule) available materials bag may be caused the acid of this compound inactivation and the effect of other natural condition to protect this compound to avoid.
Medical compounds of the present invention can comprise one or more pharmacy acceptable salts." pharmacy acceptable salt " is meant that the desirable biological activity that has kept parent compound can't cause that (referring to for example Berge, S.M. waits people (1977) J.Pharm.Sci. for the salt of any undesirable toxicological effect 66: 1-19).The example of this salt comprises acid salt and base addition salt.Acid salt comprises the salt that is derived from nontoxic mineral acid (for example hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, Hydrogen bromide, hydroiodic acid HI, phosphoric acid etc.), and the salt that is derived from non-toxic organic acid (for example paraffinic acid, hydroxyl alkane acid, aromatic acid, aliphatics and the aromatic sulphonic acid etc. of aliphatic monocarboxylic acid and dicarboxylic acid, phenyl replacement).The alkalescence additive salt comprises the salt that is derived from alkaline-earth metal, the salt of sodium, potassium, magnesium and calcium etc. for example, and the salt that is derived from non-toxic organic amine, N for example, N '-dibenzyl-ethylenediamin, N-methylglucosamine, chloroprocaine, choline, diethanolamine, quadrol and PROCAINE HCL, PHARMA GRADE etc.
Pharmaceutical composition of the present invention also can comprise pharmaceutically acceptable antioxidant.The example of pharmaceutically acceptable antioxidant comprises: (1) water soluble antioxidant, for example xitix, cysteine hydrochloride, sodium pyrosulfate, sodium metabisulfite, S-WAT etc.; (2) oil-soluble inhibitor, for example Quicifal, butylated hydroxy anisole (BHA), butylhydroxy toluene (BHT), Yelkin TTS, Tenox PG and alpha-tocopherol etc.; And (3) metal chelator, for example citric acid, ethylenediamine tetraacetic acid (EDTA) (EDTA), sorbyl alcohol, tartrate and phosphoric acid etc.
The suitable water-based that can adopt in pharmaceutical composition of the present disclosure and the example of non-aqueous carrier comprise water, ethanol, polyvalent alcohol (as glycerine, propylene glycol and polyoxyethylene glycol etc.), and their suitable mixture, vegetables oil, as sweet oil, and injectable organic ester, for example ethyl oleate.For example by use coating material (as Yelkin TTS), by keeping in the dispersed system required particulate size and, can keeping suitable flowability by using tensio-active agent.
These compositions also can comprise adjuvant, as sanitas, wetting agent, emulsifying agent and dispersion agent.Can pass through sterilizing program, and by adding the existence that different antibacterial agents and anti-mycotic agent (for example p-Hydroxybenzoate, butylene-chlorohydrin, phenol Sorbic Acid etc.) all can guarantee to prevent microorganism.Isotonic agent (as sugar and sodium-chlor etc.) comprised to enter in these compositions also may be to make us wishing.In addition, can realize that by comprising the material (as aluminum monostearate and gelatin) that postpones to absorb the prolongation of injectable drug form absorbs.
Pharmaceutically acceptable carrier comprises aseptic aqueous solution or dispersion liquid and is used for aseptic injectable solution or the sterilized powder of the instant preparation of dispersion liquid.Being used for this class medium of pharmaceutically active substances and the use of reagent is well known in the art.Except with inconsistent any conventional media of active compound or reagent, can consider that it uses in multiple pharmaceutical compositions of the present disclosure.Can also in these compositions, mix complementary active compound.
Therapeutic composition generally must be aseptic and be stable under manufacturing and storage condition.Said composition can be formulated into solution, microemulsion, liposome or other are suitable for the ordered structure of high drug level.This carrier can be solvent or dispersion medium, for example comprises water, ethanol, polyvalent alcohol (as glycerine, propylene glycol, and liquid macrogol etc.), and their suitable mixture.For example, by use dressing (as Yelkin TTS), by keeping in the dispersed system required particulate size and by using tensio-active agent can keep suitable flowability.In many cases, preferably in composition, comprise etc. and to ooze reagent, for example sugar, polyvalent alcohol (as N.F,USP MANNITOL, sorbyl alcohol), or sodium-chlor.Can realize that by in composition, comprising the reagent (for example Monostearate and gelatin) that postpones to absorb the prolongation of Injectable composition absorbs.By in appropriate solvent, a kind of or combination (as required) in the active compound of aequum and the above various ingredients of enumerating being admixed mutually, being carried out aseptic micro-filtration then and can prepare aseptic parenteral solution.Generally, prepare dispersion by active compound being incorporated into sterile carrier (it comprises basic dispersion medium and above-mentioned other required components of enumerating).For the situation of the sterilized powder that is used to prepare aseptic parenteral solution, preferred manufacturing procedure is vacuum-drying and lyophilize (freeze-drying), makes the powder of component of other hope of this active ingredient and any solution from its sterile filtration in advance.
To change along with the experimenter who is receiving treatment and concrete administering mode with the value of the active ingredient that produces single formulation with carrier substance combination.With carrier substance combination generally be the amount that produces the composition of result of treatment with the amount of the active ingredient that produces single formulation.Generally speaking, in 100% quantity, with the amount of the active ingredient of pharmaceutically acceptable carrier combinations approximately be from 0.01% to about 99% scope, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%.
Dosage is regulated so that best ideal reaction (as therapeutic response) to be provided.For example, can give the single bolus, can use broken dose several times within a certain period of time, perhaps according to reducing or increase dosage shown in the urgency level of treatment situation in proportion.To be mixed with parenteral composition particularly favourable for the unified unit form according to dosage of convenient drug administration and dosage.The dosage unit form of Shi Yonging refers to be used for the dispersive unit physically of experimenter's single dose to be treated herein; Each unit comprises the active compound of the predetermined amount of the generation ideal curative effect that can combine with required pharmaceutical carriers as calculated.The explanation of dosage unit form of the present disclosure is by the decision of following factor and directly depend on following factor: (a) unique property of active compound and specific therapeutical to be achieved, and (b) inherent limitation in the field of this active compound that is used for the treatment of individual sensitivity of preparation.
For the administration of this antibody, dosage is about 0.0001 to 100mg/kg, and more frequent be 0.01 to 25mg/kg host's body weight.For example, dosage can be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight or 10mg/kg body weight or in 1 to 10mg/kg scope.Higher dosage, 15mg/kg body weight for example, 20mg/kg body weight or 25mg/kg body weight can be used on demand.A kind of exemplary treatment scheme is defined as weekly and is administered once, whenever biweekly, per three weeks once, every around once, every month once, every three months once or per 3 to 6 months once.The present invention is anti--and the preferred dosage regimen of CD70 antibody comprises that by intravenous administration 1mg/kg body weight or 3mg/kg body weight use one of following dosage to give antibody: (i) per 4 weeks give 6 dosage, administration in per afterwards 3 months; (ii) per three all administrations; (iii) 3mg/kg body weight single administration is followed per three weeks by the administration of 1mg/kg body weight.
In certain methods, that two or more have the present invention of different binding specificities is anti--and the CD70 monoclonal antibody is by the while administration, and in this case, the dosage of the every kind of antibody that is given is all within indicated scope.Usually give antibody at a plurality of time points.Interval between the single dosage can be, for example a week, one month, every three months or 1 year.Also can be irregular at interval, as indicated by the blood levels of measuring the patient at the antibody of target antigen.In certain methods, it approximately is 1 to 1000 μ g/ml that adjustment dosage makes plasma antibody concentration, and approximately is 25 to 300 μ g/ml in certain methods.
Alternatively, antibody can be used as the sustained release preparation administration, in this case need be with the lower frequency administration.Dosage and frequency become the patient with antibody the intravital transformation period.Generally speaking, the transformation period of human antibodies is the longest, secondly is humanized antibody, chimeric antibody and non-human antibody.The dosage of administration and frequency can be preventative or curative the variations according to treatment.When prophylactic application, in very long for some time, give few relatively dosage with relative more not frequent interval.Some patients are treated in its remaining years relaying continued access.When curative application, need in relatively short interval, give higher relatively dosage sometimes and extenuate or stop, and preferably show the partially or completely improvement of disease symptoms until the patient until progression of disease.Afterwards, this patient can accept preventative dosage regimen.
For being used for preventing and/or treating with the unusual diseases associated of cell proliferation, the circulation composition of institute's administered compound is preferably about 0.001 μ M to 20 μ M, and preferably approximately 0.01 μ M is to 5 μ M.
Herein patient's oral dosage of Shuo Ming compound generally be from about 1mg/ days to about 10, in 000mg/ days the scope, more typically from about 10mg/ days to about 1,000mg/ days, and the most typically from about 50mg/ days to about 500mg/ days.Described according to weight in patients, typical dosage is in about 0.01 to about 150mg/kg/ day scope, more typically from about 0.1 to about 15mg/kg/ day, and the most typically from about 1 to about 10mg/kg/ day, for example 5mg/kg/ days or 3mg/kg/ days.
In at least some embodiments, patient's retardance or the dosage that suppresses tumor growth can be 1 μ mol/kg/ days or still less.For example, patient's dosage can be 0.9,0.8,0.7,0.6,0.5,0.45,0.3,0.2,0.15,0.1,0.09,0.08,0.07,0.06,0.05,0.04,0.03,0.02,0.01 or 0.005 μ mol/kg or littler (mole that refers to medicine).Preferably, antibody-drug conjugates is when during with the per daily dose administration, stoped the growth of tumour cell at least 5 days time.In at least some embodiments, this tumour is the tumour of the intravital people's type of SCID mouse.As an example, the SICD mouse can be CB17.SCID mouse (can be from Taconic, Germantown, NY acquisition).
Can change the actual dose level of the activeconstituents in the pharmaceutical composition of the present invention, to obtain and to the amount of the nontoxic activeconstituents of patient at the desirable therapeutic response of the effective realization of particular patient, composition and administering mode.Selected dosage level will depend on multiple pharmacokinetics factor, the activity that comprises particular composition of the present invention or its ester, salt or the acid amides of employing, route of administration, administration time, the discharge rate of used specific compound, the course of treatment, the other drug, compound and/or the material that in making up, use with the particular composition that is adopted, the patient's age of receiving treatment, sex, body weight, state, overall health and medical history, and in the well-known similar factor of medical field.
" the treatment effective dose " of anti-CD70 antibody of the present invention preferably cause palliating a disease symptom severity, prolong frequency and the time length of no disease symptoms time or prevent because the infringement or the deformity of this slight illness generation.For example, for the treatment of CD70 positive tumor, with respect to untreated experimenter, " treatment effective dose " preferred cell growth inhibiting or tumor growth are at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%.Compound suppresses the ability of tumor growth and can estimate in the animal model system of predict human tumor function.Alternatively, this characteristic of composition can be estimated by checking the cytostatic ability of this compound, and this restraining effect can be by the known measuring method of experienced practitioner at external test.The treatment significant quantity of therapeutic compound can reduce the size of tumour, perhaps alleviates experimenter's symptom.Those skilled in the art should be able to determine this value according to experimenter's size, the severity and the factors such as selected concrete composition or selected route of administration of experimenter's symptom.
Use one or more methods known in the art can carry out administration to composition of the present invention by one or more route of administration.Be to be understood that as the technician route of administration and/or mode will change with desirable result.The preferred route of administering of antibody of the present invention comprises intravenously, intramuscular, intracutaneous, intraperitoneal, subcutaneous, spinal cord or other parenteral admin approach, for example by injection or infusion.The phrase of Shi Yonging " parenteral admin " is meant in intestines and topical is other administering modes by injection usually herein, and include but not limited in intravenously, intramuscular, intra-arterial, the sheath, capsule is interior, socket of the eye is interior, intracardiac, intracutaneous, intraperitoneal, under tracheae, subcutaneous, epidermis, under the intraarticular, capsule, in the subarachnoid space, backbone, epidural and breastbone inner injection and infusion.
Alternatively, antibody of the present invention can carry out administration through non-parenteral approach, and non-parenteral approach is for example local, epidermis or mucosal route administration, and for example intranasal is interior, oral, vagina, rectum, hypogloeeis or topical routes.
Active compound available support preparation, this carrier will protect compound to avoid snap-out release, and controlled release preparation for example comprises implant, through skin patch and microencapsulation delivery system.Can use polymkeric substance biodegradable, biocompatibility, for example ethane-acetic acid ethyenyl ester, polyanhydride, polyglycolic acid, collagen, poe and poly(lactic acid).The many methods that are used to prepare this preparation patent, or well-known to those skilled in the art.Referring to, Sustained and ControlledRelease Drug Delivery Systems for example, J.R.Robinson writes, Marcel Dekker, Inc., New York, 1978.
Therapeutic composition can utilize medical apparatus as known in the art to carry out administration.For example, in preferred embodiments, therapeutic composition of the present invention can utilize the needleless hypodermic injection unit to carry out administration, for example at U.S. Patent number 5,399,163,5,383,851,5,312,335,5,064,413,4,941,880,4,790,824 or 4,596, the device disclosed in 556.The useful in the present invention implant of knowing and the example of model comprise: U.S. Patent number 4,487,603, and it has disclosed the implantable trace infusion pump that is used for discharging with controlled velocity medicine; U.S. Patent number 4,486,194, it has disclosed the therapeutic system that is used for by percutaneous drug delivery; U.S. Patent number 4,447,233, it has disclosed the medication infusion pump that is used for accurate infusion velocity delivering drugs; U.S. Patent number 4,447,224, it has disclosed the implantable infusion device that is used for continuing the variable flow rate that medicine sends; U.S. Patent number 4,439,196, it has disclosed the osmotic drug with multi-cavity compartment and has sent delivery system; And U.S. Patent number 4,475,196, it has disclosed osmotic drug and has sent delivery system.These patents are incorporated this paper by reference into.Many other this implants, delivery system and model are well known by persons skilled in the art.
In certain embodiments, human monoclonal antibody of the present invention is prepared to guarantee that it distributes in vivo suitable.For example, hemato encephalic barrier (BBB) has stoped many high-hydrophilic compounds.For guaranteeing that therapeutic compound of the present invention passes BBB (time) if desired, for example can be formulated in them in the liposome.Be used to make the method for liposome, referring to, for example, U.S. Patent number 4,522,811; 5,374,548; And 5,399,331.These liposomes may comprise one or more parts, and these parts optionally are transported in the specific cell or organ, strengthen thus the sending of target agent (referring to, V.V.Ranade J.Clin.Pharmacol.29:685 for example, (1989)).Exemplary targeting moiety comprises folate or vitamin H (referring to, people's such as Low U.S. Patent number 5,416,016 for example); Mannoside (people such as Umezawa, (1988) Biochem.Biophys.Res.Commun. 153: 1038); Antibody (people (1995) FEBS Lett. such as P.G.Bloeman 357: 140; People such as M.Owais (1995) Antimicrob.Agents Chemother. 39: 180); Tensio-active agent albumin A acceptor (people (1995) Am.J.Physiol. such as Briscoe 1233: 134); P120 (people (1994) J.Biol.Chem. such as Schreier 269: 9090); Also referring to K.Keinanen; M.L.Laukkanen (1994) FEBS Lett. 346: 123; J.J.Killion; I.J.Fidler (1994) Immunomethods 4: 273.
Purposes of the present invention and method
Antibody of the present invention, particularly human antibodies, antibody compositions, antibody-mating partner molecule conjugate compositions and method have multiple external and the in-vivo diagnostic and the therepic use of the illness that relates to diagnosis and treatment CD70 mediation.For example, can these molecules be given to cells in culture, perhaps give the human experimenter (for example in the body), so that treatment, prevention and diagnosis various disease conditions external or stripped.Term " experimenter " is intended to comprise the mankind and non-human animal as used in this." non-human animal " comprises all vertebratess, for example Mammals and nonmammalian, for example, non-human primate, sheep, dog, cat, ox, horse, chicken, Amphibians and Reptilia.Preferred experimenter comprises the human patients of suffering from by the illness of the active mediation of CD70.These methods are suitable for treating the human patients of suffering from the illness relevant with the CD70 unconventionality expression especially.When with The CD70 link coupledAntibody-mating partner molecule conjugate is during with another kind of reagent administration, and these two kinds of materials can be successively or administration simultaneously.
Under the situation of antibody specific combination CD70 of the present invention, antibody of the present invention is used in the expression that cell surface detects CD70 specifically, and, can be used for coming purifying CD70 by immunoaffinity purification.
CD70 is expressed in the various human cancers, comprises renal cell carcinoma, transitivity breast cancer, cerebral tumor, leukemia, lymphoma and nasopharyngeal carcinoma (people (2005) J Urol. such as Junker 173: 2150-3; People such as Sloan (2004) Am J Pathol. 164: 315-23; Held-Feindt and Mentlein (2002) Int JCancer 98: 352-6; People such as Hishima (2000) Am J Surg Pathol. 24: 742-6; People such as Lens (1999) Br J Haematol. 106: 491-503).Anti--CD70 antibody can use separately to suppress the growth of cancerous tumour.Alternately, following explanation, anti--CD70 antibody can use with other immunogenicity reagent, standard cancer treatments or other antibodies.
Preferred cancer (its growth can use antibody of the present invention to suppress) comprises the cancer that generally immunotherapy is responded.The limiting examples of the preferred cancer that is used for the treatment of comprises kidney (for example renal cell carcinoma), breast cancer, cerebral tumor, chronic or acute leukemia (comprising acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytoblast leukemia, chronic lymphocytoblast leukemia), lymphoma (for example, hodgkin's and non-Hodgkin lymphomas, lymphocytic lymphoma, primary CNS lymphoma, t cell lymphoma) and nasopharyngeal carcinoma.Use the example of medicable other cancers of method of the present invention to comprise: melanoma (for example, metastatic malignant melanoma), prostate cancer, colorectal carcinoma, lung cancer, osteocarcinoma, carcinoma of the pancreas, skin carcinoma, head and neck cancer, skin or intraocular malignant melanoma, uterus carcinoma, ovarian cancer, the rectum cancer, the anal field cancer, cancer of the stomach, carcinoma of testis, uterus carcinoma, carcinoma of fallopian tube, carcinoma of endometrium, cervical cancer, carcinoma of vagina, the vaginal orifice cancer, esophagus cancer, carcinoma of small intestine, the endocrine system cancer, thyroid carcinoma, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, urethral carcinoma, penile cancer, children's solid tumor, bladder cancer, kidney or carcinoma of ureter, carcinoma of renal pelvis (carcinoma of the renalpelvis), central nervous system (CNS) knurl, tumor vessel takes place, tumor of spine, brain stem glioma, pituitary adenoma, Kaposi, the epiderm-like cancer, squamous cell carcinoma, the combination of environmental induction cancer (comprising that those are by asbestos inductive cancer, for example mesothelioma) and described cancer.
In addition, expressing on the different tumour cells under the situation of CD70, human antibodies of the present invention, antibody compositions and method can be used for treating the experimenter who suffers from the tumorigenicity illness, this tumorigenicity illness for example is to be the illness of feature there to be the tumour cell of expressing CD70, for example comprise, renal cell carcinoma (RCC) is as hyaline cell RCC, glioblastoma, breast cancer, cerebral tumor, nasopharyngeal carcinoma, non-Hodgkin lymphomas (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma, primary cutaneous type (anaplastic large-celllymphomas) (ALCL), multiple myeloma, T-cell lymphoma,cutaneous, nodositas micromere lymphoma, lymphocytic lymphoma, lymphoma peripheral T cell, lennert lymphoma, immunoblastic lymphoma, T chronic myeloid leukemia/lymphoma (ATLL), adult T cell leukemia (T-ALL), center parent cell/centrocyte (entroblastic/centrocytic) is (cb/cc)) the follicular lymphoma cancer, the cytophyletic diffuse type maxicell of B lymphoma, angioimmunoblastic lymphadenopathy (AILD) sample t cell lymphoma, the lymphoma that HIV is relevant based on body cavity, embryonal carcinoma, undifferentiated nasopharyngeal carcinoma (for example schmincke tumor), the Ka Siermanshi disease, Kaposi, multiple myeloma, Walden Si Telunshi macroglobulinemia and other B cell lymphomas.
Therefore, in one embodiment, the invention provides a kind of method that in subject, suppresses growth of tumour cell, comprise anti--CD70 antibody or its antigen-binding portion thereof of this experimenter being treated significant quantity.Preferably, this antibody is human resisting-CD70 antibody (any antibody in the human as described in this Anti-Human's class of the example CD70 antibody).Additionally or alternately, this antibody may be chimeric or humanized anti--CD70 antibody.
In addition, the interaction that has also proposed CD70 and CD27 plays a role in cell-mediated autoimmune disease, as experiment systemic autoimmune encephalomyelitis (EAE) (people (2000) J.Neuroimmunol. such as Nakajima 109: 188-96).It is to be mediated by the inhibition that TNF-α generates that this effect is considered to part.In addition, the blocking-up of CD70 signal conduction suppressed the CD40 mediation the CD8 positive T cell clone's property propagation and reduce generation (people (2004) J.Immunol. such as Taraban of the positive memory T cell of CD8 173: 6542-6).So, human antibodies of the present invention, antibody compositions and method can be used for treating the experimenter with autoimmunization obstacle, are the illness of feature there to be the B cell of expressing CD70 for example, for example comprise experimental autoimmunization encephalomyelitis.Can use other autoimmunization obstacle of antibody of the present invention to comprise, but be not limited to systemic lupus erythematous (SLE), insulin-dependent diabetes mellitus (IDDM), inflammatory bowel (IBD) (comprising Crohn's disease, ulcerative colitis and celiac disease), multiple sclerosis (MS), psoriasis, autoimmune thyroiditis, rheumatoid arthritis (RA) and glomerulonephritis.In addition, antibody of the present invention can be used for suppressing or stops transplant rejection reaction or treatment graft versus host disease (GVH disease) (GVHD).
In addition, play a role in the signal conduction of the interaction that has also proposed CD70 and CD27 on the CD4 positive cell.Show some viruses for the CD27 approach sends signal, caused the destruction that neutralizing antibody reacts (people (2006) J Exp Med such as Matter 203: 2145-55).So, human antibodies of the present invention, antibody compositions and method can be used for treating the experimenter of virus infection, comprise, for example, infect human immunodeficiency virus (HIV), hepatitis (first type, B-mode and third type), simplexvirus is (as VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, Echo virus, rhinovirus, Coxsackie virus, coronavirus (cornovirus), respiratory syncytial virus, mumps virus, rotavirus, Measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue fever virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus and entomophila property encephalitis and lymphocytic choriomeningitis virus (LCMV), or be used for the treatment of HIV infection/AIDS.In addition, human antibodies of the present invention, antibody compositions and method can be used for suppressing the generation of TNF-α.
In one embodiment, antibody of the present invention (for example, human monoclonal antibody, polyspecific and bispecific molecule and composition) can be used for detecting the level of CD70 or contain the level of the cell of CD70 at surface of cell membrane, these levels can be associated with some disease symptoms afterwards.Alternately, antibody can be used for suppressing or blocking the function of CD70, this then again can with prevent from or alleviate some disease symptoms to be associated, hinted the mediators of CD70 thus for this disease.Allowing to form between antibody and the CD70 under the condition of mixture, this can contact and realize with anti--CD70 antibody by making experiment sample and check sample.In experiment sample and check sample, detect between antibody and the CD70 formed alloy and compare.
In another embodiment, can at first test antibody of the present invention (for example, human antibodies, polyspecific and bispecific molecule and composition) the combination activity relevant with external treatment or diagnostic uses.For example, use the fluidic cell that illustrates in following examples to measure and to test composition of the present invention.
Antibody of the present invention (for example, human antibodies, polyspecific and bispecific molecule, immune conjugate and composition) has in the treatment of CD70 relative disease and other purposes aspect the diagnosis.For example, human monoclonal antibody, polyspecific molecule or bispecific molecule, and immune conjugate can be used in vivo or externally excite one or more following biologic activity: suppress to express CD70 cell growth and/or kill the cell of expressing CD70; The phagolysis or the ADCC of the cell of CD70 expressed in mediation when human effector cell exists; Or the combining of blocking-up CD70 part and CD70.
In a specific embodiment, this antibody (for example, human antibodies, polyspecific and bispecific molecule and composition) is used for interior therapeutic, prevention or diagnoses the relevant disease of multiple CD70.The example of the disease that CD70 is relevant also comprises except other, the autoimmunization obstacle, experimental autoimmunization encephalomyelitis (EAE), cancer, renal cell carcinoma (RCC), as hyaline cell RCC, glioblastoma multiforme, breast cancer, cerebral tumor, nasopharyngeal carcinoma, the non-Hodgkin lymphomas, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma, primary cutaneous type (ALCL), multiple myeloma, T-cell lymphoma,cutaneous, nodositas micromere lymphoma, the lymphocyte lymphoma, lymphoma peripheral T cell, lennert lymphoma, immunoblastic lymphoma, T chronic myeloid leukemia/lymphoma (ATLL), adult T cell leukemia (T-ALL), center parent cell/centrocyte (cb/cc) follicular lymphoma cancer, the cytophyletic diffuse type large celllymphoma of B, angio-immunoblastic lymphadenopathy (AILD) sample t cell lymphoma, the lymphoma that HIV is relevant based on body cavity, embryonal carcinoma, undifferentiated type nasopharyngeal carcinoma (for example, schmincke tumor), the Ka Siermanshi disease, Kaposi, multiple myeloma, Walden Si Telunshi macroglobulinemia and other B cell lymphomas.
In vivo or the external suitable route that gives antibody compositions of the present invention (for example human monoclonal antibody, polyspecific and bispecific molecule and immune conjugate) be known in the art, and can select by those those of ordinary skill.For example, antibody compositions can carry out administration by injection (as intravenously or subcutaneous).The optimal dose of used molecule will depend on experimenter's the age and the concentration and/or the prescription of body weight and antibody compositions.
As described above, the mankind of the present invention are anti--CD70 antibody can with one or more therapeutical agents (for example cytotoxic agent, radioactivity toxic agent or immunosuppressor) co-administered.Antibody can be connected with reagent (as immunocomplex) and administration, perhaps can with this reagent separate administration.Under a kind of (separate administration) situation of back, antibody can be before this reagent, give afterwards or give simultaneously with this reagent or can carry out co-administered with other known treatment (for example anticancer therapy, as radiation).Among other things, this type of therapeutical agent also comprises antineoplastic agent such as Dx (Zorubicin), cis-platinum bleomycin sulfate, carmustine, Chlorambucil and endoxan hydroxyurea, they self only effective when or subtoxic level toxic to the patient.Cis-platinum is with 100mg/ agent intravenous administration, every around once, and Zorubicin is with 60 to 75mg/ml dosage intravenous administration, per 21 days once.The mankind of the present invention are anti--and the co-administered of CD70 antibody or their Fab and chemotherapeutic provides two kinds of carcinostatic agents, and these two kinds of carcinostatic agents work by different mechanism, and generation is at human tumor cell's cellulotoxic effect.This co-administered can solve because tumour cell produces the problem of drug resistance or antigenic change aspect, develops immunity to drugs or antigenic change can cause tumour cell and antibody not to react.
Target specific effector cell of the present invention, for example, with composition (for example, human antibodies, polyspecific molecule and bispecific molecule) continuous effector cell, also useful as therapeutics.The effector cell who is used for target can be a human leucocyte, for example, and scavenger cell, neutrophilic granulocyte or monocyte.Other cells comprise that eosinophilic granulocyte, natural killer cell and other have the cell of IgG-or IgA-acceptor.If desired, the effector cell can obtain in subject to be treated.The target-specific effector cell can be used as that the cell suspension in the acceptable solution carries out administration on physiology.The cell count that gives can be 10 8To 10 9The order of magnitude, but can change to some extent according to therapeutic purpose.Usually, this value is enough to obtain in the location of target cell (for example, expressing the tumour cell of CD70), and realizes cell killing by (for example phagolysis).Route of administration also can change.
Target-specific effector cell's therapy can be carried out with other technical tie-ups of removing target cell.For example, the antitumor therapy that uses composition of the present invention (for example, human antibodies, polyspecific molecule and bispecific molecule) and/or have an effector cell of these compositions can be united use with chemotherapy.In addition, the combination immunotherapy can be used for instructing the repulsive interaction of two kinds of different cytotoxic effect groups to tumour cell.For example, the anti--CD70 antibody that links to each other with anti-Fc γ RI or anti--CD3 can be united use with IgG-or IgA-receptor-specific wedding agent.
Bispecific molecule of the present invention and polyspecific molecule also can be used for regulating Fc γ R or the Fc γ R level on the effector cell, for example carry out capping and removing by the lip-deep acceptor of pair cell.The mixture of anti-FC receptor also can be used for this purpose.
Composition of the present invention (for example human antibodies, polyspecific molecule and bispecific molecule and immune conjugate) also can use existing under the situation of complement, said composition has the complement binding site, for example from the part of IgG1, IgG2 or the IgG3 or the IgM of conjugated complement.In one embodiment, the stripped treatment of cell mass (comprising target cell with binding reagents of the present invention and suitable effector cell) can replenish by the serum that adds complement or contain complement.The phagolysis that is coated with the target cell of binding reagents of the present invention can be improved by the combination of complement protein.In another embodiment, being coated with the target cell of composition of the present invention (for example human antibodies, polyspecific and bispecific molecule) also can be by the complement cracking.In another embodiment, composition of the present invention is activating complement not.
Composition of the present invention (for example human antibodies, polyspecific molecule and bispecific molecule and immune conjugate) also can give with complement.Therefore, the composition that comprises human antibodies, polyspecific or bispecific molecule and serum or complement within the scope of the invention.These compositions are favourable, because complement is positioned at very the position near human antibodies, polyspecific or bispecific molecule.Alternately, human antibodies of the present invention, polyspecific or bispecific molecule can with complement or serum separate administration.
Equally within the scope of the present invention be the test kit that comprises antibody compositions of the present invention (for example human antibodies, dual specific or polyspecific molecule or immune conjugate) and working instructions.This test kit may further include one or more other medicament, for example immunosuppression reagent, cytotoxin reagent or radiotoxin reagent or one or more of the present invention other human antibodies (for example, human antibodies with complementary activity, this antibody combines with epi-position in the CD70 antigen that is different from the first antibody-like).
Therefore, can additionally be given another kind of therapeutical agent by (before giving human antibodies of the present invention, simultaneously or afterwards) with the patient of antibody compositions of the present invention treatment, for example cytotoxic agent or radioactivity toxic agent, this can strengthen or increase the result of treatment of this human antibodies.
In other embodiments, can treat the experimenter with reagent extraly, this reagent is used the cytokine therapy experimenter by (for example), regulates (for example, strengthening or inhibition) Fc γ or Fc γ receptor expression or activity.The preferred cytokine that gives in polyspecific molecular therapy process comprises granulocyte colony-stimulating factor (G-CSF), rHuGM-CSF (GM-CSF), IFN-(IFN-γ) and tumour necrosis factor (TNF).
Composition of the present invention (for example human antibodies, polyspecific and bispecific molecule) also can be used for the cell of targeted expression Fc γ R or CD70, for example for this type of cell of mark.For this purposes, can with this wedding agent with can be connected by detected molecule.Therefore, the invention provides the method for the cell of stripped or external localization and expression Fc acceptor (as Fc γ R or CD70).This detectable mark can be, for example, and radio isotope, fluorescent chemicals, enzyme or enzyme cofactor.
In a specific embodiment, the invention provides and be used for detecting CD70 antigen that is present in sample or the method for measuring the antigenic amount of CD70, these methods comprise: sample and check sample are contacted with human monoclonal antibody or its antigen-binding portion thereof, and this antibody or its antigen-binding portion thereof can form between they and CD70 under the condition of mixture and be attached on the CD70 specifically.Detect the formation of mixture then, wherein compare with check sample, the difference mixture that forms in this sample has been indicated and had CD70 antigen in this sample.
In another embodiment again, immune conjugate of the present invention with compound (for example can be used for, therapeutical agent, marker, cytotoxin, radiotoxin, immunosuppressor etc.) be targeted on the cell with CD70 cell surface receptor, this application is undertaken by this compounds is connected to this antibody.For example, anti--CD70 antibody can be coupled on any cytotoxin compounds, these cytotoxin compounds are illustrated in U.S. Patent number 6,281,354 and 6,548,530, U.S. serial 60/991,300, in the U.S. Patent Application Publication No. 20030050331,20030064984,20030073852 and 20040087497 or be disclosed among the WO 03/022806, these documents all are incorporated herein by reference.Therefore, the present invention also provides and has exsomatized or the interior method (for example, using detectable mark, as radio isotope, fluorescent chemicals, enzyme or enzyme cofactor) that the cell of expressing CD70 is positioned of body.Alternately, immune conjugate can be used for killing the cell with CD70 cell surface receptor by cytotoxin or radiotoxin are targeted to CD70.
By following example the present invention is further set forth, these examples should not be interpreted as further restriction.The content of institute's drawings attached that the application quotes in the whole text and all reference, Genbank sequence, patent and disclosed patent application all is incorporated herein by reference.
Embodiment
Embodiment 1: at the human monoclonal antibody's of CD70 generation
Antigen
The immunization protocol utilization has been merged the recombinant human CD70 of dual myc-His label as antigen.Alternately, used full cellular immunization in some immunity, this full cellular immunization is used renal carcinoma cell line 786-O (ATCC accession number CRL-1932) and is used renal carcinoma cell line A-498 (ATCC accession number HTB-44) booster immunization.
Genetically modified HuMAb
Figure A20078005123101711
And KM
HCo7, the HCo12 of use HuMab transgenic mice and the KM strain preparation of HCo17 strain and transgenosis transchromosomic mice are at the complete human monoclonal antibody of CD70, every kind of equal express human antibody gene of mouse.In these mouse species, endogenous mouse κ light chain gene is as at people such as Chen (1993) EMBO J. 12: 811-820 explanation is destroyed homozygously, and this endogenous murine heavy chain gene as the quilt that illustrates of the embodiment 1 of PCT open file WO 01/09187 destroy homozygously.In addition, this mouse species carries human κ light chain transgenosis KCo5 (as at people such as Fishwild (1996) Nature Biotechnology 14: 845-851 illustrates) and human heavy chain transgene HCo7, HCo12 or HCo17 (as embodiment 2 explanations of PCT open file WO 01/09187).As explanation among the open text WO 02/43478 of PCT, KM
Figure A20078005123101721
Strain contains the SC20 transfection chromosome.
HuMab and KM immunity:
Be to produce the complete human monoclonal antibody at CD70, the full cell that is used as antigenic recombinant human CD70 or expresses CD70 on cell surface is to HuMab
Figure A20078005123101722
And KM
Figure A20078005123101723
Carry out immunity.The immunization protocol common to the HuMAb mouse is illustrated in Lonberg, people such as N. (1994) Nature 368(6474): 856-859; Fishwild, people such as D. (1996) NatureBiotechnology 14: among 845-851 and the PCT open file WO 98/24884.For the first time mouse was 6 to 16 ages in week during infusion antigen.With 5 * 10 6To 10 * 10 6Individual cell passes through intraperitoneal (IP), subcutaneous (Sc) to the HuMab mouse or fills up injection by foot and carry out immunity.
Through IP immunity twice, the antigen in too many or too much for use in 3 to 21 days then full freund's adjuvant or the Ribi adjuvant is through IP immunity (amounting to 11 immunity at the most) with the antigen in complete Freund's adjuvant or the Ribi adjuvant for transgenic mice.By getting blood monitoring immunne response behind the socket of the eye.By ELISA and FACS (following explanation) screening blood plasma, and the anti--CD70 human immunoglobulin mouse that will have enough titres is used for merging.The mouse booster immunization is put to death mouse and removed spleen after 3 days through intravenously with antigen.Typically, every kind of antigen being carried out 10 to 35 times merges.Every kind of all immune tens mouse of antigen.
Produce the HuMab of anti--CD70 antibody
Figure A20078005123101724
Or KM
Figure A20078005123101725
Selection:
In order to select the HuMab that can produce in conjunction with the antibody of CD70
Figure A20078005123101726
Or KM
Figure A20078005123101727
Screen by the serum of flow cytometry, select with the clone of the human CD70 of express recombinant and combine, and be not bonded serum with the control cells of not expressing CD70 to the mice immunized of hanging oneself.In addition, filter out and 786-O or A-498 cell bonded serum by flow cytometry.In brief, Chinese hamster ovary celI, 786-O cell or the A498 cell by will expressing CD70 with hatch the combination of having assessed anti--CD70 antibody mutually with anti--CD70 antibody of 1: 20 dilution proportion.Washed cell, and detected combination with Anti-Human's class IgG Ab of FITC mark.(Becton Dickinson, San Jose CA) have carried out flow cytometry to use the FACSCalibur flow cytometer.As Fishwild, people such as D. (1996) illustrate, combine and are not further tested with combining by ELISA of CD70 with the parental generation Chinese hamster ovary celI bonded antibody of expressing non-CD70 with the Chinese hamster ovary celI of expressing CD70.In brief, with purified recombinant C D70 fusion rotein bag in the PBS solution, Chinese hamster ovary celI 1-2 μ g/ml, that hang oneself transfection by microtiter plate, 5% chicken serum sealing 200 μ l/ holes, in the PBS/ tween (0.05%) 4 ℃ of overnight incubation, is used then in 100 μ l/ holes.The diluent of the serum of the CD70 mice immunized of hanging oneself in the future adds to every hole, and hatches 1 to 2 hour under environment thing temperature.With PBS/ tween clean plate, and then at room temperature hatched 1 hour with the anti-IgG polyclonal antibody of the goat of coupling horseradish peroxidase (HRP).After the washing, (Sigma, A-1888 0.22mg/ml) develop the color to plate, and analyze at OD 415-495 place with spectrophotometer with the ABTS substrate.To manifest, the mouse of anti--CD70 antibody of high titre is used for merging.Merge as described below, and resisting-the CD70 activity by ELISA test hybridoma supernatant liquor.
Produce the human monoclonal antibody's of anti-CD70 the generation of hybridoma:
Use based on the standard scheme of PEG or use the big chamber of Cyto Pulse cytogamy electroporation apparatus (Cyto Pulse Sciences, Inc., Glen Burnie, the electricity based on electric field MD) merges, will be from HuMab
Figure A20078005123101731
And/or KM
Figure A20078005123101732
Isolated mouse boosting cell and mouse myeloma cell line merge.For the generation of antigen-specific antibodies the hybridoma that produces is screened then.PEG with 50% (Sigma) merges the single cell suspension of splenocyte of the immune mouse of hanging oneself and the SP2/0 nonsecreting type murine myeloma cell (ATCC, CRL 1581) of 1/4th numbers.With cell with about 1 * 10 5/ hole is inoculated on the flat-bottom microtiter plates, in the DMEM high glucose medium, hatch a week afterwards, this substratum contains L-glutaminate and Sodium.alpha.-ketopropionate (Mediatech, Inc., Herndon, VA), and further contain 10% foetal calf serum (Hyclone, Logan, UT), 18% P388DI conditioned medium, 5% the Origen Hybridoma clone factor (BioVeris, Gaithersburg, VA), the L-glutaminate of 4mM, the HEPES of 5mM, 0.055mM beta-mercaptoethanol, the penicillin of 50 units/ml, 50mg/ml Streptomycin sulphate and 1 * xanthoglobulin-aminopterin-induced syndrome-thymidine (HAT) substratum (Sigma; Added HAT in back 24 hours in fusion).After one week, replaced the culture medium culturing cell of HAT with HT.Resist-each hole of CD70 mono-clonal IgG antibody screening at the mankind by FACS or ELISA (above explanation) then.After the hybridoma extensive growth, after 10 to 14 days, monitor substratum usually.Hybridoma to secretory antibody is inoculated once more, screening once more, and, if still IgG is positive, then carry out at least twice subclone by limiting dilution antagonism-CD70 monoclonal antibody.Then stable subclone is used for further sign external the cultivation so that produce a small amount of antibody in tissue culture medium (TCM).
Select hybridoma clone 2H5,10B4,8B5,18E7 and 69A7 to be used for further analysis.
Embodiment 2: the structural characterization of human monoclonal antibody 2H5,10B4,8B5,18E7,69A7 and 1F4
Use Standard PC R technology from 2H5,10B4,8B5,18E7,69A7 and 1F4 hybridoma, to obtain the heavy chain of 2H5,10B4,8B5,18E7,69A7 and 1F4 monoclonal antibody and the cDNA sequence that variable region of light chain is encoded respectively, and the dna sequencing technology of the standard of use check order.
The Nucleotide of the variable region of heavy chain of 2H5 and aminoacid sequence are respectively as shown in Figure 1A and SEQ IDNO:49 and SEQ ID NO:1.
The Nucleotide of the variable region of light chain of 2H5 and aminoacid sequence are respectively shown in Figure 1B and SEQ IDNO:55 and SEQ ID NO:7.
The 2H5 heavy chain that relatively proved of 2H5 heavy chain immunoglobulin sequence and known human racial immunity sphaeroprotein sequence of heavy chain has utilized that to plant from the mankind be the VH section of VH 3-30.3, undetermined D section and to plant from the mankind be the JH section of JH4b.2H5VH sequence and kind are that the comparison of VH 3-30.3 sequence is shown in Fig. 7.Use the definite Kabat system in CDR district that the 2H5VH sequence is further analyzed, obtain respectively as Figure 1A and 7 and SEQ ID NO:13,19 and 25 shown in heavy chain CDR1, CDR2 and the synoptic diagram in CDR3 district.
2H5 light chain immunoglobulin sequences and known human racial immunity sphaeroprotein sequence of light chain proved that relatively it is the VL section of VK L6 and to plant from the mankind be the JK section of JK 4 that the 2H5 light chain is used to from human the kind.2H5VL sequence and kind are that the comparison of VK L6 sequence is shown in Figure 11.Use the definite Kabat system in CDR district that the 2H5VL sequence is further analyzed, obtain respectively as Figure 1B and 11 and SEQ ID NO:31,37 and 43 shown in light chain CDR1, CDR2 and the synoptic diagram in CDR3 district.
The Nucleotide of the variable region of heavy chain of 10B4 and aminoacid sequence are shown in respectively in Fig. 2 A and SEQID NO:50 and 2.
The Nucleotide of the variable region of light chain of 10B4 and aminoacid sequence are shown in respectively in Fig. 2 B and SEQ IDNO:56 and 8.
The heavy chain immunoglobulin sequence of 10B4 and known human racial immunity sphaeroprotein sequence of heavy chain relatively proved the 10B4 heavy chain be used to from human plant be VH 3-30.3 the VH section, to plant from the mankind be the D section of 4-11 and to plant from the mankind be the JH section of JH 4b.10B4 VH sequence and kind are that the comparison of VH 3-30.3 sequence is shown in Fig. 7.Use the definite Kabat system in CDR district that the 10B4VH sequence is further analyzed, obtain respectively as Fig. 2 A and 7, and the synoptic diagram in heavy chain CDR1, CDR2 shown in the SEQ ID NO:14,20 and 26 and CDR3 district.
10B4 light chain immunoglobulin sequences and known human racial immunity sphaeroprotein sequence of light chain proved that relatively it is the VL section of VK L18 and to plant from the mankind be the JK section of JK 3 that the 10B4 light chain is used to from human the kind.10B4VL sequence and kind are that the comparison of VK L18 sequence is shown in Figure 12.Use the definite Kabat system in CDR district that the 10B4VL sequence is further analyzed, obtain respectively as Fig. 2 B and 12, and the synoptic diagram in light chain CDR1, CDR2 shown in the SEQ ID NO:32,38 and 44 and CDR3 district.
The Nucleotide of the variable region of heavy chain of 8B5 and aminoacid sequence are shown in respectively in Fig. 3 A and SEQ IDNO:51 and 3.
The Nucleotide of the variable region of light chain of 8B5 and aminoacid sequence are shown in respectively in Fig. 3 B and SEQ IDNO:57 and 9.
The heavy chain immunoglobulin sequence of 8B5 and known human racial immunity sphaeroprotein sequence of heavy chain proved that relatively it is V that the 8B5 heavy chain is used to from human the kind HThe VH section of 3-33, to plant from the mankind be the D section of 3-10 and to plant from the mankind be the JH section of JH 4b.8B5 VH sequence and kind are that the comparison of VH 3-33 sequence is shown in Fig. 8.Use the definite Kabat system in CDR district that the 8B5VH sequence is further analyzed, obtain respectively as Fig. 3 A and 8, and the synoptic diagram in heavy chain CDR1, CDR2 shown in the SEQ ID NO:15,21 and 27 and CDR3 district.
8B5 light chain immunoglobulin sequences and known human racial immunity sphaeroprotein sequence of light chain proved that relatively it is the VL section of VK L15 and to plant from the mankind be the JK section of JK 4 that the 8B5 light chain is used to from human the kind.8B5VL sequence and kind are that the comparison of VK L15 sequence is shown in Figure 13.Use the definite Kabat system in CDR district that the 8B5VL sequence is further analyzed, obtain respectively as Fig. 3 B and 13, and the synoptic diagram in light chain CDR1, CDR2 shown in the SEQ ID NO:33,39 and 45 and CDR3 district.
The Nucleotide of the variable region of heavy chain of 18E7 and aminoacid sequence are shown in Fig. 4 A and SEQ IDNO:52 and 4 respectively.
The Nucleotide of the variable region of light chain of 18E7 and aminoacid sequence are shown in Fig. 4 B and SEQ IDNO:58 and 10 respectively.
The heavy chain immunoglobulin sequence of 18E7 and known human racial immunity sphaeroprotein sequence of heavy chain relatively proved the 18E7 heavy chain be used to from human plant be VH 3-33 the VH section, to plant from the mankind be the D section of 3-10 and to plant from the mankind be the JH section of JH 4b.18E7VH sequence and kind are that the comparison of VH 3-33 sequence is shown in Fig. 8.Use the definite Kabat system in CDR district that the 18E7VH sequence is further analyzed, obtain respectively as Fig. 4 A and 8, and the synoptic diagram in heavy chain CDR1, CDR2 shown in the SEQ ID NO:16,22 and 28 and CDR3 district.
18E7 light chain immunoglobulin sequences and known human racial immunity sphaeroprotein sequence of light chain proved that relatively it is the VL section of VK L15 and to plant from the mankind be the JK section of JK 4 that the 18E7 light chain is used to from human the kind.18E7VL sequence and kind are that the comparison of VK L15 sequence is shown in Figure 13.Use the definite Kabat system in CDR district that the 18E7VL sequence is further analyzed, obtain respectively as Fig. 4 B and 13, and the synoptic diagram in light chain CDR1, CDR2 shown in the SEQ ID NO:34,40 and 46 and CDR3 district.
The Nucleotide of the variable region of heavy chain of 69A7 and aminoacid sequence are respectively shown in Fig. 5 A and SEQ IDNO:53 and 5.
The Nucleotide of the variable region of light chain of 69A7 and aminoacid sequence are respectively shown in Fig. 5 B and SEQ IDNO:59 and 11.
The heavy chain immunoglobulin sequence of 69A7 and known human racial immunity sphaeroprotein sequence of heavy chain proved that relatively it is V that the 69A7 heavy chain is used to from human the kind HThe VH section of 4-61, to plant from the mankind be the D section of 4-23 and to plant from the mankind be the JH section of JH 4b.69A7VH sequence and kind are that the comparison of VH 4-61 sequence is shown in Fig. 9.Use the definite Kabat system in CDR district that the 69A7VH sequence is further analyzed, obtain respectively as Fig. 5 A and 9, and the synoptic diagram in heavy chain CDR1, CDR2 shown in the SEQ ID NO:17,23 and 29 and CDR3 district.
69A7 light chain immunoglobulin sequences and known human racial immunity sphaeroprotein sequence of light chain proved that relatively it is the VL section of VK L6 and to plant from the mankind be the JK section of JK 4 that the 69A7 light chain is used to from human the kind.69A7VL sequence and kind are that the comparison of VK L6 sequence is shown in Figure 14.Use the definite Kabat system in CDR district that the 69A7VL sequence is further analyzed, obtain respectively as Fig. 5 B and 14, and the synoptic diagram in light chain CDR1, CDR2 shown in the SEQ ID NO:35,41 and 47 and CDR3 district.
The Nucleotide of the variable region of heavy chain of 1F4 and aminoacid sequence are respectively shown in Fig. 5 A and SEQ IDNO:54 and 6.
The Nucleotide of the variable region of light chain of 1F4 and aminoacid sequence are respectively shown in Fig. 5 B and SEQ IDNO:60 and 12.
The heavy chain immunoglobulin sequence of 1F4 and known human racial immunity sphaeroprotein sequence of heavy chain relatively proved the 1F4 heavy chain be used to from human plant be VH 3-23 the VH section, to plant from the mankind be the D section of 4-4 and to plant from the mankind be the JH section of JH 4b.1F4VH sequence and kind are that the comparison of VH 3-23 sequence is shown in Figure 10.Use the definite Kabat system in CDR district that the 1F4VH sequence is further analyzed, obtain respectively as Fig. 5 A and 10, and the synoptic diagram in heavy chain CDR1, CDR2 shown in the SEQ ID NO:18,24 and 30 and CDR3 district.
1F4 light chain immunoglobulin sequences and known human racial immunity sphaeroprotein sequence of light chain proved that relatively it is the VL section of VK A27 and to plant from the mankind be the JK section of JK 2 that the 1F4 light chain is used to from human the kind.1F4VL sequence and kind are that the comparison of VK A27 sequence is shown in Figure 15.Use the definite Kabat system in CDR district that the 1F4VL sequence is further analyzed, obtain respectively as Fig. 5 B and 15, and the synoptic diagram in light chain CDR1, CDR2 shown in the SEQ ID NO:36,42 and 48 and CDR3 district.
Embodiment 3: the sign of anti--CD70 human monoclonal antibody's binding specificity
Compare with combining of CD70 by the ELISA antagonism-CD70 antibody of standard, check binding specificity CD70 through immune purifying.
The CD70 bag of the band myc label of reorganization is spent the night on plate, tested the combination of its antagonism-CD70 human monoclonal antibody 2H5,10B4,8B5 and 18E7 then.Carry out the ELISA step of standard.Concentration with 1 μ g/ml adds anti--CD70 human monoclonal antibody, and carries out titration with 1: 2 serial dilution ratio.The anti-IgG of goat (Fc or κ chain the are special) polyclonal antibody that uses coupling horseradish peroxidase (HRP) is as second antibody.The results are shown in Figure 16.Anti--CD70 human monoclonal antibody 2H5,10B4,8B5 and 18E7 combine with CD70 with high specific.
Embodiment 4: with the CD70 bonded on the cancerous cell line surface that is expressed in kidney anti--sign of CD70 antibody
By flow cytometry test anti--CD70 antibody and combining at the renal cell carcinoma cell of cell surface expression CD70.
Each self-test the antibodies of renal cell carcinoma clone A-498 (ATCC accession number HTB-44), 786-O (ATCC accession number CRL-1932), ACHN (ATCC accession number CRL-1611), Caki-1 (ATCC accession number HTB-46) and Caki-2 (ATCC accession number HTB-47).By with 1 * 10 5Individual cell with concentration be the 2H5 of 1 μ g/ml hatch assess HuMAb2H5 anti--CD70 human monoclonal antibody's combination.Washed cell, and detect combination with the anti-IgG Ab of FITC mark.(Becton Dickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown in Figure 17.Anti--CD70 monoclonal antibody 2H5 combines with renal carcinoma cell line A-498,786-O, ACHN, Caki-1 and Caki-2.
Test renal cell carcinoma clone 786-O and A-498 for the HuMAb of different concns anti--combination of CD70 human monoclonal antibody 2H5,8B5,10B4 and 18E7.By with 5 * 10 5Individual cell is hatched the combination of assessing anti--CD70 human monoclonal antibody with antibody, and this antibody initial concentration is 50 μ g/ml, and carries out serial dilution with 1: 3 thinning ratio antagonist.Washed cell, and detect combination with the anti-human IgG Ab of PE mark.(Becton Dickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown among Figure 18 A (786-O) and Figure 18 B (A-498).As measured by painted average fluorescent strength (MFI), anti-CD7 monoclonal antibody 2H5,8B5,10B4 and 18E7 combine with renal carcinoma cell line 786-O and A-498 in the mode of concentration dependent.The EC of anti--CD70 monoclonal antibody 50The scope of value is to be from 3.984nM to 11.84nM from 1.844nM to 6.669nM and for A-498 clone for 786-O clone.
By with 2 * 10 5Individual cell and concentration are that 2H5 or the 69A7 of 10 μ g/ml hatched, to HuMAb2H5 and 69A7 anti--the CD70 human monoclonal antibody assesses with combining of renal cell carcinoma clone 786-O.Use the isotype control antibodies as the negative control thing.Washed cell, and detect combination with the anti-IgG Ab of FITC mark.(BectonDickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown in Figure 18 C.Two kinds of anti--CD70 monoclonal antibodies can both combine with renal carcinoma cell line 786-O.
Tested renal cell carcinoma clone 786-O to the HuMAb of different concns anti--combination of CD70 human monoclonal antibody 69A7.By with 5 * 10 5Individual cell is hatched the combination of having assessed anti--CD70 human monoclonal antibody with antibody, and this antibody initial concentration is 10 μ g/ml, and carries out serial dilution with 1: 3 thinning ratio antagonist.Washed cell, and detect combination with the anti-IgG Ab of PE mark.(Becton Dickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown in Figure 18 D.As measured by painted average fluorescent strength (MFI), anti--CD70 monoclonal antibody 69A7 combines with renal carcinoma cell line 786-O in the concentration dependent mode.Anti--CD70 monoclonal antibody 69A7 and 786-O bonded EC 50Value is 6.927nM.
These digital proofs are anti--and CD70HuMAb combines with the clone of renal cell carcinoma.
Embodiment 5: with the CD70 bonded that is expressed in the lymphoma cell line surface anti--sign of CD70 antibody
Anti--CD70 antibody and combining have been tested by flow cytometry at the lymphoma cell of cell surface expression CD70.
Tested the combination of lymphoma cell line Daudi (ATCC accession number CCL-213), HuT 78 (ATCC accession number TIB-161) and Raji (ATCC accession number CCL-86) antagonist respectively.By with 1 * 10 5Cell with concentration be the 2H5 of 1 μ g/ml hatch assess HuMAb2H5 anti--CD70 human monoclonal antibody's combination.Washed cell, and detect combination with the anti-IgG Ab of FITC mark.Use is not expressed the Jurkat clone of CD70 as the negative control thing at cell surface.(Becton Dickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown in Figure 19.As measured by painted average fluorescent strength (MFI), anti--CD70 monoclonal antibody 2H5 combines with lymphoma cell line Daudi, HuT 78 and Raji.
Test lymphoma cell line Raji and Granta 519 (DSMZ accession number 342) to the HuMAb of different concns anti--combination of CD70 human monoclonal antibody 2H5.By with 5 * 10 5Individual cell is hatched the combination of assessing anti--CD70 human monoclonal antibody with antibody, and this antibody initial concentration is 50 μ g/ml, and carries out serial dilution with 1: 3 thinning ratio antagonist.Use the isotype control antibodies as the negative control thing.Washed cell, and detect combination with the anti-human IgG Ab of PE mark.(Becton Dickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown among Figure 20 A (Raji) and the 20B (Granta 519).As measured by painted average fluorescent strength (MFI), anti--CD70 monoclonal antibody 2H5 combines with lymphoma cell line Raji and Granta 519 in the concentration dependent mode.The EC of anti--CD70 antibody 50Value is 1.332nM for the Raji cell, and is 1.330nM for Granta 519 cells.
By with 2 * 10 5Individual cell with concentration be the HuMAb of 10 μ g/ml hatch assessed HuMAb 2H5 and 69A7 anti--the CD70 human monoclonal antibody combines with the Raji lymphoma cell line.Washed cell, and detect combination with the anti-IgG Ab of FITC mark.Use the isotype control antibodies and only use second antibody as the negative control thing.(Becton Dickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown among Figure 20 C.As measured by painted average fluorescent strength (MFI), two kinds of anti--CD70 monoclonal antibodies all combine with the Raji lymphoma cell line.
The FACS that is at war with measures to illustrate the binding specificity that 69A7 compares with 2H5.The Raji cell is with exposed 69A7,2H5, the isotype control antibodies of 10 μ g/ml concentration or do not have antibody and hatch.After the washing, be that the 69A7 of the coupling FITC of 10 μ g/ml is hatched with concentration with cell.Washed cell, and detect combination with the anti-IgG Ab of FITC mark.(Becton Dickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown in Figure 20 D.Anti--CD70 antibody 69A7 and 2H5 have all hindered the combination of the 69A7 of FITC mark, and the two is shared similar in conjunction with epi-position to show 2H5 and 69A7.
Further tested the combination of Daudi lymphoma cell line and 786-O kidney cancer cell antagonist.By with 2 * 10 5Individual cell with concentration be the 69A7 of 1 μ g/ml hatch assessed HuMAb69A7 anti--CD70 human monoclonal antibody's combination.Washed cell, and detect combination with the anti-IgG Ab of FITC mark.Use is not expressed the Jurkat clone of CD70 as the negative control thing at cell surface.(Becton Dickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown in Figure 20 E.As measured by painted average fluorescent strength (MFI), anti--CD70 monoclonal antibody 69A7 combines with Daudi lymphoma cell line and 786-O renal carcinoma cell line.
These digital proofs are anti--and CD70HuMAb combines with lymphoma cell line.
Embodiment 6: the Scatchard of the binding affinity of anti--CD70 monoclonal antibody analyzes
Use the Scatchard analytical test 2H5,8B5,10B4 and 18E7 monoclonal antibody to binding affinity through the Chinese hamster ovary celI system of CD70 transfection.
Use the CD70 transfection CHO cell of standard technique, and it is grown in the RPMI substratum that contains 10% foetal calf serum (FBS) with total length.Cell is by tryptic digestion, and at binding buffer liquid (24mM Tris pH 7.2,137mM NaCl, 2.7mM KCl, 2mM glucose, 1mM CaCl based on Tris 2, 1mM MgCl 2, 0.1%BSA) the middle washing once, and in binding buffer liquid, cell is transferred to 2 * 10 6Individual cell/ml.1% skim-milk bag in the water is by Millipore plate (MAFB NOB), and storage is spent the night under 4 ℃.With 0.2ml binding buffer liquid plate is washed three times.50 microlitre damping fluids are added to separately in the maximum combined hole (total binding).25 microlitre damping fluids are added to separately in the control wells (non-specific binding).With different concns 125I-is anti--CD70 antibody with the volume of 25 μ l add to institute porose in.In control wells, add the unlabelled antibody that volume is 25 μ l different concns with 100 times of excessive amounts, and with the Chinese hamster ovary celI (2 * 10 of 25 μ l in the binding buffer liquid through the CD70 transfection 6It is porose that individual cell/ml) adds to institute.Plate placed on the shaking table hatched 2 hours at 4 ℃ with 200RPM.Hatch finish after, with 0.2ml cold lavation buffer solution (24mM Tris pH 7.2,500mM NaCl, 2.7mM KCl, 2mM glucose, 1mMCaCl 2, 1mM MgCl 2, 0.1%BSA) with Millipore plate washing 3 times.Remove filtrate and count with gamma counter.(San Diego CA), carries out balance in conjunction with assessment with single site incorporating parametric to utilize Prism software.
Use above scatchard in conjunction with mensuration, antibody is to the K of the Chinese hamster ovary celI of transfection CD70 DFor 2H5 is 2.1nM, is 5.1nM for 8B5, is 1.6nM for 10B4, is 1.5nM for 18E7.
Embodiment 7: the internalization of anti--CD70 monoclonal antibody
Use the test of Hum-Zap internalization assay method anti--ability of CD70HuMAb internalization to the kidney cancer cell of expressing CD70.The Hum-Zap assay method is tested the internalization of human first antibody, this be by to cytotoxin sapotoxin albumen mutually the combination of link coupled IgG with second antibody of avidity carry out.
To express the kidney cancerous cell line 786-O of CD70 with 1.25 * 10 4Individual cells/well (100 μ l/ hole) is inoculated in the hole and spends the night.Xiang Kongzhong adds anti--CD70HuMAb antibody 2H5,8B5,10B4 or 18E7, and initial concentration is 30nM, and carries out titration with 1: 3 serial dilution ratio.Will be to the nonspecific isotype control antibodies of CD70 as the negative control thing.(CA IT-22-25), and is hatched plate 72 hours for Advanced Targeting Systems, San Diego to add Hum-Zap with the concentration of 11nM.Use 1.0 μ Ci's then 3The H-thymidine is to plate pulse 24 hours, collects and (Packard Instruments, Meriden CT) carry out reading with Top Count Scintillation Counter.The results are shown among Figure 21.Anti--CD70 antibody 2H5,8B5,10B4 and 18E7 show, in the 786-O kidney cancer cells of expressing CD70 3The antibody concentration dependency that the H-thymidine mixes reduces.The EC of anti--CD70 antibody 2H5 50Value is 0.9nM.This digital proof anti--CD70 antibody 2H5,8B5,10B4 and 18E7 internalization to the kidney cancerous cell line.
Embodiment 8: in renal cell carcinoma clone coupling cytotoxic anti--assessment of the cell killing effect of CD70 antibody
In this embodiment, in cell proliferating determining, test the ability that the anti--CD70 monoclonal antibody that is coupled to cytotoxin D (Figure 73) is killed and wounded the positive renal cell carcinoma clone of CD70.Cytotoxin D is a kind of esterase activated prodrug that needs.
To resist-CD70HuMAb antibody 2H5,8B5,10B4 or 18E7 combine with cytotoxin D by connector, this connector is peptidyl, hydrazone or disulfide linkers for example.In the hole with 2.5 * 10 4Kidney cancerous cell line ACHN and the Caki-2 of CD70 expressed in individual cells/well (100 μ l/ hole) inoculation, and with 1.25 * 10 4The kidney cancerous cell line 786-O of individual cells/well (100 μ l/ hole) inoculation expression CD70 3 hours.Xiang Kongzhong adds anti--CD70 antibody-cytotoxin conjugate, and initial concentration is 30nM, and carries out titration with 1: 3 serial dilution ratio.Will be to the nonspecific isotype control antibodies of CD70 as the negative control thing.Plate was hatched 69 hours.Use 1.0 μ Ci's then 3The H-thymidine is to plate pulse 24 hours, collects and (PackardInstruments, Meriden CT) carry out reading with Top Count Scintillation Counter.The results are shown among Figure 22 A (Caki-2), 22B (786-O) and the 22C (ACHN).Anti--CD70 antibody 2H5,8B5,10B4 and 18E7 show, in Caki-2, the 786-O and ACHN kidney cancer cells that express CD70 3The antibody that the H-thymidine mixes-cytotoxin concentration dependent reduces.The EC of anti--CD70 antibody 50Value scope in the CAKI-2 cell is from 6nM to 76nM, and scope is from 1.6nM to 3.9nM in the 786-O cell, and scope is from 9nM to 108nM in the ACHN cell.This digital proof anti--CD70 antibody 2H5,8B5,10B4 and 18E7 when being coupled to cytotoxin, be cytotoxic to the kidney cancer cells.
Embodiment 9: the active assessment of ADCC of anti--CD70 antibody
In this embodiment, in the fluorocyte toxicity test, tested anti--CD70 monoclonal antibody and in the presence of the effector cell, killed and wounded the ability of CD70 positive cell line by antibody dependent cellular cytotoxicity (ADCC).
From whole blood, prepare human effector cell as described below.Separate by the standard Ficoll-Paque, from the whole blood of heparinization purifying human peripheral blood mononuclear cells.Cell is resuspended in the RPMI1640 substratum that contains 10%FBS and the human IL-2 of 200U/ml, and in 37 ℃ of overnight incubation.Next day collecting cell, and substratum washing 4 times, and with 2 * 10 7Individual cell/ml re-suspended cell.(Perkin Elmer, Wellesley is MA) per 1 * 10 with BATDA reagent 6Under the target cell 2.5 μ l BATDA/mL situations CD70 male target cell was hatched 20 minutes in 37 ℃.With target cell washing 4 times, centrifugal and reach 1 * 10 5The final volume of individual cell/ml.
Use the following following CD70 positive cell line of Delfia fluorescent emission analytical test to the mankind anti--antibodies specific ADCC:ARH-77 (the human B lymphoblast leukemia of CD70 monoclonal antibody; ATCC accession number CRL-1621), HuT78 (human skin lymphocyte lymphoma; ATCC accession number TIB-161), Raji (human B lymphocyte Burkitt lymphoma; ATCC accession number CCL-86) and negative control cell system (the human hodgkin's lymphomas of L540; DSMZ storage .ACC 72).Each target cell system (100 μ l are through the target cell of mark) is hatched with 50 μ l effector cells and 50 μ l antibody.The target cell of using in the whole experiment and effector cell's ratio are 1: 50.In all researchs, use human IgG1 isotype contrast as the negative control thing.In the 2000rpm pulse centrifugal and 37 ℃ hatch one hour after, collect supernatant liquor, centrifugal fast once more, and 20 μ l supernatant liquors are transferred in the flat underside, in plate, add 180 μ l Eu solution (Perkin Elmer, Wellesley MA), and carries out reading in RubyStar readout instrument (BMG Labtech).Cracking % is calculated as follows: (sample burst size-spontaneous burst size * 100)/(maximum burst size-spontaneous burst size), wherein spontaneous burst size is the fluorescence from those holes of only containing target cell, and maximum burst size is also to have used the fluorescence in those holes of 2%Triton-* processing from containing target cell.The cytotoxicity cracking % of the cell of ARH-77, HuT78, Raji and L-540 clone is shown in respectively among Figure 23 A to D.Use HuMAb anti--to have expressed the CD70 positive cells be that ARH-77, HuT78 and Raji all show antibody-mediated cytotoxicity for each of CD70 antibody 2H5 and 18E7, and negative control cell to be L-540 do not show appreciable cytotoxicity in the presence of anti--CD70 antibody.This digital proof, HuMAb is anti--and CD70 antibody demonstrated expressing the SC of CD70 positive cells.
Embodiment 10: the assessment coupling has the cell killing effect of cytotoxic resisting-CD70 antibody to human lymphoma clone
In this embodiment, in cell proliferating determining, test the ability that the anti--CD70 monoclonal antibody 2H5 that is coupled to cytotoxin C (Figure 72) kills and wounds the positive human lymphoma cell line of CD70.Cytotoxin C is a kind of esterase activated prodrug that needs.
To resist-CD70HuMAb antibody 2H5 is by connector and cytotoxin C coupling mutually, and this connector is peptidyl, hydrazone or disulfide linkers for example.The embodiment that can be coupled to the cytotoxin compounds on the antibody of the present invention is illustrated in the application of submitting to simultaneously: the U.S. serial of submitting on September 26th, 2,005 60/720, the PCT publication number WO07/038658 that on September 26th, 499 and 2006 submitted to, their content merges as a reference at this.To express human lymphoma cancerous cell line Daudi, HuT 78, Granta 519 and the Raji of CD70 with 10 5Individual cells/well (100 μ l/ hole) was inoculated in the hole 3 hours.Xiang Kongzhong adds anti--CD70 antibody-cytotoxin conjugate, and initial concentration is 30nM, and carries out titration with 1: 2 serial dilution ratio.Also HuMAb antibody 2H5-cytotoxin conjugate is tested on Jurkat cell (a kind of negative control cell of not expressing CD70 at cell surface is).Plate was hatched 72 hours.Use 0.5 μ Ci's then 3The H-thymidine is to plate pulse 8 hours, stops afterwards cultivating, and collects and carries out reading with Top Count Scintillation Counter (Packard Instruments).Figure 24 has shown the effect of 2H5-conjugate to Daudi, HuT 78, Granta 519 and Jurkat cell.Anti--CD70 antibody 2H5 has showed in Daudi, the HuT 78 and Granta 519B cell lymphoma cancer cells that express CD70 3The antibody that the H thymidine mixes-cytotoxin concentration dependent reduces, and does not have corresponding performance in the Jurkat cell.
In an independent mensuration, will express the human lymphoma cancerous cell line Raji of CD70 with 10 4Cells/well (100 μ l/ hole) was inoculated in the hole 3 hours.Xiang Kongzhong adds anti--CD70 antibody-cytotoxin conjugate, and initial concentration is 30nM, and carries out titration with 1: 3 serial dilution ratio.Use cytotoxin-conjugate isotype control antibodies thing in contrast.Plate was hatched 72 hours, washed once at 3 hours, perhaps continuous washing.Use 0.5 μ Ci's then 3The H-thymidine stops cultivating to plate pulse 8 hours afterwards, and collection is also carried out reading in Top Count Scintillation Counter (PackardInstruments).Figure 25 A and 25B illustrate respectively and adopt washing in 3 hours or continuous washing in the Raji cell 3The antibody that the H thymidine mixes-cytotoxin concentration dependent reduces.
This digital proof with the cytotoxin link coupled anti--CD70 antibody demonstrates the SC for the human lymphoma cancer cells.
Embodiment 11: use exposed and coupling cytotoxic anti--CD70 antibody is to the treatment of in-vivo tumour heteroplastic transplantation model
With coupling cytotoxic anti--CD70 antibody carries out interior therapeutic to the mouse of having implanted the renal cell carcinoma tumour, with check antibody in vivo to the influence of tumor growth.
The laboratory operation of use standard increases to A-498 (ATCC accession number HTB-44) and ACHN (ATCC accession number CRL-1611) external.6 to 8 weeks male Ncr nude mouse between ages (Taconic, Hudson, NY) every mouse in the PBS/ matrigel (Matrigel) (1: 1) that the right side abdomen has been implanted subcutaneously at 0.2ml 7.5 * 10 6ACHN or A-498 cell.Mouse is weighed, and after implantation, with electronic caliper weekly twice pair of tumour carry out three-dimensional measurement.According to length * wide * high gross tumor volume that calculates.To have average 270mm 3ACHN tumour or average 110mm 3The mouse of A498 tumour divide at random to the treatment group.Injected in mouse peritoneum at 0 day PBS vehicle, the cytotoxic isotype control antibodies of coupling or coupling cytotoxic anti--CD70HuMAb 2H5.The example that can be coupled to the cytotoxin compounds of antibody of the present invention is illustrated in the U.S. Provisional Application series number of submitting on September 26th, 2,006 60/720,499 and PCT publication number WO 07/038658, and its content merges as a reference at this.(cytotoxin A (N1), cytotoxin B (Figure 71) and cytotoxin C (Figure 72)) tests the mouse in the A-498 sample sets with three kinds of different cytotoxin compounds.The tumor growth 60 days of monitoring mouse after the administration.When tumour reaches tumour terminal point (2000mm 3) time, make mouse euthanasia.
The results are shown in Figure 26 A (A-498 tumour) and Figure 26 B (ACHN tumour).Being coupled to anti--CD70 antibody 2H5 on the cytotoxin has prolonged and has reached tumour terminal point volume (2000mm 3) mean time, and slowed down the tumor growth process.Therefore, with anti--CD70 antibody-cytotoxin conjugate interior therapeutic tumor growth had direct restraining effect.
Embodiment 12: the immunohistochemistry of 2H5
Use is from hyaline cell renal cell carcinoma (ccRCC), lymphoma and glioblastoma patient's clinical biopsy, by immunohistochemical detection the ability of anti--CD70HuMAb 2H5 identification CD70.
Used the frozen section of 5 μ m carry out immunohistochemistry measure (Ardais Inc, USA).After dry 30 minutes, with acetone fixed section (following 10 minutes of room temperature), and air-dry 5 minutes.With slide glass rinsing in PBS, and then with the PBS preincubate that contains 10% normal goats serum 20 minutes, and the 2H5 of FITCization that then is used in the 10 μ g/ml that contain 10% normal goats serum among the PBS was in incubated at room 30 minutes.Then, wash slide 3 times with PBS, and at room temperature use mouse anti FITC (10 μ g/ml DAKO) to hatch 30 minutes.Wash slide with PBS once more, and at room temperature use goat anti-mouse HRP conjugate (DAKO) to hatch 30 minutes.Wash slide 3 times with PBS again.Use diaminobenzidine (Sigma) as substrate, obtain brown colouring.After with distilled water wash, slide glass is used phenodin (hematoxyllin) counterstaining 1 minute.Subsequently, slide glass was washed for 10 seconds in mobile distilled water, and with glycergel (DAKO) sealing.Clinical examination of living tissue immunohistochemical staining has shown the positive staining in non-Hodgkin lymphomas, plasmoma, ccRcc and glioblastoma section.Have only malignant cell to be positive in each case, adjacent healthy tissues is not colored.
Embodiment 13: the generation of removing fucosylated HuMAb
The verified antibody with fucosyl residues of reduction has improved the ADCC ability of antibody.In this embodiment, produced the 2H5HuMAb that lacks fucosyl residues.
Is that (Princeton NJ) carries out electroporation to Ms704-PF for Biowa, Inc. with the heavy chain of expressing antibodies 2H5 and the carrier of light chain to the Chinese hamster ovary celI that lacks fucose transferase gene FUT 8.By ((KS) the drug resistance clone is selected in middle growth to Ex-Cell 325-PF CHO substratum CA) for JRH Biosciences, Lenexa for Invitrogen, Carlsbad containing 6mM L-glutaminate and 500 μ g/ml G418.Measure the clone that IgG is expressed in screening by standard ELISA.Produced two independent clone B8A6 and B8C11, its productive rate is in the scope of each cell 1.0 to 3.8 pik every day.
Embodiment 14: the active assessment of ADCC of removing fucosylated anti--CD70 antibody
In this embodiment, in the fluorocyte toxicity test, tested go fucosylated and non-go fucosylated anti--the CD70 monoclonal antibody kills and wounds the ability of CD70 positive cell through antibody dependent cellular cytotoxicity effect (ADCC) in the presence of the effector cell.
As above method to the mankind anti--CD70 monoclonal antibody 2H5 goes fucosylated.Following from whole blood the preparation human effector cell.Separate by the standard Ficoll-Paque, from the whole blood of heparinization purifying human peripheral blood mononuclear cells.Cell is resuspended in the RPMI1640 substratum that contains 10%FBS (substratum) and the human IL-2 of 200U/ml, and 37 ℃ of overnight incubation.Second day, collecting cell and once with substratum washing, and with 2 * 10 7The density of individual cell/ml is resuspended.With per 1 * 10 6Target cell/mL is with the concentration of 2.5 μ l BATDA, and (Perkin Elmer, Wellesley MA) were hatched 20 minutes at 37 ℃ with CD70 positive target cell and BATDA reagent in the substratum that is supplemented with 2.5mM probenecid (mensuration medium).Target cell is with the PBS washing that contains 20mM HEPES and 2.5mM probenecid four times, centrifugal and make that final volume is 1 * 10 in measuring medium 5Individual cell/ml.
Use following Delfia fluorescent emission analytical test go fucosylated and non-go the fucosylated mankind anti--CD70 monoclonal antibody 2H5 is to antibodies specific ADCC:ARH-77 (the human B lymphoblast leukemia of following CD70 positive cell line; ATCC accession number CRL-1621), MEC-1 (human chronic B cell leukemia; DSMZ accession number ACC 497), SU-DHL-6 (human B cell lymphoma, DSMZ accession number Acc572), IM-9 (human B lymphoblast; ATCC accession number CCL-159) and HuT 78 (human skin lymphocyte lymphoma; ATCC accession number TIB-161).With target cell is that ARH77 (100 μ l are through the target cell of mark) is with 50 μ l effector cells and 50 μ l 2H5 or go fucosylated 2H5 antibody to hatch.Using target cell and effector cell's ratio in the whole experiment is 1: 50.Use human IgG1 isotype contrast as the negative control thing.In the 2100rpm pulse centrifugal and 37 ℃ hatch after one hour and collect supernatant liquor, centrifugal fast once more, and 20 μ l supernatant liquors are transferred in the flat underside, Eu solution (the Perkin Elmer that in plate, adds 180 μ l, Wellesley, MA), and with Fusion Alpha TRF plate reader (Perkin Elmer) reading.Cracking % is calculated as follows: (sample burst size-spontaneous burst size * 100)/(maximum burst size-spontaneous burst size), wherein spontaneous burst size is the fluorescence from the hole of only containing target cell, and maximum burst size is from containing target cell and through the fluorescence in the hole that 3% lysol is handled.The cytotoxicity specificity cracking % of the cell of ARH-77 clone is shown in Figure 27 A to F.Express the CD70 positive cells and be ARH-77, MEC-1, SU-DHL-6, IM-9 and HuT 78 shown HuMAb anti--the antibody-mediated cytotoxicity of CD70 antibody 2H5 and with the raising of removing the relevant specificity cracked per-cent of fucosylated form of anti--CD70 antibody 2H5.In addition, shown that anti-CD16 antibody blocks the ADCC effect in MEC-1 clone.This digital proof, go fucosylated HuMAb anti--CD70 antibody demonstrated the SC to the raising of expressing the CD70 positive cells.
Embodiment 15: utilize 51Cr discharges and measures the ADCC activity of assessing anti--CD70 antibody
In this embodiment, exist 51Tested anti--CD70 monoclonal antibody during Cr discharge to measure and in the presence of the effector cell, killed and wounded the ability of CD70 positive Raji bone-marrow-derived lymphocyte through antibody dependent cellular cytotoxicity effect (ADCC).
Separate by the standard Ficoll-Paque, from the whole blood of heparinization purifying human peripheral blood mononuclear cells (effector cell).With cell with 2 * 10 6The density of individual/mL is resuspended in the RPMI1640 substratum that contains 10%FBS and the human IL-2 of 200U/ml, and in 37 ℃ of overnight incubation.Second day, collecting cell and once with substratum washing, and with 2 * 10 7The density of individual cell/ml is resuspended.In the 1ml cumulative volume with 2,000,000 target Raji cell (human B lymphocyte Burkitt lymphomas; ATCC accession number CCL-86) with 200 μ Ci 51Cr was hatched under 37 ℃ 1 hour.With the target cell washing once, be resuspended in the 1ml substratum, and under 37 ℃, hatch 30 minutes in addition.After last hatching, with the target cell washing once, and to make final volume be 1 * 10 5Cell/ml.For last ADCC measures, Raji cell that 100 μ l are labeled and 50 μ l effector cells and 50 μ l antibody are hatched.Using target cell and effector cell's ratio in the whole experiment is 1: 100.In all researchs, use human IgG1 isotype contrast as the negative control thing.In some researchs, before adding PBMC, the PBMC culture is distributed to the anti-human CD16 antibody that contains 20 μ g/mL, irrelevant mouse IgG1 antibody or does not have in the test tube of antibody to assay plate.Hatched 15 minutes use hemocyte as described above but washed cell not subsequently at 27 ℃.37 ℃ hatch 4 hours after, collect supernatant liquor and also go up counting at Cobra II auto-γ Counter (Packard Instruments) with 240-400keV reading window.Count per minute is plotted as the function of antibody concentration, and (San Diego CA), analyzes data by non-linear regression, S shape dose response (variable slope) to use Prism software.Determine cracking per-cent by following equation: cracking %=(sample CPM-does not have antibody CPM)/TritonX CPM-does not have antibody CPM) * 100.The antibody titer curve of the cytotoxicity specificity cracking % of Raji clone is shown among Figure 28.This digital proof is anti--and CD70 antibody has the ADCC effect to Raji clone.Anti--CD70 antibody is at the EC of Raji cell 50Value is 36nM.Cytotoxic bar graph in the presence of anti-CD16 antibody on the Raji cell is shown among Figure 29.This digital proof is anti--and the ADCC effect of CD70 antibody on the Raji cell depend on CD16.
Embodiment 16: assessment is anti--the ADCC activity of CD70 antibody on activated T cells
In this embodiment, in the fluorocyte toxicity test, tested and go fucosylatedly and non-to go fucosylated anti--CD70 monoclonal antibody in the presence of the effector cell, to kill and wound the ability of activated T cells through antibody dependent cellular cytotoxicity effect (ADCC).
As described above to the mankind anti--CD70 monoclonal antibody 2H5 goes fucosylated.The human effector cell of preparation as described above.Magnetic bead (purity＞90%) with anti-CD3 bag quilt carries out positive selection to human splenic t-cell.With the heat-inactivated FCS irritation cell of IL-2+10% of the pearl of anti-CD3 and CD28 bag quilt and the 25ng/ml in the Iscove substratum 6 days.Collecting cell and by the mixing of iodate third ingot (60% can referring to) mensuration viability screens (gate) and analyzes at the expression of CD70 (on viable cell approximately the 65%CD70 positive) viable cell before measuring carrying out ADCC.
Following use Delfia fluorescent emission analytical test go fucosylated and non-go the fucosylated mankind anti--CD70 monoclonal antibody 2H5 is at the special ADCC of antibody of activated T cells.Will be activated target T cell (100 μ l are through the target cell of mark) with the 2H5 of 50 μ l effector cells and 50 μ l or go fucosylated 2H5 antibody to hatch.Using target cell and effector cell's ratio in the whole experiment is 1: 50.Use human IgG1 isotype contrast as the negative control thing.In the 2100rpm pulse centrifugal and 37 ℃ hatch after one hour and collect supernatant liquor, centrifugal fast once more, and 20 μ l supernatant liquors are transferred in the flat underside, Eu solution (the Perkin Elmer that in plate, adds 180 μ l, Wellesley, MA), and with Fusion Alpha TRF plate reader (Perkin Elmer) reading.Cracking % is calculated as follows: (sample burst size-spontaneous burst size * 100)/(maximum burst size-spontaneous burst size), wherein spontaneous burst size is the fluorescence from the hole of only containing target cell, and maximum burst size is from containing target cell and with the fluorescence in the hole of 3% lysol processing.At activated T cells, the cytotoxicity specificity cracking % of cell is shown in Figure 30.The antibody-mediated cytotoxicity of activated T cells has demonstrated HuMAb anti--CD70 antibody 2H5 and with the specificity cracking per-cent that goes the relevant increase of fucosylated form of anti--CD70 antibody 2H5.Add anti-CD16 antibody in two kinds of fucosylated forms by fucosylated and non-the going of going, blocked antibody-mediated cytotoxicity at anti--CD70 antibody.The not effect of contrast IgG pair cell toxicity.This digital proof, go fucosylated HuMAb anti--CD70 antibody demonstrates the SC of the raising of activated T cells.
Embodiment 17: the blocking-up of receptor-ligand CD70-CD27 bonded is measured
In this embodiment, to use the blocking-up assay method to test anti--ability that the CD70 monoclonal antibody is blocked the interaction of CD70 and part CD27.
With 100 μ l/ holes the hole is wrapped at 4 ℃ with the anti-IgG antibody (Fc-sp.) of 2 μ g/ml and to be spent the night.In room temperature with 1% BSA/PBS with 200 μ l/ holes to hole sealing 1 hour.Vibration moving limit in limit adds the CD27-Fc-his of the 0.16 μ g/ml in 100 μ l/ holes in each hole, continue 1 hour at 37 ℃.Each hole is with 200 μ l/ hole PBS/ polysorbas20s (0.05% (v: v)) wash 5 times.Anti--CD70 antibody dilutes in 10%NHS+1%BSA/PBS, and mixes with the CD70-myc-his of 0.05 μ g/ml, at room temperature hatched 1 hour, and with the PBS/ polysorbas20 (0.05% (v: v)) wash 5 times in 200 μ l/ holes.Use the interactional antibody of known blocking-up CD70/CD27 as positive control, and use the isotype control antibodies as the negative control thing.The mixture of CD70 and anti--CD70 antibody is blocked with anti-Fc antibody, and adds 100 μ l/ hole CD70-myc-his+ antibody in the hole of containing CD27-Fc-his.Mixtures incubated is 1 hour under 37 ℃ of vibrations.In mixture, add the anti--myc-HRP (in 10%NHS+1%BSA/PBS, diluting) in 100 μ l/ holes and when 37 ℃ of vibrations, hatched 1 hour with 1: 1000.By adding 100 μ lTMB substrates, at room temperature hatch 5 to 10 minutes detection signals, add the H2SO4 of 75 μ l 0.25M then, and read the result at A450nm.The results are shown among Figure 31.This digital proof comprise some of 2H5,8B5 and 18E7 anti--CD70 antibody blocking CD70 combines with CD27's, and other antibody do not influence the interaction between CD70 and the CD27.
Embodiment 18: use of the treatment of exposed anti--CD70 antibody to the in-vivo tumour heteroplastic transplantation model
With exposed anti--CD70 antibody the mouse of having implanted the lymphoma tumour is carried out interior therapeutic, to check that antibody is in vivo to the influence of tumor growth.
The laboratory operation of use standard is in amplification in vitro ARH-77 (human B lymphoblast leukemia; ATCC accession number CRL-1621) and Raji (human B lymphocyte Burkitt lymphoma; ATCC accession number CCL-86) cell.6 to 8 weeks male Ncr nude mouse between ages (NY) every mouse has been implanted 5 * 10 in the PBS/ matrigel (1: 1) of 0.2ml at the right side abdomen for Taconic, Hudson 6ARH-77 cell or Raji cell.Mouse is weighed, and after implantation, use electronic caliper twice pair of tumour is weekly carried out three-dimensional measurement.Gross tumor volume is calculated as height * wide * long/2.To have average 80mm 3ARH-77 tumour or average 170mm 3The mouse of Raji tumour assign to the treatment group at random.With PBS vehicle, isotype control antibodies or exposed anti--CD70HuMAb 2H5 0 day through peritonaeum to the mouse administration.When tumour reaches tumour terminal point (2000mm 3) time, make mouse euthanasia.The results are shown among Figure 32 A (Raji tumour) and the 32B (ARH-77 tumour).Exposed anti--CD70 antibody 2H5 has prolonged and has reached tumour terminal point volume (2000mm 3) mean time, and slowed down the tumor growth process.Therefore, use separately anti--CD70 antibody to treat to have in the direct body and suppress effect for tumor growth.
Embodiment 19: use the cytotoxin link coupled anti--CD70 antibody is to the treatment of body endolymph struma knurl heteroplastic transplantation model
With the cytotoxin link coupled anti--CD70 antibody carries out interior therapeutic to the mouse of having implanted the lymphoma tumour, with check antibody in vivo to the influence of tumor growth.
The laboratory operation amplification in vitro ARH-77 of use standard (human B lymphoblast leukemia; ATCC accession number CRL-1621), Granta 519 (DSMZ accession number .342) and Raji (human B lymphocyte Burkitt lymphoma; ATCC accession number CCL-86) cell.6 to 8 weeks male Ncr nude mouse between ages (NY) every mouse has been implanted subcutaneously in 0.2ml PBS/ matrigel (1: 1) 5 * 10 at the right side abdomen for Taconic, Hudson 6Individual ARH-77,10 * 10 6Individual Granta 519 or 5 * 10 6Individual Raji cell.Mouse is weighed, and after implantation, use electronic caliper twice pair of tumour is weekly carried out three-dimensional measurement.Gross tumor volume is calculated as height * wide * long/2.To have average 80mm 3(ARH-770), 220mm 3(Granta 519) or 170mm 3The mouse of tumour (Raji) is assigned to the treatment group at random.With PBS vehicle, cytotoxin link coupled isotype control antibodies or cytotoxin link coupled anti--CD70HuMAb 2H5 carried out administration through peritonaeum to mouse at 0 day.Employed conjugate is by shearing the free toxin that connector discharged among the N1 in this experiment.The example that can be coupled to the cytotoxin compounds of antibody of the present invention is illustrated in the U.S. Provisional Application series number of submitting on September 26th, 2,005 60/720, the PCT publication number WO 07/038658 that on September 26th, 499 and 2006 submitted to, their content merges as a reference at this.When tumour reaches tumour terminal point (2000mm 3) time, make mouse euthanasia.The results are shown among Figure 33 A (ARH-77), 33B (Granta 519) and the 33C (Raji tumour).Being coupled to anti--CD70 antibody 2H5 on the cytotoxin has prolonged and has reached tumour terminal point volume (2000mm 3) mean time, and slowed down the tumor growth process.Therefore, the anti--treatment of CD70 antibody-cytotoxin conjugate has in the direct body the lymphoma tumor growth and suppresses effect.
Embodiment 20: the cross reactivity of anti--CD70 antibody and rhesus monkey B lymphoma cell
Also adopt facs analysis to assess the ability that anti--CD70 antibody 69A7 and rhesus monkey CD70 positive B lymphoma cell line LCL8664 (ATCC#:CRL-1805) carry out cross reaction.By with 1 * 10 5Individual cell with concentration be the 69A7 of 1 μ g/ml hatch assess HuMAb 69A7 anti--CD70 human monoclonal antibody's combination.Washed cell, and detect combination with the anti-IgG Ab of FITC mark.Use the isotype control antibodies as the negative control thing.(Becton Dickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown among Figure 34.This result has proved that anti--CD70 antibody 69A7 and the positive B lymphoma cell of monkey CD70 carry out cross reaction.
Embodiment 21: the internalization when anti--CD70 antibodies arrives the 786-O kidney cancer cell
Use immunofluorescence dyeing to test HuMab to resist-CD70 antibody 69A7 and the 2H5 internalization on being attached to these cells the time with the human renal carcinoma cell line of 786-O.From tissue culture flasks, collect 786-O cell (the per 100 μ l 1 * 10 in every hole in 96 orifice plates by handling with 0.25% trypsinase/EDTA 4Individual cell), then with FACS damping fluid (PBS+5%FBS, substratum) in every kind of HuMab of 5 μ g/ml anti--CD70 antibody hatched on ice 30 minutes together.Use human IgG1 isotype contrast as the negative control thing.After substratum washing 2 times, cell is resuspended in this substratum (every hole 100 μ l), and then has the anti-people's second antibody of goat (Jackson ImmunoResearch Lab) of PE to hatch 30 minutes on ice with the coupling of diluting with 1: 100.For morphology and immune fluorescence intensity, under fluorescent microscope (Nikon), made cell imaging immediately at 0 minute, perhaps under 37 ℃, hatch the different time.In the cell of-CD70 antibody staining anti-, observed fluorescence, but in control antibodies, do not seen fluorescence with HuMab.In these are measured, with the direct link coupled HuMab of FITC anti--CD70 antibody also obtained similar result.These results have shown on the cell surface membrane that had two kinds of anti--CD70HuMab in 0 minute and fluorescence occurred.After hatching in 30 minutes, the film fluorescence intensity significantly reduces and the increase of inner fluorescence.Time point at 120 minutes, film fluorescence is not obvious, the substitute is to have tangible fluorescence in the intracellular region chamber.This digital proof HuMab is anti--and CD70 antibody can be by the specificity internalization when the tumour cell with endogenous expression CD70 combines.
Embodiment 22: HuMAb is anti--and CD70 blocks the combination of known mouse anti-CD70 antibody
In this experiment, tested HuMAb anti--CD70 antibody 69A7 is to known mouse anti-CD70 antibody ability of blocking that combines with CD70 positive kidney 786-O cell.(Ancell, Bayport MN) were hatched on ice 20 minutes with the HuMAb 69A7 of 1,5 or 10 μ g/ml with mouse anti-CD70 antibody BU-69 of 786-O cell and 1 μ g/ml.Use IgG1 and IgG2 isotype control antibodies as the negative control thing.Washed cell twice, and detect combination with the anti-IgG Ab of FITC mark.(BectonDickinson, San Jose CA) carry out flow cytometry to use the FACSCalibur flow cytometer.The results are shown among Figure 35.Anti--CD70HuMAb 69A7 blocks the combination of mouse anti-CD70 antibody in the concentration dependent mode.
Embodiment 23: HuMAb is anti--and the CD70 inflammation-inhibiting replys
In this experiment, tested HuMAb anti--CD70 antibody 2H5 is to the inhibition of inflammatory response.With the human CD70 constructs of total length (CHO-S/mCD32/CD70 cell) to stable transfection the CHO-S cell of mouse CD32 (CHO-S/mCD32 cell) carry out transient transfection.Confirmed surface expression (data not shown goes out) by the flow cytometry that uses 2A5 and the anti-human IgG second antibody of PE link coupled.In the hole of three of the same form of 96 orifice plates, with 1 * 10 5Individual CHO-S/mCD32 or CHO-S/mCD32/CD70 cells/well, the anti-hCD3 of 1 μ g/ml (clone OKT3; BD Bioscience) and the serial dilution of HuMAb 2H5 or non-fucosylated 2H5 (2H5NF) to warp
Figure A20078005123101951
Human T Cell Enrichment Kit (Cat#15061; StemCell Technologies Inc) 1 * 10 of purifying 6The human peripheral blood CD3 positive T cell in individual/hole carries out stimulated in vitro.After having collected 3 days supernatant liquor aliquots, the secretion of having measured IFN-(INF-γ) by quantitative ELISA test kit (BD Biosciences)., hatched 8 hours the plate pulse with the 3H-thymidine of 1 μ Ci/ml, collecting cell and
Figure A20078005123101952
1450Microbeta Counter (Wallac, Inc.) upward right 3Reading is carried out in mixing of H-thymidine.Use IgG1 isotype control antibodies as the negative control thing.The results are shown in Figure 36 A to B.2H5 and 2H5NF have suppressed the propagation that CD70 stimulates altogether (Figure 36 A) fully in the dose-dependently mode.Data show that also it is specific that the inhibition of 2H5 stimulates altogether to CD70, because the not influence of propagation of the positive CHO-S/mCD3 mediation of 2H5 antagonism CD3.2H5 and 2H5NF have also suppressed the secretion (Figure 36 B) of the INF-γ that CD70 stimulates altogether fully in the dose-dependently mode.Data show that also it is specific that the inhibition of 2H5 stimulates altogether to CD70, because the not influence of INF-γ secretion of the positive CHO-S/mCD3 mediation of 2H5 antagonism CD3.Take together the common stimulation of the human T cell of the functional blocking-up of data presentation 2H5 and 2H5NF CD70.
In conjunction with CMV peptide IPSINVHHY (SEQ ID NO:90) (ProImmune, Oxford, UK) serial dilution of the B*3501 of 25ng/ml and HuMAb 2H5 exists down cultivated the human MHC I class haplotype positive peripheral blood lymphocytes of B*3501 (PBMC) 11 days, this cell at cytomegalovirus (CMV) specific T-cells reaction carried out prescreen (Astarte, Inc).By flow cytometry culture is analyzed, for the CD8 positive T cell is to dye (clone RPA-T8, BD Biosciences), be the oligopolymer dyeing (F114-4B of the peptide-MHC I class pentamer by the APC mark for the special CD8 positive T cell of peptide by the anti-CD8 of PE link coupled; ProImmune) and for viability is by lacking the dyeing of iodate third ingot.Use the isotype control antibodies as the negative control thing.The results are shown in Figure 37 A-C.2H5 has partly suppressed peptide specific CD8 positive T cell propagation, and 2H5NF and the anti-MHC I of positive control class Ab (clone W6/32; BD Bioscience) suppressed peptide specific CD8 positive T cell propagation (Figure 37 A) fully.Do not observe significantly the reducing of total cell survival (Figure 37 B).Do not observe total CD8 positive cell number purpose and significantly reduce (Figure 37 C).Take together, data show that 2H5 and 2H5NF effect are special to the CD8 positive T cell that peptide stimulates.A data represented other experiment of carrying out with same donor.
To carry out the positive PBMC (Astarte of human MHC I class haplotype B*3501 of prescreen at the special t cell responses of cytomegalovirus (CMV), Inc) carry out 11 days cultivation, this is in the presence of in conjunction with the HuMAb 2H5 of the 25ng/ml B*3501 of CMV peptide IPSINVHHY (ProImmune) (SEQ ID NO:90) and 20 μ g/ml, has or do not exist a functional blocking antibody (clone 3G8; Carry out under the situation of the serial dilution of anti-human CD16 BD Biosciences) (FcR γ III), and the number of peptide specific CD8 positive cell is analyzed by flow cytometry by above explanation then.The results are shown among Figure 38.The reverse by anti-CD16 dose-dependently to peptide specific CD8 positive T cell inhibition of proliferation of 2H5 and 2H5NF mediation shows that 2H5 and 2H5NF suppress to be that interaction by 2H5 and 2H5NF and CD16 positive effector cell mediates.Compare with 2H5, the 3G8 that need approximately have more 1000 times reverses the inhibition that 2H5NF mediates.No matter how the concentration of 3G8 does not all exist by the negative isotype contrast CD8 positive T cell inhibition of proliferation special to peptide, and for the special CD8 positive T cell inhibition of proliferation of peptide, 3G8 exists very little of the effect that does not have fully to the positive control W6/32 by functional blocking-up.
Embodiment 24: use the cytotoxin link coupled anti--CD70 antibody is to the treatment of kidney tumour heteroplastic transplantation model in the body
With the cytotoxin link coupled anti--CD70 antibody carries out interior therapeutic to the mouse of having implanted the kidney tumour, to check that antibody is in vivo to the influence of tumor growth.In this embodiment, anti--CD70 antibody 2H5 is coupled to N2.N2 is a kind of esterase activated prodrug that needs.
The laboratory operation of use standard is at amplification in vitro 786-O (ATCC accession number CRL-1932) and Caki-1 (ATCC accession number HTB-46).At 6 to 8 weeks male CB17.SCID mouse between ages (Taconic, Hudson, NY) every mouse 2,500,000 786-O or Caki-1 cell in the PBS/ matrigel (1: 1) that the right side abdomen has been implanted subcutaneously at 0.2ml.Mouse is weighed, and after implantation, use electronic caliper twice pair of tumour is weekly carried out three-dimensional measurement.Gross tumor volume is calculated as length * wide * height.To have average 200mm 3The mouse of tumour divide at random to the treatment group.0 day with PBS vehicle, cytotoxin link coupled isotype control antibodies or cytotoxin link coupled anti--CD70Hu MAb 2H5 carries out administration through peritonaeum to mouse.The example that can be coupled to the cytotoxin compounds of antibody of the present invention is illustrated in the U.S. Provisional Application series number of submitting on September 26th, 2,005 60/720,499 and in the PCT publication number WO 07/038658 that submitted on September 26th, 2006, its content merges as a reference at this.When tumour reaches gross tumor volume terminal point (2000mm 3) time, make mouse euthanasia.The results are shown among Figure 39 A (786-O) and Figure 39 B (Caki-1).Being coupled to anti--CD70 antibody 2H5 on the N2 has prolonged and has reached tumour terminal point volume (2000mm 3) mean time, and slowed down the tumor growth process.The body weight change of animal through treating is less than 10%.
Therefore, with anti--CD70 antibody-cytotoxin conjugate treatment the lymphoma tumor growth is had in the direct body and suppress effect.
Embodiment 25: use the interior therapeutic of anti--CD70 immune conjugate to the renal cell carcinoma heteroplastic transplantation model
With the cytotoxin link coupled anti--CD70 antibody carries out interior therapeutic to the mouse of having implanted the kidney tumour, to check that antibody is in vivo to the influence of tumor growth.
As described above, preparation be connected to anti--complex compound N1 of CD70 2H5 antibody of mercaptanization and the immune conjugate of N2 (referring to for example, U.S. Patent Application Publication No. 2006/0024317; And PCT application number PCT/US2006/37793).The NOD-SCID mouse is implanted subcutaneously 2.5 * 10 6Individual 786-O cell.The formation of monitoring tumour is up to reaching about 80mm through measuring mean tumour volume (using accurate calipers) 3The group of eight tumor-bearing mices is treated with one of following material of single dose: (a) vehicle contrast, (b) immune conjugate anti--CD70-N1, or (c) immune conjugate anti--CD70-N2.Give immune conjugate through intraperitoneal (i.p.) to mouse to resist-CD70-N1 and anti--CD70-N2, dosage is respectively the N1 equivalent of 0.3 μ mol/kg and the N2 equivalent of 0.1 μ mol/kg.Anti--CD70-N1 group obtains the treatment second time of same dose after 21 days in the administration first time.In 62 days experimentation, monitor tumor growth by measuring with accurate calipers.
As obvious visible in Figure 40, compare with the mouse that when only with vehicle contrast treatment, has the tumor growth of essence, with immune conjugate anti--the single dose treatment of CD70-N1 or anti--CD70-N2 causes mouse not have tumour (and still do not have up to 62 days tumour) in 15 days.
Embodiment 26: use immune conjugate anti--CD70-N2 is to the interior therapeutic of renal cell carcinoma heteroplastic transplantation model
With the cytotoxin link coupled anti--CD70 antibody carries out interior therapeutic to the mouse of having implanted the kidney tumour, to check that antibody is in vivo to the effect of tumor growth.
As in embodiment 25 explanations, prepared the immune conjugate of the complex compound N2 of the anti--CD702H5 antibody that is connected to mercaptanization.Every mouse of SCID mouse be implanted subcutaneously in 0.1ml PBS and 0.1ml matrigel 2.5 * 10 6Individual 786-O cell.The formation of monitoring tumour is up to reaching about 105mm through measuring mean tumour volume (using accurate calipers) 3The group of eight tumor-bearing mices is treated with one of following material of single dose: (a) vehicle contrast, (b) isotype contrast (c) only has anti--CD70 antibody 2H5, or (d) immune conjugate anti--CD70-N2.Intraperitoneal gives immune conjugate to mouse and resists-CD70-N2 and isotype contrast N2 (IgG-N2), and dosage is the N2 equivalent of 0.1 μ mol/kg.Give the anti--CD70 antibody protein dosage of the used N2 equivalent equivalence of immune conjugate CD70-N2 (that is, with) of 10mg/kg.In 62 days experimentation, monitor tumor growth by measuring with accurate calipers.
As obvious visible in Figure 41, with when only with contrast or with only comparing with anti--mouse of having the tumor growth of essence during the CD70 Antybody therapy, with immune conjugate anti--treatment of the single low dosage of CD70-N2 causes mouse to have the detectable tumour of bottom line (and keeping this situation up to 62 days) in 10 days.
Embodiment 27: in the body renal cell carcinoma xenotransplantation to immune conjugate anti--dose response of CD70-N2
With the cytotoxin link coupled anti--CD70 antibody carries out interior therapeutic to the mouse of having implanted the kidney tumour, to check that antibody is in vivo to the influence of tumor growth.
As explanation in embodiment 25, prepare the immune conjugate of the complex compound N2 of the anti--CD70 2H5 antibody that is connected to mercaptanization.Every mouse of SCID mouse be implanted subcutaneously in 0.1ml PBS and 0.1ml matrigel 2.5 * 10 6Individual 786-O cell.The formation of monitoring tumour is up to reaching about 280mm through measuring mean tumour volume (using accurate calipers) 3With (a) vehicle contrast or (b) immune conjugate anti--group of eight tumor-bearing mices of CD70-N2 treatment.Through intraperitoneal give the immune conjugate of one of every group of following dosage of mouse anti--the N2 equivalent of CD70-N2:0.03 μ mol/kg, 0.01 μ mol/kg or 0.005 μ mol/kg.In experimentation, monitor tumor growth by measuring with accurate calipers.
As obvious visible in Figure 42, the immune conjugate of beat all low dosage is anti--and CD70-N2 causes gross tumor volume to reduce, and gross tumor volume reduces to take place in the mode of dose-dependently.
Embodiment 28: immune conjugate is anti--and CD70-N2 is in vivo to the effectiveness of another kind of renal cell carcinoma heteroplastic transplantation model
With the cytotoxin link coupled anti--CD70 antibody carries out interior therapeutic to the mouse of having implanted the kidney tumour, to check that antibody is in vivo to the influence of tumor growth.
As explanation in embodiment 25, prepared the immune conjugate of the complex compound N2 of the anti--CD702H5 antibody that is connected to mercaptanization.Every mouse of SCID mouse be implanted subcutaneously in 0.1ml PBS and 0.1ml matrigel 2.5 * 10 6Individual Caki-1 cell.The formation of monitoring tumour is up to reaching about 105mm through measuring mean tumour volume (using accurate calipers) 3Group with eight tumor-bearing mices of one of following material of single dose treatment: (a) vehicle contrast, (b) isotype contrast (c) only has anti--CD70 antibody 2H5, or (d) immune conjugate anti--CD70-N2.Give immune conjugate through intraperitoneal to mouse and resist-CD70-N2 and isotype contrast N2, dosage is the N2 equivalent of 0.3 μ mol/kg.Give the anti--CD70 antibody protein dosage of the used N2 equivalent equivalence of immune conjugate CD70-N2 (that is, to) of 11.5mg/kg.In 62 days experimentation, monitor tumor growth by measuring with accurate calipers.
As obvious visible in Figure 43, with when only with contrast or only compare with anti--mouse of having the tumor growth of essence during the CD70 Antybody therapy, with immune conjugate anti--the single dose treatment of CD70-N2 causes mouse to have the detectable tumour of bottom line, continues to much about 40 days.Therefore, anti--CD70 immune conjugate is effectively resisted multiple kidney model.
Embodiment 29: immune conjugate is anti--the CD70-N2 effectiveness in the lymphoma model in vivo
With the cytotoxin link coupled anti--CD70 antibody carries out interior therapeutic to the mouse of having implanted the lymphoma tumour, to check that antibody is in vivo to the influence of tumor growth.
As explanation in embodiment 25, prepared the immune conjugate of the complex compound N2 of the anti--CD702H5 antibody that is connected to mercaptanization.Every mouse of SCID mouse be implanted subcutaneously in 0.1ml PBS and 0.1ml matrigel 1.0 * 10 7Individual Raji cell.The formation of monitoring tumour is up to reaching about 50mm through measuring mean tumour volume (using accurate calipers) 3Group with eight tumor-bearing mices of one of following material of single dose treatment: (a) vehicle contrast, (b) isotype contrast, or (c) immune conjugate anti--CD70-N2.Intraperitoneal gives immune conjugate to mouse and resists-CD70-N2, and dosage is the N2 equivalent of 0.3 μ mol/kg.In 60 days experimentation, monitor tumor growth by measuring with accurate calipers.
As obvious visible in Figure 44, with when only with contrast or only compare with anti--mouse of having the tumor growth of essence during the CD70 Antybody therapy, with immune conjugate anti--the single dose treatment of CD70-N2 causes mouse to have the detectable tumour of bottom line, continues to much about 40 days.Therefore, anti--CD70 immune conjugate is also effectively resisted lymphoma.
Embodiment 30: immune conjugate is anti--safety research of CD70-N2
With the immune conjugate of one of following dosage anti--CD70-N2 treats BALB/c mouse through intraperitoneal: the N2 equivalent of 0.1 μ mol/kg, 0.3 μ mol/kg, 0.6 μ mol/kg or 0.9 μ mol/kg.Measured the weight of mouse initial 12 day every day, and after this measured termly after administration 60 days.When mouse lose weight surpass initial body weight 20% the time, make mouse euthanasia.The data of marking and drawing in Figure 45 are mean body weights of every group.
As obvious visible in Figure 45, when the N2 equivalent that is lower than 0.9 μ mol/kg with dosage carried out administration, anti--CD70-N2 immune conjugate was tolerated well and is safe.Therefore, immune conjugate anti--dosage (in the scope of the N2 equivalent of about 0.005 to 0.3 μ mol/kg) that CD70-N2 has shown effect will have good security feature.
Embodiment 31: immune conjugate is anti--the further safety research of CD70-N2
Immune conjugate is anti--and another safety research of CD70-N2 carries out in than Ge Er dog male.Immune conjugate and the situation that medicine is only arranged have been compared.Two than Ge Er dog in through intravenously give the immune conjugate of N2 equivalent of 0.18 μ mol/kg anti--medicine that N2 is only arranged (in the N2 structure, not having connector) of CD70-N2 and 0.15 μ mol/kg.After administration, per hour monitor dog in 4 hours, and do twice clinical observation every day, continue 28 days.Measure body weight every day after administration 8 days, and after this measure weekly.The administration last stage carry out twice and administration after carried out in 3,7,14 and 28 days standard hematology, solidify and clinical chemistry testing.The results are shown in Figure 46 A to D.Because toxic clinical indication, 8 angels do not have a dog euthanasia in the medicine group after administration.As shown in Figure 46 A to D, tolerated anti--CD70-N2 immune conjugate well through the dog of treatment.
Embodiment 32: the ADCC of the anti--antibody-mediated activated of CD70 human B cell
In this research, tested HuMAb anti--CD70 antibody and non-fucosylated form mediation be to the ability of the ADCC effect of human B cell.Freezing human splenocyte is thawed and the B cell is carried out negative purifying by magnetic bead.With purified B cell with 2 * 10 6/ ml cultivates in the RPMI+10%FBS that is supplemented with NEAA, Sodium.alpha.-ketopropionate, β-ME and penicillin/streptomycin.The B cell is activated 3 days by the anti-CD40 of the LPS of 10 μ g/ml and 5 μ g/ml.Collecting cell, washing and an aliquot dye with the non-fucosylated 2H5 of vitamin H link coupled (2H5NF-bio)+streptavidin-APC.Ficoll-Paque by standard separates, and is purified into human peripheral blood mononuclear effector cell from the whole blood of heparinization, and in the presence of the IL-2 of 50U/ml overnight incubation.Activated B cell is with per 1 * 10 6The Na of individual cell 100 μ Ci 2 51CrO 4(Perkin Elmer, Wellesley MA) carry out mark, continue 1 hour.Ratio with 1: 100 in the presence of the serial dilution of 2H5 and 2H5NF (non-fucosylated) is added to the effector cell in the target cell that is labeled.In addition, in the presence of the muroid of 20 μ g/ml anti-CD16 antibody 3G8 or mouse isotype control antibodies, measure tester at 10 μ g/ml.37 ℃ carry out hatching for 4 times continue 4 hours after, with cell centrifugation and the Cobra II with reading window of 240 to 400KeV automatically-γ readout instrument (PerkinElmer) goes up supernatant liquor carried out reading.Specificity cracking percentage calculation is: (experiment burst size-spontaneous burst size)/(maximum burst size-spontaneous burst size) * 100, wherein: (i) no effect cell and the target cell that do not have an antibody contrast as spontaneous burst size and (ii) the target cell in the presence of 3% lysol stain remover contrast and effector cell as maximum burst size.Mark and draw special cracking per-cent at antibody concentration, and use GraphPad Prism TM3.0 software (San Diego, CA), by non-linear regression, analyze data by S shape dose response (variable slope).
Data are shown among Figure 47.2H5NF combines with about 60% the B of activated cell.2H5NF and 2H5 have all induced the cracking of activated human B cell, but 2H5NF is than 2H5 stronger (about 10 times) and more effective.Anti-CD16 antagonist inductive cracked reverses and confirms that antibody-mediated cracked mechanism of action is the cell-mediated ADCC of NK.Therefore, 2H5 and 2H5NF all mediate the human ADCC of activated B cell.
Embodiment 33: anti--CD70 antibody is to the vitro inhibition of the human CD4 positive T cell propagation of CMV antigenic stimulation
This studies have shown that anti--CD70 antibody mediates by the cracking ability of the positive human T cell of the CD70 of antigen activates (these cells are key factors of inflammatory process in autoimmunization and inflammatory diseases) by the effector cell in the natural human PBMC culture that is present in through stimulating through ADCC.
With the CMV male through the donor of prescreen with 1 * 10 6Individual cell/ml is incubated in the AIM-V substratum that is supplemented with 10% heat-inactivated FCS on 24 well culture plates, and stimulates in the presence of biotinylated 2H5, the 2H5NF of 2 μ g/ml or hIgG1nf contrast antibody with the CMV lysate of 5.0 μ g/ml.The 9th day collecting cell and use hematimeter and trypanblue exclusion method by the aliquot counting having been determined the number of every milliliter of viable cell in every kind of culture.Cell is washed in the dyeing damping fluid and seal with 5% human serum.Biotinylated 2H5,2H5NF or hIgG1nf are joined in isopyknic cell, and final concentration is 20 μ g/ml.Cell was hatched 30 minutes, and washing is also dyeed with anti-CD4-FITC and PE link coupled streptavidin.Cell is hatched 30 minutes, washed twice is also then fixed and is changed thoroughly with the BDCytofix/Cytoperm test kit again.Cell washed twice in the perm/wash damping fluid is also carried out cell inner dyeing with anti-INF γ-APC (BD Clone B27).Cell was hatched 30 minutes, wash and be resuspended in the dyeing damping fluid.Analyze at expressing pair cell in CD70 surface expression and the INF-γ born of the same parents on the CD4 positive cell of living through screening by flow cytometry.(per-cent that the per-cent by the CD70 positive in CD4 screens or INF γ positive cell of cell/ml) multiply by total CD4 positive cell multiply by overall number ((the %CD70 positive or the INF γ positive) * (the %CD4 positive) * (total viable cell/ml)) of every milliliter of viable cell to the positive and CD4 positive/INF γ male number in the CD4 positive under every kind of condition/CD70 again.
These data are shown among Figure 48.2H5 and 2H5NF at the 9th day 2 μ g/ml have reduced by the CMV activated CD70 positive/CD4 positive cell of 67% and 97% respectively.Two kinds of antibody all are effectively, but 2H5NF than 2H5 more effectively by being present in the ADCC that the positive effector cell of CD16 in the normal human blood mediates the Ag activated CD4 positive/CD70 positive T cell.
Embodiment 34: relative with the human CD70 antibody of the positive renal carcinoma cell line 786-0 of CD70 bonded 1F4,1F4NF and 2H5NF in conjunction with feature
This research the combine feature of anti--CD70 antibody with natural expression CD70 male human cancer cell line 786-0 cell.Mankind kidney cell's gland cell system 786-0 is grown into to be converged, collect with trypsinase, wash in the damping fluid in dyeing, and with final concentration be 30,10,3,1,0.4,0.1,0.04 and 1F4,1F4NF, 2H5NF, hIgG1-NF or the hIgG4 of 0.01ug/ml hatch.Cell was hatched 30 minutes washed twice in the dyeing damping fluid, and with goat F (ab) on ice ' 2-Anti-Human class-IgG (Fc)-PE conjugate dyeed 30 minutes.Washed cell also is resuspended in dyeing and is used for flow cytometry in the damping fluid.
Data are shown in Figure 49.2H5NF is to carry out combination than 1F4 and the low concentration of 1F4NF.For the CD70 of n cell surface expression, 2H5NF has the binding affinity that is better than 1F4 and 1F4NF.1F4 and 1F4NF combine with 786-0 clone equally well, do not show the influence of specificity in conjunction with feature because of the NF isotype.
Embodiment 35: 1F4 and 1F4NF mediate the relative capacity of ADCC on CD70 positive lymphomas clone ARH77
In this research, anti--CD70 antibody of having tested fucosylated and non-fucosylated (nf) mediates the relative ability of ADCC on CD70 positive lymphomas clone ARH77.Ficoll-Paque by standard separates be purified into human peripheral blood mononuclear effector cell from the whole blood of heparinization, and in the presence of 50U/ml IL-2 overnight incubation.With per 1 * 10 6The Na of individual cell 100 μ Ci 2 51CrO 4(Perkin Elmer, Wellesley MA) carry out mark to the ARH77 cell, continue 1 hour.Ratio with 1: 100 in the presence of the serial dilution thing of 2H5 and 2H5nf is added to the effector cell in the target cell of mark.In addition, 5 μ g/ml testers are measured.After hatching 4 hours under 37 ℃, the automatic γ readout instrument of Cobra II (Perkin Elmer) of cell centrifugation and the reading window by having 240-400KeV is carried out reading to supernatant liquor.Specificity cracking percentage calculation is: (experiment burst size-spontaneous burst size)/(maximum burst size-spontaneous burst size) * 100 wherein: (i) target cell of no effect cell and no antibody contrast as spontaneous burst size and (ii) the target cell in the presence of 3% lysol stain remover contrast and effector cell as maximum burst size.
Data are shown among Figure 50.1F4 and 1F4NF mediate ADCC on the positive ARH77 cell of CD70, and 1F4NF is the ADCC mediation person stronger than 1F4.
Embodiment 36: suppress in the anti--body of CD70 cytotoxin E tumor growth
-CD70-cytotoxin E conjugate anti-in order to prove be as the extensive use of target therapeutic agent of the different tumour cells of opposing, three kinds of renal cell carcinoma heteroplastic transplantation models of SCID mouse and two kinds of lymphoma models is used to test effect in the body of anti--CD70-cytotoxin E conjugate.The cytotoxin conjugate of CD70 antibody 2H5 refers to resist-CD70-cytotoxin E herein, it comprises the reorganization 2H5 that is connected to cytotoxin E (Figure 74) and resists-CD70 antibody, cytotoxin E is discussed in the U. S. application series number of submitting on December 28th, 2,006 60/882, in 461, its full content is incorporated in this as a reference specially.Cytotoxin E is a prodrug form, and not only needs to discharge from antibody for activating, and also needs the shearing of 4 ' carbamate groups to discharge active part.
Anti-in order to prove-activity of CD70-cytotoxin E on 786-O cell heteroplastic transplantation model, will be at 0.1ml PBS and 0.1ml Matrigel TMIn 2,500,000 786-O cell skins implant in every SCID mouse down, and reach 110mm when tumour 3Mean size the time, by anti--CD70-cytotoxin E (with the dosage of 0.005,0.03 or 0.1 μ mol/kg body weight) group of 8 mouse is treated through the peritoneal injection single dose.In addition, only with vehicle or only control group is injected with anti--CD70 antibody (to be equivalent to dosage) or with the isotype control antibodies that is connected to cytotoxin E (with the dosage of 0.03 and 0.1 μ mol/kg) with anti--employed those dosage of CD70-cytotoxin E of 0.03 and 0.1 μ mol/kg.Write down the weight of gross tumor volume (LWH/2) and mouse in the whole experiment, experimentation was continued medication back 61 days.The results are shown in Figure 51.In this concrete mouse heteroplastic transplantation model (it be immunocompromised (immunocompomised)) and under the dosage of explanation, do not demonstrate influence (that is, not suppressing tumor growth) to gross tumor volume with exposed CD70 Antybody therapy.The isotype contrast is to growth of tumor also almost not influence.On the contrary, anti--CD70-cytotoxin E conjugate has clearly shown the dose-dependently antitumor curative effect.Even at 0.03 μ mol/kg, the curative effect of this specificity conjugate shows maximumly.
Next the activity of anti--CD70-cytotoxin E has the proof of obtaining in the heteroplastic SCID mouse of A498 tumour lotus.With the A498 cell (at 0.1ml PBS and 0.1ml Matrigel TMIn 5,000,000 cell/mouse) be implanted subcutaneously in the SCID mouse, and reach 110mm when tumour 3Mean size the time, by anti--CD70 cytotoxin E (with the dosage of 0.03,0.1 or 0.3 μ mol/kg body weight) group of eight mouse is treated through the peritoneal injection single dose.In addition, only control group is injected with vehicle.In the whole experiment of this research, write down the weight of gross tumor volume (LWH/2) and mouse, made experimentation continue to proceed to after the administration about 60 days.The results are shown in Figure 52.These results prove, and anti--CD70-cytotoxin E conjugate treats kidney in this model be powerful, and this treatment is a dose-dependently.
Next the activity of anti--CD70-cytotoxin E has the proof of obtaining in the heteroplastic SCID mouse of Caki-1 tumour lotus.With Caki-1 cell (0.1ml PBS and 0.1ml Matrigel TMIn 2,500,000 cell/mouse) be implanted subcutaneously in the SCID mouse, and reach 150mm when tumour 3Mean size the time, by anti--CD70 cytotoxin E (with 0.03,0.1 or 0.3 μ mol/kg body weight) group of 8 mouse is treated through the peritoneal injection single dose.Also use extra group, carried out administration at interval in 14 days, the effect of research repeated doses treatment by anti--CD70-cytotoxin-E conjugate with 0.1 μ mol/kg of two dosage.In addition, only control group is injected with vehicle.In the whole experiment of this research, write down the weight of gross tumor volume (LWH/2) and mouse, experimentation was carried out after administration 62 days.The results are shown in Figure 53.It is effectively that these results prove anti--CD70-cytotoxin E conjugate has the kidney of the mouse of caki-1 tumour to the treatment lotus, and this treatment is a dose-dependently.
Anti-in order to prove-activity of CD70-cytotoxin E in the lymphoma model, have lotus and to have carried out treatment research in the heteroplastic SCID mouse of subcutaneous Raji.With the Raji cell (at 0.1ml PBS and 0.1ml Matrigel TMIn 1,000 ten thousand cell/mouse) be implanted subcutaneously in the SCID mouse, and reach 250mm when tumour 3Mean size the time, treat through the group that anti--CD70 cytotoxin E (with 0.03,0.1 or 0.3 μ mol/kg body weight) of peritoneal injection single dose forms 8 mouse.In addition, only use vehicle, or control group is injected with the isotype control antibodies that is connected to cytotoxin E (with 0.1 or 0.3 μ mol/kg body weight).In the whole experiment of this research, write down the weight of gross tumor volume (LWH/2) and mouse, continued to carry out after this experimentation administration about 60 days.The results are shown in Figure 54.These results prove, and anti--CD70-cytotoxin E conjugate treats in this model that lymphoma also is effectively, and this treatment is a dose-dependently.
Use Daudi xenotransplantation to carry out second lymphoma Study of model.With the Daudi cell (at 0.1ml PBS and 0.1ml Matrigel TMIn 1,000 ten thousand cell/mouse) be implanted subcutaneously in the SCID mouse, and reach 70mm when tumour 3Mean size the time, by anti--CD70 cytotoxin E (with 0.1 or 0.3 μ mol/kg body weight) group of 8 mouse is treated through the peritoneal injection single dose.In addition, only with vehicle, only control group is injected with anti--CD70 antibody or with isotype control antibodies cytotoxin E conjugate (with 0.1 or 0.3 μ mol/kg body weight).In the whole experiment of this research, write down the weight of gross tumor volume (LWH/2) and mouse, continued to carry out after this experimentation administration about 60 days.The results are shown in Figure 55.In this concrete mouse heteroplastic transplantation model (it is immunocompromised) and under illustrated dosage, do not demonstrate influence (that is, not suppressing tumor growth) to gross tumor volume with exposed CD70 Antybody therapy.By contrast, anti--CD70-cytotoxin E conjugate is effectively to lymphoma in this model, and this treatment is a dose-dependently.
Can in a plurality of species, to observe effect in order proving, the heteroplastic transplantation model of nude rat to be tested.In this model, whole body has been implanted subcutaneously Caki-1 cell (1,000 ten thousand cell/mouse in 0.2ml RPMI-1640) by the nude rat of gamma-radiation, and reaches 100mm when the tumour mean size 3The time, by anti--CD70-cytotoxin E (with 0.1 or 0.3 μ mol/kg body weight) group of rat is treated through the peritoneal injection single dose.Alternately carry out multiple doses treatment, wherein accept the dosage of 0.3 μ mol/kg body weight, totally 3 dosage the 8th, 15 and 22 day rat.In addition, only with vehicle, only with single dose or with identical multiple doses scheme control group is injected with anti--CD70 antibody or isotype control antibodies cytotoxin E conjugate (with the dosage of 0.3 μ mol/kg body weight).In the whole process of this research, write down gross tumor volume (LW 2/ 2) and the weight of rat.The results are shown in Figure 56.In this concrete rat heteroplastic transplantation model (it is immunocompromised) and under illustrated dosage, do not demonstrate influence (that is, not suppressing tumor growth) to gross tumor volume with exposed CD70 Antybody therapy.By contrast, anti--CD70-cytotoxin E conjugate has shown significant antitumous effect.Improved effect with the multiple doses treatment, and the body weight of animal has been had no significant effect.Even use the repeated doses scheme, this isotype contrast conjugate demonstrates the influence minimum to tumor growth.
In three different animal species, tested the security of anti--CD70 conjugate.The groups of 5 normal balb/c mouse is carried out administration (intraperitoneal) with anti--CD70-cytotoxin E, and dosage is 0.1,0.3,0.6 and 0.9 μ mol/kg body weight, and compares in 60 days with the animal of only injecting vehicle and to monitor the weight of animals.In the process of this research, the control animal weight increase 10 to 20%.The mouse that carries out administration with anti--CD70 cytotoxin E is presented under the lower dosage that this conjugate is tolerated usually well and is very little to the influence of body weight.Exist the toxicity of tangible dose-dependently to increase, high dosage causes the temporary transient reduction of animal body weight before recovery.Yet when dosage surpassed the curative effect required dosage of heteroplastic transplantation model, this conjugate was still tolerated well.The results are shown in Figure 57.
Also in dog and monkey, tested toxicity.The group of three dogs is carried out administration with the dosage of 0.1,0.2,0.3,0.4 and 0.6 μ mol/kg body weight, and the group of two monkeys is carried out administration with the dosage of 0.2,0.4,0.6 and 0.8 μ mol/kg body weight.Paying special attention to total white blood cell count(WBC) and platelet count in every research, is toxic special sensitive telltales of anti--CD70 antibody-cytotoxin E conjugate because it is believed that these.In dog, do not observe Cytometric noticeable change, reach 0.6 μ mol/kg body weight up to dosage.The temporary transient decline of platelet count occurred at this dosage, and white blood cell count(WBC) has reduced also.Under any dosage, the variation of observed these parameters is very little in monkey.Two research all supports to resist-and the toxic dose of CD70 conjugate in animal be significantly higher than the effect dosage in xenograft models.The results are shown among Figure 58 (result of dog) and 59 (results of monkey).
Embodiment 37: suppress tumor growth in vivo by anti--CD70-cytotoxin F
In this embodiment, in two kinds of kidneys and a kind of lymphoma heteroplastic transplantation model, proved the effect of anti--CD70-cytotoxin F.The cytotoxin conjugate of CD70 antibody 2H5 is called CD70-cytotoxin F at this, and it comprises the reorganization 2H5 that is connected on the cytotoxin F (Figure 75) and resists-CD70 antibody.Cytotoxin F is a kind of esterase activated prodrug that needs.
Anti-in order to prove-activity of CD70-cytotoxin F in the xenotransplantation of 786-O cell, every mouse of SCID mouse has been implanted subcutaneously at 0.1ml PBS and 0.1ml Matrigel TMIn 2,500,000 786-O cells, and reach 110mm when tumour 3Mean size the time, by anti--CD70-cytotoxin F (with the dosage of 0.005,0.03 or 0.1 μ mol/kg body weight) group of 8 mouse is treated through the peritoneal injection single dose.In addition, only control group is injected with vehicle or with the isotype control antibodies that is connected to cytotoxin F (with the dosage of 0.03 μ mol/kg and 0.1 μ mol/kg).In the whole process of this research, write down the weight of gross tumor volume (LWH/2) and mouse, make this research proceed to after the administration 62 days.The results are shown in Figure 60.In this concrete mouse heteroplastic transplantation model (it is immunocompromised) and under illustrated dosage, do not demonstrate influence (that is, not suppressing tumor growth) to gross tumor volume with the treatment of exposed CD70 antibody.In this experiment, isotype contrast conjugate is also almost to not influence of growth of tumor, and the mouse of anti--CD70-cytotoxin F treatment clearly shows the antitumor efficacy of dose-dependently.Even at 0.03 μ mol/kg, the performance of the curative effect of this specificity conjugate is maximum.
Lotus the activity that has next proved anti--CD70-cytotoxin F in the heteroplastic SCID mouse of Caki-1 tumour is arranged.With the Caki-1 cell (at 0.1ml PBS and 0.1ml Matrigel TMIn 2,500,000 cell/mouse) be implanted subcutaneously in the SCID mouse, and reach 120mm when tumour 3Mean size the time, by anti--CD70 cytotoxin F (with the dosage of 0.03,0.1 or 0.3 μ mol/kg body weight) group of 8 mouse is treated through the peritoneal injection single dose.In addition, only control group is injected with vehicle.The weight of record gross tumor volume and mouse in the whole experiment of this research continues to proceed to after the administration 62 days.The results are shown in Figure 61.It is powerful that these results prove anti--CD70-cytotoxin F conjugate has in the mouse of caki-1 tumour lotus, and this treatment is a dose-dependently.
Anti-in order to prove-activity of CD70-cytotoxin F in the lymphoma model, in having the SCID mouse of Raji heterograft, subcutaneous lotus carried out treatment research.With the Raji cell (at 0.1mlPBS and 0.1ml Matrigel TMIn 1,000 ten thousand cell/mouse) be implanted subcutaneously in the SCID mouse, and reach 250mm when tumour 3Mean size the time, by anti--CD70 cytotoxin F (with the dosage of 0.03,0.1 or 0.3 μ mol/kg body weight) group of 8 mouse is treated through the peritoneal injection single dose.In addition, only control group is injected with vehicle or with the isotype control antibodies that is connected to cytotoxin F (with the dosage of 0.1 or 0.3 μ mol/kg body weight).In the whole experiment of this research, write down the weight of gross tumor volume (LWH/2) and mouse, continue to proceed to after the administration about 60 days.The results are shown in Figure 62.That these results prove is anti--CD70-cytotoxin F conjugate opposing lymphoma also is powerful, and this treatment is a dose-dependently.
Embodiment 38: suppress tumor growth in vivo by anti--CD70-toxin G
In this embodiment, in the heteroplastic transplantation model of two kidneys, proved the effect of anti--CD70-cytotoxin G.The cytotoxin conjugate of CD70 antibody 2H5 is called CD70-cytotoxin G at this, and it comprises the reorganization 2H5 that is connected to cytotoxin G (Figure 76) and resists-CD70 antibody.Cytotoxin G is a kind of esterase activated prodrug that needs.
Anti-in order to prove-activity of CD70-cytotoxin G in the xenotransplantation of 786-O cell, every mouse of SCID mouse has been implanted subcutaneously at 0.1ml PBS and 0.1ml Matrigel TMIn 2,500,000 786-O cells, and reach 110mm when tumour 3Mean size the time, by anti--CD70-cytotoxin G (with the dosage of 0.005,0.03 or 0.1 μ mol/kg body weight) group of 8 mouse is treated through the peritoneal injection single dose.In addition, only control group is injected with vehicle or with the isotype control antibodies that is connected to cytotoxin G (with the dosage of 0.03 μ mol/kg and 0.1 μ mol/kg).In the whole process of this research, write down the weight of gross tumor volume (LWH/2) and mouse, continue to proceed to after the administration 61 days.The results are shown among Figure 63.These results proof in this test anti--CD70 antibody itself or isotype contrast conjugate to almost not influence of growth of tumor, and with resist-mouse that CD70-cytotoxin G treats clearly shows the antitumor efficacy of dose-dependently.
Lotus the activity that has next proved anti--CD70-cytotoxin G in the heteroplastic SCID mouse of Caki-1 tumour is arranged.With the Caki-1 cell (at 0.1ml PBS and 0.1ml Matrigel TMIn 2,500,000 cell/mouse) be implanted subcutaneously in the SCID mouse, and reach 120mm when tumour 3Mean size the time, by anti--CD70 cytotoxin G (with the dosage of 0.03,0.1 or 0.3 μ mol/kg body weight) group of 8 mouse is treated through the peritoneal injection single dose.In addition, only control group is injected with vehicle.In the whole experiment of this research, write down the weight of gross tumor volume (LWH/2) and mouse, continue to proceed to after the administration 61 days.The results are shown in Figure 64.These results prove anti--CD70-cytotoxin G conjugate has lotus that the opposing kidney is powerful in the mouse of caki-1 tumour, and this treatment is a dose-dependently.
Embodiment 39: suppress tumor growth in vivo by anti--CD70-toxin H
In this embodiment, in the heteroplastic transplantation model of two kidneys, proved the effect of anti--CD70-cytotoxin H.The cytotoxin conjugate of CD70 antibody 2H5 is called CD70-cytotoxin H at this, and it comprises the reorganization 2H5 that is connected on the cytotoxin H (Figure 77) and resists-CD70 antibody.
Lotus the activity that has proved anti--CD70-cytotoxin H in the heteroplastic SCID mouse of A498 tumour is arranged.With the A498 cell (at 0.1ml PBS and 0.1ml Matrigel TMIn 5,000,000 cell/mouse) be implanted subcutaneously in the SCID mouse, and reach 110mm when tumour 3Mean size the time, by anti--CD70 cytotoxin H (with 0.1 μ mol/kg body weight) group of 8 mouse is treated through the peritoneal injection single dose.In addition, only control group is injected with vehicle.In the whole experiment of this research, write down the weight of gross tumor volume (LWH/2) and mouse, continue to proceed to after the administration about 60 days.The results are shown among Figure 65.These results prove, and anti--CD70-cytotoxin H conjugate opposing kidney is powerful.
Anti-in order to prove-activity of CD70-cytotoxin H in the xenotransplantation of Caki-1 cell, with subcutaneous 0.1ml PBS and the 0.1ml Matrigel of being implanted in of every mouse of SCID mouse TMIn 2,500,000 Caki-1 cells, and reach 130mm when tumour 3Mean size the time, by anti--CD70-cytotoxin H (with the dosage of 0.03,0.1 or 0.3 μ mol/kg body weight) group of 8 mouse is treated through the peritoneal injection single dose.In addition, only control group is injected with vehicle or with the isotype control antibodies that is connected to cytotoxin H (with the dosage of 0.1 μ mol/kg and 0.3 μ mol/kg).In the whole process of this research, write down the weight of gross tumor volume (LWH/2) and mouse, continue to proceed to after the administration 61 days.The results are shown in Figure 66.In this concrete mouse heteroplastic transplantation model (it is immunocompromised) and under illustrated dosage, do not demonstrate influence (that is, not suppressing tumor growth) to gross tumor volume with exposed CD70 Antybody therapy.Isotype contrast conjugate is to growth of tumor also almost not influence in this experiment.On the contrary, anti--CD70-cytotoxin H conjugate clearly illustrates the antitumor efficacy of dose-dependently.
Embodiment 40: suppress tumor growth in vivo by anti--CD70-toxin I
In this embodiment, in the heteroplastic transplantation model (the 786-O cell in the SCID mouse and the Caki-1 cell in the nude rat) of two kidneys, proved the effect of anti--CD70-cytotoxin I.The cytotoxin conjugate of CD70 antibody 2H5 is called CD70-cytotoxin I at this, and it comprises the reorganization 2H5 that is connected on the cytotoxin I (Figure 78) and resists-CD70 antibody.
Lotus the activity that has proved anti--CD70-cytotoxin I in the heteroplastic SCID mouse of 786-O tumour is arranged.With the 786-O cell (at 0.1ml PBS and 0.1ml Matrigel TMIn 2,500,000 cell/mouse) be implanted subcutaneously in the SCID mouse, and reach 170mm when tumour 3Mean size the time, by anti--CD70 cytotoxin I (with the dosage of 0.005 μ mol/kg body weight) group of 6 mouse is treated through the peritoneal injection single dose.In addition, only control group is injected with vehicle.In the whole process of this research, write down the weight of gross tumor volume (LWH/2) and mouse.The results are shown among Figure 67.Even it also is powerful that these result's proofs anti--CD70-cytotoxin I conjugate under low dosage is resisted kidney.
Can to observe this effect in order proving in a plurality of species, to have tested the heteroplastic transplantation model in the nude rat.In this model, Caki-1 cell (1,000 ten thousand cell/rats in 0.2ml RPMI-1640) is implanted subcutaneously in the nude rat, and reaches 100mm when tumour 3Mean size the time, by anti--CD70-cytotoxin I (with the dosage of 0.3 μ mol/kg body weight) group of rat is treated through the peritoneal injection single dose.In addition, only with vehicle, only control group is injected with anti--CD70 antibody or the isotype control antibodies cytotoxin I conjugate that is used as single dose (with the dosage of 0.3 μ mol/kg body weight).In the whole process of this research, write down gross tumor volume (LW 2/ 2) and the weight of rat.The results are shown among Figure 68.These result's proofs only have CD70 antibody to almost not influence of tumor growth, and isotype contrast conjugate does not show the influence to tumor growth.Yet anti--CD70-cytotoxin I conjugate has shown remarkable antitumor effect.Tumor regression is achieved.Therefore, anti--CD70-cytotoxin I conjugate has shown antitumor action in a plurality of species.
Embodiment 41: suppress tumor growth in vivo by anti--CD70-toxin J
In this embodiment, in the heteroplastic transplantation model (the 786-O cell in the SCID mouse) of kidney, proved the effect of anti--CD70-cytotoxin J.The cytotoxin conjugate of CD70 antibody 2H5 is called CD70-cytotoxin J at this, and it comprises the reorganization 2H5 that is connected on the cytotoxin J (Figure 79) and resists-CD70 antibody.Cytotoxin J need shear a kind of prodrug of activated by glucuronidase.
Lotus the activity that has proved anti--CD70-cytotoxin J in the heteroplastic SCID mouse of 786-O tumour is arranged.With the 786-O cell (at 0.1ml PBS and 0.1ml Matrigel TMIn 2,500,000 cell/mouse) be implanted subcutaneously in the SCID mouse, and reach 170mm when tumour 3Mean size the time, by anti--CD70 cytotoxin J (with the dosage of 0.03 μ mol/kg body weight) group of 6 mouse is treated through the peritoneal injection single dose.In addition, only control group is injected with vehicle.In the whole process of this research, write down the weight of gross tumor volume (LWH/2) and mouse.The results are shown among Figure 69.It is powerful that these result's proofs anti--CD70-cytotoxin J conjugate in this model is resisted kidney.
Embodiment 42: by anti--T cell proliferation that CD70 antibody function blocking-up CD70 stimulates altogether
This embodiment has illustrated by anti--CD70 antibody 1F4IgG1,1F4IgG4,2H5,2H5F (ab ') 2The analysis and the sign of the T cell proliferation that stimulates altogether with the functional blocking-up of 2H5Fab CD70.
Use MACS CD3 microballon from cryopreserved PBMC, to isolate human CD3 male T cell, and then mouse CD32 and human CD70 stable transfection in the presence of Chinese hamster ovary celI that mitomycin C handles, with cell with 2 * 10 6Individual cell/ml cultivates in the heat-inactivated FCS of RPMI-1640 perfect medium+10%.Cell stimulated 3 days with the anti-CD3 of 1 μ g/ml (clone OKT3), added 1 μ Ci/ hole 3The H-thymidine continues 6 hours, and collecting cell.Propagation is measured as the CPM that measures by scintillation counting technique mixes.
This data presentation, 1F4 and 2H5 antibody can be blocked the caused propagation of being undertaken by the T cell of the anti-CD3 stimulation of the mankind of CD27 signal conduction of CD70 mediation in the dose-dependently mode.It is active that these data show that also the functional blocking-up atypia ground that is undertaken by 2H5 needs the multimerization of the cell surface CD70 of IgG1Fc district mediation to influence blocking-up, and 1F4 does not typically need.Referring to Figure 70.For 2H5IgG1, by 2H5F (ab ') 2Functional blocking-up effect reduce and the functional blocking-up of 2H5Fab is active lacks fully, proved the uncommon feature of 2H5 bonded epi-position.By contrast, it is active that 1F4IgG4 active CD70 multimerization that has proved that 1F4 bonded epi-position does not typically need IgG1 Fc district to mediate of functional blocking-up of equivalence for 1F4IgG1 influences blocking-up, and the active antibody of functional blocking-up is observed usually as having.
Therefore, these data presentation, for the human t cell activation of functional blocking-up CD70 mediation, 2H5 is in conjunction with having the epi-position unusual and characteristic that possibility is unique.The quality and the effectiveness that can also help in addition, the ADCC, internalization, avidity etc. of 2H5IgG1 or 2H5NF mediation by 2H5 bonded epi-position.
The ability of the T cell proliferation that is stimulated by the anti-CD3 of the mankind that the CD27 signal conduction of antibody 1F4 and 2H5 blocking-up CD70 mediation causes is relevant with the treatment of any inflammation sign, and wherein the function of CD70 plays a role in disease process.
The sequence identifier tabulation
??SEQ??ID?NO： Sequence ??SEQ??ID?NO： Sequence
??1 VH amino acid 2H5 ??31 VK CDR1 amino acid 2H5
??2 VH amino acid/11 0B4 ??32 VK CDR1 amino acid/11 0B4
??3 VH amino acid 8B5 ??33 VK CDR1 amino acid 8B5
??4 VH amino acid/11 8E7 ??34 VK CDR1 amino acid/11 8E7
??5 VH amino acid 69A7 ??35 VK CDR1 amino acid 69A7 and 69A7Y
??6 VH amino acid/11 F4 ??36 VK CDR1 amino acid/11 F4
??7 VK amino acid 2H5 ??37 VK CDR2 amino acid 2H5
??8 VK amino acid/11 0B4 ??38 VK CDR2 amino acid/11 0B4
??9 VK amino acid 8B5 ??39 VK CDR2 amino acid 8B5
??10 VK amino acid/11 8E7 ??40 VK CDR2 amino acid/11 8E7
??11 VK amino acid 69A7 and 69A7Y ??41 VK CDR2 amino acid 69A7 and 69A7Y
??12 VK amino acid/11 F4 ??42 VK CDR2 amino acid/11 F4
??13 VH CDR1 amino acid 2H5 ??43 VK CDR3 amino acid 2H5
??14 VH CDR1 amino acid/11 0B4 ??44 VK CDR3 amino acid/11 0B4
??15 VH CDR1 amino acid 8B5 ??45 VK CDR3 amino acid 8B5
??16 VH CDR1 amino acid/11 8E7 ??46 VK CDR3 amino acid/11 8E7
??17 VH CDR1 amino acid 69A7 and 69A7Y ??47 VK CDR3 amino acid 69A7 and 69A7Y
??18 VH CDR1 amino acid/11 F4 ??48 VK CDR3 amino acid/11 F4
??19 VH CDR2 amino acid 2H5 ??49 VH Nucleotide 2H5
??20 VH CDR2 amino acid/11 0B4 ??50 VH Nucleotide 10B4
??21 VH CDR2 amino acid 8B5 ??51 VH Nucleotide 8B5
??22 VH CDR2 amino acid/11 8E7 ??52 VH Nucleotide 18E7
??23 VH CDR2 amino acid 69A7 and 69A7Y ??53 VH Nucleotide 69A7
??24 VH CDR2 amino acid/11 F4 ??54 VH Nucleotide 1F4
??25 VH CDR3 amino acid 2H5 ??55 VK Nucleotide 2H5
??26 VH CDR3 amino acid/11 0B4 ??56 VK Nucleotide 10B4
??27 VH CDR3 amino acid 8B5 ??57 VK Nucleotide 8B5
??28 VH CDR3 amino acid/11 8E7 ??58 VK Nucleotide 18E7
??29 VH CDR3 amino acid 69A7 ??59 VK Nucleotide 69A7 and 69A7Y
??30 VH CDR3 amino acid/11 F4 ??60 VK Nucleotide 1F4
??61 VH 3-30.3 kind is an amino acid ??69 JH 4b kind is an amino acid
??62 VH 3-33 kind is an amino acid ??70 JK is an amino acid for 4 kinds
??63 VH 4-61 kind is an amino acid ??71 JK is an amino acid for 3 kinds
??64 VH 3-23 kind is an amino acid ??72 JK is an amino acid for 2 kinds
??65 VK L6 kind is an amino acid ??73 VH amino acid 69A7Y
??66 VK L18 kind is an amino acid ??74 VH Nucleotide 69A7Y
??67 VK L15 kind is an amino acid ??75 VH CDR3 amino acid 69A7Y
??68 VK A27 kind is an amino acid ??76 Human CD70 (P32970)
??77 The peptide connector
??78 The peptide connector
??79 The peptide connector
??80 The peptide connector
??81 The peptide connector
??82 The peptide connector
??83 The peptide connector
??84 The peptide connector
??85 The peptide connector
??86 The peptide connector
??87 The peptide connector
??88 The peptide connector
??89 The peptide connector
??90 The cytomegalovirus peptide
??91 The peptide connector
??92 The peptide connector
Sequence table
＜110〉Medarex Inc.
MA section is proper
JA Te Leite
The DJ gold
C handkerchief grace
The J Caldarelli
M Ya Manaka
KA is prosperous peaceful
＜120〉in conjunction with human antibodies of CD70 and uses thereof
<130>077375.0533
<150>US?60/870,091
<151>2006-12-14
<150>US?60/915,314
<151>2007-05-01
<150>US?60/991,702
<151>2007-11-30
<160>92
<170>PatentIn?version?3.4
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＜213〉mankind
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Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?Arg
1???????????????5???????????????????10??????????????????15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20??????????????????25??????????????????30
Ile?Met?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35??????????????????40??????????????????45
Ala?Val?Ile?Ser?Tyr?Asp?Gly?Arg?Asn?Lys?Tyr?Tyr?Ala?Asp?Ser?Val
50??????????????????55??????????????????60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65??????????????????70??????????????????75??????????????????80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85??????????????????90??????????????????95
Ala?Arg?Asp?Thr?Asp?Gly?Tyr?Asp?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr
100?????????????????105?????????????????110
Leu?Val?Thr?Val?Ser?Ser
115
<210>2
<211>119
<212>PRT
＜213〉mankind
<400>2
Gln?Ile?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?Arg
1???????????????5???????????????????10??????????????????15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Gly?Tyr?Tyr
20??????????????????25??????????????????30
Ala?Met?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35??????????????????40??????????????????45
Ala?Val?Ile?Ser?Tyr?Asp?Gly?Ser?Ile?Lys?Tyr?Tyr?Ala?Asp?Ser?Val
50??????????????????55??????????????????60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65??????????????????70??????????????????75??????????????????80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85??????????????????90??????????????????95
Ala?Arg?Glu?Gly?Pro?Tyr?Ser?Asn?Tyr?Leu?Asp?Tyr?Trp?Gly?Gln?Gly
100?????????????????105?????????????????110
Thr?Leu?Val?Thr?Val?Ser?Ser
115
<210>3
<211>118
<212>PRT
＜213〉mankind
<400>3
Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?Arg
1???????????????5???????????????????10??????????????????15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Thr?Ser?Gly?Phe?Thr?Phe??Ser?Asp?Tyr
20??????????????????25??????????????????30
Gly?Met?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35??????????????????40??????????????????45
Ala?Val?Ile?Trp?Tyr?Asp?Gly?Ser?Asn?Lys?Tyr?Tyr?Ala?Asp?Ser?Val
50??????????????????55??????????????????60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Lys?Thr?Leu?Ser
65??????????????????70??????????????????75??????????????????80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85??????????????????90??????????????????95
Ala?Arg?Asp?Ser?Ile?Met?Val?Arg?Gly?Asp?Tyr?Trp?Gly?Gln?Gly?Thr
100?????????????????105?????????????????110
Leu?Val?Thr?Val?Ser?Ser
115
<210>4
<211>118
<212>PRT
＜213〉mankind
<400>4
Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?Arg
1???????????????5???????????????????10??????????????????15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Asp?His
20??????????????????25??????????????????30
Gly?Met?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35??????????????????40??????????????????45
Ala?Val?Ile?Trp?Tyr?Asp?Gly?Ser?Asn?Lys?Tyr?Tyr?Ala?Asp?Ser?Val
50??????????????????55??????????????????60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65??????????????????70??????????????????75??????????????????80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85??????????????????90??????????????????95
Ala?Arg?Asp?Ser?Ile?Met?Val?Arg?Gly?Asp?Tyr?Trp?Gly?Gln?Gly?Thr
100?????????????????105?????????????????110
Leu?Val?Thr?Val?Ser?Ser
115
<210>5
<211>122
<212>PRT
＜213〉mankind
<400>5
Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu
1???????????????5???????????????????10??????????????????15
Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Val?Ser?Ser?Asp
20??????????????????25??????????????????30
Tyr?Tyr?Tyr?Trp?Ser?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu
35??????????????????40??????????????????45
Trp?Leu?Gly?Tyr?Ile?Tyr?Tyr?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro?Ser
50??????????????????55??????????????????60
Leu?Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe
65??????????????????70??????????????????75??????????????????80
Ser?Leu?Lys?Leu?Arg?Ser?Val?Thr?Thr?Ala?Asp?Thr?Ala?Val?Tyr?Tyr
85??????????????????90??????????????????95
Cys?Ala?Arg?Gly?Asp?Gly?Asp?Tyr?Gly?Gly?Asn?Cys?Phe?Asp?Tyr?Trp
100?????????????????105?????????????????110
Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115?????????????????120
<210>6
<211>120
<212>PRT
＜213〉mankind
<400>6
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1???????????????5???????????????????10??????????????????15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ile?Tyr
20??????????????????25??????????????????30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35??????????????????40??????????????????45
Ser?Ala?Ile?Ser?Asp?Ser?Gly?Gly?Arg?Thr?Tyr?Phe?Ala?Asp?Ser?Val
50??????????????????55??????????????????60
Arg?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Ser
65??????????????????70??????????????????75??????????????????80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85??????????????????90??????????????????95
Ala?Lys?Val?Asp?Tyr?Ser?Asn?Tyr?Leu?Phe?Phe?Asp?Tyr?Trp?Gly?Gln
100?????????????????105?????????????????110
Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115?????????????????120
<210>7
<211>107
<212>PRT
＜213〉mankind
<400>7
Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Leu?Ser?Pro?Gly
1???????????????5???????????????????10??????????????????15
Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Tyr
20??????????????????25??????????????????30
Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile
35??????????????????40??????????????????45
Tyr?Asp?Ala?Ser?Asn?Arg?Ala?Thr?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly
50??????????????????55??????????????????60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Glu?Pro
65??????????????????70??????????????????75??????????????????80
Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Arg?Thr?Asn?Trp?Pro?Leu
85??????????????????90??????????????????95
Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys
100?????????????????105
<210>8
<211>107
<212>PRT
＜213〉mankind
<400>8
Ala?Ile?Gln?Leu?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1???????????????5???????????????????10??????????????????15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Ala
20??????????????????25??????????????????30
Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Phe?Leu?Ile
35??????????????????40??????????????????45
Tyr?Asp?Ala?Ser?Ser?Leu?Glu?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50??????????????????55??????????????????60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65??????????????????70??????????????????75??????????????????80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Phe?Asn?Ser?Tyr?Pro?Phe
85??????????????????90??????????????????95
Thr?Phe?Gly?Pro?Gly?Thr?Lys?Val?Asp?Ile?Lys
100?????????????????105
<210>9
<211>107
<212>PRT
＜213〉mankind
<400>9
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1???????????????5???????????????????10??????????????????15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Trp
20??????????????????25??????????????????30
Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Glu?Lys?Ala?Pro?Lys?Ser?Leu?Ile
35??????????????????40??????????????????45
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50??????????????????55??????????????????60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65??????????????????70??????????????????75??????????????????80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Ser?Tyr?Pro?Leu
85??????????????????90??????????????????95
Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys
100?????????????????105
<210>10
<211>107
<212>PRT
＜213〉mankind
<400>10
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1???????????????5???????????????????10??????????????????15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Trp
20??????????????????25??????????????????30
Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Glu?Lys?Ala?Pro?Lys?Ser?Leu?Ile
35??????????????????40??????????????????45
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50??????????????????55??????????????????60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65??????????????????70??????????????????75??????????????????80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Ser?Tyr?Pro?Leu
85??????????????????90??????????????????95
Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys
100?????????????????105
<210>11
<211>107
<212>PRT
＜213〉mankind
<400>11
Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Leu?Ser?Pro?Gly
1???????????????5???????????????????10??????????????????15
Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Tyr
20??????????????????25??????????????????30
Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile
35??????????????????40??????????????????45
Phe?Asp?Ala?Ser?Asn?Arg?Ala?Thr?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly
50??????????????????55??????????????????60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Glu?Pro
65??????????????????70??????????????????75??????????????????80
Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Arg?Ser?Asn?Trp?Pro?Leu
85??????????????????90??????????????????95
Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys
100?????????????????105
<210>12
<211>108
<212>PRT
＜213〉mankind
<400>12
Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly
1???????????????5???????????????????10??????????????????15
Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Ser?Ser
20??????????????????25??????????????????30
Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu
35??????????????????40??????????????????45
Ile?Tyr?Gly?Ala?Ser?Ser?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser
50??????????????????55??????????????????60
Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Leu?Glu
65??????????????????70??????????????????75??????????????????80
Pro?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Ser?Ser?Pro
85??????????????????90??????????????????95
Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys
100?????????????????105
<210>13
<211>5
<212>PRT
＜213〉mankind
<400>13
Ser?Tyr?Ile?Met?His
1???????????????5
<210>14
<211>5
<212>PRT
＜213〉mankind
<400>14
Tyr?Tyr?Ala?Met?His
1???????????????5
<210>15
<211>5
<212>PRT
＜213〉mankind
<400>15
Asp?Tyr?Gly?Met?His
1???????????????5
<210>16
<211>5
<212>PRT
＜213〉mankind
<400>16
Asp?His?Gly?Met?His
1???????????????5
<210>17
<211>7
<212>PRT
＜213〉mankind
<400>17
Ser?Asp?Tyr?Tyr?Tyr?Trp?Ser
1???????????????5
<210>18
<211>5
<212>PRT
＜213〉mankind
<400>18
Ile?Tyr?Ala?Met?Ser
1???????????????5
<210>19
<211>17
<212>PRT
＜213〉mankind
<400>19
Val?Ile?Ser?Tyr?Asp?Gly?Arg?Asn?Lys?Tyr?Tyr?Ala?Asp?Ser?Val?Lys
1???????????????5???????????????????10??????????????????15
Gly
<210>20
<211>17
<212>PRT
＜213〉mankind
<400>20
Val?Ile?Ser?Tyr?Asp?Gly?Ser?Ile?Lys?Tyr?Tyr?Ala?Asp?Ser?Val?Lys
1???????????????5???????????????????10??????????????????15
Gly
<210>21
<211>17
<212>PRT
＜213〉mankind
<400>21
Val?Ile?Trp?Tyr?Asp?Gly?Ser?Asn?Lys?Tyr?Tyr?Ala?Asp?Ser?Val?Lys
1???????????????5???????????????????10??????????????????15
Gly
<210>22
<211>17
<212>PRT
＜213〉mankind
<400>22
Val?Ile?Trp?Tyr?Asp?Gly?Ser?Asn?Lys?Tyr?Tyr?Ala?Asp?Ser?Val?Lys
1???????????????5???????????????????10??????????????????15
Gly
<210>23
<211>16
<212>PRT
＜213〉mankind
<400>23
Tyr?Ile?Tyr?Tyr?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro?Ser?Leu?Lys?Ser
1???????????????5???????????????????10??????????????????15
<210>24
<211>17
<212>PRT
＜213〉mankind
<400>24
Ala?Ile?Ser?Asp?Ser?Gly?Gly?Arg?Thr?Tyr?Phe?Ala?Asp?Ser?Val?Arg
1???????????????5???????????????????10??????????????????15
Gly
<210>25
<211>9
<212>PRT
＜213〉mankind
<400>25
Asp?Thr?Asp?Gly?Tyr?Asp?Phe?Asp?Tyr
1???????????????5
<210>26
<211>10
<212>PRT
＜213〉mankind
<400>26
Glu?Gly?Pro?Tyr?Ser?Asn?Tyr?Leu?Asp?Tyr
1???????????????5???????????????????10
<210>27
<211>9
<212>PRT
＜213〉mankind
<400>27
Asp?Ser?Ile?Met?Val?Arg?Gly?Asp?Tyr
1???????????????5
<210>28
<211>9
<212>PRT
＜213〉mankind
<400>28
Asp?Ser?Ile?Met?Val?Arg?Gly?Asp?Tyr
1???????????????5
<210>29
<211>12
<212>PRT
＜213〉mankind
<400>29
Gly?Asp?Gly?Asp?Tyr?Gly?Gly?Asn?Cys?Phe?Asp?Tyr
1???????????????5???????????????????10
<210>30
<211>11
<212>PRT
＜213〉mankind
<400>30
Val?Asp?Tyr?Ser?Asn?Tyr?Leu?Phe?Phe?Asp?Tyr
1???????????????5???????????????????10
<210>31
<211>11
<212>PRT
＜213〉mankind
<400>31
Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Tyr?Leu?Ala
1???????????????5???????????????????10
<210>32
<211>11
<212>PRT
＜213〉mankind
<400>32
Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Ala?Leu?Ala
1???????????????5???????????????????10
<210>33
<211>11
<212>PRT
＜213〉mankind
<400>33
Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Trp?Leu?Ala
1???????????????5???????????????????10
<210>34
<211>11
<212>PRT
＜213〉mankind
<400>34
Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Trp?Leu?Ala
1???????????????5???????????????????10
<210>35
<211>11
<212>PRT
＜213〉mankind
<400>35
Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Tyr?Leu?Ala
1???????????????5???????????????????10
<210>36
<211>12
<212>PRT
＜213〉mankind
<400>36
Arg?Ala?Ser?Gln?Ser?Ile?Ser?Ser?Ser?Tyr?Leu?Ala
1???????????????5???????????????????10
<210>37
<211>7
<212>PRT
＜213〉mankind
<400>37
Asp?Ala?Ser?Asn?Arg?Ala?Thr
1???????????????5
<210>38
<211>7
<212>PRT
＜213〉mankind
<400>38
Asp?Ala?Ser?Ser?Leu?Glu?Ser
1???????????????5
<210>39
<211>7
<212>PRT
＜213〉mankind
<400>39
Ala?Ala?Ser?Ser?Leu?Gln?Ser
1???????????????5
<210>40
<211>7
<212>PRT
＜213〉mankind
<400>40
Ala?Ala?Ser?Ser?Leu?Gln?Ser
1???????????????5
<210>41
<211>7
<212>PRT
＜213〉mankind
<400>41
Asp?Ala?Ser?Asn?Arg?Ala?Thr
1???????????????5
<210>42
<211>7
<212>PRT
＜213〉mankind
<400>42
Gly?Ala?Ser?Ser?Arg?Ala?Thr
1???????????????5
<210>43
<211>9
<212>PRT
＜213〉mankind
<400>43
Gln?Gln?Arg?Thr?Asn?Trp?Pro?Leu?Thr
1???????????????5
<210>44
<211>9
<212>PRT
＜213〉mankind
<400>44
Gln?Gln?Phe?Asn?Ser?Tyr?Pro?Phe?Thr
1???????????????5
<210>45
<211>9
<212>PRT
＜213〉mankind
<400>45
Gln?Gln?Tyr?Asn?Ser?Tyr?Pro?Leu?Thr
1???????????????5
<210>46
<211>9
<212>PRT
＜213〉mankind
<400>46
Gln?Gln?Tyr?Asn?Ser?Tyr?Pro?Leu?Thr
1???????????????5
<210>47
<211>9
<212>PRT
＜213〉mankind
<400>47
Gln?Gln?Arg?Ser?Asn?Trp?Pro?Leu?Thr
1???????????????5
<210>48
<211>9
<212>PRT
＜213〉mankind
<400>48
Gln?Gln?Tyr?Gly?Ser?Ser?Pro?Tyr?Thr
1???????????????5
<210>49
<211>354
<212>DNA
＜213〉mankind
<400>49
caggtgcagc?tggtggagtc?tgggggaggc?gtggtccagc?ctgggaggtc?cctgagactc?????60
tcctgtgcag?cctctggatt?taccttcagt?agctatatta?tgcactgggt?ccgccaggct????120
ccaggcaagg?ggctggagtg?ggtggcagtt?atatcatatg?atggaagaaa?caaatactac????180
gcagactccg?tgaagggccg?attcaccatc?tccagagaca?attccaagaa?cacgctgtat????240
ctgcaaatga?acagcctgag?agctgaggac?acggctgtgt?attactgtgc?gagagatacg????300
gatggctacg?attttgacta?ctggggccag?ggaaccctgg?tcaccgtctc?ctca??????????354
<210>50
<211>357
<212>DNA
＜213〉mankind
<400>50
caaatacaac?tggtggagtc?tgggggaggc?gtggtccagc?ctgggaggtc?cctgagactc????60
tcctgtgcag?cctctggatt?caccttcggt?tactatgcta?tgcactgggt?ccgccaggct????120
ccaggcaagg?ggctggagtg?ggtggcagtt?atatcatatg?atggaagcat?taaatactac????180
gcagactccg?tgaagggccg?attcaccatc?tccagagaca?attccaagaa?cacgctgtat????240
ctgcaaatga?acagcctgag?agctgaggac?acggctgtgt?attactgtgc?gagagagggc????300
ccttacagta?actaccttga?ctactggggc?cagggaaccc?tggtcaccgt?ctcctca???????357
<210>51
<211>354
<212>DNA
＜213〉mankind
<400>51
caggtgcagc?tggtggagtc?tgggggaggc?gtggtccagc?ctgggaggtc?cctgagactc?????60
tcctgtgcga?cgtctggatt?caccttcagt?gactatggca?tgcactgggt?ccgccaggct????120
ccaggcaagg?ggctggagtg?ggtggcagtt?atatggtatg?atggaagtaa?taaatactat????180
gcagactccg?tgaagggccg?attcaccatc?tccagagaca?attccaagaa?aacgctgtct????240
ctgcaaatga?acagcctgag?agccgaggac?acggctgtgt?attactgtgc?gagagattct????300
attatggttc?ggggggacta?ctggggccag?ggaaccctgg?tcaccgtctc?ctca??????????354
<210>52
<211>354
<212>DNA
＜213〉mankind
<400>52
caggtgcagc?tggtggagtc?tgggggaggc?gtggtccagc?ctgggaggtc?cctgagactc?????60
tcctgtgcag?cgtctggatt?caccttcagc?gaccatggca?tgcactgggt?ccgccaggct????120
ccaggcaagg?ggctggagtg?ggtggcagtt?atatggtatg?atggaagtaa?taaatactat????180
gcagactccg?tgaagggccg?attcaccatc?tccagagaca?attccaagaa?cacgctgtat????240
ctgcaaatga?acagcctgag?agccgaggac?acggctgtgt?attactgtgc?gagagattct????300
attatggttc?ggggggacta?ctggggccag?ggaaccctgg?tcaccgtctc?ctca??????????354
<210>53
<211>366
<212>DNA
＜213〉mankind
<400>53
caggtgcagc?tgcaggagtc?gggcccagga?ctggtgaagc?cttcggagac?cctgtccctc?????60
acctgcactg?tctctggtgg?ctccgtcagc?agtgattatt?actactggag?ctggatccgg????120
cagcccccag?ggaagggact?ggagtggctt?gggtatatct?attacagtgg?gagcaccaac????180
tacaacccct?ccctcaagag?tcgagtcacc?atatcagtag?acacgtccaa?gaaccagttc????240
tccctgaagc?tgaggtctgt?gaccactgcg?gacacggccg?tgtattactg?tgcgagaggg????300
gatggggact?acggtggtaa?ctgttttgac?tactggggcc?agggaaccct?ggtcaccgtc????360
tcctca???????????????????????????????????????????????????????????????366
<210>54
<211>360
<212>DNA
＜213〉mankind
<400>54
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgagactc?????60
tcctgtgcag?cctctggatt?cacctttagc?atctatgcca?tgagctgggt?ccgccaggct????120
ccagggaagg?ggctggagtg?ggtctcagct?attagtgata?gtggtggtcg?cacatacttc????180
gcagactccg?tgaggggccg?gttcaccatc?tccagagaca?attccaagaa?cacgctgtct????240
ctgcaaatga?acagcctgag?agccgaggac?acggccgtat?attactgtgc?gaaggtcgac????300
tacagtaact?acctattctt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcctca????360
<210>55
<211>321
<212>DNA
＜213〉mankind
<400>55
gaaattgtgt?tgacacagtc?tccagccacc?ctgtctttgt?ctccagggga?aagagccacc?????60
ctctcctgca?gggccagtca?gagtgttagc?agctacttag?cctggtacca?acagaaacct????120
ggccaggctc?ccaggctcct?catctatgat?gcatccaaca?gggccactgg?catcccagcc????180
aggttcagtg?gcagtgggtc?tgggacagac?ttcactctca?ccatcagcag?cctagagcct????240
gaagattttg?cagtttatta?ctgtcagcag?cgtaccaact?ggccgctcac?tttcggcgga????300
gggaccaagg?tggagatcaa?a??????????????????????????????????????????????321
<210>56
<211>321
<212>DNA
＜213〉mankind
<400>56
gccatccagt?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?cagagtcacc?????60
atcacttgcc?gggcaagtca?gggcattagc?agtgctttag?cctggtatca?gcagaaacca????120
gggaaagctc?ctaagttctt?gatctatgat?gcctccagtt?tggaaagtgg?ggtcccatca????180
aggttcagcg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?cctgcagcct????240
gaagattttg?caacttatta?ctgtcaacag?tttaatagtt?acccattcac?tttcggccct????300
gggaccaaag?tggatatcaa?a??????????????????????????????????????????????321
<210>57
<211>321
<212>DNA
＜213〉mankind
<400>57
gacatccaga?tgacccagtc?tccatcctca?ctgtctgcat?ctgtaggaga?cagagtcacc?????60
atcacttgtc?gggcgagtca?gggtattagc?agctggttag?cctggtatca?gcagaaacca????120
gagaaagccc?ctaagtccct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca????180
aggttcagcg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?cctgcagcct????240
gaagattttg?caacttatta?ctgccaacag?tataatagtt?acccgctcac?tttcggcgga????300
gggaccaagg?tggagatcaa?a??????????????????????????????????????????????321
<210>58
<211>321
<212>DNA
＜213〉mankind
<400>58
gacatccaga?tgacccagtc?tccatcctca?ctgtctgcat?ctgtaggaga?cagagtcacc?????60
atcacttgtc?gggcgagtca?gggtattagc?agctggttag?cctggtatca?gcagaaacca????120
gagaaagccc?ctaagtccct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca????180
aggttcagcg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?cctgcagcct????240
gaagattttg?caacttatta?ctgccaacag?tataatagtt?acccgctcac?tttcggcgga????300
gggaccaagg?tggagatcaa?a??????????????????????????????????????????????321
<210>59
<211>321
<212>DNA
＜213〉mankind
<400>59
gaaattgtgt?tgacacagtc?tccagccacc?ctgtctttgt?ctccagggga?aagagccacc?????60
ctctcctgca?gggccagtca?gagtgttagc?agctacttag?cctggtacca?acagaaacct????120
ggccaggctc?ccaggctcct?catctttgat?gcatccaaca?gggccactgg?catcccagcc????180
aggttcagtg?gcagtgggtc?tgggacagac?ttcactctca?ccatcagcag?cctagagcct????240
gaagattttg?cagtttatta?ctgtcagcaa?cgtagcaact?ggccgctcac?tttcggcgga????300
gggaccaagg?tggagatcaa?a??????????????????????????????????????????????321
<210>60
<211>324
<212>DNA
＜213〉mankind
<400>60
gaaattgtgt?tgacgcagtc?tccaggcacc?ctgtctttgt?ctccagggga?aagagccacc?????60
ctctcctgca?gggccagtca?gagtattagc?agcagctact?tagcctggta?ccagcagaaa????120
cctggccagg?ctcccaggct?cctcatctat?ggtgcatcca?gcagggccac?tggcatccca????180
gacaggttca?gtggcagtgg?gtctgggaca?gacttcactc?tcaccatcag?cagactggag????240
cctgaagatt?ttgcagtgta?ttactgtcag?cagtatggta?gctcaccgta?cacttttggc????300
caggggacca?agctggagat??caaa??????????????????????????????????????????324
<210>61
<211>98
<212>PRT
＜213〉mankind
<400>61
Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?Arg
1???????????????5???????????????????10??????????????????15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20??????????????????25??????????????????30
Ala?Met?His?Trp?Val?Arg?Gln?Ala??Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35??????????????????40??????????????????45
Ala?Val?Ile?Ser?Tyr?Asp?Gly?Ser?Asn?Lys?Tyr?Tyr?Ala?Asp?Ser?Val
50??????????????????55??????????????????60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65??????????????????70??????????????????75??????????????????80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85??????????????????90??????????????????95
Ala?Arg
<210>62
<211>98
<212>PRT
＜213〉mankind
<400>62
Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?Arg
1???????????????5???????????????????10??????????????????15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20??????????????????25??????????????????30
Gly?Met?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35??????????????????40??????????????????45
Ala?Val?Ile?Trp?Tyr?Asp?Gly?Ser?Asn?Lys?Tyr?Tyr?Ala?Asp?Ser?Val
50??????????????????55??????????????????60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65??????????????????70??????????????????75??????????????????80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85??????????????????90??????????????????95
Ala?Arg
<210>63
<211>99
<212>PRT
＜213〉mankind
<400>63
Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu
1???????????????5???????????????????10??????????????????15
Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Val?Ser?Ser?Gly
20??????????????????25??????????????????30
Ser?Tyr?Tyr?Trp?Ser?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu
35??????????????????40??????????????????45
Trp?Ile?Gly?Tyr?Ile?Tyr?Tyr?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro?Ser
50??????????????????55??????????????????60
Leu?Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe
65??????????????????70??????????????????75??????????????????80
Ser?Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr
85??????????????????90??????????????????95
Cys?Ala?Arg
<210>64
<211>98
<212>PRT
＜213〉mankind
<400>64
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1???????????????5???????????????????10??????????????????15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20??????????????????25??????????????????30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35??????????????????40??????????????????45
Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50??????????????????55??????????????????60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65??????????????????70??????????????????75??????????????????80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85??????????????????90??????????????????95
Ala?Lys
<210>65
<211>94
<212>PRT
＜213〉mankind
<400>65
Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Leu?Ser?Pro?Gly
1???????????????5???????????????????10??????????????????15
Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Tyr
20??????????????????25??????????????????30
Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile
35??????????????????40??????????????????45
Tyr?Asp?Ala?Ser?Asn?Arg?Ala?Thr?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly
50??????????????????55??????????????????60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Glu?Pro
65??????????????????70??????????????????75??????????????????80
Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Arg?Ser?Asn?Trp
85??????????????????90
<210>66
<211>95
<212>PRT
＜213〉mankind
<400>66
Ala?Ile?Gln?Leu?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1???????????????5???????????????????10??????????????????15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Ala
20??????????????????25??????????????????30
Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35??????????????????40??????????????????45
Tyr?Asp?Ala?Ser?Ser?Leu?Glu?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50??????????????????55??????????????????60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65??????????????????70??????????????????75??????????????????80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Phe?Asn?Ser?Tyr?Pro
85??????????????????90??????????????????95
<210>67
<211>95
<212>PRT
＜213〉mankind
<400>67
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1???????????????5??????????????????10??????????????????15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Trp
20??????????????????25??????????????????30
Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Glu?Lys?Ala?Pro?Lys?Ser?Leu?Ile
35??????????????????40??????????????????45
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50??????????????????55??????????????????60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65??????????????????70??????????????????75??????????????????80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Ser?Tyr?Pro
85??????????????????90??????????????????95
<210>68
<211>96
<212>PRT
＜213〉mankind
<400>68
Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly
1???????????????5???????????????????10??????????????????15
Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Ser
20??????????????????25??????????????????30
Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu
35??????????????????40??????????????????45
Ile?Tyr?Gly?Ala?Ser?Ser?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser
50??????????????????55??????????????????60
Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Leu?Glu
65??????????????????70??????????????????75??????????????????80
Pro?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Ser?Ser?Pro
85??????????????????90??????????????????95
<210>69
<211>15
<212>PRT
＜213〉mankind
<400>69
Tyr?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
1???????????????5???????????????????10??????????????????15
<210>70
<211>12
<212>PRT
＜213〉mankind
<400>70
Leu?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys
1??????????????5???????????????????10
<210>71
<211>12
<212>PRT
＜213〉mankind
<400>71
Phe?Thr?Phe?Gly?Pro?Gly?Thr?Lys?Val?Asp?Ile?Lys
1???????????????5???????????????????10
<210>72
<211>12
<212>PRT
＜213〉mankind
<400>72
Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys
1???????????????5???????????????????10
<210>73
<211>122
<212>PRT
＜213〉mankind
<400>73
Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu
1???????????????5???????????????????10??????????????????15
Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Val?Ser?Ser?Asp
20??????????????????25??????????????????30
Tyr?Tyr?Tyr?Trp?Ser?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu
35??????????????????40??????????????????45
Trp?Leu?Gly?Tyr?Ile?Tyr?Tyr?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro?Ser
50??????????????????55??????????????????60
Leu?Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe
65??????????????????70??????????????????75??????????????????80
Ser?Leu?Lys?Leu?Arg?Ser?Val?Thr?Thr?Ala?Asp?Thr?Ala?Val?Tyr?Tyr
85??????????????????90??????????????????95
Cys?Ala?Arg?Gly?Asp?Gly?Asp?Tyr?Gly?Gly?Asn?Tyr?Phe?Asp?Tyr?Trp
100?????????????????105?????????????????110
Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115?????????????????120
<210>74
<211>366
<212>DNA
＜213〉mankind
<400>74
caggtgcagc?tgcaggagtc?gggcccagga?ctggtgaagc?cttcggagac?cctgtccctc??????60
acctgcactg?tctctggtgg?ctccgtcagc?agtgattatt?actactggag?ctggatccgg?????120
cagcccccag?ggaagggact?ggagtggctt?gggtatatct?attacagtgg?gagcaccaac?????180
tacaacccct?ccctcaagag?tcgagtcacc?atatcagtag?acacgtccaa?gaaccagttc?????240
tccctgaagc?tgaggtctgt?gaccactgcg?gacacggccg?tgtattactg?tgcgagaggg?????300
gatggggact?acggtggtaa?ctattttgac?tactggggcc?agggaaccct?ggtcaccgtc?????360
tcctca????????????????????????????????????????????????????????????????366
<210>75
<211>12
<212>PRT
＜213〉mankind
<400>75
Gly?Asp?Gly?Asp?Tyr?Gly?Gly?Asn?Tyr?Phe?Asp?Tyr
1???????????????5???????????????????10
<210>76
<211>193
<212>PRT
＜213〉mankind
<400>76
Met?Pro?Glu?Glu?Gly?Ser?Gly?Cys?Ser?Val?Arg?Arg?Arg?Pro?Tyr?Gly
1???????????????5???????????????????10??????????????????15
Cys?Val?Leu?Arg?Ala?Ala?Leu?Val?Pro?Leu?Val?Ala?Gly?Leu?Val?Ile
20??????????????????25??????????????????30
Cys?Leu?Val?Val?Cys?Ile?Gln?Arg?Phe?Ala?Gln?Ala?Gln?Gln?Gln?Leu
35??????????????????40??????????????????45
Pro?Leu?Glu?Ser?Leu?Gly?Trp?Asp?Val?Ala?Glu?Leu?Gln?Leu?Asn?His
50??????????????????55??????????????????60
Thr?Gly?Pro?Gln?Gln?Asp?Pro?Arg?Leu?Tyr?Trp?Gln?Gly?Gly?Pro?Ala
65??????????????????70??????????????????75??????????????????80
Leu?Gly?Arg?Ser?Phe?Leu?His?Gly?Pro?Glu?Leu?Asp?Lys?Gly?Gln?Leu
85??????????????????90??????????????????95
Arg?Ile?His?Arg?Asp?Gly?Ile?Tyr?Met?Val?His?Ile?Gln?Val?Thr?Leu
100?????????????????105?????????????????110
Ala?Ile?Cys?Ser?Ser?Thr?Thr?Ala?Ser?Arg?His?His?Pro?Thr?Thr?Leu
115?????????????????120?????????????????125
Ala?Val?Gly?Ile?Cys?Ser?Pro?Ala?Ser?Arg?Ser?Ile?Ser?Leu?Leu?Arg
130?????????????????135?????????????????140
Leu?Ser?Phe?His?Gln?Gly?Cys?Thr?Ile?Ala?Ser?Gln?Arg?Leu?Thr?Pro
145?????????????????150?????????????????155?????????????????160
Leu?Ala?Arg?Gly?Asp?Thr?Leu?Cys?Thr?Asn?Leu?Thr?Gly?Thr?Leu?Leu
165?????????????????170?????????????????175
Pro?Ser?Arg?Asn?Thr?Asp?Glu?Thr?Phe?Phe?Gly?Val?Gln?Trp?Val?Arg
180?????????????????185?????????????????190
Pro
<210>77
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>77
Ala?Leu?Ala?Leu
1
<210>78
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>78
Ala?Leu?Ala?Leu
1
<210>79
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>79
Gly?Phe?Leu?Gly
1
<210>80
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>80
Pro?Arg?Phe?Lys
1
<210>81
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>81
Thr?Arg?Leu?Arg
1
<210>82
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>82
Ser?Lys?Gly?Arg
1
<210>83
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>83
Pro?Asn?Asp?Lys
1
<210>84
<211>6
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>84
Pro?Val?Gly?Leu?Ile?Gly
1???????????????5
<210>85
<211>5
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>85
Gly?Pro?Leu?Gly?Val
1???????????????5
<210>86
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>86
Gly?Pro?Leu?Gly?Ile?Ala?Gly?Gln
1???????????????5
<210>87
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>87
Pro?Leu?Gly?Leu
1
<210>88
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>88
Gly?Pro?Leu?Gly?Met?Leu?Ser?Gln
1???????????????5
<210>89
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>89
Gly?Pro?Leu?Gly?Leu?Trp?Ala?Gln
1???????????????5
<210>90
<211>9
<212>PRT
＜213〉cytomegalovirus
<400>90
Ile?Pro?Ser?Ile?Asn?Val?His?His?Tyr
1???????????????5
<210>91
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>91
Leu?Leu?Gly?Leu
1
<210>92
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉peptide linker
<400>92
Ala?Leu?Ala?Leu
1

Claims (63)

1. antibody-mating partner molecule conjugate comprises the antigen-binding portion thereof of isolating human monoclonal antibody or this antibody, and the mating partner molecule, wherein the human CD70 of this antibodies and show at least a in the following characteristic:
(a) with 1 * 10 -7M or littler K DIn conjunction with human CD70; And
(b) in conjunction with the renal cell carcinoma tumor cell line;
(c) in conjunction with lymphoma cell line;
(d) expressed the cell internalizing of CD70;
(e) show antibody dependent cellular cytotoxicity (ADCC) at the cell of expressing CD70; And
(f) when suppressing to express the growth of the cell of CD70 mutually during coupling in vivo with cytotoxin, wherein this mating partner molecule is a therapeutical agent.
2. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this antibody show characteristic (a) and (b), (c), (d), (e) and (f) at least two kinds.
3. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this antibody show characteristic (a) and (b), (c), (d), (e) and (f) at least three kinds.
4. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this antibody show characteristic (a) and (b), (c), (d), (e) and (f) at least four kinds.
5. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this antibody show characteristic (a) and (b), (c), (d), (e) and (f) at least five kinds.
6. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this antibody show characteristic (a) and (b), (c), (d), (e) and (f) in all six kinds.
7. antibody as claimed in claim 1-mating partner molecule conjugate, this conjugate is with 5.5 * 10 -9M or littler avidity combine with human CD70.
8. antibody as claimed in claim 1-mating partner molecule conjugate, this conjugate is with 3 * 10 -9M or littler avidity combine with human CD70.
9. antibody as claimed in claim 1-mating partner molecule conjugate, this conjugate is with 2 * 10 -9M or littler avidity combine with human CD70.
10. antibody-mating partner molecule conjugate, comprise isolating monoclonal antibody or its antigen-binding portion thereof, and the mating partner molecule, this monoclonal antibody or its antigen-binding portion thereof are in conjunction with the epi-position on the human CD70 that is discerned by reference antibody, and wherein this reference antibody comprises:
(a) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:1; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:7;
(b) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:2; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:8;
(c) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:3; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:9;
(d) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:4; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:10;
(e) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:5; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:11;
(f) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:73; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:11; Perhaps
(g) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:6; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:12;
Wherein this mating partner molecule is a therapeutical agent.
11. antibody as claimed in claim 10-mating partner molecule conjugate, wherein this reference antibody comprises:
Variable region of heavy chain comprises aminoacid sequence SEQ ID NO:1; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:7.
12. antibody as claimed in claim 10-mating partner molecule conjugate, wherein this reference antibody comprises:
Variable region of heavy chain comprises aminoacid sequence SEQ ID NO:2; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:8.
13. antibody as claimed in claim 10-mating partner molecule conjugate, wherein this reference antibody comprises:
Variable region of heavy chain comprises aminoacid sequence SEQ ID NO:3; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:9.
14. antibody as claimed in claim 10-mating partner molecule conjugate, wherein this reference antibody comprises:
Variable region of heavy chain comprises aminoacid sequence SEQ ID NO:4; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:10.
15. antibody as claimed in claim 10-mating partner molecule conjugate, wherein this reference antibody comprises:
Variable region of heavy chain comprises aminoacid sequence SEQ ID NO:5; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:11.
16. antibody as claimed in claim 10-mating partner molecule conjugate, wherein this reference antibody comprises:
Variable region of heavy chain comprises aminoacid sequence SEQ ID NO:73; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:11.
17. antibody as claimed in claim 10-mating partner molecule conjugate, wherein this reference antibody comprises:
Variable region of heavy chain comprises aminoacid sequence SEQ ID NO:6; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:12.
18. antibody-mating partner molecule conjugate comprises isolating monoclonal antibody or its antigen-binding portion thereof, and the mating partner molecule, this monoclonal antibody or its antigen-binding portion thereof comprise variable region of heavy chain, and this variable region of heavy chain is human V H3-30.3 gene, human V H3-33 gene, human V H4-61 gene or human V HThe product of 3-23 gene or derived from this gene, wherein this antibodies specific ground is in conjunction with CD70, and wherein this mating partner molecule is a therapeutical agent.
19. antibody-mating partner molecule conjugate comprises isolating monoclonal antibody or its antigen-binding portion thereof, and the mating partner molecule, this monoclonal antibody or its antigen-binding portion thereof comprise variable region of light chain, and this variable region of light chain is human V KL6 gene, human V KL18 gene, human V KL15 gene, human V KL6 gene or human V KThe product of 27 genes or derived from this gene, wherein this antibodies specific ground is in conjunction with CD70, and wherein this mating partner molecule is a therapeutical agent.
20. antibody-mating partner molecule conjugate comprises isolated antibody or its antigen-binding portion thereof, and the mating partner molecule, this antibody or its antigen-binding portion thereof comprise:
(a) variable region of heavy chain, this variable region of heavy chain are human V HThe product of 3-33 gene or derived from this gene; And variable region of light chain, this variable region of light chain is human V KThe product of L15 gene or derived from this gene;
(b) variable region of heavy chain, this variable region of heavy chain are human V H3-30.3 the product of gene or derived from this gene; And variable region of light chain, this variable region of light chain is human V KThe product of L6 gene or derived from this gene; Wherein this antibodies specific ground is in conjunction with human CD70;
(c) variable region of heavy chain, this variable region of heavy chain are human V H3-30.3 the product of gene or derived from this gene; And variable region of light chain, this variable region of light chain is human V KThe product of L-18 gene or derived from this gene; Wherein this antibodies specific ground is in conjunction with human CD70;
(d) variable region of heavy chain, this variable region of heavy chain are human V HThe product of 4-61 gene or derived from this gene; And variable region of light chain, this variable region of light chain is human V KThe product of L6 gene or derived from this gene; Wherein this antibodies specific ground is in conjunction with human CD70; Perhaps
(e) variable region of heavy chain, this variable region of heavy chain are human V HThe product of 3-23 gene or derived from this gene; And variable region of light chain, this variable region of light chain is human V KThe product of A27 gene or derived from this gene; Wherein this antibodies specific ground is in conjunction with human CD70,
Wherein this mating partner molecule is a therapeutical agent.
21. antibody as claimed in claim 1-mating partner molecule conjugate comprises:
(a) variable region of heavy chain CDR1 comprises SEQ ID NO:13;
(b) variable region of heavy chain CDR2 comprises SEQ ID NO:19;
(c) variable region of heavy chain CDR3 comprises SEQ ID NO:25;
(d) variable region of light chain CDR1 comprises SEQ ID NO:31;
(e) variable region of light chain CDR2 comprises SEQ ID NO:37; And
(f) variable region of light chain CDR3 comprises SEQ ID NO:43.
22. antibody as claimed in claim 1-mating partner molecule conjugate comprises:
(a) variable region of heavy chain CDR1 comprises SEQ ID NO:14;
(b) variable region of heavy chain CDR2 comprises SEQ ID NO:20;
(c) variable region of heavy chain CDR3 comprises SEQ ID NO:26;
(d) variable region of light chain CDR1 comprises SEQ ID NO:32;
(e) variable region of light chain CDR2 comprises SEQ ID NO:38; And
(f) variable region of light chain CDR3 comprises SEQ ID NO:44.
23. antibody as claimed in claim 1-mating partner molecule conjugate comprises:
(a) variable region of heavy chain CDR1 comprises SEQ ID NO:15;
(b) variable region of heavy chain CDR2 comprises SEQ ID NO:21;
(c) variable region of heavy chain CDR3 comprises SEQ ID NO:27;
(d) variable region of light chain CDR1 comprises SEQ ID NO:33;
(e) variable region of light chain CDR2 comprises SEQ ID NO:39; And
(f) variable region of light chain CDR3 comprises SEQ ID NO:45.
24. antibody as claimed in claim 1-mating partner molecule conjugate comprises:
(a) variable region of heavy chain CDR1 comprises SEQ ID NO:16;
(b) variable region of heavy chain CDR2 comprises SEQ ID NO:22;
(c) variable region of heavy chain CDR3 comprises SEQ ID NO:28;
(d) variable region of light chain CDR1 comprises SEQ ID NO:34;
(e) variable region of light chain CDR2 comprises SEQ ID NO:40; And
(f) variable region of light chain CDR3 comprises SEQ ID NO:46.
25. antibody as claimed in claim 1-mating partner molecule conjugate comprises:
(a) variable region of heavy chain CDR1 comprises SEQ ID NO:17;
(b) variable region of heavy chain CDR2 comprises SEQ ID NO:23;
(c) variable region of heavy chain CDR3 comprises SEQ ID NO:29;
(d) variable region of light chain CDR1 comprises SEQ ID NO:35;
(e) variable region of light chain CDR2 comprises SEQ ID NO:41; And
(f) variable region of light chain CDR3 comprises SEQ ID NO:47.
26. antibody as claimed in claim 1-mating partner molecule conjugate comprises:
(a) variable region of heavy chain CDR1 comprises SEQ ID NO:17;
(b) variable region of heavy chain CDR2 comprises SEQ ID NO:23;
(c) variable region of heavy chain CDR3 comprises SEQ ID NO:75;
(d) variable region of light chain CDR1 comprises SEQ ID NO:35;
(e) variable region of light chain CDR2 comprises SEQ ID NO:41; And
(f) variable region of light chain CDR3 comprises SEQ ID NO:47.
27. antibody as claimed in claim 1-mating partner molecule conjugate comprises:
(a) variable region of heavy chain CDR1 comprises SEQ ID NO:18;
(b) variable region of heavy chain CDR2 comprises SEQ ID NO:24;
(c) variable region of heavy chain CDR3 comprises SEQ ID NO:30;
(d) variable region of light chain CDR1 comprises SEQ ID NO:36;
(e) variable region of light chain CDR2 comprises SEQ ID NO:42; And
(f) variable region of light chain CDR3 comprises SEQ ID NO:48.
28. antibody-mating partner molecule conjugate comprises isolating monoclonal antibody or its antigen-binding portion thereof, and the mating partner molecule, this isolating monoclonal antibody or its antigen-binding portion thereof comprise:
(a) variable region of heavy chain comprises the aminoacid sequence that is selected from SEQ ID NO:1 to 6 and 73; And
(b) variable region of light chain comprises the aminoacid sequence that is selected from SEQ ID NO:7 to 12;
Wherein this antibodies specific ground is in conjunction with human CD70 albumen, and wherein this mating partner molecule is a therapeutical agent.
29. antibody as claimed in claim 28-mating partner molecule conjugate comprises:
(a) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:1; And
(b) variable region of light chain comprises aminoacid sequence SEQ ID NO:7.
30. antibody as claimed in claim 28-mating partner molecule conjugate comprises:
(a) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:2; And
(b) variable region of light chain comprises aminoacid sequence SEQ ID NO:8.
31. antibody as claimed in claim 28-mating partner molecule conjugate comprises:
(a) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:3; And
(b) variable region of light chain comprises aminoacid sequence SEQ ID NO:9.
32. antibody as claimed in claim 28-mating partner molecule conjugate comprises:
(a) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:4; And
(b) variable region of light chain comprises aminoacid sequence SEQ ID NO:10.
33. antibody as claimed in claim 28-mating partner molecule conjugate comprises:
(a) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:5; And
(b) variable region of light chain comprises aminoacid sequence SEQ ID NO:11.
34. antibody as claimed in claim 28-mating partner molecule conjugate comprises:
(a) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:73; And
(b) variable region of light chain comprises aminoacid sequence SEQ ID NO:11.
35. antibody as claimed in claim 28-mating partner molecule conjugate comprises:
(a) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:6; And
(b) variable region of light chain comprises aminoacid sequence SEQ ID NO:12.
36. antibody-mating partner molecule conjugate, comprise isolating monoclonal antibody or its antigen-binding portion thereof, and the mating partner molecule, this monoclonal antibody or the combination of its antigen-binding portion thereof are by the epi-position on the human CD70 albumen of following antibody recognition, and described antibody comprises:
(a) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:1; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:7;
(b) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:2; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:8;
(c) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:3; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:9;
(d) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:4; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:10;
(e) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:5; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:11;
(f) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:73; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:11; Perhaps
(g) variable region of heavy chain comprises aminoacid sequence SEQ ID NO:6; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:12,
Wherein this mating partner molecule is a therapeutical agent.
37. a composition comprises antibody as claimed in claim 1-mating partner molecule conjugate and pharmaceutically acceptable carrier.
38. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this therapeutical agent is a cytotoxin.
39. a composition comprises antibody as claimed in claim 38-mating partner molecule conjugate and pharmaceutically acceptable carrier.
40. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this therapeutical agent is a radio isotope.
41. a composition comprises antibody as claimed in claim 40-mating partner molecule conjugate and pharmaceutically acceptable carrier.
42. suppress to express the method for the growth of tumour cell of CD70, comprise that the tumour cell with this expression CD70 contacts with antibody as claimed in claim 1-mating partner molecule conjugate, make the growth of tumour cell of this expression CD70 be suppressed like this.
43. method as claimed in claim 42, wherein the tumour cell of this expression CD70 is kidney tumor cell or lymphoma cell.
44. method as claimed in claim 42, wherein the tumour cell of this expression CD70 is from being selected from renal cell carcinoma or lymphadenomatous cancer.
45. a treatment experimenter method for cancer comprises giving this experimenter antibody as claimed in claim 1-mating partner molecule conjugate, and this experimenter's cancer is obtained medical treatment.
46. method as claimed in claim 45, wherein this cancer is renal cell carcinoma or lymphoma.
47. method as claimed in claim 45, wherein this cancer is to be selected from: renal cell carcinoma (RCC), hyaline cell RCC, glioblastoma, non-Hodgkin lymphomas (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma, primary cutaneous type (ALCL), multiple myeloma, T-cell lymphoma,cutaneous, nodositas micromere lymphoma, lymphocytic lymphoma, lymphoma peripheral T cell, lennert lymphoma, immunoblastic lymphoma, T chronic myeloid leukemia/lymphoma (ATLL), adult T cell leukemia (T-ALL), center parent cell/centrocyte (cb/cc)) follicular lymphoma cancer, the cytophyletic diffuse type maxicell of B lymphoma, angio-immunoblastic lymphadenopathy (AILD) sample t cell lymphoma, the lymphoma that HIV is relevant based on body cavity, embryonal carcinoma, undifferentiated nasopharyngeal carcinoma, schmincke tumor, the Ka Siermanshi disease, Kaposi sarcoma, multiple myeloma, Wal Dan Sitelunshi macroglobulinemia and B cell lymphoma.
48. treatment or prevention experimenter's the method for autoimmune disease comprises giving this experimenter antibody as claimed in claim 1-mating partner molecule conjugate, this experimenter's autoimmune disease is obtained medical treatment or prevents.
49. treatment or prevention experimenter's the method for inflammation comprises giving this experimenter antibody as claimed in claim 1-mating partner molecule conjugate, this experimenter's inflammation is obtained medical treatment or prevents.
50. the method for treatment experimenter's virus infection comprises giving this experimenter antibody as claimed in claim 1-mating partner molecule conjugate, and this experimenter's virus infection is obtained medical treatment.
51. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this mating partner molecule is coupled on this antibody by chemical linkers.
52. antibody as claimed in claim 51-mating partner molecule conjugate, wherein this chemical linkers is selected from peptidyl connector, hydrazine connector and disulfide linkers.
53. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this renal cell carcinoma tumor cell line is selected from 786-O, A-498, ACHN, Caki-1 and Caki-2 clone.
54. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this lymphoma cell line is a B cell tumour clone.
55. antibody as claimed in claim 54-mating partner molecule conjugate, wherein this B cell tumour clone is selected from Daudi, HuT 78, Raji and Granta 519 clones.
56. antibody as claimed in claim 1-mating partner molecule conjugate, wherein this antibody or its antigen-binding portion thereof right and wrong are fucosylated.
57. the antigen-binding portion thereof of isolating monoclonal antibody or this antibody comprises: variable region of heavy chain comprises aminoacid sequence SEQ ID NO:6; And variable region of light chain, comprise aminoacid sequence SEQ IDNO:12.
58. isolating monoclonal antibody or its antigen-binding portion thereof, this monoclonal antibody or the combination of its antigen-binding portion thereof are by the epi-position on the human CD70 albumen of following antibody recognition, and described antibody comprises: variable region of heavy chain comprises aminoacid sequence SEQ ID NO:6; And variable region of light chain, comprise aminoacid sequence SEQ ID NO:12.
59. the antigen-binding portion thereof of an isolating monoclonal antibody or this antibody comprises:
(a) variable region of heavy chain CDR1 comprises SEQ ID NO:18;
(b) variable region of heavy chain CDR2 comprises SEQ ID NO:24;
(c) variable region of heavy chain CDR3 comprises SEQ ID NO:30;
(d) variable region of light chain CDR1 comprises SEQ ID NO:36;
(e) variable region of light chain CDR2 comprises SEQ ID NO:42; And
(f) variable region of light chain CDR3 comprises SEQ ID NO:48.
60. antibody as claimed in claim 57, wherein this antibody or its antigen-binding portion thereof right and wrong are fucosylated.
61. an isolated nucleic acid molecule, this nucleic acid molecule is encoded to antibody as claimed in claim 57 or its antigen-binding portion thereof.
62. an expression vector comprises nucleic acid molecule as claimed in claim 61.
63. a host cell comprises expression vector as claimed in claim 62.
CNA2007800512313A 2006-12-14 2007-12-13 In conjunction with human antibodies of CD70 and uses thereof Pending CN101605906A (en)

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US60/870,091 2006-12-14
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US60/991,702 2007-11-30

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