CN101326197B - Human monoclonal antibodies to O8E - Google Patents

Human monoclonal antibodies to O8E Download PDF

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CN101326197B
CN101326197B CN200680046065.3A CN200680046065A CN101326197B CN 101326197 B CN101326197 B CN 101326197B CN 200680046065 A CN200680046065 A CN 200680046065A CN 101326197 B CN101326197 B CN 101326197B
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antibody
variable region
seq
sequence
antigen
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CN101326197A (en
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A·J·科尔曼
M·J·塞尔比
L-S·路
A·威特
黄海春
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E.R. expensive precious & Sheng Si limited liability company is executed
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Medarex LLC
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Abstract

The present disclosure provides isolated monoclonal antibodies, particularly human monoclonal antibodies that specifically bind to O8E with high affinity. Nucleic acid molecules encoding the antibodies of this disclosure, expression vectors, host cells and methods for expressing the antibodies of this disclosure are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of this disclosure are also provided. This disclosure also provides methods for treating cancer.

Description

The human monoclonal antibodies of anti-O8E
The cross reference of related application
The application requires the right of priority of the U.S. Provisional Application sequence number 60/748,914 of submitting on December 8th, 2005 and the U.S. Provisional Application sequence number 60/824,593 of submitting on September 5th, 2006, and the full content of above-mentioned application is incorporated herein by reference.
Technical field
The application relates generally to immunology and biology field.The nucleic acid of the anti-O8E monoclonal antibody of people more specifically, provided herein, the anti-O8E monoclonal antibody of encoding human, the method for preparing the anti-O8E monoclonal antibody of people and the treatment method take the Growth of Cells of expressing O8E as the disorders such as cancers of feature.
Background technology
Mammary cancer and ovarian cancer are respectively American Women's because of the second of cancer mortality and the 4th 's reason (American Cancer Society (2005) Cancer facts and figures).American Cancer Society estimates, the U.S. in 2005 will have near 40,000 women and die from mammary cancer, have about 16,000 stars to die from ovarian cancer.The superficial epithelium tumour accounts for more than 80% of all malignants tumor of ovary, comprise serous tumor, mucinous tumors, endometrioid tumors and clear cell carcinoma (people such as Seidman. " Blaustein ' s Pathology of the Female Genital Tract " 791-4 (Kurman, editor, 5 thEd.New York, Springer-Verlag, 2002).Ovarian cancer appears at late period usually, and this moment, metastatic disease was diffused into zone and position, distant place (Pettersson, (1994) Int.Fed.Of Gyn.and Obstetrics, Vol.22; With people such as Heintz. (2001) J.Epidermiol.Biostat.6:107-38).Therefore, the survival possibility of mammary cancer is apparently higher than ovarian cancer, and patient with breast cancer's 5 annual survival rates are better than in fact ovarian cancer patients.
B7 sample molecule belongs to immunoglobulin (Ig) (Ig) superfamily.The outer part of the born of the same parents of B7 sample molecule contains single IgV and IgC territory, and the total approximately amino acid identity of 20%-40%.B7 sample molecule plays a crucial role in the control of antigen-specific immune response and fine setting.O8E is also referred to as B7H4, B7x and B7S1, a member of B7 family, (the people such as Carreno is considered to work in the pungency of t cell response and inhibition are regulated, (2002) people such as Ann.Rev.Immunol.20:29-53 and Khoury, (2004) Immunity 20:529-538).People O8E is mapped on karyomit(e) 1, it is comprised of six exons and five introns, across 66kb, wherein exon 6 is used for alternative splicing, and produce two different transcripts (people such as Choi. (2003) J.Immunol.171:4650-4654).
O8E by with the T cell on receptors bind bring into play its physiologic function, inducing cell cycle arrest and suppress cytokine secretion, cytotoxicity development and CD4 then +And CD8 +The T cell produce cytokine (people such as Prasad. (2003) Immunity 18:863-873; The people such as Sica. (2003) Immunity 18:849-861; The people such as Wang. (2004) Microbes Infect.6:759-66; With people such as Zang. (2003) Proc.Natl.Acad.Sci.U.S.A.100:10388-10392).The attenuator that O8E may be inflammatory response has been proposed, and can work in antigen specific immune and antitumor downward of replying (people such as Zang. (2003) Proc.Natl.Acad Sci.U.S.A.100:10388-10392; The people such as Prasad (2003) Immunity 18:863-873; The people such as Sica. (2003) Immunity 18:849-861; The people such as Choi (2003) J.Immunol.171:4650-4654; With people such as Carreno. (2003) TrendsImmunol 24:524-7).
O8E mRNA rather than protein expression detected in the normal somatic cell tissue of broad range, these tissues comprise liver, skeletal muscle, kidney, pancreas and small intestine (people such as Sica. the people such as (2003) Immunity 18:849-61 and Choi. (2003) J.Immunol.171:4650-4).After stimulating T cell, B cell, monocyte and dendritic cell, O8E is derivable; Yet the expression that immunohistochemical analysis is disclosed in several surrounding tissues is extremely low, and an exception is positive dyeing (the same) in some ovarian cancer and lung cancer.In addition, O8E equal overexpression as one man in primary and metastatic breast cancer, with tumor grade or by stages irrelevant, point out this protein have in mammary cancer biology crucial effect (people such as Tringler. (2005) Clinical Cancer Res.11:1842-48).Referring to United States Patent (USP) 6,962,980; 6,699,664; 6,468,546; 6,488,931; 6,670,463; With 6,528,253, described patent all is incorporated herein by reference in full.
The treatment of advanced breast cancer and ovarian cancer can be used multiple therapy methods, the traditional chemical therapy, hormonotherapy (aromatase inhibitor, luliberin analogue), bis phosphoric acid salt and the signal transduction inhibitor (Smith (2002) Lancet, 260:790-2) that comprise radiotherapy, use cytotoxic antitumor agents.Yet many patients are relatively poor to replying of above-mentioned arbitrary methods for the treatment of, perhaps do not reply at all regrettably.Therefore, need evaluation for new molecular marked compound and the therapeutical agent of mammary cancer and ovarian cancer.Therefore, O8E has represented a kind of valuable cancer therapy target, and described cancer comprises ovarian cancer and mammary cancer and the multiple other diseases that is expressed as feature with O8E.
Summary of the invention
The invention provides with O8E (a/k/a B7H4, B7S1 and B7x) in conjunction with and show the monoclonal antibody of separating, particularly human sequence's monoclonal antibody of many desired characteristic.These characteristics comprise with people O8E high-affinity is combined.The method of the disease of using antibody of the present invention and the multiple O8E mediation of combination treatment also is provided.
In one aspect, the present invention relates to a kind of monoclonal antibody or its antigen-binding portion thereof of separation, wherein this antibody:
(a) with 1 * 10 -7M or lower K DBe combined with people O8E; And
(b) be combined with people's Chinese hamster ovary celI of O8E transfection.
In certain embodiments, this antibody and mammary gland cell tumor clone such as clone SKBR3 (ATCC preserving number HTB-30) combination.
This antibody is generally people's antibody, but in alternate embodiment, and this antibody can be also, for example, and murine antibody, chimeric antibody or humanized antibody.
In one embodiment, this antibody is with 5 * 10 -8M or lower K DBe combined with people O8E, with 2 * 10 -8M or lower K DBe combined with people O8E, with 1 * 10 -8M or lower K DBe combined with people O8E, with 5 * 10 -9M or lower K DBe combined with people O8E, with 4 * 10 -9M or lower K DBe combined with people O8E, with 3 * 10 -9M or lower K DBe combined with people O8E, perhaps with 2 * 10 -9M or lower K DBe combined with people O8E.
In another embodiment, this antibody is combined rear by these cell internalizings at the O8E that expresses on SKBR3 mammary gland cell tumor cell.
In another embodiment, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, wherein this antibody is combined O8E with reference antibody cross competition, wherein said reference antibody:
(a) with 1 * 10 -7M or lower K DBe combined with people O8E; And
(b) be combined with people's Chinese hamster ovary celI of O8E transfection.
In various embodiments, described reference antibody comprises:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:1; With
(b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:6.
Perhaps described reference antibody comprises:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:2; With
(b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:7;
Perhaps described reference antibody comprises:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:3; With
(b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:8;
Perhaps described reference antibody comprises:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:4; With
(b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:9;
Perhaps described reference antibody comprises:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:5; With
(b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:10.
On the other hand, the present invention relates to a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises generation certainly or derives from people V HThe variable region of heavy chain of 4-34 gene (its protein represents with SEQID NO:51 at this), wherein this antibody and O8E specific binding.The present invention also provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, and it comprises generation certainly or derives from people V HThe variable region of heavy chain of 3-53 gene (its protein represents with SEQ ID NO:52 at this), wherein this antibody and O8E specific binding.The present invention also provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, and it comprises generation certainly or derives from people V HThe variable region of heavy chain of 3-9/D3-10/JH6b gene (its protein represents with SEQ ID NO:53 at this), wherein this antibody and O8E specific binding.
The present invention further provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises generation certainly or derives from people V KThe variable region of light chain of A27 gene (its protein represents with SEQ IDNO:54 at this), wherein this antibody and O8E specific binding.The present invention further provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises generation certainly or derives from people V KThe variable region of light chain of L6/JK1 gene (its protein represents with SEQ ID NO:55 at this), wherein this antibody and O8E specific binding.
On the other hand, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises:
(a) people V HThe variable region of heavy chain of 4-34,3-53 or 3-9 gene; With
(b) people V KA27 or V KThe variable region of light chain of L6,
Wherein this antibody and O8E specific binding.
In a related embodiment, this antibody comprises people V HThe variable region of heavy chain of 4-34 gene and people V KThe variable region of light chain of A27 gene.In another related embodiment, this antibody comprises people V HThe variable region of heavy chain of 3-53 gene and people V KThe variable region of light chain of A27 gene.In another related embodiment, this antibody comprises people V HThe variable region of heavy chain of 3-9 gene and people V KThe variable region of light chain of L6 gene.
On the other hand, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises:
The variable region of heavy chain that comprises CDR1, CDR2 and CDR3 sequence; With the variable region of light chain that comprises CDR1, CDR2 and CDR3 sequence, wherein:
(a) variable region of heavy chain CDR3 sequence comprises and is selected from SEQ ID NO:21,22,23,24 and 25 aminoacid sequence and the conservative aminoacid sequence of modifying thereof;
(b) variable region of light chain CDR3 sequence comprises and is selected from SEQ ID NO:36,37,38,39 and 40 aminoacid sequence and the conservative aminoacid sequence of modifying thereof;
(c) this antibody is with 1 * 10 -7M or lower K DBe combined with people O8E;
(d) with by people's Chinese hamster ovary celI of O8E transfection be combined.
Typically, variable region of heavy chain CDR2 sequence comprises and is selected from SEQ ID NO:16,17,18,19 and 20 aminoacid sequence and the conservative aminoacid sequence of modifying thereof; And variable region of light chain CDR2 sequence comprises and is selected from SEQ ID NO:31,32,33,34 and 35 aminoacid sequence and the conservative aminoacid sequence of modifying thereof.Typically, variable region of heavy chain CDR1 sequence comprises and is selected from SEQ ID NO:11,12,13,14 and 15 aminoacid sequence and the conservative aminoacid sequence of modifying thereof; And variable region of light chain CDR1 sequence comprises and is selected from SEQ ID NO:26,27,28,29 and 30 aminoacid sequence and the conservative aminoacid sequence of modifying thereof.
A kind of concrete combination comprises:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:11;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:16;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:21;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:26;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:31; With
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:36.
Another kind of concrete combination comprises:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:12;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:17;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:22;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:27;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:32; With
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:37.
Another kind of concrete combination comprises:
(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:18;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:23;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:28;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:33; With
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:38.
Another kind of concrete 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:19;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:24;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:29;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:34; With
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:39.
Another kind of concrete 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:20;
(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:30;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:35; With
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:40.
Other concrete antibody of the present invention or its antigen-binding portion thereof comprise:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:1; With
(b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:6.
Another kind of concrete combination comprises:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:2; With
(b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:7.
Another kind of concrete combination comprises:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:3; With
(b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:8.
Another kind of concrete combination comprises:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:4; With
(b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:9.
Another kind of concrete combination comprises:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:5; With
(b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:10.
In another aspect of this invention, provide and be combined antibody or its antigen-binding portion thereof of O8E with above-mentioned any antibody competition.
Antibody of the present invention can be, for example, and as the full length antibody of IgG1, IgG2 or IgG4 isotype.Perhaps, these antibody can be antibody fragments, as Fab, Fab ' or Fab ' 2Fragment or single-chain antibody (for example scFv).
The present invention also provides a kind of immune conjugate, and it comprises antibody of the present invention or its antigen-binding portion thereof that is connected with therapeutical agents such as cytotoxin or radio isotope.The present invention also provides a kind of bispecific molecule, and it comprises antibody of the present invention or its antigen-binding portion thereof that is connected with the second funtion part, and this second funtion part has the binding specificity different from this antibody or its antigen-binding portion thereof.
The composition that comprises antibody of the present invention or its antigen-binding portion thereof or immune conjugate or bispecific molecule and medicine acceptable carrier also is provided.
The present invention also comprises the nucleic acid molecule of coding antibody of the present invention or its antigen-binding portion thereof, and the expression vector that comprises these nucleic acid, comprises the host cell of these expression vectors, and uses the method that these host cells prepare anti-O8E antibody.And, the invention provides the genetically modified transgenic mice of a kind of human immunoglobulin heavy chain of containing and light chain, wherein this mouse is expressed antibody of the present invention, and by the hybridoma of this mouse preparation, wherein this hybridoma produces antibody of the present invention.
On the other hand, the invention provides a kind for the treatment of or the prevention method take the growth of tumour cell of expressing O8E as the disease of feature, comprise to the experimenter and use effective treatment or prevent of the present invention anti-O8E people's antibody of the amount of this disease.Described disease can be cancer, for example the mammary gland cell cancer.
On the other hand, the invention provides a kind of method for the treatment of autoimmune disease, comprise of the present invention anti-O8E people's antibody of using the amount of effective treatment autoimmune disease to the experimenter.
By following detailed description and embodiment, other features and advantages of the present invention will be apparent, and this detailed description and embodiment should not be construed as restrictive.The content that runs through the patent application of all reference, Genbank item, patent and the announcement of quoting in the application all is incorporated herein by reference herein especially.
Description of drawings
Figure 1A shows nucleotide sequence (SEQ IDNO:41) and the aminoacid sequence (SEQ ID NO:1) of 1G11 human monoclonal antibodies variable region of heavy chain.Delineate out CDR1 (SEQ ID NO:11), CDR2 (SEQ ID NO:16) and CDR3 (SEQ ID NO:21) and distinguished, and pointed out the kind system source of V and J.
Figure 1B shows nucleotide sequence (SEQ IDNO:46) and the aminoacid sequence (SEQ ID NO:6) of 1G11 human monoclonal antibodies variable region of light chain.Delineate out CDR1 (SEQ ID NO:26), CDR2 (SEQ ID NO:31) and CDR3 (SEQ ID NO:36) and distinguished, and pointed out the kind system source of V and J.
Fig. 2 A shows nucleotide sequence (SEQ IDNO:42) and the aminoacid sequence (SEQ ID NO:2) of 2A7 human monoclonal antibodies variable region of heavy chain.Delineate out CDR1 (SEQ ID NO:12), CDR2 (SEQ ID NO:17) and CDR3 (SEQ ID NO:22) and distinguished, and pointed out the kind system source of V, D and J.
Fig. 2 B shows nucleotide sequence (SEQ IDNO:47) and the aminoacid sequence (SEQ ID NO:7) of 2A7 human monoclonal antibodies variable region of light chain.Delineate out CDR1 (SEQ ID NO:27), CDR2 (SEQ ID NO:32) and CDR3 (SEQ ID NO:37) and distinguished, and pointed out the kind system source of V and J.
Fig. 3 A shows nucleotide sequence (SEQ IDNO:43) and the aminoacid sequence (SEQ ID NO:3) of 2F9 human monoclonal antibodies variable region of heavy chain.Delineate out CDR1 (SEQ ID NO:13), CDR2 (SEQ ID NO:18) and CDR3 (SEQ ID NO:23) and distinguished, and pointed out the kind system source of V, D and J.
Fig. 3 B shows nucleotide sequence (SEQ IDNO:48) and the aminoacid sequence (SEQ ID NO:8) of 2F9 human monoclonal antibodies variable region of light chain.Delineate out CDR1 (SEQ ID NO:28), CDR2 (SEQ ID NO:33) and CDR3 (SEQ ID NO:38) and distinguished, and pointed out the kind system source of V and J.
Fig. 4 A shows nucleotide sequence (SEQ IDNO:44) and the aminoacid sequence (SEQ ID NO:4) of 12E1 human monoclonal antibodies variable region of heavy chain.Delineate out CDR1 (SEQ ID NO:14), CDR2 (SEQ ID NO:19) and CDR3 (SEQ ID NO:24) and distinguished, and pointed out the kind system source of V, D and J.
Fig. 4 B shows nucleotide sequence (SEQ IDNO:49) and the aminoacid sequence (SEQ ID NO:9) of 12E1 human monoclonal antibodies variable region of light chain.Delineate out CDR1 (SEQ ID NO:29), CDR2 (SEQ ID NO:34) and CDR3 (SEQ ID NO:39) and distinguished, and pointed out the kind system source of V and J.
Fig. 5 A shows nucleotide sequence (SEQ IDNO:45) and the aminoacid sequence (SEQ ID NO:5) of 13D12 human monoclonal antibodies variable region of heavy chain.Delineate out CDR1 (SEQ ID NO:15), CDR2 (SEQ ID NO:20) and CDR3 (SEQ ID NO:25) and distinguished, and pointed out the kind system source of V, D and J.
Fig. 5 B shows nucleotide sequence (SEQ IDNO:50) and the aminoacid sequence (SEQ ID NO:10) of 13D12 human monoclonal antibodies variable region of light chain.Delineate out CDR1 (SEQ ID NO:30), CDR2 (SEQ ID NO:35) and CDR3 (SEQ ID NO:40) and distinguished, and pointed out the kind system source of V and J.
Fig. 6 shows that the weight chain variable region amino acid sequence of 1G11 and 13D 12 and ethnic group are V HThe comparison of 4-34 aminoacid sequence (SEQ ID NO:51).
Fig. 7 shows that the weight chain variable region amino acid sequence of 2A7 and 2F9 and ethnic group are V HThe comparison of 3-53 aminoacid sequence (SEQ ID NO:52).
Fig. 8 shows that the weight chain variable region amino acid sequence of 12E1 and combination ethnic group are V HThe comparison of 3-9/D3-10/JH6b aminoacid sequence (SEQ ID NO:53).
Fig. 9 shows that the light chain variable region amino acid sequence of 1G11,2A7,2F9 and 13D12 and ethnic group are V KThe comparison of A27 aminoacid sequence (SEQ ID NO:54).
Figure 10 shows that the light chain variable region amino acid sequence of 12E1 and combination ethnic group are V KThe comparison of L6/JK1 aminoacid sequence (SEQ ID NO:55).
Figure 11 A and 11B show the ELISA experimental result, prove human monoclonal antibodies and the O8E specific binding of anti-human O8E.Figure 11 A shows the anti-O8E antibody sandwich of employment elisa plate, then adds the O8E albumen of purifying and the result that detects with the anti-O8E antiserum(antisera) of rabbit.Figure 11 B shows with the coated elisa plate of anti-mouse Fc antiserum(antisera), then add monoclonal anti C9 (0.6 μ g/ml), then Penta-O8E albumen titration shown in use, then use the result of the 1 anti-O8E antibody test of μ g/ml people.
Figure 12 shows the fluidic cell experimental result, proves that anti-O8E human monoclonal antibodies 2A7 is combined with the Chinese hamster ovary celI of O8E transfection.
Figure 13 shows the fluidic cell experimental result, proves that O8E is at SKOV3 and the HEK cells of SKBR3 breast cancer cell and O8E transfection.
Figure 14 shows the result of Hum-Zap internalization experiment, proves that the human monoclonal antibodies of anti-human O8E can internalization arrive O8E +In Chinese hamster ovary celI.
Figure 15 shows the result of Hum-Zap internalization experiment, proves that the human monoclonal antibodies of anti-human O8E can internalization arrive O8E +In the SKBR3 cell.
Figure 16 shows the result of using the anti-O8E monoclonal antibody of various human (comprising 1G11,2A7,2F9 and 13D12) to carry out epitope mapping research.
Figure 17 shows cytotoxicity (ADCC) measurement result that antibody relies on, and reference's monoclonal anti O8E antibody kills MCF-7 SKBR3 in the mode that depends on ADCC.
Figure 18 shows cytotoxicity (ADCC) measurement result that antibody relies on, and reference's monoclonal anti O8E antibody kills the SKOV3 cell of O8E transfection in the mode that depends on ADCC.
Figure 19 shows cytotoxicity (ADCC) measurement result that antibody relies on, and reference's monoclonal anti O8E antibody kills MCF-7 SKBR3 in the mode that depends on concentration and ADCC.
Figure 20 shows the result of studying in the body that the SCID mouse is carried out, and represents that anti-O8E antibody is to the tumor growth restraining effect of HEK-B7H4 tumour.
Detailed Description Of The Invention
The present invention relates to the monoclonal antibody of separating with high-affinity specific binding O8E (a/k/a B7H4, B7S1 and B7x), particularly human sequence's monoclonal antibody.In certain embodiments, antibody sources of the present invention is sequence in specific heavy chain and light chain kind, and/or comprises certain structural features, as comprises the CDR district of specific amino acid sequence.The invention provides the antibody of separation, the method for preparing this antibody, the immune conjugate that contains this antibody and bispecific molecule and contain the pharmaceutical composition of antibody of the present invention, immune conjugate or bispecific molecule.The invention still further relates to and utilize this antibody (for example) detection O8E and treatment to express the method for relevant disorders such as cancers with O8E.Correspondingly, the present invention also provides and uses anti-O8E Antybody therapy kinds cancer of the present invention, for example treats the method for mammary gland cell cancer, metastatic breast cancer, gonad cell cancer, Metastatic carcinoma in the ovary and renal cell carcinoma.
For the present invention is more readily understood, some terms have at first been defined.Being defined in the detailed Description Of The Invention content of other illustrates.
Term " O8E ", " B7H4 ", " B7x " and " B7S1 " in this article can Alternates, comprise variant, isotype (isoforms), homologue, straight homologues (ortholog) and the paralog thing (paralog) of people O8E.For example, the O8E specific antibody in some cases can with the O8E cross reaction from species beyond the mankind.In other embodiments, people O8E specific antibody may be fully specific for people O8E, and may not show the cross reactivity of species or other types.Term " people O8E " refers to human sequence O8E, is the complete amino acid sequence (SEQ ID NO:56) of the people O8E of NP_078902 as the Genbank accession number.O8E is also referred to as in the art, for example, and BL-CAM, B3, Leu-14 and Lyb-8.People O8E sequence may be different from the people O8E of SEQ ID NO:56, for example, has conservative sudden change or has sudden change in non-conservative district, and CD22 has substantially the same biological function with the people O8E of SEQ ID NO:56.For example, the biological function of people O8E is that have in the extracellular domain of O8E can be by the epi-position of antibodies specific combination of the present invention, perhaps the biological function of people O8E comprises, for example, suppressor T cell propagation, inhibition cytokine produce, suppress the cell cycle generation or be combined with φt cell receptor.
Concrete people O8E sequence usually on aminoacid sequence the people O8E at least 90% with SEQ ID NO:56 identical, and contain and can determine that when comparing with the O8E aminoacid sequence of other species (for example mouse) this aminoacid sequence is human sequence's amino-acid residue.In some cases, people O8E is may at least 95% or at least 96%, 97%, 98% or 99% identical with the O8E of SEQ ID NO:56 on aminoacid sequence.In certain embodiments, the demonstration of people O8E sequence is no more than 10 amino acid whose differences with the O8E of SEQ ID NO:56.In certain embodiments, people O8E may show that O8E with SEQ ID NO:56 is no more than 5 or be no more than 4,3,2 or 1 amino acid whose differences.Percentage identity can as described hereinly be measured.
Term " immunne response " refers to the effect of the soluble large molecule (comprising antibody, cytokine and complement) that for example lymphocyte, antigen presenting cell, phagocytic cell, granulocyte and above-mentioned cell or liver produce, this effect cause selective injury, destruction or remove the pathogenic agent of invading from human body, by the cell or tissue of pathogenic infection, cancer cells, or (in the situation that autoimmunization or pathologic inflammation) normal cell or tissue.
Biochemical relationship between the multi-signal transduction molecule that " signal transduction pathway " refers to work signal another part from the part of a cell to a cell transmits.Phrase used herein " cell surface receptor " comprises, for example, can receive signal and stride across molecule and the molecular complex that cytoplasmic membrane is propagated sort signal.An example of " cell surface receptor " of the present invention is the O8E acceptor.
Term mentioned in this article " antibody " comprises complete antibody and any Fab (i.e. " antigen-binding portion thereof ") or strand." antibody " refer to comprise by disulfide linkage interconnection together at least two weights (H) chain and the glycoprotein of two light (L) chains, or its antigen-binding portion thereof.Every heavy chain (is abbreviated as V at this by variable region of heavy chain H) and the CH composition.CH is by three domain Cs H1, C H2And C H3Form.Every light chain (is abbreviated as V at this by variable region of light chain L) and the constant region of light chain composition.Constant region of light chain is by a domain C LForm.V HAnd V LThe district can further be further divided into the hypervariable region, is called complementary determining region (CDR), and CDR is dispersed in the more conservative zone that is called as framework region (FR).Each V HAnd V LForm by three CDR and four FR, they are arranged to carboxyl terminal in the following order from aminoterminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.The variable region of heavy chain and light chain contain can with the binding domains of AI.The constant region of antibody can the mediated immunity sphaeroprotein and the combination of host tissue or the factor, and this host tissue or the factor comprise first composition (C1q) of immune various cell (for example effector cell) and classical complement system.
" antigen-binding portion thereof " of term antibody used herein (or " antibody moiety ") refers to keep the one or more fragments with the antibody of the ability of antigen (for example O8E) specific binding.The antigen combined function that has proved antibody can be exercised by the fragment of full length antibody.In " antigen-binding portion thereof " of term antibody, the example of included binding fragment comprises: (i) Fab fragment, and namely by V L, V H, C LAnd C H1The unit price fragment that structural domain forms; (ii) F (ab ') 2Fragment namely is included in the bivalent fragment of two Fab fragments that the hinge area place connects by disulfide linkage; (iii) Fab ' fragment, it is that the Fab with hinge area part (sees Fundamental Immunology (Paul ed., 3rd ed.1993) basically; (iv) the Fd fragment that is formed by VH and CH1 structural domain; (v) by the V of antibody single armed LAnd V HThe Fv fragment that structural domain forms; (vi) by V HThe dAb fragment (Ward etc. (1989) Nature 341:544-546) that structural domain forms; (vii) complementary determining region (CDR) that separates; (Viii) nano antibody (nanobody) namely, contains the variable region of heavy chain of single variable domain and two constant domains.In addition, although two structural domain V of Fv fragment LAnd V HBy independent genes encoding, but they can utilize recombination method to link together by synthetic linker, and this linker makes them can make protein chain, wherein a V LAnd V HDistrict's pairing consists of monovalent molecule and (is called scFv (scFv); Referring to, such as (1988) Science 242:423-426 such as Bird; With (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883 such as Huston).This single-chain antibody is also included within term antibody " antigen-binding portion thereof ".These antibody fragments are with well known to a person skilled in the art that routine techniques obtains, and use the method identical with complete antibody that the practicality of these fragments is screened.
" antibody of separation " used herein refers to substantially not contain the antibody (for example, substantially not containing the antibody of being combined with the antigen-specific except O8E with the antibody that separates of O8E specific binding) of other antibody with different antigen-specifiies.But, with the antibody that separates of O8E specific binding with may have cross reactivity such as other antigens such as O8E molecule from other species.And the antibody of separation can not contain other cell materials and/or chemical substance substantially.
Term used herein " monoclonal antibody " or " monoclonal antibody combination " refer to the preparation of single molecular antibody molecule.Monoclonal antibody combination shows single binding specificity and the affinity to defined epitope.
Term used herein " people's antibody " or " human sequence's antibody " comprise the antibody with following variable region, and in this variable region, it is immunoglobulin sequences that framework region and CDR district all are derived from ethnic group.And if this antibody contains constant region, also to be derived from ethnic group be immunoglobulin sequences to this constant region.People's antibody can comprise modification afterwards, comprises natural or synthetic modification.It not is to be the amino-acid residue (for example, by external random or site-specific mutagenesis or the sudden change introduced by somatic mutation in body) of immunoglobulin sequences coding by ethnic group that people's antibody of the present invention can comprise.But term used herein " people's antibody " does not comprise that the CDR sequence of the kind system that wherein is derived from another Mammals kind such as mouse has been transplanted to the antibody on people's frame sequence.
Term " human monoclonal antibodies " (can comprise term " sequence " after " people ") refers to show the antibody of single binding specificity, and it has wherein framework region and CDR district and all is derived from the variable region that ethnic group is immunoglobulin sequences.In one embodiment, human monoclonal antibodies is produced by hybridoma, this hybridoma comprises the B cell that merges with immortalized cell, and this B cell obtains from have the genetically modified genomic transgenic nonhuman animal of the people's of containing heavy chain transgenosis and light chain (for example transgenic mice).
term used herein " recombinant human antibody " comprises by recombination method and preparing, express, everyone antibody that produces or separate, for example: (a) from for the antibody that separates human immunoglobulin gene's transgenosis or trans-chromosome animal (for example mouse) or hybridoma prepared therefrom (hereinafter further describing), (b) antibody from separating through the host cell of translation table intelligent antibody such as transfectoma, (c) antibody that separates from restructuring combination people antibody library, (d) by comprising human immunoglobulin gene's sequence montage any other method preparation for other DNA sequence dnas, express, the antibody that produces or separate.These recombinant human antibodies have wherein framework region and CDR district and all are derived from the variable region that ethnic group is immunoglobulin sequences.These recombinant human antibodies can experience vitro mutagenesis (perhaps, when the transgenic animal of end user Ig sequence, experience body endosome cell mutation), so the V of recombinant antibodies but in certain embodiments, HAnd V LAlthough the aminoacid sequence in district is that to be derived from ethnic group be V HAnd V LSequence and associated sequence, but may not be in the repertoire (repertoire) that the natural people's of being present in antibody kind is in vivo.
Term used herein " isotype " refers to the antibody isotype (for example IgM or IgG1) by the weight chain constant area gene coding.
Phrase " antibody of identification antigen " and " antigen-specific antibodies " are used interchangeably with term " antibody of being combined with antigen-specific " at this.
Term " people's antibody derivatives " refers to any modified forms of people's antibody, for example the conjugate of antibody and other reagent or antibody.
The CDR sequence that term " humanized antibody " refers to wherein to derive from the kind system of another Mammals kind such as mouse has been transplanted to the antibody on people's frame sequence.Also can carry out other framework region in people's frame sequence modifies.
Term " chimeric antibody " refers to that wherein variable region sequences derives from species and the constant region sequence derives from the antibody of another species, and for example wherein variable region sequences derives from mouse antibodies and the constant region sequence derives from the antibody of people's antibody.
The antibody of term used herein " with people O8E specific binding " refers to 1 * 10 -7M or lower, more preferably 5 * 10 -8M or lower, more preferably 3 * 10 -8M or lower, more preferably 1 * 10 -9M or lower, even more preferably 5 * 10 -9M or lower K DThe antibody of being combined with people O8E.
Term used herein " substantially not in conjunction with " protein or the cell meaning are not with this protein or Cell binding or not with high-affinity and this protein or Cell binding, that is, and and with 1 * 10 -6M or higher, more preferably 1 * 10 -5M or higher, more preferably 1 * 10 -4M or higher, more preferably 1 * 10 -3M or higher, even more preferably 1 * 10 -2M or higher K DWith this protein or Cell binding.
Term " K used herein Assoc" or " K a" refer to the association rate of specific antibodies-AI, and term " K used herein dis" or " K d" refer to the dissociation rate of specific antibodies-AI.Term " K used herein D" referring to dissociation constant, it is by K dWith K aRatio obtains (is K d/ K a), and be expressed as volumetric molar concentration (M).The K of antibody DValue can be measured with the method that this area is set up.Measure antibody K DA kind of preferred method be to use surperficial plasmon resonant method, preferably use bio-sensor system, as Biacore System.
" high-affinity " of term IgG antibody used herein refers to that antibody is for the K of target antigen DBe 1 * 10 -7M or lower, more preferably 5 * 10 -8M or lower, more preferably 1 * 10 -9M or lower, even more preferably 5 * 10 -9M or lower.But for other antibody isotypes, " high-affinity " is different in conjunction with possibility.For example, for the IgM isotype, " high-affinity " is in conjunction with referring to that antibody has 10 -6M or lower, more preferably 10 -7M or lower, even more preferably 10 -8M or lower K D
Term used herein " experimenter " comprises anyone or non-human animal.Term " non-human animal " comprises all vertebratess, and for example Mammals and nonmammalian are as non-human primate, sheep, dog, cat, horse, ox, chicken, amphibian animal, fish, reptile etc.
Term used herein " O8E " has synonymous with term " B7H4 ", " B7S1 ", " B7x ", and these terms differently appear in scientific and technical literature.The public can obtain the aminoacid sequence of O8E (B7H4), with reference to GenBank accession number AAZ17406, AAS13400, AAP37283, CAI12739 and CAI12737, and with reference to the people such as Prasad (2003) Immunity 18:863-873; The people such as Sica. (2003) Immunity 18:849-861; With U.S. Patent No. 6,891,030, all be incorporated herein by reference in full.
Describe in further detail in the application's all respects chapters and sections below.
Anti-O8E antibody
Antibody of the present invention is characterised in that specific functional features or the characteristic of antibody.For example, these antibody and people O8E specific binding.Typically, antibody of the present invention is combined with O8E with high-affinity, for example K DBe 1 * 10 -7M or lower.Anti-O8E antibody of the present invention typically shows following one or more feature:
(a) with 1 * 10 -7M or lower K DBe combined with people O8E;
(b) be combined with people's Chinese hamster ovary celI of O8E transfection.
Typically, this antibody is with 5 * 10 -8M or lower K DBe combined with people O8E, with 2 * 10 -8M or lower K DBe combined with people O8E, with 5 * 10 -9M or lower K DBe combined with people O8E, with 4 * 10 -9M or lower K DBe combined with people O8E, with 3 * 10 -9M or lower K DBe combined with people O8E, with 2 * 10 -9M or lower K DBe combined with people O8E, perhaps with 1 * 10 -9M or lower K DBe combined with people O8E.
Evaluation antibody is known to the standard test of the binding ability of O8E in the art, comprises, for example ELISA, Western engram analysis, RIA and flow cytometry.Suitable test is described in detail in an embodiment.The binding kinetics of antibody (for example binding affinity) also can be by standard test well known in the art (as ELISA, Scatchard and Biacore
Figure S2006800460653D00171
Systems analysis) estimate.As the another one example, antibody of the present invention can with breast cancer tumour clone for example SKBR3 clone be combined.
Monoclonal antibody 1G11,2A7,2F9,12E1 and 13D12
The exemplary antibody of the present invention comprises as separating and carry out human monoclonal antibodies 1G11,2A7,2F9,12E1 and the 13D12 of structural characterization as described in embodiment 1 and 2.The V of 1G11,2A7,2F9,12E1 and 13D12 HAminoacid sequence is presented at respectively in SEQ ID NO:1,2,3,4 and 5.The V of 1G11,2A7,2F9,12E1 and 13D12 LAminoacid sequence is presented at respectively in SEQID NO:6,7,8,9 and 10.
Because each in these antibody can both be combined with O8E, these V HAnd V LSequence can " be mixed and mate ", thereby produces other anti-O8E binding molecule of the present invention.O8E can be with above reaching detecting in conjunction with test (for example FACS or ELISA) described in embodiment with these combinations that " mix and mate " antibody.Preferably, work as V HAnd V LWhen chain mixes and mates, from specific V H/ V LThe V of pairing HSequence is replaced by V similar on structure HSequence.Equally, preferably, from specific V H/ V LThe V of pairing LSequence is replaced by V similar on structure LSequence.
Therefore, in one aspect, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises:
(a) comprise the variable region of heavy chain that is selected from SEQ ID NO:1,2,3,4 and 5 aminoacid sequence; With
(b) comprise the variable region of light chain that is selected from SEQ ID NO:6,7,8,9 and 10 aminoacid sequence; Wherein this antibody and O8E, preferred people O8E specific binding.
Preferred heavy chain and light chain combination comprise:
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:1; (b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:6; Or
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:2; (b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:7; Or
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:3; (b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:8; Or
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:4; (b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:9; Or
(a) comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:5; (b) comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:10.
On the other hand, the invention provides the heavy chain that comprises 1G11,2A7,2F9,12E 1 and 13D12 and the antibody of light chain CDR1, CDR2 and CDR3 or its combination.The V of 1G11,2A7,2F9,12E 1 and 13D 12 HThe aminoacid sequence of CDR1 is shown in SEQ ID NO:11,12,13,14 and 15.The V of 1G11,2A7,2F9,12E 1 and 13D 12 HThe aminoacid sequence of CDR2 is shown in SEQ ID NO:16,17,18,19 and 20.The V of 1G11,2A7,2F9,12E1 and 13D 12 HThe aminoacid sequence of CDR3 is shown in SEQ ID NO:21,22,23,24 and 25.The V of 1G11,2A7,2F9,12E1 and 13D 12 KThe aminoacid sequence of CDR1 is shown in SEQ ID NO:26,27,28,29 and 30.The V of 1G11,2A7,2F9,12E1 and 13D 12 KThe aminoacid sequence of CDR2 is shown in SEQ ID NO:31,32,33,34 and 35.The V of 1G11,2A7,2F9,12E1 and 13D12 KThe aminoacid sequence of CDR3 is shown in SEQ ID NO:36,37,38,39 and 40.The CDR district Kabat (Kabat of system, (1991) Sequences of Proteins of ImmunologicalInterest such as E.A., the 5th edition, U.S.Department of Health and Human Services, NIH publication number 91-3242) delineate out.
Above-mentionedly all list in following table 1 and sequence table at this amino acid and nucleotide sequence that is named as people's antibody of 1G11,2A7,2F9,12E1 and 13D12.
The sequence of the heavy chain of table 1. people antibody 1G11,2A7,2F9,12E1 and 13D12 and variable region of light chain and constant region and corresponding CDR
SEQ ID NO：15 The aminoacid sequence of 13D12 human monoclonal antibodies variable region of heavy chain CDR1 GYYWS
SEQ ID NO：16 The aminoacid sequence of 1G11 human monoclonal antibodies variable region of heavy chain CDR2 EINHSGTTNYNPSLKS
SEQ ID NO：17 The aminoacid sequence of 2A7 human monoclonal antibodies variable region of heavy chain CDR2 VIYGSGRTYYADSVKG
SEQ ID NO：18 The aminoacid sequence of 2F9 human monoclonal antibodies variable region of heavy chain CDR2 VIYGSGRTDCADSVKG
SEQ ID NO：19 The aminoacid sequence of 12E1 human monoclonal antibodies variable region of heavy chain CDR2 GISWNSGSIGYADSVKG
SEQ ID NO：20 The aminoacid sequence of 13D12 human monoclonal antibodies variable region of heavy chain CDR2 KINHSGSTNYNPSLKS
SEQ ID NO：21 The aminoacid sequence of 1G11 human monoclonal antibodies variable region of heavy chain CDR3 LSSWSNWAFEY
SEQ ID NO：22 The aminoacid sequence of 2A7 human monoclonal antibodies variable region of heavy chain CDR3 DTYAMDV
SEQ ID NO：23 The aminoacid sequence of 2F9 human monoclonal antibodies variable region of heavy chain CDR3 DGDYGMDV
SEQ ID NO：24 The aminoacid sequence of 12E1 human monoclonal antibodies variable region of heavy chain CDR3 LYGSGSSDFYYYGMDV
SEQ ID NO：25 The aminoacid sequence of 13D12 human monoclonal antibodies variable region of heavy chain CDR3 ELRYFENYYYGMDV
SEQ ID NO：26 The aminoacid sequence of 1G11 human monoclonal antibodies variable region of light chain CDR1 RASQSVSSTYLA
SEQ ID NO：27 The aminoacid sequence of 2A7 human monoclonal antibodies variable region of light chain CDR1 RASQSVSSSYLA
SEQ ID NO：28 The aminoacid sequence of 2F9 human monoclonal antibodies variable region of light chain CDR1 RASQSVSSSYLA
SEQ ID NO：29 The aminoacid sequence of 12E1 human monoclonal antibodies variable region of light chain CDR1 RASQSVSSYLA
SEQ ID NO：30 The aminoacid sequence of 13D12 human monoclonal antibodies variable region of light chain CDR1 RASQSVSSSYLA
SEQ ID NO：31 The aminoacid sequence of 1G11 human monoclonal antibodies variable region of light chain CDR2 GASRRAT
SEQ ID NO：32 The aminoacid sequence of 2A7 human monoclonal antibodies variable region of light chain CDR2 GASSRAT
SEQ ID NO：33 The aminoacid sequence of 2F9 human monoclonal antibodies variable region of light chain CDR2 GASSRAT
SEQ ID NO：34 The aminoacid sequence of 12E1 human monoclonal antibodies variable region of light chain CDR2 DASNRAT
SEQ ID NO：35 The aminoacid sequence of 13D12 human monoclonal antibodies variable region of light chain CDR2 GASSRAT
SEQ ID NO：36 The aminoacid sequence of 1G11 human monoclonal antibodies variable region of light chain CDR3 QQYGSSPLT
SEQ ID NO：37 The aminoacid sequence of 2A7 human monoclonal antibodies variable region of light chain CDR3 QQYGSSPMYT
SEQ ID NO：38 The aminoacid sequence of 2F9 human monoclonal antibodies variable region of light chain CDR3 QQYGSSPLYT
SEQ ID NO：39 The aminoacid sequence of 12E1 human monoclonal antibodies variable region of light chain CDR3 QQRRT
Figure S2006800460653D00211
SEQ ID NO：47 The nucleotide sequence of 2A7 human monoclonal antibodies variable region of light chain GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCA GAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAA CCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCA GCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG GTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAG CCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTA GCTCACCCATGTACACTTTTGGCCAGGGGACCAAGCTGGA GATCAAA
SEQ ID NO：48 The nucleotide sequence of 2F9 human monoclonal antibodies variable region of light chain GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCA GAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAA CCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCA GCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG GTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAG CCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTA GCTCACCTCTGTACACTTTTGGCCAGGGGACCAAGCTGGA GATCAAA
SEQ ID NO：49 The nucleotide sequence of 12E1 human monoclonal antibodies variable region of light chain GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCA GAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCT GGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACA GGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTC TGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCT GAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGGACGT TCGGCCAAGGGACCAAGGTGGAAATCAAA
SEQ ID NO：50 The nucleotide sequence of 13D12 human monoclonal antibodies variable region of light chain GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCA GAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAA CCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCA GCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG GTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAG CCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTA GCTCACCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAAT CAAA
SEQ ID NO：51 Ethnic group is V HThe aminoacid sequence of 4-34 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQP PGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSL KLSSVTAADTAVYYCAR
SEQ ID NO：52 Ethnic group is V HThe aminoacid sequence of 3-53 EVQLVESGGGLIQPGGSLRLSCAASGFTVSSNYMSWVRQA PGKGLEWVSVIYSGGSTYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAR
SEQ ID NO：53 Ethnic group is V HThe aminoacid sequence of 3-9/D3-10/JH6b EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQA PGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLY LQMNSLRAEDTALYYCAKDYGSGSYYYYYGMDVWGQGTTV TVSS
SEQ ID NO：54 Ethnic group is V kThe aminoacid sequence of A27 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK PGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSP
SEQ ID NO：55 Ethnic group is V kThe aminoacid sequence of L6/JK1 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWTFGQGTKVEIK
Because these antibody all can be combined with O8E, and antigen-binding specificity mainly provides by CDR1,2 and 3 districts, V HCDR1, CDR 2 and CDR 3 sequences and V KCDR1, CDR 2 and CDR 3 sequences can " mix and mate " that (namely the CDR from different antibodies can mix and mate, but each antibody must contain V HCDR1, CDR 2 and CDR 3 and V KCDR 1, CDR 2 and CDR 3), thus other anti-O8E binding molecule of the present invention produced.O8E with these " mix and mate " antibody combination can with above reach described in embodiment in conjunction with test (for example FACS, ELISA, Biacore
Figure S2006800460653D00231
Systems analysis) detect.Preferably, work as V HWhen the CDR sequence is mixed and is mated, from specific V HThe CDR1 of sequence, CDR2 and/or CDR3 sequence are replaced by CDR sequence similar on structure.Equally, work as V KWhen the CDR sequence is mixed and is mated, from specific V KBe replaced by CDR sequence similar on structure the CDR1 of sequence, CDR2 and/or CDR3 sequence preference.It is obvious to the skilled person that by with one or more V HAnd/or V LThe CDR region sequence replaces with from similar sequence on the structure of the CDR sequence of monoclonal antibody 1G11 disclosed herein, 2A7,2F9,12E1 and 13D12, can produce new V HAnd V LSequence.
Therefore, in yet another aspect, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises:
(a) comprise the variable region of heavy chain CDR1 that is selected from SEQ ID NO:11,12,13,14 and 15 aminoacid sequence;
(b) comprise the variable region of heavy chain CDR2 that is selected from SEQ ID NO:16,17,18,19 and 20 aminoacid sequence;
(c) comprise the variable region of heavy chain CDR3 that is selected from SEQ ID NO:21,22,23,24 and 25 aminoacid sequence;
(d) comprise the variable region of light chain CDR1 that is selected from SEQ ID NO:26,27,28,29 and 30 aminoacid sequence;
(e) comprise the variable region of light chain CDR2 that is selected from SEQ ID NO:31,32,33,34 and 35 aminoacid sequence; With
(f) comprise the variable region of light chain CDR3 that is selected from SEQ ID NO:36,37,38,39 and 40 aminoacid sequence; Wherein this antibody and O8E, preferably with people O8E specific binding.
In a preferred embodiment, this antibody comprises:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:11;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:16;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:21;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:26;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:31; With
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:36.
In another preferred embodiment, this antibody comprises:
(a) comprise the variable region of heavy chain CDR1 of SEQ ID NO:12;
(b) comprise the variable region of heavy chain CDR2 of SEQ ID NO:17;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:22;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:27;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:32; With
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:37.
In another preferred embodiment, this antibody comprises:
(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:18;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:23;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:28;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:33; With
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:38.
In another preferred embodiment, this antibody 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:19;
(c) comprise the variable region of heavy chain CDR3 of SEQ ID NO:24;
(d) comprise the variable region of light chain CDR1 of SEQ ID NO:29;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:34; With
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:39.
In another preferred embodiment, this antibody 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:20;
(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:30;
(e) comprise the variable region of light chain CDR2 of SEQ ID NO:35; With
(f) comprise the variable region of light chain CDR3 of SEQ ID NO:40.
Known in this field, do not rely on CDR1 and/or CDR2 territory, independent CDR3 territory namely can determine antibody for the binding specificity of isogeneic, and can predictably produce based on common CDR3 sequence and have the specific Multiple Antibodies of identical combination.Referring to, for example, Klimka etc., British J.of Cancer 83 (2): 252-260 (2000) (having described the heavy chain variable domain CDR3 generation humanization that only uses mouse-anti-CD30 antibody Ki-4 resists-CD30 antibody); Beiboer etc., J.Mol Biol.296:833-849 (2000) (described only use parent mouse MOC-31 anti--heavy chain CDR3 sequence generation restructuring Glycoproteins in Epithelial-2 (EGP-2) antibody of EGP-2 antibody); Rader etc., Proc.Natl.Acad.Sci U.S.A.95:8910-8915 (1998) (has described use mouse-anti beta 2 integrin alpha vβ 3The lineup source anti-alpha 2 integrin α in the heavy chain of antibody LM609 and light chain variable CDR3 territory vβ 3Antibody, wherein each member's antibody contains different sequences outside the CDR3 territory, and can be with parent's murine antibody in conjunction with identical epi-position, its avidity is the same with parent's murine antibody high or higher); Barbas etc., J.Am.Chem.Soc.116:2161-2162 (1994) (disclose CDR3 territory to antigen in conjunction with most important contribution is provided); Barbas etc., Proc, Natl.Acad.Sci.U.S.A.92:2529-2533 (1995) (has described the Fab (SI-1 of three kinds of anti-human placenta dnas, SI-40 and SI-32) the transplanting of heavy chain CDR3 sequence on the heavy chain of anti-tetanus toxoid Fab, replaced thus the heavy chain CDR3 that exists, and proved that independent CDR3 provides binding specificity); With Ditzel etc., J.Immunol.157:739-749 (1996) (described and transplanted research, wherein only be enough to keep the binding specificity of parent Fab to monospecific IgG Toxoid,tetanus in conjunction with the heavy chain CDR3 of Fab p313 antibody transfer parent polyspecific Fab LNA3).Above-mentioned each reference all is incorporated herein by reference in full.
Therefore, in some aspects, the invention provides the monoclonal antibody that comprises one or more heavy chains from non-human antibody such as mouse or large murine antibody and/or light chain CDR3 territory, wherein this monoclonal antibody can with the O8E specific binding.In certain embodiments, these antibody that comprise one or more heavy chains from the non-human antibody and/or light chain CDR3 territory of the present invention can be competed combination with corresponding parent non-human antibody (a); (b) reservation function characteristic; (c) in conjunction with identical epi-position; And/or (d) has a similar binding affinity.
In other respects, the invention provides the monoclonal antibody that comprises one or more heavy chains from the first antibody (as the people's antibody that obtains from the non-human animal) and/or light chain CDR3 territory, wherein this first antibody can with the O8E specific binding, and wherein replaced lacking to the CDR3 territory in people's antibody of the binding specificity of O8E from the CDR3 territory of this first antibody, thus produce can with second people's antibody of O8E specific binding.In certain embodiments, the antibody that comprises one or more heavy chains from the first antibody and/or light chain CDR3 territory of the present invention can be competed combination with the corresponding the first antibody of parent (a); (b) reservation function characteristic; (c) in conjunction with identical epi-position; And/or (d) has a similar binding affinity.
Has the antibody that specific kind is sequence
In certain embodiments, antibody of the present invention comprises from specific kind and is the variable region of heavy chain of heavy chain immunoglobulin gene and/or is the variable region of light chain of light chain immunoglobulin gene from specific kind.
For example, in a preferred embodiment, the present invention relates to a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises generation certainly or derives from people V HThe variable region of heavy chain of 4-34 gene, wherein this antibody and O8E specific binding.In another preferred embodiment, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises generation certainly or derives from people V HThe variable region of heavy chain of 3-53 gene, wherein this antibody and O8E specific binding.In another preferred embodiment, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof or fragment of separation, it comprises generation certainly or derives from people V HThe variable region of heavy chain of 3-9/D3-10/JH6b gene, wherein this antibody and O8E specific binding.
In another preferred embodiment, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises generation certainly or derives from people V KThe variable region of light chain of A27 gene, wherein this antibody and O8E specific binding.In another preferred embodiment, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof or fragment of separation, it comprises generation certainly or derives from people V KThe variable region of light chain of L6/JK1 gene, wherein this antibody and O8E specific binding.
In another preferred embodiment, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, wherein this antibody:
(a) comprise generation certainly or derive from people V H4-34 gene, people V H3-53 gene or combination people V HThe variable region of heavy chain of 3-9/D3-10/JH6b (described gene encode respectively the aminoacid sequence shown in SEQ ID NO:51,52 and 53);
(b) comprise generation certainly or derive from people V KA27 gene or combination people V KThe variable region of light chain of L6/JK1 gene (described gene encode respectively the aminoacid sequence shown in SEQ ID NO:54 and 55); And
(c) this antibody and O8E, preferred people O8E specific binding.
Has respectively V H4-34 and V kThe V of A27 HAnd V KThe example of antibody be 1G11 and 13D 12.Has respectively V H3-53 and V kThe V of A27 HAnd V KThe example of antibody be 2A7 and 2F9.Has respectively V H3-9/D3-10/JH6b and V KThe V of L6/JK1 HAnd V KThe example of antibody be 13D12.
In this article, if a kind of variable region of people's antibody is to obtain from the system of end user's racial immunity globulin gene, this people's antibody comprises " produce from " or " deriving from " specific kind is heavy chain or the variable region of light chain of sequence.Such system comprises the transgenic mice with target antigen immunity carrier immunoglobulin gene, perhaps is illustrated in human immunoglobulin gene library on phage with the target antigen examination." produce from " or " deriving from " ethnic group are that people's antibody of immunoglobulin sequences can be identified like this: the aminoacid sequence that aminoacid sequence and the ethnic group of this people's antibody is immunoglobulin (Ig) compares, and the ethnic group that is chosen on sequence close to this human antibody sequence (the highest % identity is namely arranged) is immunoglobulin sequences.People's antibody of " produce from " or " deriving from " particular person racial immunity sphaeroprotein sequence and this kind are that sequence is compared and may be comprised amino acid difference, for example have a mind to introduce due to the somatic mutation of natural generation or rite-directed mutagenesis the amino acid difference that causes.But, people's antibody of selecting is that the aminoacid sequence of immunoglobulin gene coding is usually at least 90% identical with ethnic group on aminoacid sequence, and contains when confirming when comparing that with the racial immunity sphaeroprotein aminoacid sequence (for example the mouse kind is sequence) of other species this people's antibody belongs to the amino-acid residue of human antibodies.In some cases, people's antibody is can at least 95% or even at least 96%, 97%, 98% or 99% identical with the aminoacid sequence of this racial immunity globulin gene coding on aminoacid sequence.Typically, deriving from the performance of people's antibody and this ethnic group that specific ethnic group is sequence is that the aminoacid sequence that immunoglobulin gene is encoded is no more than 10 amino acid whose differences.In some cases, this people's antibody may show aminoacid sequence with this racial immunity globulin gene coding and be no more than 5 or even surpass 4,3,2 or 1 amino acid whose differences.
Homologous antibody
In another embodiment, the heavy chain that antibody of the present invention comprises and variable region of light chain contain the aminoacid sequence with the amino acid sequence homologous of preferred antibody described herein, and wherein this antibody has kept the required function characteristic of the anti-O8E antibody of the present invention.
For example, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises variable region of heavy chain and variable region of light chain, wherein:
(a) variable region of heavy chain comprises and the aminoacid sequence that is selected from SEQ ID NO:1,2,3,4 and 5 aminoacid sequence 80% homology at least;
(b) variable region of light chain comprises and the aminoacid sequence that is selected from SEQ ID NO:6,7,8,9 and 10 aminoacid sequence 80% homology at least;
(c) with 1 * 10 -7M or lower K DBe combined with people O8E; With
(d) be combined with people's Chinese hamster ovary celI of O8E transfection.
In the other embodiment, V HAnd/or V LAminoacid sequence can with above-mentioned sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% homology.Has the V with above-mentioned sequence HAnd V LThe V of district height (namely 80% or higher) homology HAnd V LThe antibody in district can followingly obtain: then mutagenesis (for example site-directed mutagenesis or PCR mediation mutagenesis) coding SEQ ID NO:41,42,43,44,45,46,47,48,49 and 50 nucleic acid molecule are changed the function that antibody keeps (namely (c) and (d) described function) with what function test described herein detected coding.
As used herein, the percentage homology between two aminoacid sequences is equal to two percentage identity between sequence.After the length of consideration for the number in the room of carrying out the required introducing of best comparison between two sequences and each room, the percentage identity between two sequences is the function (being the sum * 100 in the number/site of % homology=same loci) of the number of the total same loci of these two sequences.The determining and to realize with mathematical algorithm described in following non-limiting example of sequence comparison between two sequences and percentage identity.
Percentage identity between two aminoacid sequences can be with the E.Meyers and the W.Miller (Comput.Appl.Biosci. that are integrated in ALIGN program (2.0 version), 4:11-17 (1988)) algorithm is determined, it uses PAM120 weight residue table, 12 room length point penalty, 4 gap penalty.In addition, percentage identity between two aminoacid sequences also can be determined with the Needleman the GAP program that is integrated into GCG software package (can obtain from http://www.gcg.com) and the algorithm of Wunsch (J.Mol.Biol.48:444-453 (1970)), it uses Blossum 62 matrixes or PAM250 matrix, 16,14,12,10,8,6 or 4 room weight, and 1,2,3,4,5 or 6 length weight.
In addition or alternately, protein sequence of the present invention can be further used as " search sequence " and be used for carrying out the retrieval of public database, for example identifies correlated series.This retrieval can be carried out with the XBLAST program (2.0 version) of (1990) J.Mol.Biol.215:403-10 such as Altschul.The retrieval of BLAST protein can be carried out with the XBLAST program, score=50, and word length=3 are with the aminoacid sequence of acquisition with antibody molecule homology of the present invention.In order to obtain to adopt as described room BLAST of (1997) Nucleic Acids Res.25 (17): 3389-3402 such as Altschul for the relatively room comparison of purpose.When adopting BLAST and room blast program, can use the default parameter of program (for example XBLAST and NBLAST) separately.Referring to http://www.ncbi.nlm.nih.gov.
Has the conservative antibody of modifying
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 one or more in these CDR sequences comprise specific amino acid sequence or its conservative modification the based on preferred antibody described herein (for example 1G11,2A7,2F9,12E1 or 13D12), and wherein this antibody keeps the required function characteristic of the anti-O8E antibody of the present invention.Therefore, the invention provides a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it 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:
(a) variable region of heavy chain CDR3 sequence comprises and is selected from SEQ ID NO:21,22,23,24 and 25 aminoacid sequence and the conservative aminoacid sequence of modifying thereof,
(b) variable region of light chain CDR3 sequence comprises and is selected from SEQ ID NO:36,37,38,39 and 40 aminoacid sequence and the conservative aminoacid sequence of modifying thereof,
(c) with 1 * 10 -7M or lower K DBe combined with people O8E; With
(d) be combined with people's Chinese hamster ovary celI of O8E transfection.
In a preferred embodiment, variable region of heavy chain CDR2 sequence comprises and is selected from SEQ IDNO:16,17,18,19 and 20 aminoacid sequence and its conservative aminoacid sequence of modifying; And variable region of light chain CDR2 sequence comprises and is selected from SEQ ID NO:31,32,33,34 and 35 aminoacid sequence and its conservative aminoacid sequence of modifying.In another preferred embodiment, variable region of heavy chain CDR1 sequence comprises and is selected from SEQ ID NO:11,12,13,14 and 15 aminoacid sequence and its conservative aminoacid sequence of modifying; And variable region of light chain CDR1 sequence comprises and is selected from SEQ ID NO:26,27,28,29 and 30 aminoacid sequence and its conservative aminoacid sequence of modifying.
In various embodiments, antibody can be, for example, and people's antibody, humanized antibody or chimeric antibody.
Term used herein " conserved sequence modification " refer to not can remarkably influenced or change contain binding characteristic amino acid modified of the antibody of this aminoacid sequence.Conservative modification like this comprises amino-acid substitution, interpolation and disappearance.Can by standard technique well known in the art, as the mutagenesis of site-directed mutagenesis and PCR mediation, introduce modification in antibody of the present invention.Conservative amino acid replacement is that amino-acid residue is replaced with the amino-acid residue with similar side chain.Family with amino-acid residue of similar side chain defines in the art.these families comprise: have basic side chain (Methionin for example, arginine, Histidine), acid side-chain (aspartic acid for example, L-glutamic acid), neutral polar side chain (glycine for example, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine, tryptophane), non-polar sidechain (L-Ala for example, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met)), β-branched building block (Threonine for example, α-amino-isovaleric acid, Isoleucine) and aromatic side chains (tyrosine for example, phenylalanine, tryptophane, Histidine) amino acid.Therefore, the one or more amino-acid residues in the CDR district of antibody of the present invention can be replaced into other amino-acid residues from identical side chain family, and can detect the function that the antibody of change keeps.
Antibody with the identical epi-position of anti-O8E antibodies of the present invention
In another embodiment, the invention provides with any O8E monoclonal antibody of the present invention in conjunction with the antibody of the identical epi-position on people O8E (namely can be combined with any monoclonal antibody cross competition of the present invention O8E antibody).In preferred embodiments, the reference antibody for cross competition research can be that monoclonal antibody 1G11 (has the V as shown in SEQ ID NO:1 and 6 respectively HAnd V LSequence), or monoclonal antibody 2A7 (have the V as shown in SEQ ID NO:2 and 7 respectively HAnd V LSequence), or monoclonal antibody 2F9 (have the V as shown in SEQ ID NO:3 and 8 respectively HAnd V LSequence), or monoclonal antibody 12E1 (have the V as shown in SEQ IDNO:4 and 9 respectively HAnd V LSequence), or monoclonal antibody 13D12 (have the V as shown in SEQ ID NO:5 and 10 respectively HAnd V LSequence).These cross competition antibody can be identified with the ability of 1G11,2A7,2F9,12E1 or 13D12 cross competition in measuring at standard O8E according to them.For example, can utilize BIAcore
Figure S2006800460653D00311
Systems analysis, ELISA mensuration or flow cytometry prove the cross competition with antibody of the present invention.The ability that tested antibody suppression (for example) 1G11,2A7,2F9,12E1 or 13D12 are combined with people O8E proves, this test antibody can be with 1G11,2A7,2F9,12E1 or 13D12 competition in conjunction with people O8E, therefore with 1G11,2A7,2F9,12E1 or 13D12 in the same manner in conjunction with the identical epi-position on people O8E.In a preferred embodiment, be human monoclonal antibodies in conjunction with the antibody of the identical epi-position on people O8E in the same manner with 1G11,2A7,2F9,12E1 or 13D 12.These human monoclonal antibodies can as preparation as described in embodiment with separate.
The antibody of engineered antibody and modification
Antibody of the present invention further can utilize has one or more V disclosed herein HAnd/or V LThe antibody of sequence prepares as parent material, and to build a kind of antibody of modification, the antibody of this modification is with respect to comparing the characteristic that can have change with initial antibody.Can (be V by modifying one or two variable region HAnd/or V L) for example in one or more CDR district and/or the one or more residues in one or more framework region build antibody.In addition, perhaps, can build antibody by the residue of modifying in constant region, for example change the effector function of this antibody.
The variable region through engineering approaches of one type that can carry out is that CDR transplants.Antibody is mainly to interact by the amino-acid residue that is arranged in six heavy chains and light chain complementary determining region (CDR) and target antigen.For this reason, the sequence outer than CDR of the aminoacid sequence in CDR is more diversified between each antibody.Because the CDR sequence is responsible for most of antibody-AIs, therefore can express the specific natural recombinant antibodies that has the characteristic of antibody of simulation by building following expression vector: this expression vector comprises from the specific natural CDR sequence that has antibody, this CDR sequence be transplanted on frame sequence from the different antibodies with different qualities (referring to, for example, Riechmann, L. etc. (1998) Nature 332:323-327; Jones, P. etc. (1986) Nature 321:522-525; Queen, C. etc. (1989) Proc.Natl.Acad.Sci.U.S.A.86:10029-10033; The United States Patent (USP) 5,225,539 of Winter, and the United States Patent (USP) 5,530,101 of Queen etc.; 5,585,089; 5,693,762 and 6,180,370).
therefore, another embodiment of the invention relates to a kind of monoclonal antibody or its antigen-binding portion thereof of separation, it comprises and contains CDR1, the variable region of heavy chain of CDR2 and CDR3 sequence, this CDR1, CDR2 and CDR3 sequence comprise respectively and are selected from SEQ ID NO:11, 12, 13, 14 and 15, SEQ ID NO:16, 17, 18, 19 and 20, with SEQ ID NO:21, 22, 23, 24 and 25 aminoacid sequence, and comprise and contain CDR1, the variable region of light chain of CDR2 and CDR3 sequence, this CDR1, CDR2 and CDR3 sequence comprise respectively and are selected from SEQ ID NO:26, 27, 28, 29 and 30, SEQ ID NO:31, 32, 33, 34 and 35, with SEQ IDNO:36, 37, 38, 39 and 40 aminoacid sequence.Therefore, these antibody contain the V of monoclonal antibody 1G11,2A7,2F9,12E1 or 13D12 HAnd V LThe CDR sequence, but the frame sequence different from these antibody may be contained.
These frame sequences can kind be to obtain the public DNA database of antibody gene sequence or the reference delivered from comprising.For example, the kind of people's heavy chain and chain variable region gene is that DNA sequence dna can be found in following resource: " VBase " ethnic group is that sequence library (can be from the Internet Www.mrc-cpe.cam.ac.uk/vbaseObtain), and Kabat, E.A. etc. (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH publication number 94-61242; Tomlinson, I.M. etc. (1992) " The Repertoire of Human Germline V HSequences Reveals about Fifty Groups of VH Segments with DifferentHypervariable Loops " J.Mol.Biol.227:776-798; And Cox, J.P.L. etc. (1994) " A Directory of Human Germ-line V HSegments Reveals a Strong Bias intheir Usage " Eur.J.Immunol.24:827-836; Its content all is incorporated herein by reference.As another example, the kind of people's heavy chain and chain variable region gene is that DNA sequence dna can be found in the Genbank database.For example, the following heavy chain kind of finding in HCo7 HuMAb mouse is that sequence can obtain according to following Genbank accession number: 1-69 (NG_0010109, NT_024637 and BC070333), 3-53 (NG_0010109 and NT_024637) and 3-7 (NG_0010109 and NT_024637).As another example, the following heavy chain kind of finding in VK HCo12 HuMAb mouse is that sequence can obtain according to following 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), 4-34 (CAJ556644) and 3-23 (AJ406678).
Use a kind of sequence similarity search method that is called as breach BLAST the protein sequence database of antibody protein sequence and compiling is compared (people such as Altschul. (1997) Nucleic Acids Research 25:3389-3402), the method is well known to a person skilled in the art.BLAST is a kind of heuritic approach, and wherein between antibody sequence and database sequence, the comparison of statistically significant may contain the section of height scoring of parallelism word to (HSP).Extend or prune the section that can't improve its scoring and hit (hit) to being called as.In brief, the nucleotide sequence (http://vbase.mrc-cpe.cam.ac.uk/vbase1/list2.php) in translation VBASE source keeps between FR1 and FR3 framework region and comprises the zone of FR1 and FR3.The mean length of database sequence is 98 residues.Remove the tumor-necrosis factor glycoproteins that mates fully on the protein total length.The blastp program of default canonical parameter is adopted in use except the low-complexity strainer of closing, and the substitution matrix of BLOSUM62, and protein is carried out the BLAST retrieval, filters front 5 and produces hitting of sequences match.Translate nucleotide sequence in whole 6 frameworks, the framework that does not contain terminator codon in the coupling section of database sequence is considered to potential hitting.Verify with blast program tblastx subsequently, the antibody sequence in whole 6 frameworks of this program translation, and these translations and the VBASE nucleotide sequence of dynamic translation in whole 6 frameworks are compared.
Identity be between antibody sequence and Protein Data Bank on the sequence total length all aminoacid coupling.Positive (identity+replacements coupling) is not identical, but the amino-acid substitution that is guided by the BLOSUM62 substitution matrix.If antibody sequence is with identical identity and two database sequences match, the strongest positive hitting is confirmed as matching sequence and hits.
The preferred frame sequence that is used for antibody of the present invention structurally is similar to the frame sequence that is used by selected antibody of the present invention, for example, is similar to the V that the preferred monoclonal antibody of the present invention is used H4-34 frame sequence (SEQ ID NO:51) and/or V H3-53 frame sequence (SEQ IDNO:52) and/or V H3-9/D3-10/JH6b frame sequence (SEQ ID NO:53) and/or V KA27 frame sequence (SEQ ID NO:54) and/or combination V KL6/JK1 frame sequence (SEQ IDNO:55).V HCDR1, CDR 2 and CDR 3 sequences and V KThe source racial immunity globulin gene that CDR1, CDR 2 and CDR3 sequence can be transplanted to this frame sequence has on the framework region of identical sequence, and perhaps can be transplanted to this kind be that sequence is compared on the framework region that contains one or more sudden changes to the CDR sequence.For example, have been found that in some cases, with the residue in framework region suddenly change for the antigen binding capacity that keeps or strengthen antibody be favourable (referring to, for example, the United States Patent (USP) 5,530,101 of Queen etc.; 5,585,089; 5,693,762 and 6,180,370).
It is sudden change V that the variable region of another kind of type is modified HAnd/or V KAmino-acid residue in CDR1, CDR2 and/or CDR3 district, thus one or more binding characteristics (for example avidity) of target antibody improved.Can carry out the mutagenesis of site-directed mutagenesis or PCR mediation, suddenly change to introduce, the impact of antagonist combination, perhaps other objective function characteristics, can estimate with the external or in vivo test that provides in described herein and embodiment.Preferred introducing (as indicated above) is conservative modifies.These sudden changes can be amino-acid substitution, interpolation or disappearance, but preferred displacement.And, generally change 1,2,3,4 or 5 residue that is no more than in the CDR district.
Therefore, in another embodiment, the invention provides anti-O8E monoclonal antibody or its antigen-binding portion thereof of separation, its variable region of heavy chain that comprises contains: (a) V HThe CDR1 district, it comprises and is selected from SEQ ID NO:11,12,13,14 and 15 aminoacid sequence, or compares the aminoacid sequence with 1,2,3,4 or 5 amino-acid substitution, disappearance or interpolation with 15 with SEQID NO:11,12,13,14; (b) V HThe CDR2 district, it comprises and is selected from SEQ ID NO:16,17,18,19 and 20 aminoacid sequence, or compares the aminoacid sequence with 1,2,3,4 or 5 amino-acid substitution, disappearance or interpolation with 20 with SEQ ID NO:16,17,18,19; (c) V HThe CDR3 district, it comprises and is selected from SEQ ID NO:21,22,23,24 and 25 aminoacid sequence, or compares the aminoacid sequence with 1,2,3,4 or 5 amino-acid substitution, disappearance or interpolation with 25 with SEQ ID NO:21,22,23,24; (d) V KThe CDR1 district, it comprises and is selected from SEQ ID NO:26,27,28,29 and 30 aminoacid sequence, or compares the aminoacid sequence with 1,2,3,4 or 5 amino-acid substitution, disappearance or interpolation with 30 with SEQID NO:26,27,28,29; (e) V KThe CDR2 district, it comprises and is selected from SEQ ID NO:31,32,33,34 and 35 aminoacid sequence, or compares the aminoacid sequence with 1,2,3,4 or 5 amino-acid substitution, disappearance or interpolation with 35 with SEQ ID NO:31,32,33,34; (f) V KThe CDR3 district, it comprises and is selected from SEQ ID NO:36,37,38,39 and 40 aminoacid sequence, or compares the aminoacid sequence with 1,2,3,4 or 5 amino-acid substitution, disappearance or interpolation with 40 with SEQ ID NO:36,37,38,39.
Engineered antibody of the present invention for example comprises in order to improve the antibody characteristic its V HAnd/or V KInterior framework residue has carried out the antibody of modifying.Carrying out such framework modification is generally in order to reduce the immunogenicity of antibody.For example, a kind of method is to be sequence with one or more framework residues " reverse mutation " for corresponding the kind.More particularly, can to contain the kind from derivative this antibody be the different framework residue of sequence to the antibody of experience somatic mutation.Be sequence by comparing the antibody frame sequence with the kind that derives this antibody, can identify these residues.
For example, for 1G11, V HAmino-acid residue #71 (FR3 in) be L-Ala, and at corresponding V HThe 4-34 kind is that in sequence, this residue is α-amino-isovaleric acid.Being configuration in order to make the framework region sequence revert to its kind, can by the mutagenesis of (for example) site-directed mutagenesis or PCR mediation, be sequence (for example, the V of 1G11 with this somatic mutation " reverse mutation " for planting HThe residue #71 of FR3 can be α-amino-isovaleric acid from L-Ala " reverse mutation ").The antibody of " reverse mutation " is also included within the present invention like this.
Another example is, for 1G11, and V HAmino-acid residue #81 (FR3 in) be arginine, and at corresponding V HThe 4-34 kind is that in sequence, this residue is Methionin.Be configuration in order to make the framework region sequence revert to its kind, for example, can be with the V of 1G11 HThe residue #81 of FR3 be Methionin from arginine " reverse mutation ".The antibody of " reverse mutation " is also included within the present invention like this.
Another example is, for 13D12, and V HAmino-acid residue #83 (FR3 in) be l-asparagine, and at corresponding V HThe 4-34 kind is that in sequence, this residue is Serine.Be configuration in order to make the framework region sequence revert to its kind, for example, can be with the V of 13D12 HThe residue #83 of FR3 be Serine from l-asparagine " reverse mutation ".The antibody of " reverse mutation " is also included within the present invention like this.
Another example is, for 2A7, and V HAmino-acid residue #67 (FR3 in) be α-amino-isovaleric acid, and at corresponding V HThe 3-53 kind is that in sequence, this residue is phenylalanine.Be configuration in order to make the framework region sequence revert to its kind, for example, can be with the V of 2A7 HThe residue #67 of FR3 be phenylalanine from α-amino-isovaleric acid " reverse mutation ".The antibody of " reverse mutation " is also included within the present invention like this.
Another example is, for 2F9, and V HAmino-acid residue #28 (FR1 in) be Isoleucine, and at corresponding V HThe 3-53 kind is that in sequence, this residue is Threonine.Be configuration in order to make the framework region sequence revert to its kind, for example, can be with the V of 2F9 HThe residue #28 of FR1 be Threonine from Isoleucine " reverse mutation ".The antibody of " reverse mutation " is also included within the present invention like this.
Another example is, for 12E1, and V HAmino-acid residue #23 (FR1 in) be α-amino-isovaleric acid, and at corresponding V HThe 3-9 kind is that in sequence, this residue is L-Ala.Be configuration in order to make the framework region sequence revert to its kind, for example, can be with the V of 12E1 HThe residue #23 of FR1 be L-Ala from α-amino-isovaleric acid " reverse mutation ".The antibody of " reverse mutation " is also included within the present invention like this.
Another example is, for 1G11, and V kAmino-acid residue #7 (FR1 in) be phenylalanine, and at corresponding V kThe A27 kind is that in sequence, this residue is Serine.Be configuration in order to make the framework region sequence revert to its kind, for example, can be with the V of 1G11 kThe residue #7 of FR1 be Serine from phenylalanine " reverse mutation ".The antibody of " reverse mutation " is also included within the present invention like this.
Another example is, for 1G11, and V kAmino-acid residue #47 (FR2 in) be α-amino-isovaleric acid, and at corresponding V kThe A27 kind is that in sequence, this residue is leucine.Be configuration in order to make the framework region sequence revert to its kind, for example, can be with the V of 1G11 kThe residue #47 of FR2 be leucine from α-amino-isovaleric acid " reverse mutation ".The antibody of " reverse mutation " is also included within the present invention like this.
The framework of another kind of type is modified the one or more residues that relate in framework region and even one or more CDR district and is suddenlyd change, and removing t cell epitope, thereby reduces the potential immunogenicity of this antibody.The method is also referred to as " disimmunity ", is record in detail in 20030153043 United States Patent (USP) in the publication No. of Carr etc.
Thereby engineered antibody of the present invention also comprise wherein by change t cell epitope on antibody interactional amino acid modified to amino-acid residue modify those antibody of improving or reducing immunogenic response (referring to, for example U.S. Patent No. 6,835,550; 6,897,049 and 6,936249).
Except the modification of carrying out in framework region or CDR district, perhaps as its replacement scheme, also antibody of the present invention can be transform as in the Fc district and comprise modification, generally in order to change one or more functional performances of this antibody, as serum half-life, complement fixation, Fc receptors bind and/or antigen dependent cellular cytotoxicity.In addition, also can with antibody chemically modified of the present invention (for example one or more chemical parts can be connected on this antibody), perhaps modify its glycosylation of change, to change one or more functional performances of this antibody.These embodiments are all described in detail hereinafter.In the Fc district, the numbering of residue is the numbering of the EU index of Kabat.
In one embodiment, modify the hinge area of CH1, the number of cysteine residues in this hinge area is changed, for example increase or reduce.The method record in detail in No. 5,677,425, the U.S. Patent No. of Bodmer etc.The number that changes halfcystine in the CH1 hinge area is to promote the assembling of light chain and heavy chain for (for example), or improves or reduce the stability of antibody.
In another embodiment, the Fc hinge area of antagonist suddenlys change, to reduce the biological half-life of this antibody.More specifically, introduce one or more amino acid mutations to the CH2-CH3 structural domain interface region of Fc-hinge fragment, the combination that makes this antibody and SP (SpA) weakens than the combination of natural Fc-hinge arrangement territory and SpA.The method record in detail in No. 6,165,745, the U.S. Patent No. of Ward etc.
In another embodiment, modified antibodies is to improve its biological half-life.Can make and in all sorts of ways.For example, as described in the U.S. Patent No. 6,277,375 of Ward, can introduce one or more following sudden changes: T252L, T254S, T256F.Perhaps, as the U.S. Patent No. 5,869 of Presta etc., 046 and 6,121,022 is described, in order to improve biological half-life, this antibody can change in CH1 or CL district, makes it to contain the receptor binding domain of remedying from two rings of IgG Fc district CH2 structural domain.
In some other embodiment, by being that different amino-acid residues changes the Fc district with at least one radical amino acid replacement, to change the effector function of antibody.For example, can be selected from amino-acid residue 234,235,236,237,297,318,320, one or more amino-acid substitutions of 322 are different amino-acid residues, make the avidity of this antibody pairing effect part change, but keep the antigen binding capacity of parental antibody.The reformed effect part of its avidity can be, for example, and the C1 composition of Fc acceptor or complement.The method is in the U.S. Patent No. 5,624,821 and 5,648 of Winter etc., describes in more detail in 260.
In another embodiment, can be selected from amino-acid residue 329, one or more amino-acid substitutions of 331 and 322 are different amino-acid residues, make the C1q Binding change of this antibody and/or CDC (CDC) reduce or eliminate.The method is described in the U.S. Patent No. 6,194,551 of Idusogie etc. in more detail.
In another embodiment, change the one or more amino-acid residues in amino acid sites 231 and 239, thereby change the ability of this antibody complement-fixing.The method further describes in the PCT of Bodmer etc. announces WO 94/29351.
in another embodiment, for the ability that improves antibody-mediated antibody dependent cellular cytotoxicity (ADCC) and/or improve antibody to the avidity of Fc γ acceptor, modify the Fc district by modifying one or more amino acid in following site: 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.The method further describes in the PCT of Presta announces WO 00/42072.And, the upper binding site for Fc γ RI, Fc γ RII, Fc γ RIII and FcRn of human IgG1 is mapped, and described the combination with improvement variant (referring to, Shields, R.L. etc. (2001) J.Biol.Chem.276:6591-6604).The specific sudden change at site 256,290,298,333,334 and 339 places shows the combination that has improved with Fc γ RIII.In addition, following combination mutant shows the combination that has improved with Fc γ RIII: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.
In another embodiment, change the glycosylation of antibody.The antibody (i.e. this antibody deficiency glycosylation) that for example, can prepare sugar based.For example, in order to improve antibody to the avidity of antigen, can change glycosylation.Such carbohydrate modification can be realized by one or more glycosylation sites that (for example) changes in antibody sequence.For example, can carry out one or more amino-acid substitutions, one or more variable regions framework glycosylation site is disappeared, thereby eliminate the glycosylation of this site.This sugar basedization can improve antibody to the avidity of antigen.This method is in the U.S. Patent No. 5,714,350 and 6,350 of Co etc., describes in more detail in 861.
In addition or alternately, can prepare the antibody that type of glycosylation changes, as the low fucosylation antibody of fucosido residue reduced number, or the antibody that increases of decile GlcNac structure.The glycosylation pattern of verified this change has improved the ADCC ability of antibody.This carbohydrate modification can be expressed antibody by (for example) and be realized in the host cell that glycosylation mechanism changes.The cell that glycosylation mechanism changes is existing the description in the art, can be used as host cell, expresses therein recombinant antibodies of the present invention, thereby produces the antibody that glycosylation changes.For example, clone Ms704, Ms705 and Ms709 lack fucosyl transferase gene, FUT8 (α (1,6) fucosyl transferase gene) lacks Fucose in the carbohydrate of antibody at them of therefore expressing in Ms704, Ms705 and Ms709 clone.By using two kinds of FUT8 genes in the directed CHO/DG44 of destruction of alternative carrier cell, produce Ms704, Ms705 and Ms709FUT8 -/-Clone (referring to the U.S. Patent application No.20040110704 of Yamane etc. and Yamane-Ohnuki etc. (2004) Biotechnol Bioeng87:614-22).Another example is, the EP 1,176 of Hanai etc., 195 have described the clone of the FUT8 gene with function destruction, and this genes encoding fucosyl transferase is owing to having reduced or eliminated α (1,6) key involved enzyme, the antibody of expressing in this clone shows as low fucosylation.Hanai etc. have described also that to be used on the N-Acetyl-D-glucosamine in the binding antibody Fc district enzymic activity of interpolation Fucose low or do not have the clone of enzymic activity, and for example rat myeloma cell is YB2/0 (ATCC CRL 1662).The PCT announcement WO 03/035835 of Presta has put down in writing a kind of variation Chinese hamster ovary celI and has been, the Lec13 cell, its ability that Fucose is connected on the carbohydrate of Asn (297)-connection reduces, also cause the antibody of expressing in this host cell for low fucosylation (referring to, Shields, R.L. etc. (2001) J.Biol.Chem.277:26733-26740).The PCT announcement WO 99/54342 of Umana etc. has put down in writing the glycosyltransferase of expressing the glycoprotein modification, and (for example β (1,4) through engineering approaches clone-N-acetylglucosaminyl transferase III (GnTIII)), the antibody of therefore expressing in this project clone shows as decile GlcNac structure to be increased, cause the ADCC activity of antibody to improve (referring to, Umana etc. (1999) Nat.Biotech.17:176-180).In addition, the fucosyl residues of antibody can downcut with fucosidase.For example, the fucosidase alpha-L-fucosidase from antibody remove fucosyl residues (Tarentino, A.L. etc. (1975) Biochem.14:5516-23).
It is PEGization that the another kind to antibody described herein that the present invention relates to is modified.For example, for biology (for example serum) transformation period of improving antibody, can be with this antibody PEGization.For a kind of antibody of PEGization, generally under one or more PEG groups and condition that antibody or antibody fragment are connected, this antibody or its fragment and polyoxyethylene glycol (PEG) reactive ester or the aldehyde derivatives as PEG reacted.Preferably, by carry out PEGization with acylation reaction or the alkylated reaction of reactive PEG molecule (or similar reaction water-soluble polymkeric substance).Term used herein " polyoxyethylene glycol " comprises any PEG form of other protein of derivatize, as list (C1-C10) alkoxyl group-or aryloxy-polyoxyethylene glycol or polyoxyethylene glycol-maleimide.In certain embodiments, antibody that will PEGization is a kind of antibody of sugar based.The method of protein PEGization is known in the art, and can be used for antibody of the present invention.Referring to, for example, the EP 0 401 384 of the EP 0 154 316 of Nishimura etc. and Ishikawa etc.
The antibody engineering method
As mentioned above, can utilize and have V disclosed herein HAnd V KThe anti-O8E antibody of sequence is by modifying V HAnd/or V KSequence or the constant region that is attached thereto produce new anti-O8E antibody.Therefore, in another aspect of this invention, utilize the constitutional features of anti-O8E antibody of the present invention (for example 1G11,2A7,2F9,12E1 or 13D12), produce anti-O8E antibody relevant on structure, on this structure, relevant antibody keeps at least a functional performance of antibody of the present invention, as being combined with people O8E.As mentioned above, for example, one or more CDR district of 1G11,2A7,2F9,12E1 or 13D12 or its sudden change can be made up with known framework region and/or other CDR restructuring, thereby produce the of the present invention anti-O8E antibody of other recombined engineering.The modification of other types comprises the described modification of above part.The parent material that is used for engineering method is one or more V provided herein HAnd/or V KSequence, or its one or more CDR district.In order to produce engineered antibody, not necessarily must actual prepare (namely being expressed as protein) and have one or more V provided herein HAnd/or V KSequence, or the antibody in its one or more CDR district.But with information contained in this sequence as parent material, produce by derivative " s-generation " sequence of original series, then preparation should " s-generation " sequence, and it is expressed as protein.
Therefore, in another embodiment, the invention provides a kind of method for preparing anti-O8E antibody, comprising:
(a) provide: (i) variable fragments of heavy chain sequence, its comprise be selected from SEQ ID NO:11,12,13,14 and 15 CDR1 sequence, be selected from SEQ ID NO:16,17,18,19 and 20 CDR2 sequence and/or be selected from SEQ ID NO:21,22,23,24 and 25 CDR3 sequence; And/or (ii) variable region of light chain antibody sequence, its comprise be selected from SEQ ID NO:26,27,28,29 and 30 CDR1 sequence, be selected from SEQ ID NO:31,32,33,34 and 35 CDR2 sequence and/or be selected from SEQ ID NO:36,37,38,39 and 40 CDR3 sequence;
(b) change at least one interior amino-acid residue of variable fragments of heavy chain sequence and/or variable region of light chain antibody sequence, thereby produce the antibody sequence of at least one change; With
The antibody sequence that (c) will change is expressed as protein.
The antibody sequence that can utilize the standard molecular biological technique preparation and express described change.
Typically, kept a kind of, some or all functional performances of anti-O8E antibody described herein by the antibody of the antibody sequence coding that changes, this functional performance includes but not limited to:
(i) with 1 * 10 -7M or lower K DBe combined with people O8E;
(ii) be combined with people's Chinese hamster ovary celI of O8E transfection.
The functional performance of the antibody that changes can be estimated with (as be shown in the examples) standard test used in the art and/or described herein (for example flow cytometry, combination are measured).
In some embodiment of the engineering method of antibody of the present invention, can be along all or part of anti-O8E antibody coding sequence random or selectivity introduce sudden change, and can be in conjunction with active and/or other functional performances as described here, the anti-O8E antibody of the modification that screening obtains.Mutation method is described in the art.For example, the PCT of Short announcement WO 02/092780 has put down in writing and has utilized saturation mutagenesis, synthetic being linked and packed or their combination results and the method for screening antibodies sudden change.In addition, the PCT of Lazar etc. announces WO 03/074679 and has also put down in writing the method for utilizing the calculating sifting method to optimize the plysiochemical character of antibody.
The encode nucleic acid molecule of antibody of the present invention
Another aspect of the present invention relates to the nucleic acid molecule of the antibody of the present invention of encoding.This nucleic acid may reside in intact cell, cell lysate, or with partial purification or basically pure form exist.When by comprise that alkali/SDS is processed, when the standard technique of the aobvious band of CsCl, column chromatography, agarose gel electrophoresis and additive method well known in the art and other cellular constituents or other pollutents (for example other nucleus or protein) separation and purification, nucleic acid is " separation " or " basically pure ".Referring to, the ed. such as F.Ausubel (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, NewYork.Nucleic acid of the present invention can be for example DNA or RNA, and can contain or can not contain intron sequences.In a preferred embodiment, this nucleic acid is the cDNA molecule.
Nucleic acid of the present invention can utilize standard molecular biological technique to obtain.For hybridoma (for example, the antibody of the hybridoma by the preparation of the transgenic mice of carrier's immunoglobulin gene as described further below) expressing, the light chain of antibody of coding hybridoma preparation and the cDNA of heavy chain can obtain with standard pcr amplification or cDNA clone technology.For the antibody (for example using display technique of bacteriophage) that obtains, can reclaim one or more nucleic acid of this antibody of coding from the library from the immunoglobulin gene library.
The preferred nucleic acid molecule of the present invention is the V of coding 1G11,2A7,2F9,12E1 or 13D12 monoclonal antibody HAnd V LThe nucleic acid molecule of sequence.The V of coding 1G11,2A7,2F9,12E1 and 13D12 HThe DNA sequence dna of sequence is respectively shown in SEQ ID NO:41,42,43,44 and 45.The V of coding 1G11,2A7,2F9,12E1 and 13D12 LThe DNA sequence dna of sequence is respectively shown in SEQ ID NO:46,47,48,49 and 50.
In case obtain coding V HAnd V LThe DNA fragmentation of fragment can further operate these DNA fragmentations by the standard recombinant dna technology, for example variable region gene is converted into full length antibody chain gene, Fab fragment gene or scFv gene.In these operations, coding V LOr V HDNA fragmentation effectively be connected with another DNA fragmentation of coding another protein such as antibody constant region or flexible connection body.Term " effectively connection " meaning is that two DNA fragmentations link together as used herein, makes the aminoacid sequence of these two DNA fragmentations codings keep meeting reading frame.
By the V that will encode HDNA and encoding heavy chain constant region (CH1, CH2 another DNA molecular of being connected with CH3 effectively is connected, can be with the coding V that separates HThe DNA in district is converted into the total length heavy chain gene.The sequence of people's weight chain constant area gene be known in the art (referring to, for example, Kabat, (1991) Sequences of Proteins of Immunologicl Interest such as E.A., the 5th edition, U.S.Department of Health and Human Services, NIH publication number 94-61242), comprise that these regional DNA fragmentations can obtain by the standard pcr amplification.CH can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably IgG1 or IgG4 constant region.For Fab fragment heavy chain gene, coding V HDNA can effectively be connected with another DNA molecular of encoding heavy chain CH1 constant region.
By the V that will encode LDNA effectively be connected with another DNA molecular of coding constant region of light chain CL, can be with the coding V that separates LThe DNA in district is converted into full-length light chains gene (and Fab light chain gene).The sequence of people's constant region of light chain gene be known in the art (referring to, for example, Kabat, (1991) Sequences of Proteins of ImmunologiclInterest such as E.A., the 5th edition, U.S.Department of Health and Human Services, NIH publication number 94-61242), comprise that these regional DNA fragmentations can obtain by the standard pcr amplification.In preferred embodiments, constant region of light chain can be κ or λ constant region, but is most preferably the κ constant region.
In order to produce the scFv gene, V will encode HAnd V LDNA fragmentation and coding flexible connection body encoding amino acid sequence (Gly for example 4-Ser) 3The another one fragment effectively connect, make V HAnd V LSequence can be expressed as continuous single chain protein matter, its V LAnd V HThe district by this flexible connection body connect (referring to, such as (1988) Science 242:423-426 such as Bird; Huston etc. (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883; McCafferty etc. (1990) Nature 348:552-554).
The generation of monoclonal antibody of the present invention
Monoclonal antibody of the present invention (mAb) can prepare by multiple technologies, comprises conventional monoclonal anti body method, for example, and the described standard body hybridoma technique of Kohler and Milstein (1975) Nature 256:495.Although the preferred body hybridoma technique in principle, can use the other technologies of preparation monoclonal antibody, for example the virus of bone-marrow-derived lymphocyte or oncogenic transformation.
Preferred animal system for the preparation of hybridoma is the mouse system.Producing hybridoma with mouse is a kind of program of perfect foundation.Immune programme for children is well known in the art with separating the technology by immune spleen cell that is used for merging.Fusion partner (for example rat bone marrow tumour cell) and fusion program are also known.
Chimeric or humanized antibody of the present invention can prepare based on the sequence of the mouse monoclonal antibody that obtains as mentioned above.The DNA of encoding heavy chain and light chain immunoglobulin (Ig) can obtain from the inhuman hybridoma of target, and the Application standard Protocols in Molecular Biology transform it as and contains the human normal immunoglobulin sequence.For example, in order to produce chimeric antibody, can utilize method well known in the art the mouse variable region to be connected on human constant region (referring to, for example, the U.S. Patent No. 4,816,567 of Cabilly etc.).In order to produce humanized antibody, can utilize method well known in the art mouse CDR district is inserted in people's framework (referring to, for example, the U.S. Patent No. 5,225,539 of Winter, and the U.S. Patent No. 5,530,101 of Queen etc.; 5,585,089; 5,693,762 and 6,180,370).
In a preferred embodiment, antibody of the present invention is human monoclonal antibodies.This anti-O8E human monoclonal antibodies can produce with the transgenosis of carrying groups of people's immunity system rather than mouse system or transchromosomic mice.These transgenosiss and transchromosomic mice are included in this and are known as respectively the HuMab mouse With the KM mouse
Figure S2006800460653D00442
Mouse, and be commonly referred to as " people Ig mouse " at this.
The HuMab mouse
Figure S2006800460653D00443
(Medarex, Inc.) comprise people's heavy chain (μ and γ) that coding do not reset and human immunoglobulin gene's minigene seat of κ light chain immunoglobulin sequences, with the orthomutation that makes endogenous μ and κ chain gene seat inactivation (referring to, for example, (1994) Nature368 (6474) such as Lonberg: 856-859).Therefore, this mouse shows as Mouse IgM or κ expresses reduction, and in response to immunity, the people's heavy chain that imports and the classification conversion of light chain transgenosis experience and somatic mutation, thereby generation high-affinity human IgG κ monoclonal antibody (Lonberg, N. etc. (1994), the same; Summary Lonberg, N. (1994) Handbook of Experimental Pharmacology113:49-101; Lonberg, N. and Huszar, D. (1995) Intern.Rev.Immunol.Vol.13:65-93, and Harding, F. and Lonberg, N. (1995) Ann.N.Y.Acad.Sci764:536-546).The preparation of HuMab mouse and use, and the genomic modification that carries of this mouse are described in detail in document below: Taylor, L. etc. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. etc. (1993) International Immunology 5:647-656; Tuaillon etc. (1993) Proc.Natl.Acad.Sci USA 90:3720-3724; Choi etc. (1993) Nature Genetics 4:117-123; Chen, J. etc. (1993) EMBO is J.12:821-830; Tuaillon etc. (1994) J.Immunol.152:2912-2920; Taylor, L. etc. (1994) International Immunology 6:579-591; And Fishwild, D. etc. (1996) Nature Biotechnology 14:845-851, the content of these documents is incorporated herein by reference in full.Further referring to, the U.S. Patent No. 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; With 5,770,429; U.S. Patent No. 5,545,807 with Surani etc.; The PCT publication No. WO 92/03918 of Lonberg and Kay, WO 93/12227, and WO 94/25585, and WO 97/13852, WO 98/24884 and WO 99/45962; PCT publication No. WO 01/14424 with Korman etc.
In another embodiment, can use the mouse of carrier's immunoglobulin sequences on transgenosis and transfection chromosome, for example the mouse of carrier's heavy chain transgenosis and people's light chain transfection chromosome, produce people's antibody of the present invention.This mouse is referred to herein as " KM mouse
Figure S2006800460653D00451
", in announcing WO 02/43478, the PCT of Ishida etc. describes in detail.
Moreover the alternative transgenic animal system of expressing the human immunoglobulin gene can obtain in the art, can be used for producing anti-O8E antibody of the present invention.For example, can use the alternative transgenosis system of a kind of Xenomouse of being known as (Abgenix, Inc.), this mouse is in the U.S. Patent No. 5,939,598 such as Kucherlapati etc.; 6,075,181; 6,114,598; Describe in 6,150,584 and 6,162,963.
And the alternative trans-chromosome animal system of expressing the human immunoglobulin gene can obtain in the art, can be used for producing anti-O8E antibody of the present invention.For example, can use the mouse of carrier's heavy chain transfection chromosome and people's light chain transfection chromosome, it is known as " TC mouse "; This mouse is described in (2000) the Proc.Natl.Acad.Sci.USA 97:722-727 such as Tomizuka.As the another one example, the ox (Kuroiwa etc. (2002) Nature Biotechnology 20:889-894) of carrier's heavy chain and light chain transfection chromosome has been described in this area, it can be used for producing anti-O8E antibody of the present invention.
Human monoclonal antibodies of the present invention also can use the phage display method preparation for examination human immunoglobulin gene library.This phage display method for separating of people's antibody is set up in the art.Referring to, for example, the U.S. Patent No. 5,223,409 of Ladner etc.; 5,403,484; With 5,571,698; The U.S. Patent No. 5,427,908 and 5,580,717 of Dower etc.; The U.S. Patent No. 5,969,108 and 6,172,197 of McCafferty etc.; U.S. Patent No. 5,885,793 with Griffiths etc.; 6,521,404; 6,544,731; 6,555,313; 6,582,9130,31,32,33,34,35 and 36,593,081.
Human monoclonal antibodies of the present invention also can be with SCID mouse preparation, in this SCID mouse reconstruct people's immunocyte, therefore can produce people's antibody response when immunity.This mouse is in the U.S. Patent No. 5,476,996 and 5,698 such as Wilson etc., describes in 767.
The immunity of people Ig mouse
When end user Ig mouse produces people's antibody of the present invention, according to Lonberg, N. etc. (1994) Nature 368 (6474): 856-859; Fishwild, D. etc. (1996) Nature Biotechnology14:845-851; Described with PCT announcement WO 98/24884 and WO 01/14424, with this mouse of goods immunity of the clone of expressing O8E, O8E antigen purifying or enrichment and/or restructuring O8E or O8E fusion rotein.Preferably, for the first time when immunity mouse be 6-16 age in week.For example, can use preparation (5-50 μ g) the intraperitoneal immunity people Ig mouse of O8E antigen purifying or restructuring.
Produce in the detailed procedure embodiment 1 below of the complete human monoclonal antibodies of anti-O8E and describe.Use the empirical evidence of various antigen accumulation, antigen intraperitoneal (IP) immunity in initial use Freund's complete adjuvant, when then using every other week the antigen intraperitoneal immunity (totally six times at most) in Freund's incomplete adjuvant, transgenic mice produces and replys.But, find that the adjuvant outside freund's adjuvant is also effective.In addition, find when there is no adjuvant, full cell has hyperimmunization originality.For example use in the immunization protocol process and get the plasma sample monitoring immunne response that blood obtains after eye socket.Screen blood plasma by ELISA, merge (as described in Example 1) with having the mouse that enough anti-O8E human normal immunoglobulins tires.With antigen, mouse is carried out the intravenously booster immunization, put to death afterwards in 3 days and the taking-up spleen.Expect that each immunity may need 2-3 fusion.The general immune 6-24 mouse of each antigen.Usually HCo7 and HCo12 use.The generation of HCo7 and HCo12 mouse system is respectively in U.S. Patent No. 5,770,429 and the embodiment 2 of the open WO01/09187 of PCT in describe.In addition, HCo7 and HCo12 transgenosis can be hybridized, and produce a kind of mouse with two kinds of different people heavy chain transgenosiss (HCo7/HCo12).Alternately or in addition, as described in WO 02/43478 as open in PCT, can use the KM mouse
Figure S2006800460653D00471
System.
Produce the preparation of the hybridoma of human monoclonal antibodies of the present invention
In order to prepare the hybridoma that produces human monoclonal antibodies of the present invention, from by separating Morr. cell and/or lymph-node cell the mouse of immunity, and merge with suitable immortalized cell system (for example mouse myeloma cell line).Screen according to the generation of antigen-specific antibodies the hybridoma that obtains.For example, can use 50%PEG, will be from being merged by the non-secretion murine myeloma cell of the Sp2/0 of the single cell suspension of the splenic lymphocyte of immune mouse and 1/3rd quantity (ATCC, CRL 1581).Perhaps, can use the electro fusion method based on electric field, use the large chamber cytogamy of Cyto Pulse electroporation apparatus (Cyto Pulse Sciences, Inc., GlenBurnie, MD), will be merged by the Sp2/0 murine myeloma cell of the single-cell suspension liquid of the splenic lymphocyte of immune mouse and equivalent.With cell with about 1 * 10 5Density be inoculated in flat-bottom microtiter plates, then hatched in selective medium for two weeks, this selective medium is being added with 5mM HEPES, 0.055mM beta-mercaptoethanol, 50mg/ml gentamicin and 1 * HAT (Sigma; CRL P-7185) DMEM (Mediatech, CRL 10013, contain high concentration glucose, L-glutaminate and Sodium.alpha.-ketopropionate) in contain 10% foetal calf serum (Hyclone, Logan, UT), 10%P388DI (ATCC, CRL TIB-63) condition matrix, 3-5%origen (IGEN).Approximately 1-2 is after week, culturing cell in the substratum of having replaced HAT with HT.Then by ELISA or FACS according to human monoclonal IgM and IgG antibody screening each hole.Then can use the O8E positive antibody of O8E recombinant protein screening positive clone by ELISA, perhaps, for example, use the cell O8E positive antibody of the cell screening positive colony of CHO-O8E transfection for example of expressing O8E by FACS.In case hybridoma growth widely occurs, usually can observe substratum after 10-14 days.With the hybridoma of secretory antibody plating again, screening again, if remain positive for human IgG, can be by limiting dilution with monoclonal antibody twice of subclone at least.Then at the stable subclone of vitro culture, be used for characterizing to produce a small amount of antibody in tissue culture medium (TCM).
For the Purification of Human monoclonal antibody, the hybridoma of selection can be grown in two liters of rotation shaking flasks that are used for the monoclonal antibody purifying.Filtering supernatant, concentrated, use afterwards albumin A-sepharose (Pharmacia, Piscataway, N.J.) to carry out affinity chromatography.The IgG that elutes guarantees purity by gel electrophoresis and high performance liquid chromatography inspection.Change buffered soln into PBS, determine concentration with 1.43 optical extinction coefficient according to OD280.Monoclonal antibody can be divided into equal portions and preserve under-80 ℃.
Produce the preparation of the transfectoma of monoclonal antibody of the present invention
Utilize the combination (for example, Morrison, S. (1985) science 229:1202) of (for example) recombinant DNA technology well known in the art and gene transfection method, also can produce antibody of the present invention in the host cell transfectoma.
For example, in order to express antibody or its antibody fragment, can be by standard molecular biological technique (for example, use the hybridoma of expressing target antibody to carry out pcr amplification or cDNA clone), obtain the DNA of encoding part or full-length light chains and heavy chain, and this DNA is inserted in expression vector, make gene and transcribe and be connected control sequence and effectively be connected.In context, term " the effectively connect " meaning is that antibody gene is connected in carrier, makes transcribing and translating the control sequence performance they regulate the expectation function of transcribing and translating of this antibody gene in carrier.Select the expression vector and the expression control sequenc that are complementary with expression host cell used.Light chain of antibody gene and heavy chain of antibody gene can be inserted in carrier separately, perhaps, more generally, two genes are inserted in same expression vector.By standard method, antibody gene is inserted into (for example, the complementary restriction site on the antibody gene fragment is connected with carrier, the perhaps if there is no work of restriction site, flush end connection) in expression vector.In the CH of isotype by being inserted into the expectation of encoding and the expression vector of constant region of light chain, make V HC in section and carrier HSection effectively connects, and V KC in section and carrier LSection effectively connects, and can utilize the light chain of antibody described herein and the full length antibody gene that variable region of heavy chain produces any antibody isotype.In addition, perhaps alternately, recombinant expression vector can be encoded and is conducive to the signal peptide of secretory host cell antibody chain.Can with the antibody chain gene clone in carrier, make signal peptide and the N-terminal of this antibody chain gene be connected with meeting reading frame.Signal peptide can be immunoglobulin (Ig) signal peptide or allos the signal peptide signal peptide of the protein of NIg (that is, from).
Except the antibody chain gene, the adjusting sequence that recombinant expression vector of the present invention is also expressed in host cell with this antibody chain gene of control.Term " adjusting sequence " comprises other expression controlling elementss (for example, polyadenylation signal) of promotor, enhanser and the genetic transcription of control antibody chain or translation.Such adjusting sequence is for example described in Goeddel (Gene ExpressionTechnology.Methods in Enzymology 185, Academic Press, San Diego, CA (1990)).It will be appreciated by those skilled in the art that the design of expression vector, comprise the selection of regulating sequence, depend on such as the selection of the host cell that will transform, the factors such as protein expression level of expectation.Being used for preferred adjusting sequence that mammalian host cell expresses comprises and instructs protein at the viral element of mammalian cell high level expression, for example derive from promotor and/or the enhanser of cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (for example, adenovirus major late promoter (AdMLP)) and polyomavirus.Alternately can use non-viral adjusting sequence, for example ubiquitin promotor or beta-globin promotor.In addition, regulatory element also can be by forming from the sequence such as different sourcess such as SR α promoter systems, SR α promoter systems contains from the long terminal repeat of the sequence of SV40 early promoter and people's 1 type T chronic myeloid leukemia virus (Takebe, Y. etc. (1988) Mol.Cell.Biol.8:466-472).
Except antibody chain gene and adjusting sequence, recombinant expression vector of the present invention can also carry other sequence, for example regulates sequence (for example, replication orgin) and selected marker that carrier copies in host cell.The selected marker is conducive to screening vector and has imported wherein host cell (referring to, for example, the U.S. Patent No. 4,399,216,4,634,665 and 5,179,017 of Axel etc.).For example, the selected marker brings resistance generally for the host cell that has imported carrier, for example G418, Totomycin or methotrexate resistance.Preferred selected marker comprises Tetrahydrofolate dehydrogenase (DHFR) gene (being used for methotrexate in the dhfr-host cell selects/amplification) and neo gene (being used for G418 selects).
In order to express light chain and heavy chain, be transfected in host cell by the expression vector of standard technique with encoding heavy chain and light chain.The various forms of term " transfection " comprises and is usually used in foreign DNA is imported various technology in protokaryon or eukaryotic host cell, for example, and electroporation, calcium phosphate precipitation, the transfection of DEAE-dextran etc.Although can express antibody of the present invention in theory in protokaryon or eukaryotic host cell, but preferably in eukaryotic cell, most preferably express this antibody in mammalian host cell, because such eukaryotic cell, mammalian cell particularly more may be assembled and secrete correct folding and have immunocompetent antibody than prokaryotic cell prokaryocyte.It is reported, the prokaryotic expression antibody gene can't produce active antibody (Boss, M.A. and Wood, C.R. (1985) Immunology Today 6:12-13) by high productivity.
Comprise that for the preferred mammal host cell of expressing recombinant antibodies of the present invention Chinese hamster ovary (Chinese hamster ovary celI) (comprises Urlaub and Chasin, (1980) the dhfr-CHO cell of describing in Proc.Natl.Acad.Sci.USA 77:4216-4220, use together with the DHFR selected marker, for example, as described in R.J.Kaufman and P.A.Sharp (1982) Mol.Biol.159:601-621), NSO myeloma cell, COS cell and SP2 cell.Especially, the another kind of preferred expression system that is used for NSO myeloma cell is the disclosed GS gene expression system of WO 87/04462, WO89/01036 and EP 338,841.When the recombinant expression vector with the encoding antibody gene imports in mammalian host cell, by the time that the host cell cultivation is enough to antibody is expressed in host cell, or more preferably, cultivating is enough to make the time of antibody-secreting in the substratum of host cell growth, and produces antibody.But the application standard method of purifying protein reclaims antibody from substratum.
The sign that antibody is combined with antigen
By (for example) flow cytometry, can detect the combination of antibody of the present invention and OgE.In brief, fresh results are expressed the cell of O8E from tissue culture flasks, preparation single-cell suspension liquid.The cell suspending liquid of expressing O8E directly dyes with first antibody with first antibody dyeing or after fixing with the PBS solution of 1% paraformaldehyde.Approximately 100 ten thousand cells are resuspended in the PBS that contains 0.5%BSA and 50-200 μ g/ml first antibody, hatch on ice 30 minutes.Cell is with containing 0.1%BSA, 0.01%NaN 3The PBS washed twice, be resuspended in 100 μ l1: in the mountain goat anti-human igg (Jackson ImmunoResearch, West Grove, PA) of FITC-couplings of 100 dilutions, hatched again on ice 30 minutes.Cell is washed twice again, is resuspended in the 0.5ml lavation buffer solution, in the upper analysis of fluorescence dyeing of FACSCalibur cell counter (Becton-Dickinson, San Jose, CA).
Perhaps, can detect by standard ELISA the combination of antibody of the present invention and O8E.In brief, with the coated microtiter plate of the solution of purifying O8E in PBS of 0.25 μ g/ml, then with 5% bovine serum albumin sealing in PBS.Add the diluent (for example, from the diluent of the blood plasma of O8E immune mouse) of antibody in each hole, and hatched under 37 ℃ 1-2 hour.Wash culture plate with PBS/Tween, hatched under 37 ℃ 1 hour together afterwards and with second reagent (for example, for people's antibody, being mountain goat anti-human igg Fc specific polyclonal reagent) of alkaline phosphatase coupling.After washing, culture plate develops the color with pNPP substrate (1mg/ml), and analyzes under OD405-650.Preferably, merge with the mouse that shows high-titer.
Elisa assay as above also can be used for screening the hybridoma that shows with the original positive reaction of O8E immunity.The hybridoma that to be combined with the O8E high affinity carries out subclone, and further characterizes.Select to keep the reactive clone of parent cell (by ELISA or FACS) from each hybridoma, preparation 5-10 bottle cell bank is kept under-140 ℃, is used for antibody purification.
For the anti-O8E antibody of purifying, the hybridoma of selection is grown in two liters of rotation shaking flasks that are used for the monoclonal antibody purifying.Then filtering supernatant and concentrated uses albumin A-sepharose (Pharmacia, Piscataway, NJ) to carry out affinity chromatography.Check that by gel electrophoresis and high performance liquid chromatography the IgG of wash-out is to guarantee purity.Change buffered soln into PBS, and use 1.43 optical extinction coefficient according to OD 280Determine concentration.Monoclonal antibody is divided into equal portions and preserves under-80 ℃.
In order to determine that whether the anti-O8E monoclonal antibody of selecting is combined with unique epi-position, can use the business to sell reagent (Pierce, Rockford, IL) with every kind of antibody biotinylation.Can use the coated elisa plate of O8E as above, use the research that is at war with of unlabelled monoclonal antibody and biotinylation monoclonal antibody.Can use the combination of streptavidin-alkaline phosphatase probe in detecting biotinylation mAb.Perhaps, as the following examples further as described in, can use the radiolabeled antibody Journal of Sex Research that is at war with, and can use the unlabelled competition antibody of Scatchard analyzing and testing.
Isotype for the antibody determining to be purified can carry out isotype ELISA with specific isotype antibody is had specific reagent.For example, in order to determine the isotype of human monoclonal antibodies, spend the night with the hole of the coated microtiter plate of the 1 anti-human immunoglobulin (Ig) of μ g/ml under 4 ℃.After the 1%BSA sealing, dull and stereotyped isotype contrast with 1 μ g/ml or test monoclonal antibody still less or purifying at room temperature reacted 1-2 hour.These holes then with the probe reaction of human IgG1 or the coupling of people IgM specific alkaline phosphatase.Make as mentioned above dull and stereotyped colour developing and analyze.
Can further detect the reactivity of anti-O8E human IgG and O8E antigen by the Western blotting.In brief, preparation O8E and carry out SDS-PAGE.After electrophoresis, the antigen that separates is transferred on nitrocellulose filter, with 10% foetal calf serum sealing, and detected with monoclonal antibody to be detected.The combination of human IgG can detect with anti-human IgG alkaline phosphatase, and develops the color with BCIP/NBT substrate sheet (Sigma Chem.Co., St.Louis, Mo.).
The physical properties of antibody
Antibody of the present invention can be further various physical propertiess by anti-O8E antibody characterize.Can utilize various tests to detect and/or distinguish different classes of antibody based on these physical propertiess.
In certain embodiments, antibody of the present invention can contain one or more glycosylation sites in light chain or variable region of heavy chain.In the variable region, the existence of one or more glycosylation sites may change (Marshall etc. (1972) Annu Rev Biochem 41:673-702 because the antigen Binding change causes the immunogenicity raising of antibody or the pK of antibody; Gala FA and MorrisonSL (2004) J Immunol 172:5489-94; Wallick etc. (1988) J Exp Med168:1099-109; Spiro RG (2002) Glycobiology 12:43R-56R; Parekh etc. (1985) Nature 316:452-7; Mimura etc. (2000) Mol Immunol 37:697-706).Known glycosylation is containing motif place's generation of N-X-S/T sequence.The glycosylation of variable region can detect with the Glycoblot test, and this tests cutting antibody, produces Fab, then utilizes the test of measuring periodate oxidation and Schiff's base formation to detect glycosylation.Perhaps, the glycosylation of variable region also can detect with Dionex light chromatography (Dionex-LC), and the method is downcut sugar from Fab becomes monose, and analyzes the content of various sugar.In some cases, preferably do not contain the glycosylated anti-O8E antibody in variable region.This can not contain the antibody of glycosylation motif or utilize the residue in standard technique sudden change glycosylation motif well known in the art to realize by being chosen in the variable region.
In a preferred embodiment, antibody of the present invention does not contain l-asparagine isomerization site.Deacylated tRNA amine or different aspartic acid effect can occur on N-G or D-G sequence respectively.Deacylated tRNA amine or different aspartic acid effect cause producing different aspartic acid, and different aspartic acid is by producing the stability that the structure of twisting together has reduced antibody at the side chain carboxyl group end rather than on main chain.The generation of different aspartic acid can be tested to measure with iso-quant, and this test utilizes reversed-phase HPLC to detect different aspartic acid.
Every kind of antibody has unique iso-electric point (pI), but antibody falls in 6 to 9.5 pH scope usually.The pI of IgG1 antibody generally falls in the pH scope of 7-9.5, and the pI of IgG4 antibody generally falls in the pH scope of 6-8.Antibody also can have the pI outside this scope.Although this effect is usually unknown, infer that pI falls within antibody outside normal range and may have certain solution under condition in vivo and fold and unstable.Iso-electric point can be tested to measure with capillary isoelectric focusing, and this test produces the pH gradient, and can utilize laser focusing to improve accuracy (Janini etc. (2002) Electrophoresis 23:1605-11; Ma etc. (2001) Chromatographia 53:S75-89; Hunt etc. (1998) J Chromatogr A800:355-67).In some cases, preferred pI value falls into the anti-O8E antibody in normal range.This can be by selecting pI to be positioned at the antibody of normal range or by utilizing the charged surface residue of standard technique sudden change well known in the art to realize.
The melting temperature (Tm) indication thermostability (Krishnamurthy R and ManningMC (2002) Curr Pharm Biotechnol 3:361-71) of every kind of antibody.Higher thermostability represents that higher overall antibody stability is arranged in vivo.The fusing point of antibody can be used such as dsc (Chen etc. (2003) Pharm Res 20:1952-60; Ghirlando etc. (1999) ImmunolLett 68:47-52) etc. technology is measured.T M1Expression antibody begins to separate folding temperature.T M2Expression antibody is separated folding temperature fully.Usually, the T of antibody of the present invention preferably M1Greater than 60 ℃, be preferably greater than 65 ℃, even more preferably greater than 70 ℃.In addition, the thermostability of antibody also can utilize circular dichroism to measure (Murray etc. (2002) J.Chromatogr Sci40:343-9).
In a preferred embodiment, select the not antibody of fast degradation.Breaking of anti-O8E antibody can be measured with capillary electrophoresis well known in the art (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).
In a further preferred embodiment, select to have the antibody of minimum congregation.Gathering can trigger pharmacokinetic property undesirable immunne response and/or change or disadvantageous.Usually, antibody have 25% or gathering still less be acceptable, preferred 20% or still less, even more preferably 15% or still less, even more preferably 10% or still less, even more preferably 5% or still less.Gathering can be measured with several technology well known in the art, comprises size-exclusion column (SEC), high performance liquid chromatography (HPLC) and light scattering method, is used for identifying monomer, dimer, tripolymer or polymer.
Immune conjugate
On the other hand, the present invention relates to anti-O8E antibody or its fragment with therapeutic part couplings such as cytotoxin, medicine (such as immunosuppressor) or radiotoxin.These conjugates are referred to herein as " immune conjugate ".Comprise that one or more cytotoxic immune conjugates are known as " immunotoxin ".Cytotoxin or cytotoxic agent comprise any reagent to cell harmful (for example killer cell).Example comprises: taxol, cytochalasin B, Gramicidin D, Ethidum Eremide, ipecamine, mitomycin, etioposide, teniposide, vincristine(VCR), vinealeucoblastine(VLB), colchicine, Zorubicin, daunorubicin, dihydroxyl anthracin diketone, mitoxantrone, Plicamycin, dactinomycin, 1-dehydrogenation testosterone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Proprasylyte and tetracycline and their analogue or homologue.therapeutical agent also comprises, for example: metabolic antagonist (for example, methotrexate, Ismipur, 6-thioguanine, cytosine arabinoside, 5 FU 5 fluorouracil, Dacarbazine (decarbazine)), alkylating agent (for example, mustargen, Chlorambucil (thioepa chlorambucil), melphalan, carmustine (BSNU) and lomustine (CCNU), endoxan, busulfan, the dibromo mannitol, streptozocin, ametycin and suitable-dichloro diamines close (DDP) cis-platinum of platinum (II)), the anthramycin class (for example, gentle red rhzomorph (being called in the past daunomycin) and Zorubicin), microbiotic (for example, dactinomycin (being called in the past actinomycin), bleomycin, Plicamycin and Antramycin (AMC)), and antimitotic agent (for example, vincristine(VCR) and vinealeucoblastine(VLB)).
Can comprise a times ganmycin, calicheamycin, maytansine, auristatin and their derivative with other preferred example of the therapeutic cells toxin of antibody coupling of the present invention.An example of calicheamycin antibody coupling matter is to can be used as (the Mylotarg that commodity are buied TMWyeth-Ayerst).
Can utilize the linker technology of this area use with cytotoxin and antibody coupling of the present invention.Be used for the linker that example with the linker type of cytotoxin and antibody coupling includes but not limited to hydrazone, thioether, ester, disulphide and contains peptide.Can select, for example, the linker that is easily cut or easily cut by proteolytic enzyme by low pH in the lysosome compartment, this proteolytic enzyme are for example preferential proteolytic enzyme of expressing in tumor tissues, as kethepsin (for example cathepsin B, C, D).
About cytotoxic type, for coupling therapeutical agent and the linker of antibody and the further discussion of method, referring to Saito, G. etc. (2003) Adv.Drug Deliv.Rev.55:199-215; Trail, P.A. etc. (2003) Cancer.Immunol.Immunother.52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, T.M. (2002) Nat.Rev.Cancer2:750-763; 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.
Antibody of the present invention also can with the radio isotope coupling, produce radioactive cytotoxic drugs, also be known as the radioimmunoassay conjugate.The radioisotopic example of the antibody coupling that can use with diagnosis or therapeutic includes but not limited to iodine 131, iodine 125, indium 111, yttrium 90And lutetium 177The method for preparing the radioimmunoassay conjugate is set up in the art.The example of radioimmunoassay conjugate can be used as commodity and obtains, and comprises Zevalin TM(IDECPharmaceuticals) and Bexxar TM(Corixa Pharmaceuticals), and can utilize similar method to use antibody of the present invention to prepare the radioimmunoassay conjugate.
Antibody coupling matter of the present invention can be used for modifying specific biologically, and drug moiety should not be construed as the chemotherapeutic that is confined to classics.For example, drug moiety can be to have bioactive protein or the polypeptide that needs.Such protein comprises, for example: have toxin or its active fragments of enzymic activity, as toxalbumin, ricin A, Pseudomonas exotoxin or diphtheria toxin; Protein is as tumour necrosis factor or interferon-γ; Or the biologically instrumentality, as lymphokine, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), rHuGM-CSF (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF ") or other somatomedins.
Well-known with the technology of this therapeutic part and antibody coupling, referring to, for example, Arnon etc., " Monoclonal Antibodies For Immunotargeting Of Drugs InCancer Therapy ", in Monoclonal Antibodies And Cancer Therapy, Reisfeld etc. (ed.), pp.243-56 (Alan R.Liss, Inc.1985); Hellstrom etc., " Antibodies For Drug Delivery ", in Controlled Drug Delivery (2nd Ed.), Robinson etc. (ed.), pp.623-53 (Marcel Dekker, Inc.1987); Thorpe, " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review ", in Monoclonal Antibodies ' 84:Biological And Clinical Applications, Pinchera etc. (ed.), pp.475-506 (1985); " Analysis; Results; And FutureProspective Of The Therapeutic Use Of Radiolabeled Antibody InCancer Therapy ", in Monoclonal Antibodies For Cancer Detection AndTherapy, Baldwin etc. (ed.), pp.303-16 (Academic Press 1985), with Thorpe etc., " The Preparation And Cytotoxic Properties Of Antibody-ToxinConjugates; " Immunol.Rev., 62:119-58 (1982).
Bispecific molecule
On the other hand, the present invention relates to comprise the bispecific molecule of anti-O8E antibody of the present invention or its fragment.Antibody of the present invention or its antigen-binding portion thereof can be derivatized or be connected on another functional molecular, on another peptide or protein (for example part of another antibody or acceptor), can binding sites different from least two or the bispecific molecule of target molecule combination thereby generate.In fact antibody of the present invention can be derivatized or be connected on more than one other functional moleculars, thus generate can from the polyspecific molecule of different binding sites more than two and/or target molecule combination; Such polyspecific molecule is also included within term used herein " bispecific molecule ".In order to produce bispecific molecule of the present invention, antibody of the present invention can or be connected (as by chemical coupling, gene fusion, non-covalent combination etc.) with one or more other binding molecules such as other antibody, antibody fragment, peptide in conjunction with stand-in are functional, thereby obtains bispecific molecule.
Therefore, the present invention includes bispecific molecule, it has at least a the first binding specificity for O8E and for the second binding specificity of the second target epi-position.In particular of the present invention, the second target epi-position is the Fc acceptor, as people Fc γ RI (CD64) or people Fc α acceptor (CD89).Therefore, the present invention includes can with effector cell (as monocyte, scavenger cell or polymorphonuclear cell (the PMN)) combination of expressing Fc γ R or Fc α R, the bispecific molecule that can be combined with the target cell of expressing O8E again.The cell that these bispecific molecules will be expressed O8E is directed at the effector cell, and it is active to trigger the receptor-mediated effector cell of Fc, as the generation of cytotoxicity (ADCC), release of cytokines or the superoxide anion of the phagolysis of the cell of expressing O8E, antibody dependent cellular mediation.
Bispecific molecule is in one embodiment of the invention of polyspecific molecule therein, and except anti--Fc binding specificity and anti-O8E binding specificity, this molecule also can comprise the 3rd binding specificity.In one embodiment, the 3rd binding specificity is anti-enhancement factor (EF) part, for example is combined with the surface protein that participates in cellular cytoxicity activity thereby strengthens molecule for the immunne response of target cell." anti-enhancement factor part " can be to be combined with specific moleculars such as antigen or acceptor, thereby causes in conjunction with antibody, functional antibodies fragment or the part of determinant to the effect enhancing of Fc acceptor or target cell antigen." anti-enhancement factor part " can be combined with Fc acceptor or target cell antigen.Alternately, anti-enhancement factor part can be combined with a kind of entity, and this entity is different from the entity of the first and second binding specificity institutes combination.For example, anti-enhancement factor part can be combined with cytotoxic T cell (as via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immunocytes, this cell causes the immunne response enhancing for target cell).
In one embodiment, bispecific molecule of the present invention comprises at least one antibody or its antibody fragment as binding specificity, comprises (for example) Fab, Fab ', F (ab ') 2, Fv, Fd, dAb or scFv.This antibody can be also light chain or heavy chain homodimer, or any its minimal segment, and as described in the U.S. Patent No. 4,946,778 of Ladner etc. in Fv or strand construct, the content of this patent is incorporated herein by reference.
In one embodiment, provided by monoclonal antibody for the binding specificity of Fc γ acceptor, the combination of this monoclonal antibody is not blocked by immunoglobulin G while (IgG).Term used herein " IgG acceptor " refers to be positioned at any one of 8 γ-chain genes on karyomit(e) 1.These genes encodings are 12 cross-films or soluble receptors isotype altogether, and these isotypes are grouped into 3 Fc γ acceptor classification: Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16).In a preferred embodiment, Fc γ acceptor is people's high-affinity Fc γ RI.People Fc γ RI is the molecule of 72kDa, and monomer I gG is shown high-affinity (10 8-10 9M -1).
Some preferably the Preparation and characterization of anti--Fc γ monoclonal antibody described in PCT application WO 88/00052 and U.S. Patent number 4,954,617 by Fanger etc., herein its content whole is incorporated herein by reference.These antibody are combined with the epi-position of Fc γ RI, Fc γ RII or Fc γ RIII in the site different from the Fc γ binding site of acceptor, thereby it is in conjunction with substantially not blocked by the IgG of physiology level.Can be used in the present invention specific anti--Fc γ RI antibody is mAb 22, mAb32, mAb 44, mAb 62 and mAb 197.The hybridoma that produces mAb 32 can obtain from American type culture collection, and the ATCC preserving number is HB9469.In the other embodiment, anti--Fc γ receptor antibody is the humanization form (H22) of monoclonal antibody 22.The generation of H22 antibody and be characterized in Graziano, R.F. etc. (1995) J.Immunol 155 (10): the PCT of 4996-5002 and Tempest etc. announces in WO 94/10332 and describes.The clone of generation H22 antibody is deposited in American type culture collection with the title of HA022CL1, and preserving number is CRL11177.
In the other preferred embodiment, by providing with the antibody of people IgA acceptor such as Fc-α acceptor (Fc α RI (CD89)) combination, the combination of this antibody is not preferably blocked by human immunoglobulin A (IgA) to the binding specificity of Fc acceptor.Term " IgA acceptor " comprises the gene product (Fc α RI) that is positioned at a α-gene on karyomit(e) 19.The alternative splicing cross-film isotype of the several 55-110kDa of known this genes encoding.Fc α RI (CD89) constitutive expression on monocyte/macrophage, acidophilia and neutrophilic granulocyte, but constitutive expression in non-effector cell colony not.Fc α RI all has medium avidity (approximately 5 * 10 to IgA1 and IgA2 7M -1), this avidity increases (Morton, H.C. etc. (1996) Critical Reviews in Immunology 16:423-440) after being exposed to such as the cytokine of G-CSF or GM-CSF.Described 4 kinds of Fc α RI-monoclonal antibody specifics, they are confirmed as A3, A59, A62 and A77, and they are combined (Monteiro, R.C. etc. (1992) J.Immunol.148:1764) with Fc α RI outside the IgA ligand binding domain.
Fc α RI and Fc γ RI are the preferred triggering acceptors for bispecific molecule of the present invention, because their (1) mainly are expressed on immune effector cell such as monocyte, PMN, scavenger cell and dendritic cell; (2) high level expression (as each cell expressing 5,000-100,000); (3) be the medium of cytotoxicity (as ADCC, phagolysis); (4) be directed at the antigen presentation of enhancing of their antigen (comprising autoantigen).
Preferred human monoclonal antibodies, but other antibody that can use in bispecific molecule of the present invention comprise, for example, mouse, chimeric and Humanized monoclonal antibodies.
The binding specificity that can form by using method coupling as known in the art as anti--FcR and anti-O8E binding specificity, prepares bispecific molecule of the present invention.For example, each binding specificity of bispecific molecule can generate separately, then coupling each other.When binding specificity is protein or peptide, can use multiple coupling agent or linking agent to carry out covalent coupling.The example of linking agent comprises albumin A, carbodiimide, N-succinimido-S-acetyl-thioacetate (SATA), 5,5 '-dithio two (2-nitrobenzoic acid) (DTNB), adjacent phenylene bismaleimides (oPDM), N-succinimido-3-(2-pyridine dithio) propionic salt (SPDP) and sulfosuccinic acylimino 4-(N-maleimide amino methyl) cyclohexyl-1-carboxylate salt (sulfo-SMCC) (referring to, for example, (1984) J.Exp.Med.160:1686 such as Karpovsky; Liu, MA etc. (1985) Proc.Natl.Acad.Sci.USA 82:8648).Additive method comprises Paulus (1985) Behring Ins.Mitt.No.78,118-132); The described methods of (1987) J.Immunol.139:2367-2375 such as Brennan etc. (1985) Science 229:81-83 and Glennie.Preferred coupling agent is SATA and sulfo-SMCC, and both all can obtain from PierceChemical Co. (Rockford, IL).
When binding specificity is antibody, the sulfydryl bonding of the C-end hinge area that they can be by two heavy chains and coupling.In an especially preferred embodiment, hinge area is modified so that it contains odd number before coupling preferred 1 sulfhydryl residue.
Alternately, two kinds of binding specificities can be encoded in identical carrier, and express in identical host cell and assembling.When bispecific molecule is mAb * mAb, mAb * Fab, Fab * F (ab ') 2Or during part * Fab fusion rotein, the method is useful especially.Bispecific molecule of the present invention can be to comprise a single-chain antibody and the single chain molecule in conjunction with determinant, or comprises two in conjunction with the strand bispecific molecule of determinant.Bispecific molecule can comprise at least two single chain molecules.Prepare the method for bispecific molecule for example at United States Patent (USP) 5,260,203, United States Patent (USP) 5,455,030, United States Patent (USP) 4,881, and 175, United States Patent (USP) 5,132,405, United States Patent (USP) 5,091,513, United States Patent (USP) 5,476, and 786, United States Patent (USP) 5,013,653, United States Patent (USP) 5,258,498 and United States Patent (USP) 5,482,858 in describe.
Bispecific molecule is combined with its specific target target and can be confirmed by for example enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), facs analysis, biological assay (as growth-inhibiting) or Western engram analysis.Each in these tests has to the target mixture existence that specific labelled reagent (as antibody) detects specific objective protein-antibody complex by using usually.For example, can utilize enzyme len antibody or the antibody fragment of identification and specific binding antibody-FcR mixture to detect the FcR-antibody complex.Perhaps, these mixtures can utilize any in multiple other immunoassays to detect.For example, but antagonist carries out radio-labeling and use in radioimmunoassay (RIA) (referring to, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course onRadioligand Assay Techniques, The Endocrine Society, is incorporated herein by reference in March, 1986).Can pass through such as the means of using gamma counter or scintillometer or by radioautograph method detection of radioactive isotropic substance.
Pharmaceutical composition
On the other hand, the invention provides a kind of composition, pharmaceutical composition for example, it contains monoclonal antibody of the present invention or its antigen-binding portion thereof with a kind of or combination formulated together of medicine acceptable carrier.Such composition can comprise (for example two or more are different) antibody of the present invention or immune conjugate or bispecific molecule a kind of or combination.For example, pharmaceutical composition of the present invention can contain in conjunction with the different epi-positions on target antigen or antibody combination (or immune conjugate or bispecific molecule) with complementary activity.
Pharmaceutical composition of the present invention also can be used in combination therapy, namely with other medicament couplings.For example, combination therapy can comprise antibody combined at least a other anti-inflammatory agent or the immunosuppressor of anti-O8E of the present invention.The example of the therapeutical agent that can use in the combination therapy application one of antibody of the present invention is below described in saving in more detail.
" medicine acceptable carrier " used herein comprises physiology compatible any He all solvents, dispersion medium, dressing, antibacterial agent and anti-mycotic agent, isotonic agent and absorption delay agent etc.Preferably, this carrier is suitable for intravenously, intramuscular, subcutaneous, parenteral, backbone or epidermis and uses (as by injection or infusion).According to route of administration, can be that antibody, immune conjugate or bispecific molecule are wrapped in a kind of material with active compound, avoid making the acid of this compound inactivation and the effect of other natural conditions to protect this compound.
Pharmaceutical composition of the present invention can comprise the acceptable salt of one or more medicines." the acceptable salt of medicine " has referred to keep the required biological activity of parental generation compound and has not caused the salt (referring to as Berge, S.M. etc. (1977) J.Pharm.Sci.66:1-19) of any toxicological action of not expecting.The example of such salt comprises acid salt and base addition salt.Acid salt comprises those by the derivative salt of the nontoxicity mineral acids such as all example hydrochloric acids, nitric acid, phosphoric acid, sulfuric acid, Hydrogen bromide, hydroiodic acid HI, phosphorous acid, and by the derivative salt of the nontoxicity organic acids such as paraffinic acid, hydroxyl alkane acid, aromatic acid, aliphatic series and aromatic sulfonic acid such as mono carboxylic acid of aliphatic series and dicarboxylic acid, phenyl substituted.Base addition salt comprises that those are by the derivative salt such as the alkaline-earth metal such as sodium, potassium, magnesium, calcium, and by such as N, the salt that the nontoxicity organic amines such as N '-dibenzyl-ethylenediamin, N-METHYL-ALPHA-L-GLUCOSAMINE, chloroprocaine, choline, diethanolamine, quadrol, PROCAINE HCL, PHARMA GRADE are derivative.
Pharmaceutical composition of the present invention also can contain the acceptable antioxidant of medicine.The example of the acceptable antioxidant of medicine comprises: (1) water soluble antioxidant, as xitix, cysteine hydrochloride, sodium pyrosulfate, Sodium Pyrosulfite, S-WAT etc.; (2) oil-soluble inhibitor is as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), Yelkin TTS, Tenox PG, alpha-tocopherol etc.; (3) metal chelator is as citric acid, ethylenediamine tetraacetic acid (EDTA) (EDTA), Sorbitol Powder, tartrate, phosphoric acid etc.
Can be used for suitable water-based in pharmaceutical composition of the present invention or the example of non-aqueous carrier and comprise water, ethanol, polyvalent alcohol (as glycerine, propylene glycol, polyoxyethylene glycol etc.), and suitable mixture, vegetables oil such as sweet oil, and injection organic ester such as ethyl oleate.For example by using capsulating material such as Yelkin TTS, in the situation that dispersion liquid is by keeping required granular size, and by the application surface promoting agent, can keep suitable mobility.
These compositions also can contain adjuvant, as sanitas, wetting agent, emulsifying agent and dispersion agent.Can be by above-mentioned sterilizing program or by comprising various antibacterial agents such as p-Hydroxybenzoate, chlorobutanol, phenol Sorbic Acid and anti-mycotic agent guarantees to prevent from existing microorganism.Also may comprise isotonic agent in composition, for example, sugar, sodium-chlor etc.In addition, by comprising the delay absorption agent, for example aluminum monostearate and gelatin, can realize the absorption that the injection-type medicine extends.
The medicine acceptable carrier comprises aseptic aqueous solution or dispersion liquid and is used for the powder agent of interim preparation aseptic parenteral solution or dispersion liquid.These are used for the medium of pharmaceutically active substances and the use of reagent is well known in the art.Except any and the inconsistent conventional media of active compound or reagent scope, comprise its application in pharmaceutical composition of the present invention.Can also mix additional active compound in composition.
Therapeutic composition must be generally aseptic and stable under preparation and storage requirement.Composition can be mixed with the ordered structure of solution, microemulsion, liposome or other suitable high drug levels.Carrier can be solvent or the dispersion agent that contains water for example, ethanol, polyvalent alcohol (for example, glycerine, propylene glycol and liquid polyethylene glycol etc.) and suitable mixture thereof.For example, by using dressing, for example Yelkin TTS, in the situation that dispersion liquid passes through the required granular size of maintenance, and by using tensio-active agent, can keep suitable mobility.Under many circumstances, preferably comprise isotonic agent in composition, for example, sugar, polyvalent alcohol be mannitol, Sorbitol Powder or sodium oxide for example.By add the delay absorption agent in composition, for example Monostearate and gelatin, can realize the absorption that the injection-type medicine extends.
By active compound is sneaked in suitable solvent with the amount of needs, and add as required a kind of or its combination in the above composition of enumerating, follow aseptic micro-filtration, can prepare aseptic parenteral solution.Usually, by being incorporated in the sterile carrier that contains basic dispersion medium and other required compositions listed above, active compound prepares dispersion agent.For the sterilized powder agent for the preparation of aseptic parenteral solution, preferred preparation method is vacuum-drying and lyophilize (freeze-drying), obtains by the solution of its sterile filtration in advance the powder that activeconstituents adds any extra required composition.
Can be from the amount of the activeconstituents of solid support material combination preparation single dose form according to the experimenter who treats and specific administration mode and different.Can be generally the amount that produces the composition of result for the treatment of with the amount of the activeconstituents of solid support material combination preparation single dose form.Usually, in 100%, the scope of this amount is about 0.01% to about 99% activeconstituents, and preferably approximately 0.1% is to about 70%, and most preferably about 1% to about 30% activeconstituents, combined with the medicine acceptable carrier.
Regulate dosage, so that the reaction (for example, therapeutic response) of best expectation to be provided.For example, single bolus can be used, the dosage that separates several times can be used in time, perhaps required according to the emergency situation for the treatment of situation, can reduce in proportion or increase dosage.Particularly advantageous is that parenteral composition is mixed with easy administration and the uniform dosage unit form of dosage.Dosage unit form used herein refers to be suitable as the experimenter's that unitary dose is used for treating the discontinuous unit of physics; Each unit contains the active compound of predetermined amount, as calculated the active compound of this predetermined amount result for the treatment of required with the pharmaceutical carrier combination results that needs.Illustrating of dosage unit form of the present invention is defined in and directly depends on the unique property of (a) active compound and the particular treatment effect that will reach, and (b) intrinsic for this restriction that is used for the treatment of the active compound of individual sensitivity of preparation in this area.
For the administration of antibody, dosage range is approximately 0.0001 to 100mg/kg, is more typically 0.01 to 25mg/kg receptor'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 the 1-10mg/kg scope.Can use higher dosage when needing, for example 15mg/kg body weight, 20mg/kg body weight or 25mg/kg body weight.The example of a treatment plan need to be administered once weekly, often biweekly, every three weeks once, every surrounding once, per month once, per March once or every 3-6 month once.The preferred dosage regimen of anti-O8E antibody of the present invention comprises through intravenously and give 1mg/kg body weight or 3mg/kg body weight, and this antibody uses the administration of one of following dosage: (i) once totally 6 times, then every 3 months once every 4 weeks; (ii) every 3 weeks once; (iii) the 3mg/kg body weight once, every 3 all 1mg/kg body weight then.
In certain methods, use simultaneously two or more the anti-O8E monoclonal antibodies of the present invention with different binding specificities, in this case, the dosage of every kind of antibody drops in described scope.Antibody is multiple dosing when being necessary usually.Interval between single-dose can be, for example, weekly, per month, every three months or every year.The interval can be also irregular, for example determines by the blood level of measuring the antibody of anti-target antigen in the patient.In certain methods, regulate dosage to reach the approximately plasma antibody concentration of 1-1000 μ g/ml, be about 25-300 μ g/ml in certain methods.
In additive method, one or more anti-O8E monoclonal antibodies of the present invention and for example anti-CTLA-4 and/or anti-PD-1 administration simultaneously of the antibody with different binding specificities, in this case, the dosage of every kind of antibody drops in described scope.
Alternately, antibody also can be used as extended release preparation and comes administration, needs in this case the lower administration of frequency.Dosage and frequency be the transformation period in the patient and difference according to antibody.Usually, people's antibody shows the longest transformation period, is humanized antibody, chimeric antibody and non-human antibody afterwards.Dosage and frequency are preventative or curative and different according to processing.In prophylactic application, in long-time with more frequently the interval give relatively low dosage.Some patient continues to accept processing in the remaining years.In therapeutic is used, sometimes need to give with shorter interval higher dosage, until the progress of disease alleviates or stop, preferred until the patient shows as disease symptoms partially or completely improves.Afterwards, can be with Prevention scheme to patient's administration.
In pharmaceutical composition of the present invention, the actual dose level of activeconstituents may change, obtaining effectively to realize the required therapeutic response to particular patient, composition and administering mode, and to the amount of the avirulent activeconstituents of patient.The dosage level of selecting depends on multi-medicament dynamic metabolism factor, the activity that comprises particular composition of the present invention or its ester, salt or the acid amides of application, route of administration, administration time, the discharge rate of the specific compound of using, the time length for the treatment of is with other drug, compound and/or the material of the particular composition combined utilization of using, the patient's who receives treatment age, sex, body weight, situation, general health situation and medical history, and known similar factor in medical field.
" the treatment effective dose " of anti-O8E antibody of the present invention preferably causes the seriousness of disease symptoms to reduce, and the frequency of disease asymptomatic stage and time length increase, and perhaps prevents because of painful damage or the anergy that causes of disease.For example, for the treatment of O8E+ tumour, with respect to the experimenter who does not receive treatment, " treatment effective dose " preferably suppresses Growth of Cells or tumor growth at least about 20%, more preferably at least about 40%, more preferably at least about 60%, more preferably at least about 80%.Compound suppresses the ability of tumor growth and can estimate in the animal model system of prediction to the curative effect of human tumor.Perhaps, also can estimate this performance of said composition by checking the cytostatic ability of compound, this inhibition can be by well known to a person skilled in the art that test is at external test.The therapeutic compound for the treatment of significant quantity can reduce tumor size, perhaps otherwise alleviates experimenter's symptom.Those skilled in the art can determine this amount according to following factor, as experimenter's size, the seriousness of experimenter's symptom and particular composition or the route of administration of selection.
Composition of the present invention can utilize one or more methods well known in the art by one or more route of administration administrations.It will be appreciated by those skilled in the art that route of administration and/or mode according to the result of expecting and difference.The preferred route of administering of antibody of the present invention comprises intravenously, intramuscular, intracutaneous, intraperitoneal, subcutaneous, backbone or other administered parenterally approach, for example injection or infusion.Phrase used herein " administered parenterally " refers to the mode of administration except intestines and topical, normally injection, include but not limited in intravenously, intramuscular, intra-arterial, sheath, in capsule, interior, intracardiac, the intracutaneous of socket of the eye, intraperitoneal, under tracheae, subcutaneous, epidermis, under intraarticular, capsule, under arachnoid membrane, in backbone, epidural and breastbone inner injection and infusion.
Alternately, antibody of the present invention also can be by the outer administration of parenteral, as local, epidermis or mucosal route administration, for example, in nose, per os, vagina, rectum, hypogloeeis or part.
Active compound can prepare together with the carrier of protecting compound not to be fast released, and for example controlled release preparation, comprise implant, transdermal patch and microcapsule delivery system.Can use biodegradable, biocompatible polymkeric substance, for example ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poe and poly(lactic acid).The a lot of methods that prepare such preparation are subjected to patent protection or are generally conventionally known to one of skill in the art.Referring to, for example, Sustained andcontrolled Release Drug Delivery Systems, J.R.Robinson, ed., MarcelDekker, Inc., New York, 1978.
Therapeutic composition can be used medical treatment device administration well known in the art.For example, in a preferred embodiment, therapeutic composition of the present invention can be used the administration of needleless hypodermic injection unit, as in U.S. Patent No. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; Or disclosed device in 4,596,556.The example that can be used for known implant of the present invention and module comprises: U.S. Patent No. 4,487,603, this patent disclose the implantable micro-infusion pump that is used for the controllable rate dispersion medicine; U.S. Patent No. 4,486,194, this patent disclose the therapeutic system that is used for by percutaneous drug delivery; U.S. Patent No. 4,447,233, this patent disclose the medical infusion pump that is used for accurate infusion rates delivering drugs; U.S. Patent No. 4,447,224, this patent disclose the implantable infusion device of unsteady flow that is used for continuous delivering drugs; U.S. Patent No. 4,439,196, this patent disclose the osmotic drug delivery system with multi-cavity compartment: and U.S. Patent No. 4,475,196, this patent discloses a kind of osmotic drug delivery system.These patents are incorporated herein by reference.Many other such implants as well known to those skilled in the art, delivery system and module.
In certain embodiments, human monoclonal antibodies of the present invention can be formulated as the correct distribution of guaranteeing in vivo.For example, blood brain barrier (BBB) has stoped many highly hydrophilic compounds.Can stride across BBB (if when needing) in order to ensure therapeutic compound of the present invention, they can be formulated in as in liposome.As for the method for preparing liposome, referring to, for example, United States Patent (USP) 4,522,811; 5,374,548; With 5,399,331.Liposome comprises and can optionally be transported specific cells or intraorganic one or more part, thereby the enhancing directed drug delivery (referring to, for example, V.V.Ranade (1989) J.Clin.Pharmacol.29:685).The example of bearing portion comprises folic acid or vitamin H (referring to, for example, the United States Patent (USP) 5,416,016 of Low etc.); Mannoside (Umezawa etc. (1988) Biochem.Biophys.Res.Commun.153:1038); Antibody (P.G.Bloeman etc. (1995) FEBS Lett.357:140; M.Owais etc. (1995) Antimicrob.Agents Chemother.39:180); Tensio-active agent albumin A acceptor (Briscoe etc. (1995) Am.J.Physiol.1233:134); P120 (Schreier etc. (1994) J.Biol.Chem.269:9090); Also referring to K.Keinanen; M.L.Laukkanen (1994) FEBS Lett.346:123; J.J.Killion; I.J.Fidler (1994) Immunomethods4:273.
Application of the present invention and method
Antibody of the present invention (particularly people's antibody), antibody compositions and method have with (for example) and detect O8E, treat cancer or strengthen immunne response relevant many in vitro and in vivo diagnosis and treatment application by blocking O8E.In a preferred embodiment, antibody of the present invention is people's antibody.For example, these molecules can be applied to the cell in external or isolated culture, and perhaps (for example) is applied to the human experimenter in vivo, thus treatment, prevention and diagnosis various diseases or enhancing immunity in multiple situation.
Term used herein " experimenter " comprises people and non-human animal.Term " non-human animal " comprises all vertebratess, for example Mammals and nonmammalian, for example non-human primate, sheep, dog, cat, ox, horse, chicken, amphibian animal and reptile.Preferred experimenter comprises suffering from the human patients that the disease relevant with the O8E expression maybe needs to strengthen immunne response.These methods are particularly suitable for treating suffers from the human patients of expressing relevant disease with abnormal O8E.These methods are particularly suitable for also treating that suffer from can be by strengthening the human patients of the disease that the cell-mediated immunne response of T treats.In order to realize that antigen specific immune strengthens, anti-O8E antibody can be used together with target antigen.Together with another medicine during administration, these two kinds of medicines can be in succession or administration simultaneously when anti-O8E antibody.
In view of antibody of the present invention and O8E specific binding, antibody of the present invention can be used for the expression of O8E on the specific detection cell surface, and can be used for by immunoaffinity purification method purifying O8E.
Cancer
O8E expresses in multiple human cancer, described cancer comprise mammary gland cell cancer, metastatic breast cancer, gonad cell cancer, Metastatic carcinoma in the ovary and renal cell carcinoma (people such as Tringler. (2005) Clinical Cancer Res.11:1842-48; The people such as Salceda (2005) Exp Cell Res.306:128-41; The people such as Tringler. (2006) Gynecol Oncol.100:44-52; The people such as Krambeck. (2006) Proc Natl Acad Sci USA 103:10391-6; The people such as Chen. (2006) Kidney Int. waits to publish; The people such as Sun (2006) Lung Cancer 53:143-51; The people such as Bignotti. (2006) Gynecol Oncol.103:405-16; The people such as Kryczek (2006) J ExpMed 203:871-81; The people such as Simon. (2006) Cancer Res.66:1570-5).Anti-O8E antibody can use separately, is used for suppressing the growth of cancerous tumour.Perhaps, as mentioned below, anti-O8E antibody also can use together with other immunogenic agents, standard cancer treatments or other antibody.
Find that B and T lymphocyte attenuator (BTLA) are the acceptors of O8E, and inhibited to immunne response, be similar to cytotoxic lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) (Carreno and Collins (2003) Trends Immunol 24:524-7).O8E by suppressor T cell breed, cytokine produces and the cell cycle produces negative regulator T cellular immunization work people (2003) J Immunol.171:4650-4 such as () Choi.O8E-Ig fusion rotein suppressor T cell activation, and antibody to the blocking-up of O8E can strengthen immunne response in the patient (people such as Sica. (2003) Immunity 18:849-61).
In one aspect, the application relates to the anti-O8E antibody of use and treats in vivo the experimenter, and the growth of cancerous tumour is suppressed.Anti-O8E antibody can use separately, is used for suppressing the growth of cancerous tumour.Perhaps, as mentioned below, anti-O8E antibody can use together with other immunogenic agents, standard cancer treatments or other antibody.
Therefore, in one embodiment, the invention provides the method for growth of tumour cell in a kind of experimenter of inhibition, comprise anti-O8E antibody or its antigen-binding portion thereof to experimenter's administering therapeutic significant quantity.Preferably, this antibody is the anti-O8E antibody of people (the anti-human O8E antibody of as described herein anyone).In addition or alternately, this antibody can be chimeric or the anti-O8E antibody of humanization.
Its growth can comprise with the preferred cancer of antibody suppression of the present invention generally has the cancer of replying to immunotherapy.The non-limitative example of medicable preferred cancer comprises mammary cancer (for example mammary gland cell cancer), ovarian cancer (for example gonad cell cancer) and renal cell carcinoma (RCC).can comprise melanoma (for example metastatic malignant melanoma) with the example of other cancers of method of the present invention treatment, prostate cancer, colorectal carcinoma, lung cancer, osteocarcinoma, carcinoma of the pancreas, skin carcinoma, brain tumor, chronic or acute leukemia (comprises acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia, lymphocytic leukemia), lymphoma (for example He Jiejin lymphomas and non_hodgkin lymphoma, the lymphocyte lymphoma, primary CNS lymphoma, t cell lymphoma), nasopharyngeal carcinoma, head and neck cancer, skin or intraocular malignant melanoma, uterus carcinoma, the rectum cancer, cancer of the anal region, 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, childhood solid tumor, bladder cancer, kidney or carcinoma of ureter, carcinoma of renal pelvis, central nervous system (CNS) tumour, tumor vessel occurs, tumor of spine, the brain stem neurospongioma, pituitary adenoma, Kaposi sarcoma, epidermis shape cancer, squamous cell carcinoma, the cancer of ambient induced (cancer that comprises Induced by Asbestos, for example mesothelioma), and the combination of described cancer.
Randomly, anti-O8E antibody can be united use people such as (, J.Immunol.173:4919-28 (2004)) He with the tumour antigen (comprising recombinant protein, peptide and carbohydrate molecule) of immunogenic agents such as cancer cells, purifying, cell with the cell of the gene transfection of coding immunostimulating cytokine.The non-limitative example of operable tumor vaccine comprises the peptide of melanoma antigen, for example the tumour cell of express cell factor GM-CSF after the peptide of gp100, MAGE antigen, Trp-2, MART1 and/or tyrosine oxidase or transfection.
In the mankind, some tumour shows as immunogenicity, for example melanoma.The threshold value that improves the T cell activation is blocked in expection by O8E, can correspondingly activate tumour in the host.
When making up with vaccination regimen, the O8E blocking-up may be the most effective.Proposed many experimental strategies of inoculating for tumour (referring to, Rosenberg, " Development of CancerVaccines " ASCO Educational Book Spring:60-62 (2000); Logothetis, ASCO Educational Book Spring:300-302 (2000); Khayat, ASCOEducational Book Spring:414-428 (2000); Foon, ASCO EducationalBook Spring:730-738 (2000); Referring to Restifo and Sznol, Cancer Vaccines, Ch.61, the people such as pp.3023-3043 in DeVita. (ed.) Cancer:Principles andPractice of Oncology, the 5th edition (1997)).In strategy, use to prepare vaccine from body or allogeneic tumor cell therein.Preferably, when the transduction tumour cell made its expression of GM-CSF, these cell vaccines were the most effective.Verified for tumor inoculation, GM-CSF is a kind of strong activator (the people .Proc.Natl.Acad.Sci U.S.A.90:3539-43 (1993) such as Dranoff) of antigen presentation.
Genetic expression in various tumours and extensive gene expression pattern research cause having defined so-called tumour specific antigen (Rosenberg, Immunity 10:281-7 (1999)).In many cases, these tumour specific antigens are in tumour and at the differentiation antigen of the cells that produces this tumour, for example melanocyte antigen gp100, MAGE antigen and Trp-2.The more important thing is, many such antigens may be the targets of the tumour-specific T cell found in the host.In order to produce the immunne response to these protein, the O8E blocking-up can be used together with the recombinant protein of expressing in collecting tumour and/or peptide.These protein are considered as autoantigen by immunity system usually, therefore to its tolerance.Tumour antigen also can comprise the protein Telomerase, it is that fringes of chromosome is synthetic needed, express in the human cancer more than 85%, and only express people such as (, Science 266:2011-2013 (1994)) Kim in a limited number of bodily tissues.(can utilize multiple means to protect these bodily tissues to exempt from immune attack).Tumour antigen can be also owing to changing protein sequence or produce the somatic mutation of two kinds of fusion roteins (as the bcr-abl in Philadelphia chromosome) between irrelevant sequence and " neoantigen " of expressing or from the idiotype of B cell tumour in cancer cells.
Other tumor vaccines can comprise the protein from the virus relevant with human cancer, and described virus is for example human papillomavirus (HPV), hepatitis virus (HBV and HCV) and Ka Boxi bleb sarcoma virus (KHSV).Another form of the tumour specific antigen that can use together with O8E blocking-up is the heat shock protein(HSP) (HSP) of the purifying that separates from tumor tissues itself.These heat shock protein(HSP)s contain the fragment from the protein of tumour cell, and these HSP are sending to cause aspect tumour immunity very effectively (Suot and SrivastavaScience 269:1585-1588 (1995)) to antigen presenting cell; The people .Science 278:117-120 (1997) such as Tamura).
Dendritic cell (DC) are effective antigen presenting cells, can be used for causing antigen-specific and reply.The DC generation of can exsomatizing, and be loaded with range protein and peptide antigen and tumour cell extract (Nestle, the people such as F.. (1998) Nature Medicine 4:328-332).Also can by hereditary means transduction DC, make it express these tumour antigens.For immune purpose, also once DC and tumour cell were directly merged (Kugler, the people such as A.. (2000) Nature Medicine6:332-336).As a kind of inoculation method, the DC immunity can be blocked effective combination with PD-1, activates stronger antitumor replying.
The O8E blocking-up also can be united use with standard cancer treatments.The O8E blocking-up can effectively be united with the chemotherapy scheme.In these situations, can reduce chemotherapeutics dosage (Mokyr, the people such as M.. (1998) Cancer Research.58:5301-5304).An example of this associating is that the antibody combined Dacarbazine of anti-O8E (decarbazine) is used for the treatment of kinds cancer.Another example of this associating is that the antibody combined interleukin-2 of anti-O8E (IL-2) is used for the treatment of kinds cancer.The O8E blocking-up is that the necrocytosis meeting that the cytotoxic effect of most of chemotherapy compounds causes causes the tumour antigen level in the antigen presentation approach to raise with the principles of science of chemotherapy combined utilization.May there be radiotherapy, operation and hormone to deprive by other combination therapys that necrocytosis and O8E blocking-up are worked in coordination with.These schemes all produce tumour antigen and originate in the host.Angiogenesis inhibitor also can be blocked the associating use with O8E.The inhibition of vasculogenesis causes death of neoplastic cells, and death of neoplastic cells can add to host antigen with tumour antigen and present in approach.
The O8E blocking antibody also can with the bi-specific antibody that makes the effector cell who expresses Fc α or Fc γ acceptor be directed to tumour unite use (referring to, for example, United States Patent(USP) Nos. 5,922,845 and 5,837,243).The bi-specific antibody two kinds of different antigens that can be used for leading.For example, anti-Fc acceptor/tumor-resistant antigen (for example Her-2/neu) bi-specific antibody has been used for scavenger cell is directed to tumor locus.This guiding can more effectively activate tumour-specific and reply.Utilize the O8E blocking-up can strengthen the T cell part that these are replied.In addition, can utilize the bi-specific antibody in conjunction with tumour antigen and dendritic cell specific cell surface marker that antigen directly is delivered to DC.
Tumour is escaped host's immunosurveillance by number of mechanisms.The inhibitive ability of immunity protein of expressing by the deactivation tumour can overcome many such mechanism.Especially comprise TGF-β (Kehrl, J. wait the people. (1986) J.Exp.Med.163:1037-1050), IL-10 (Howard, M. and O ' Garra, A. (1992) Immunology Today 13:198-200) and FasL (Hahne, the people such as M.. (1996) Science 274:1363-1365).Antibody for these materials can be united use with anti-PD-1, offsetting the effect of immunosuppressor, and is conducive to host's tumor immune response.
Other antibody that can be used for activating host immune response can be united use with anti-O8E.Comprise the molecule on the surface of dendritic cells that activates DC function and antigen presentation.Anti-cd40 antibody can effectively replace the T cell helper activity (Ridge, the people such as J.. (1998) Nature 393:474-478), and can use together with O8E antibody.Activate for T cell co-stimulatory molecules such as CTLA-4 (for example United States Patent (USP) 5,811,097), OX-40 (Weinberg, the people such as A.. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I. wait the people. (1997) NatureMedicine 3:682-685 (1997), PD-1 (people such as del Rio. (2005) Eur J Immunol.35:3545-60) and ICOS (Hutloff, the people such as A.. (1999) Nature 397:262-266) antibody the T cell activation level of raising also can be provided.
The current tumour that is used for treating multiple hematopoiesis source of bone marrow transplantation.Graft versus host disease is the consequence of this treatment, but can be from graft to acquisition treatment benefit the replying of tumour.O8E blocking-up can be used for improving the validity of the tumour-specific T cell that donor transplants.
Also there are several experimental therapy schemes to comprise exsomatize activation and expansion of antigen specific T-cells, and with these cell adoptive transfers in the receptor, to produce T cells with antigenic specificity (Greenberg, R. and Riddell, S. (1999) Science for tumour 285: 546-51).Also can utilize these methods activation for the t cell response of infectious agent such as CMV.Stripped activation expection under anti-O8E antibody exists can improve frequency and the activity of the T cell of adoptive transfer.
In view of O8E expresses on kinds of tumor cells, people's antibody of the present invention, antibody compositions and method can be used for treating suffers from the experimenter who causes tumor disease, described disease is for example that to have the tumour cell of expressing O8E be the disease of feature, comprise, for example, mammary cancer (for example mammary gland cell cancer), ovarian cancer (for example gonad cell cancer) and kidney.other can comprise melanoma (for example metastatic malignant melanoma) with the example of the cancer of method of the present invention treatment, prostate cancer, colorectal carcinoma and lung cancer, osteocarcinoma, carcinoma of the pancreas, skin carcinoma, head and neck cancer, skin or intraocular malignant melanoma, uterus carcinoma, the rectum cancer, cancer of the anal region, 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, Hokdkin disease, non_hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma, primary cutaneous type (ALCL), multiple myeloma, cutaneous T cell lymphoma, nodositas SCC 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, B is diffuse large cell lymphoma, angioimmunoblastic lymphadenopathy (AILD)-sample t cell lymphoma, HIV relevant body cavity lymphoma, embryonal carcinoma, Differentiated Nasopharyngeal Carcinoma (for example schmincke's tumor) not, castleman's disease, Kaposi sarcoma, multiple myeloma, Walden Si Telun macroglobulinemia and other B cell lymphomas, esophagus cancer, carcinoma of small intestine, the endocrine system cancer, thyroid carcinoma, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, urethral carcinoma, penile cancer, chronic or acute leukemia (comprises acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia, lymphocytic leukemia), childhood solid tumor, the lymphocyte lymphoma, bladder cancer, kidney or carcinoma of ureter, carcinoma of renal pelvis, central nervous system (CNS) tumour, primary CNS lymphoma, glioblastoma multiforme, brain tumor, nasopharyngeal carcinoma, tumor vessel occurs, tumor of spine, the brain stem neurospongioma, pituitary adenoma, Kaposi sarcoma, epidermis shape cancer, squamous cell carcinoma, t cell lymphoma, the cancer of ambient induced (cancer that comprises Induced by Asbestos), and the combination of described cancer.The present invention also can be used for treating metastatic carcinoma.
Therefore, in one embodiment, the invention provides the method for growth of tumour cell in a kind of experimenter of inhibition, comprise anti-O8E antibody or its antigen-binding portion thereof to experimenter's administering therapeutic significant quantity.Typically, this antibody is the anti-O8E antibody of people (the anti-human O8E antibody of as described herein anyone).In addition or alternately, this antibody can be chimeric or the anti-O8E antibody of humanization.
Transmissible disease
Additive method of the present invention is used for treating the patient who once was exposed to particular toxin or pathogenic agent.Therefore, another aspect of the present invention provides the method for a kind of patient's for the treatment of transmissible disease, comprises to the patient using anti-O8E antibody or its antigen-binding portion thereof, to treat this patient's transmissible disease.Preferably, this antibody is the anti-human O8E antibody of people (the anti-O8E antibody of as described herein anyone).In addition or alternately, this antibody can be chimeric or humanized antibody.
Be similar to the application for tumour as above, antibody-mediated O8E blocking-up can be used separately or unite use as supplementary means and vaccine, stimulates the immunne response to pathogenic agent, toxin and autoantigen.This methods for the treatment of especially effectively example of pathogenic agent comprises the pathogenic agent that the pathogenic agent that there is no at present effective vaccine or conventional vaccine can not be fully effective.Include but not limited to HIV, hepatitis (first, second and the third type) virus, influenza virus, simplexvirus, giardia lamblia (Giardia), malaria bacterium, leishmania, streptococcus aureus (Staphyiococcusaureus), blue pus organism (Pseudomonas Aeruginosa).The PD-1 blocking-up is particularly useful for resisting the infection of setting up as the pathogenic agent of HIV, and they compare the antigen that presents change with course of infection.When anti-human O8E administration, these new epi-positions are considered to exotic, thereby produce the strong t cell response of the negative signal weakening that is not passed through O8E.
some examples that cause the pathogenic virus of the infection that available the inventive method is treated comprise HIV, hepatitis (first, second and the third type)) virus, simplexvirus (VZV for example, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, Echo virus, rhinovirus, Coxsackie virus, coronavirus, 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 arboviruses encephalitis.
some examples that cause the pathogenic bacteria of the infection that available the inventive method is treated comprise chlamydozoan, the rickettsia bacterium, mycobacterium, staphylococcus, suis, pneumococcus, meningococcus and gonococcus (conococci), klebsiella, Bacillus proteus, Serratia, pseudomonas, legionella, diphtheria corynebacterium, Salmonellas, genus bacillus, cholera bacteria, tetanolysin, Clostridium botulinum, anthrax bacillus, plague bacillus, Leptospira (leptospirosis) and Lyme disease bacterium.
some examples that cause the pathogenic epiphyte of the infection that available the inventive method is treated comprise candiyeast (Candida albicans, candida krusei, glabrata, candida tropicalis etc.), novel Cryptococcus (Cryptococcus neoformans), aspergillus (Aspergillus fumigatus, aspergillus niger etc.), Mucor (Mucor, colter is mould, head mold), middle gram sporothrix (Sporothrix schenkii), Blastomyces dermatitidis (Blastomyces dermatitidis), Paracoccidioides brasiliensis (Paracoccidioides brasiliensis), posadasis spheriforme (Coccidioides immitis) and histoplasma capsulatum (Histoplasma capsulatum).
the more parasitic examples of pathogenicity bo that cause the infection of available the inventive method treatment comprise entamoeba histolytica (Entamoeba histolytica), colon intestines basket worm (Balantidiumcoli), Fu Shi Nai Geli protozoon (Naegleriafowleri), the kind of Acanthamoeba (Acanthamoeba sp.), Lan Shi giardia lamblia (Giardia iambia), the kind of Cryptosporidium (Cryptosporidium sp.), Pneumocystis carinii (Pneumocystis carinii), Plasmodium vivax (Plasmodium vivax), vole babesia (Babesia microti), Bruce trypanosome (Trypanosoma brucei), Oswaldocruzia (Trypanosoma cruzi), Leishmania donovani (Leishmania donovani), toxoplasma gondii (Toxoplasma gondi), ancylostoma braziliense (Nippostrongylus brasiliensis).
In all aforesaid methods, O8E blocking-up can with other forms of immunotherapy such as cytokine therapy (for example Interferon, rabbit, GM-CSF, G-CSF, IL-2) or bi-specific antibody treatment associating use, provide the tumour antigen of enhancing to present (referring to, for example, Holliger (1993) Proc.Natl.Acad.Sci.USA 90:6444-6448; Poljak (1994) Structure2:1121-1123).
Autoimmune response
Anti-O8E antibody can cause and enlarge autoimmune response.In fact, use tumour cell and peptide vaccine inducing antitumor to reply to have disclosed many antitumor replying and relate to anti-autoreactivity (in the same people such as van Elsas, observe depigmentation in the B16 melanoma that anti-CTLA-4+GM-CSF-modifies, have in the mouse of Trp-2 inoculation depigmentation (Overwijk, the people such as W.. (1999) Proc.Natl.Acad.Sci.U.S.A.96.2982-2987); The TRAMP tumour-cell vaccine causes autoimmunization prostatitis (Hurwitz, A. (2000) are the same), the melanoma peptide antigen inoculation and the vitiligo (Rosenberg that observe in people's clinical trial, SA and White, DE (1996) J.Immunother Emphasis TumorImmunol 19 (1): 81-4).
Therefore, in order to design vaccination regimen, with the immunne response of effective generation for oneself protein matter, be used for disease treatment, can consider to use the various oneself protein matter of anti-O8E blocking-up associating.For example, alzheimer's disease relates to the improper accumulation of A β peptide in the amyloid beta deposition thing in brain; Can remove these amyloid beta deposition things (people such as Schenk, (1999) Nature for the antibody response of amyloid 400: 173-177).
Other oneself protein matter also can be used as target, as are used for the treatment of the IgE of transformation reactions and asthma, and are used for the TNF α of rheumatoid arthritis.At last, can utilize anti-O8E antibody antibody induction to the antibody response of multiple hormone.Neutralizing antibody can be used for contraception to replying of reproductive hormone.The reply immune target that also can be considered to possible of neutralizing antibody to the required hormone of specific tumors growth and other dissolubility factors.
The similar approach that is used for as mentioned above anti-O8E antibody can be used for the inductive treatment systemic autoimmune replys, and treats the patient of other autoantigens such as amyloid beta deposition thing (comprising the A β in alzheimer's disease) cytokine such as TNF α and IgE improper accumulation.
Vaccine
By anti-O8E antibody and target antigen (for example vaccine) administration altogether, can utilize anti-O8E antibody stimulator antigen specific immune response.Therefore, on the other hand, the invention provides in a kind of experimenter of enhancing the method to the immunne response of antigen, comprise to this experimenter and using: (i) antigen; (ii) anti-O8E antibody or its antigen-binding portion thereof make in the experimenter immunne response to antigen strengthen.Preferably, antibody is the anti-human O8E antibody of people (for example anyone anti-O8E antibody as herein described).In addition or alternately, antibody can be chimeric or humanized antibody.Antigen can be, for example, and tumour antigen, virus antigen, bacterial antigens or from the antigen of pathogenic agent.The non-limitative example of these antigens is included in those that mention in above chapters and sections, as above-mentioned tumour antigen (or tumor vaccine) or from the antigen of virus, bacterium or above-mentioned other pathogenic agent.
Be known in the art with the external suitable route of using antibody compositions of the present invention (for example human monoclonal antibodies, polyspecific and bispecific molecule and immune conjugate) in vivo, and can be selected by those skilled in the art.For example, antibody compositions can be by injection (for example intravenously or subcutaneous) administration.The optimal dose of the molecule that uses will depend on experimenter's age and concentration and/or the preparation of body weight and antibody compositions.
As previously mentioned, the anti-O8E antibody of people of the present invention can with one or more other treatment agent for example cytotoxic agent, radioactivity toxic agent or immunosuppressor co-administered.Antibody can be connected with therapeutical agent (as immunocomplex), perhaps can separate administration with therapeutical agent.For latter's (separately administration), antibody can be before therapeutical agent, afterwards or administration simultaneously, also can with other known treatment such as anticancer therapy for example radiotherapy jointly use.These therapeutical agents especially comprise, antineoplastic agent, as Dx (Zorubicin), cis-platinum, bleomycin sulfate, carmustine, Chlorambucil, Dacarbazine and endoxan, hydroxyurea, they itself only effective when the patient is had toxicity or subtoxic level.Cis-platinum is with the dosage intravenous administration of 100mg/ agent, and every 4 week 1 time, Zorubicin is with the dosage intravenous administration of 60-75mg/ml, every 21 days 1 time.The co-administered of the anti-O8E antibody of people of the present invention or its Fab and chemotherapeutics provides two kinds of carcinostatic agents, and they work by the different mechanism to human tumor cells generation cytotoxic effect.This co-administered can solve owing to developing resistance or tumor-cell antigen and sexually revise the problem that (this will make their antagonists there is no reactivity) causes.
Also comprise medicine box in scope of the present invention, this medicine box comprises antibody compositions of the present invention (for example people's antibody, polyspecific or bispecific molecule, or immune conjugate) and operation instruction.This medicine box may further include at least a other reagent or one or more other people's antibody of the present invention (for example, have and replenish active people's antibody, the epi-position on the O8E antigen of its institute's combination is different from the first antibody).Medicine box generally comprises the label of explanation test kit content target purposes.Term " label " comprises and is attached on test kit or test kit provides or any written or material record that otherwise provides with test kit.
Combination therapy
In one embodiment, the invention provides a kind of method of overmedication proliferative disease, comprise to the patient and use O8E antibody and CTLA-4 and/or PD-1 antibody.In further embodiment, anti-O8E antibody is with inferior therapeutic dose administration, and anti-CTLA-4 and/or PD-1 antibody are with inferior therapeutic dose administration, perhaps all with inferior therapeutic dose administration.In the another one embodiment, the invention provides a kind of method that changes the adverse events relevant with using immunostimulant overmedication proliferative disease, comprise anti-CTLA-4 and/or the anti-PD-1 antibody of using anti-O8E antibody and inferior therapeutic dose to the patient.In certain embodiments, the patient is the people.In certain embodiments, anti-CTLA-4 antibody is human sequence's monoclonal antibody 10D1, and anti-PD-1 antibody is human sequence's monoclonal antibody, as 17D8,2D3,4H1,5C4 and 4A11.Human sequence's monoclonal antibody 10D1 has separated and has carried out structural characterization, and as U.S. Patent No. 6,984,720 is described.Human sequence's monoclonal antibody 17D8,2D3,4H1,5C4 and 4A11 have separated and have carried out structural characterization, patent No.60/679 as interim in the U.S., and 466 is described.
Anti-O8E antibody of the present invention, anti-CTLA-4 antibody and anti-PD-1 monoclonal antibody (mAbs) and human sequence's antibody can produce by multiple technologies, comprise conventional monoclonal anti body method, for example, the described standard body hybridoma technique of Kohler and Milstein (1975) Nature 256:495.Can use the technology of any preparation monoclonal antibody, for example the virus of bone-marrow-derived lymphocyte or oncogenic transformation.A kind of preferred animal system for the preparation of hybridoma is the mouse system.Producing hybridoma with mouse is a kind of program of perfect foundation.Immune programme for children is well known in the art with separating the technology by immune spleen cell that is used for merging.Fusion partner (for example rat bone marrow tumour cell) and fusion program be also known (referring to, for example, Hariow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor New York).
By the blocking-up of O8E and PD-1 and/or CTLA-4, the antibody combination can be used for strengthening the immunne response for the hyperplasia disease.In a preferred embodiment, antibody of the present invention is people's antibody, and for example, these molecules can perhaps for example be applied to the human experimenter in vivo at external or stripped cell in being applied to cultivation, to strengthen immunity in multiple situation.Therefore, in one aspect, the invention provides the method for a kind of experimenter's of change immunne response, comprise the combination of using antibody combination of the present invention or its antigen-binding portion thereof to the experimenter, experimenter's immunne response is changed.Preferably, reply enhancing, stimulation or rise.In the another one embodiment, the invention provides a kind of method that changes the adverse events relevant with using immunostimulating therapeutical agent overmedication proliferative disease, comprise anti-CTLA-4 or the anti-PD-1 antibody of using anti-O8E antibody and inferior therapeutic dose to the experimenter.
Antibody can strengthen in the patient immunne response to cancer cells to the blocking-up of O8E, PD-1 and CTLA-4.Its growth can utilize the cancer of antibody suppression of the present invention to comprise generally has the cancer of replying to immunotherapy.The representative example of the cancer of available conjoint therapy treatment of the present invention comprises melanoma (for example metastatic malignant melanoma), kidney, prostate cancer, mammary cancer, colorectal carcinoma and lung cancer.other can comprise osteocarcinoma with the example of the cancer of method of the present invention treatment, carcinoma of the pancreas, skin carcinoma, head and neck cancer, skin or intraocular malignant melanoma, uterus carcinoma, ovarian cancer, the rectum cancer, cancer of the anal region, 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, Hokdkin disease, non_hodgkin lymphoma, esophagus cancer, carcinoma of small intestine, the endocrine system cancer, thyroid carcinoma, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, urethral carcinoma, penile cancer, chronic or acute leukemia (comprises acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia, lymphocytic leukemia), childhood solid tumor, the lymphocyte lymphoma, bladder cancer, kidney or carcinoma of ureter, carcinoma of renal pelvis, central nervous system (CNS) tumour, primary CNS lymphoma, tumor vessel occurs, tumor of spine, the brain stem neurospongioma, pituitary adenoma, Kaposi sarcoma, epidermis shape cancer, squamous cell carcinoma, t cell lymphoma, the cancer of ambient induced (cancer that comprises Induced by Asbestos), the combination of described cancer.The present invention also can be used for treating metastatic carcinoma.
In certain embodiments, the combination of therapeutic antibodies described herein can be used as the single composition administration simultaneously in the medicine acceptable carrier, and perhaps as the composition that separates administration simultaneously, every kind of antibody is in a kind of medicine acceptable carrier.In another embodiment, the combination of therapeutic antibodies administration in succession.For example, anti-O8E antibody and the administration in succession of anti-PD-1 antibody, for example at first administration of anti-O8E, then anti-PD-1 administration, perhaps at first administration of anti-PD-1, then anti-O8E administration.In addition, if in succession give once above combination therapy, when each administration, the order of administration can be put upside down in succession, perhaps keeps identical sequence, and administration can be made up with the while administration in succession, or its arbitrary combination.For example, the administration for the first time of anti-O8E antibody and the combination of anti-PD-1 antibody can be simultaneously, and administration for the second time can be carried out in succession, at first anti-O8E, anti-PD-1 then, administration for the third time can be carried out in succession, at first anti-PD-1, anti-O8E then, by that analogy.Another representational dosage regimen can comprise it being administration in succession for the first time, i.e. at first anti-PD-1, and anti-O8E then, and administration afterwards can be carried out simultaneously.
Randomly, anti-O8E and anti-CTLA-4 and/or anti-PD-1 antibody can be further united use people such as (, J.Immunol.173:4919-28 (2004)) He with the tumour antigen (comprising recombinant protein, peptide and carbohydrate molecule) of immunogenic agents such as cancer cells, purifying, cell with by the cell of the gene transfection of coding immunostimulating cytokine.The non-limitative example of operable tumor vaccine comprises the peptide of melanoma antigen, for example the tumour cell (hereinafter further describing) of express cell factor GM-CSF after the peptide of gp100, MAGE antigen, Trp-2, MARTI and/or tyrosine oxidase or transfection.
The O8E of combination and PD-1 and/or CTLA-4 blocking-up can further be made up with vaccination regimen.Designed many experimental strategies of inoculating for tumour (referring to, Rosenberg, S. (2000) Development of Cancer Vaccines, ASCO Educational BookSpring:60-62; Logothetis, C, 2000, ASCO Educational Book Spring:300-302; Khayat, D. (2000) ASCO Educational Book Spring:414-428; Foon, K. (2000) ASCO Educational Book Spring:730-738; Referring to Restifo and Sznol, Cancer Vaccines, Ch.61, the people (eds.) such as pp.3023-3043 in DeVita, 1997, Cancer:Principles and Practice of Oncology. the 5th edition).In such strategy, use to prepare vaccine from body or allogeneic tumour cell.These cell vaccines show the most effective when the transduction tumour cell makes its expression of GM-CSF.Verified for tumor inoculation, GM-CSF is the strong activator (people (1993) the Proc.NatlAcad.Sci U.S.A.90:3539-43 such as Dranoff) of antigen presentation.
Genetic expression in various tumours and extensive gene expression pattern research cause having defined so-called tumour specific antigen (Rosenberg, Immunity 10:281-7 (1999)).In many cases, these tumour specific antigens are in tumour and at the differentiation antigen of the cells that produces this tumour, for example melanocyte antigen gp100, MAGE antigen and Trp-2.The more important thing is, many such antigens may be the targets of the tumour-specific T cell found in the host.In certain embodiments, use O8E that antibody compositions as herein described makes up and PD-1 and/or CTLA-4 blocking-up to use together with the recombinant protein of expressing in collecting tumour and/or peptide, producing the immunne response to these protein.These protein are considered as autoantigen by immunity system usually, therefore to its tolerance.Tumour antigen also can comprise the protein Telomerase, it is that fringes of chromosome is synthetic needed, express in the human cancer more than 85%, and only express people such as (, Science266:2011-2013 (1994)) Kim in a limited number of bodily tissues.(can utilize multiple means to protect these bodily tissues to exempt from immune attack).Tumour antigen can be also owing to changing protein sequence or produce the somatic mutation of two kinds of fusion roteins (as the bcr-abl in Philadelphia chromosome) between irrelevant sequence and " neoantigen " of expressing or from the idiotype of B cell tumour in cancer cells.
Other tumor vaccines can comprise the protein from the virus relevant with human cancer, and described virus is for example human papillomavirus (HPV), hepatitis virus (HBV and HCV) and Ka Boxi bleb sarcoma virus (KHSV).Another form of the tumour specific antigen that can use together with O8E blocking-up is the heat shock protein(HSP) (HSP) of the purifying that separates from tumor tissues itself.These heat shock protein(HSP)s contain the fragment from the protein of tumour cell, and these HSP are sending to cause aspect tumour immunity very effectively (Suot and SrivastavaScience 269:1585-1588 (1995)) to antigen presenting cell; The people such as Tamura. (1997) Science278:117-120).
Dendritic cell (DC) are effective antigen presenting cells, can be used for causing antigen-specific and reply.The DC generation of can exsomatizing, and be loaded with range protein and peptide antigen and tumour cell extract (Nestle, the people such as F.. (1998) Nature Medicine 4:328-332).Also can by hereditary means transduction DC, make it express these tumour antigens.For immune purpose, also once DC and tumour cell were directly merged (Kugler, the people such as A.. (2000) Nature Medicine6:332-336).As a kind of inoculation method, the DC immunity can with O8E and the effective combination of PD-1 and/or CTLA-4 blocking-up of combination, activate stronger antitumor replying.
The O8E of combination and PD-1 and/or CTLA-4 blocking-up also can further be united use with standard cancer treatments.For example, the O8E of combination and PD-1 and/or CTLA-4 blocking-up can effectively be united with the chemotherapy scheme.In these situations, observe as the combination of using anti-O8E and anti-CTLA-4 and/or anti-PD-1 antibody, can reduce with the dosage that combines other chemotherapeutics of administration of the present invention (Mokyr, the people such as M.. (1998) Cancer Research.58:5301-5304).O8E and PD-1 and/or CTLA-4 blocking-up are that the necrocytosis meeting that the cytotoxic effect of most of chemotherapy compounds causes causes the tumour antigen level in the antigen presentation approach to raise with the principles of science of chemotherapy combined utilization.Can comprise that radiotherapy, operation or hormone deprive by necrocytosis and O8E and PD-1 and/or other collaborative combination therapys of CTLA-4 blocking-up.These schemes all produce tumour antigen and originate in the host.Angiogenesis inhibitor also can with O8E and PD-1 and/or the CTLA-4 blocking-up associating use of combination.The inhibition of vasculogenesis causes death of neoplastic cells, and death of neoplastic cells can add to host antigen with tumour antigen and present in approach.
The combination of O8E and PD-1 and/or CTLA-4 blocking antibody also can with the bi-specific antibody that the effector cell that will express Fc α or Fc γ acceptor is directed to tumour unite use (referring to, for example, United States Patent(USP) Nos. 5,922,845 and 5,837,243).The bi-specific antibody two kinds of different antigens that can be used for leading.For example, anti-Fc acceptor/tumor-resistant antigen (for example Her-2/neu) bi-specific antibody has been used for scavenger cell is directed to tumor locus.This guiding can more effectively activate tumour-specific and reply.Utilize combination O8E and PD-1 and/or CTLA-4 blocking-up can strengthen the T cell part that these are replied.In addition, can utilize the bi-specific antibody in conjunction with tumour antigen and dendritic cell specific cell surface marker that antigen directly is delivered to DC.
In another example, the combination of anti-PD-1 and anti-CTLA-4 antibody can be used together with anti-tumour antibody, described anti-tumour antibody such as Rituxan
Figure S2006800460653D00811
(Rituximab), Trastuzumab (Herceptin
Figure S2006800460653D00812
) (Herceptin), Bexxar (tositumomab), Zevalin
Figure S2006800460653D00814
(ibritumomab), Campath
Figure S2006800460653D00815
(A Lun pearl monoclonal antibody), Lymphocide
Figure S2006800460653D00816
(epratuzumab), Avastin
Figure S2006800460653D00817
(Avastin) and Tarceva
Figure S2006800460653D00818
(erlotinib) etc.For example, do not wish to be bound by theory, use anticancrin or can cause cancer cells (for example tumour cell) death with the treatment of toxin conjugated anticancrin, this will strengthen the immunne response that is mediated by O8E, CTLA-4 or PD-1.In an exemplary embodiment, overmedication proliferative disease (for example cancerous tumour) can comprise anticancrin and anti-O8E and anti-PD-1 and/or anti-CTLA-4 antibody while or in succession unite use, perhaps its arbitrary combination, this can strengthen host's anti-tumor immune response.
Tumour is escaped host's immunosurveillance by number of mechanisms.The inhibitive ability of immunity protein of expressing by the deactivation tumour can overcome many such mechanism.Especially comprise TGF-β (Kehrl, J. wait the people. (1986) J.Exp.Med.163:1037-1050), IL-10 (Howard, M. and O ' Garra, A. (1992) Immunology Today 13:198-200) and FasL (Hahne, the people such as M.. (1996) Science 274:1363-1365).In the another one example, can be further unite use with anti-O8E and anti-PD-1 and/or anti-CTLA-4 for the antibody of these materials, offsetting the effect of immunosuppressor, and be conducive to host's tumor immune response.
Other antibody that can be used for activating host immune response can use with the combinatorial association of anti-O8E and anti-PD-1 and/or anti-CTLA-4.Comprise the molecule on the surface of dendritic cells that activates DC function and antigen presentation.Anti-cd40 antibody can effectively replace T cell helper activity (Ridge, J. wait the people. (1998) Nature 393:474-478), and demonstration use together with anti-CTLA-4 effectively (Ito, the people such as N.. (2000) Immunobiology 201 (5) 527-40).Activate for T cell co-stimulatory molecules such as OX-40 (Weinberg, the people such as A.. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I. wait the people. (1997) Nature Medicine 3:682-685 (1997), PD-1 (people such as del Rio. (2005) Eur J Immunol.35:3545-60) and ICOS (Hutloff, the people such as A.. (1999) Nature 397:262-266) antibody the T cell activation level of raising also can be provided.
The current tumour that is used for treating multiple hematopoiesis source of bone marrow transplantation.Graft versus host disease is the consequence of this treatment, but can be from graft to acquisition treatment benefit the replying of tumour.Combination O8E and PD-1 and/or CTLA-4 blocking-up can be used for improving the validity of the tumour-specific T cell that donor transplants.
Also there are several experimental therapy schemes to comprise exsomatize activation and expansion of antigen specific T-cells, and with these cell adoptive transfers in the receptor, to produce T cells with antigenic specificity (Greenberg, R. and Riddell, S. (1999) Science for tumour 285: 546-51).Also can utilize these methods activation for the t cell response of infectious agent such as CMV.Stripped activation expection under anti-O8E and anti-PD-1 and/or the existence of anti-CTLA-4 antibody can improve frequency and the activity of the T cell of adoptive transfer.
As described herein, organ may show Ia adverse events after immunostimulating treatment Antybody therapy, for example the gi tract (diarrhoea and colitis) after anti-CTLA-4 treatment and skin (rash with itch) event.For example, after anti-CTLA-4 Antybody therapy, also observe the gi tract Immune interrelation adverse events of non-colon in esophagus (duodenitis) and ileum (ileitis).
In certain embodiments, the invention provides a kind of method that changes the adverse events relevant with using immunostimulant overmedication proliferative disease, comprise the anti-CTLA-4 antibody of using anti-O8E antibody and inferior therapeutic dose to the patient.For example, method of the present invention provides a kind of method that reduces the sickness rate of colitis that immunostimulating treatment antibody brings out or diarrhoea by use the nonabsorbable steroid to the patient.All have colitis that this antibody brings out and the danger of diarrhoea occur because accept any patient of immunostimulating treatment antibody, whole patient colony is fit to the method according to this invention treatment.Treat inflammatory bowel (IBD) and prevent that IBD from increasing the weight of although used steroid, it does not also have to be used for preventing that (reduction sickness rate) is not diagnosed as the patient's of IBD IBD.To steroid, important side effect that even the nonabsorbable steroid is relevant hindered its prophylactic applications.
In further embodiment, the combination of O8E and PD-1 and/or CTLA-4 blocking-up (being the immunostimulating treatment anti-O8E of antibody and anti-PD-1 and/or anti-CTLA-4) can further be made up with using any nonabsorbable steroid.Therefore " nonabsorbable steroid " used herein is a kind of glucocorticosteroid, and it shows first pass metabolism widely, and in liver after metabolism, the bioavailability of this steroid is lower, namely lower than about 20%.In one embodiment of the invention, the nonabsorbable steroid is budesonide.Budesonide is a kind of glucocorticosteroid of local action, and it is in the extensive metabolism of oral rear quilt, mainly by liver metabolism.ENTOCORT EC (Astra-Zeneca) be a kind of for optimizing the oral budesonide formulation that depends on pH and time of developing to ileum and whole colon administration.ENTOCORT EC
Figure S2006800460653D00832
Be approved for treatment in the U.S. and involve ileum and/or colon ascendens light to moderate Crohn disease.ENTOCORTEC
Figure S2006800460653D00833
The oral dosage commonly used for the treatment of Crohn disease is 6-9mg/ days.ENTOCORT EC
Figure S2006800460653D00834
Absorbed by intestinal mucosa and keep after discharging in intestines.In case it arrives target tissue by intestinal mucosa, ENTOCORT EC
Figure S2006800460653D00835
Be just the metabolite with insignificant glucocorticoid activity by the extensive metabolism of the cytochrome p450 system in liver.Therefore, bioavailability lower (about 10%).The low bioavailability of budesonide causes its treatment ratio higher than the lower glucocorticosteroid of other first pass metabolism degree.Budesonide produces less side effect, comprises that the Hypothalamus-pituitary lower than the glucocorticosteroid of general action suppresses.Yet, ENTOCORT EC
Figure S2006800460653D00836
Long term administration can cause the whole body glucocorticoid efficiency, suppresses as hyperadrenalism and suprarenal gland.Referring to PDR 58 thEd.2004; 608-610.
In further embodiment, combination O8E and PD-1 and/or CTLA-4 blocking-up (being the immunostimulating treatment anti-O8E of antibody and anti-PD-1 and/or anti-CTLA-4) can further be made up with salicylate with the combination of nonabsorbable steroid.Salicylate comprises the 5-ASA agent, for example: sulfasalazine (AZULFIDINE
Figure S2006800460653D00837
, Pharmacia ﹠amp; UpJohn); Olsalazine (DIPENTUM
Figure S2006800460653D00838
, Pharmacia ﹠amp; UpJohn); Balsalazide (COLAZAL
Figure S2006800460653D00839
, SalixPharmaceuticals, Inc.); And mesalazine (ASACOL
Figure S2006800460653D00841
, Procter ﹠amp; GamblePharmaceuticals; PENTASA , Shire US; CANASA
Figure S2006800460653D00843
, AxcanScandipharm, Inc.; ROWASA
Figure S2006800460653D00844
, Solvay).
The method according to this invention, salicylate and anti-O8E and anti-PD-1 and/or anti--CTLA-4 antibody and nonabsorbable steroid Combined Preparation can comprise any overlapping or administration in succession of salicylate and nonabsorbable steroid, and purpose is to reduce the sickness rate of the colitis of immunostimulating antibody induction.Therefore, for example, the method of sickness rate that reduces the colitis of immunostimulating antibody induction according to the present invention comprises simultaneously or sequential application salicylate and nonabsorbable steroid (for example, using salicylate after 6 hours in the administration of nonabsorbable steroid) or its arbitrary combination.Further, according to the present invention, salicylate and nonabsorbable steroid can be by identical administration (for example, be oral) or by different approaches administration (for example salicylate oral and the administration of nonabsorbable steroid per rectum), this approach can be different from the route of administration of anti-O8E and anti-PD-1 and anti--CTLA-4 antibody.
Have the complement binding site (as from IgG1 ,-2 or-3 or the complement bound fraction of IgM) composition of the present invention (for example people's antibody, polyspecific and bispecific molecule and immune conjugate), also can use under the existence of complement.In one embodiment, the cell colony that contains target cell with wedding agent of the present invention and suitable effector cell's ex vivo treatment can be realized by the serum that adds complement or contain complement.The combination of complement proteins can improve the phagolysis with the coated target cell of wedding agent of the present invention.In another embodiment, also can be by the complement cracking with the coated target cell of composition of the present invention (for example people's antibody, polyspecific and bispecific molecule).In another embodiment, composition of the present invention activating complement not.
Composition of the present invention (for example people's antibody, polyspecific and bispecific molecule and immune conjugate) also can be used together with complement.Therefore, the composition that contains people's antibody, polyspecific or bispecific molecule and serum or complement also within the scope of the invention.These compositions are favourable, because complement and people's antibody, polyspecific or bispecific molecule close proximity.Alternately, people's antibody of the present invention, polyspecific or bispecific molecule and complement or serum also can separate administration.
Therefore, use in addition another kind of therapeutical agent can for patient with antibody compositions treatment of the present invention (before people's antibody administration of the present invention, while or afterwards), as cytotoxic agent or radioactivity toxic agent, this therapeutical agent can strengthen or increase the result for the treatment of of people's antibody.
In the other embodiment, can in addition with the expression of regulating (for example enhancer or inhibitor) Fc γ or Fc γ acceptor or active pharmacological agent experimenter, for example use the cytokine therapy experimenter.The preferred cell factor of using in polyspecific molecular therapy process comprises granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimutaing factor (GM-CSF), interferon-γ (IFN-γ) and tumour necrosis factor (TNF).
Composition of the present invention (for example people's antibody, polyspecific and bispecific molecule) also can be used for leading and express the cell of Fc γ R or O8E, for example these cells of mark.For this application, can be in connection with agent and minute sub-connection that can be detected.Therefore, the invention provides and exsomatize or in the method for the cell of external localization and expression Fc acceptor such as Fc γ R or O8E.Detectable can be, for example radio isotope, fluorescent chemicals, enzyme or enzyme cofactor.
In a particular, the invention provides the existence of O8E antigen in test sample or measure the method for O8E antigen amount, be included in make under the condition that allows to form between antibody or its part and O8E mixture sample contact with control sample can with human monoclonal antibodies or its antigen-binding portion thereof of O8E specific binding.Then detect complex formation, wherein sample is compared the existence of O8E antigen in the difference indication sample that mixture forms with control sample.
In other embodiments, the invention provides the method for the disease of O8E mediation in the treatment experimenter.
In another embodiment, by compound is connected with antibody, can utilize immune conjugate of the present invention with this compound (such as therapeutical agent, marker, cytotoxin, radiation toxin, immunosuppressor etc.) target to the cell with O8E cell surface receptor.For example, anti-O8E antibody can with as U.S. Patent application 10/160,972,10/161,233,10/161,234,11/134,826,11/134,685 and U.S. Provisional Patent Application No.60/720,499 described UPT and/or U.S. Patent No. 6,81,354 and 6,548,530, the open Nos.20030050331,20030064984 of United States Patent (USP), 20030073852 and 20040087497 described or disclosed any toxin compound couplings of WO03/022806.Therefore, the present invention also is provided for exsomatizing or the method for the cell of localization and expression O8E (for example using detectable, as radio isotope, fluorescent chemicals, enzyme or enzyme cofactor) in vivo.Alternately, immune conjugate passes through cytotoxin or radiates the toxin target to O8E, also can kill and wound the cell with O8E cell surface receptor.
The present invention further sets forth by the following examples, these embodiment should be interpreted as further restriction.All the content of accompanying drawing and whole reference of quoting in this application, patent and publication application all is incorporated herein by reference.
Embodiment
Embodiment 1
The generation of anti-O8E human monoclonal antibodies
The present embodiment discloses the generation with the human monoclonal antibodies of people O8E (a/k/a B7H4, B7S1 and B7x) specific binding.
Antigen
Utilize standard restructuring transfection method, with O8E transfection CHO and HEK-293 cell, as immunizing antigen.In addition, independent recombinant human O8E is also as immunizing antigen.
Transgenosis HuMAb mouse
Figure S2006800460653D00861
With the KM mouse
Figure S2006800460653D00862
With the transgenosis HuMAb mouse of expressing human immunoglobulin gene
Figure S2006800460653D00863
The complete human monoclonal antibody of HCo7 and HCo12 system and the anti-O8E of transgenosis transchromosomic mice KM system's preparation.In each of these mouse systems, endogenous mouse κ light chain gene is destroyed as described in J.12:811-820 as (1993) EMBO such as Chen with isozygotying, and announced as PCT and the endogenous mouse heavy chain gene has been destroyed as described in the embodiment 1 of WO 01/09187 with isozygotying.These mouse are all to carry as the described human kappa light chain transgenosis of (1996) Nature Biotechnology 14:845-851 KCo5 such as Fishwild.The HCo7 frenulum is just like United States Patent(USP) Nos. 5,545,806; 5,625,825; With 5,545,807 described HCo7 people's heavy chain transgenosiss.The HCo12 frenulum is announced the HCo12 people's heavy chain transgenosis described in WO01/09187 embodiment 2 just like PCT.The KM mouse
Figure S2006800460653D00864
System contains the described SC20 transfection chromosome just like PCT announcement WO 02/43478.
The immunity of HuMAb and KM:
In order to produce the complete human monoclonal antibody of anti-O8E, with cell, the cell of HEK-293-O8E transfection and/or the restructuring O8E protein immunization HuMAb mouse of purifying of CHO-O8E transfection With the KM mouse
Figure S2006800460653D00872
Be used for the HuMab mouse
Figure S2006800460653D00873
General immunization protocol at Lonberg, N. etc. (1994) Nature 368 (6474): 856-859; Fishwild describes in the open WO 98/24884 of D. etc. (1996) NatureBiotechnology 14:845-851 and PCT.Mouse is 6-16 age in week when infusion antigen for the first time.Utilize the immune HuMAb mouse of restructuring O8E protein product (5-50 μ g) of purifying TMWith the KM mouse TM
Transgenic mice is used in antigen intraperitoneal (IP) or subcutaneous (Sc) immunity twice in complete Freund's adjuvant, then is used in antigen intraperitoneal or subcutaneous inoculation 3-21 days (can reach at most 11 immunity altogether) in incomplete Freund's adjuvant.By blood sampling monitoring immunne response after eye socket.By ELISA, blood plasma is screened (as mentioned below), merge with having the mouse that enough anti-O8E human normal immunoglobulins tire.Through intravenously, mouse is carried out booster immunization with antigen, put to death mouse after 3 days and 2 days, and take out spleen.Every kind of antigen generally carries out 10-35 time and merges.Tens mouse of every kind of antigen immune.
Produce the HuMAb mouse of anti-O8E antibody
Figure S2006800460653D00874
Or KM mouse
Figure S2006800460653D00875
Selection
In order to select to produce the HuMAb mouse of the antibody that can be combined with O8E TMOr KM mouse TM, by as Fishwild, the described flow cytometry of D. etc. (1996) (the same) detects from by the serum of immune mouse.In brief, the purification of Recombinant O8E fusion rotein that is used in concentration in PBS and is 1-2 μ g/ml is with the coated microtiter plate of amount in 100 μ l/ holes, and then 4 ℃ of lower overnight incubation use 5% chicken serum in PBS/Tween (0.05%) with 200 μ l/ hole sealings.To add in each hole from the plasma extender of the mouse of O8E immunity, at room temperature hatched 1-2 hour.Wash plate with PBS/Tween, then at room temperature hatched 1 hour with the anti-human IgGFc polyclonal antibody of goat of horseradish peroxidase (HRP) coupling.After washing, culture plate is with ABTS substrate (Sigma, A-1888,0.22mg/ml) colour developing, and with spectrophotometer in the analysis of OD 415-495 place.Merge with the mouse that shows high resistance O8E antibody titer.The as mentioned below fusion, and active by the anti-O8E of ELISA and FACS detection hybridoma supernatant liquor.
Produce the generation of the hybridoma of anti-O8E human monoclonal antibodies
PEG will be from the HuMAb mouse according to the standard program use TMAnd/or KM mouse TMThe mouse boosting cell of middle separation and mouse myeloma cell line merge.Then screen according to the generation of antigen-specific antibodies the hybridoma that obtains.Use 50%PEG (Sigma) will be from being merged by the SP2/0 nonsecreting type murine myeloma cell (ATCC CRL 1581) of the splenocyte single cell suspension of immune mouse and 1/4 quantity.With cell with approximately 1 * 10 5The density in/hole is inoculated on flat-bottom microtiter plates, then hatches about two weeks in selective medium, and this selective medium is being added with 5mM HEPES, 0.055mM beta-mercaptoethanol, 50mg/ml gentamicin and 1 * HAT (Sigma; CRL P-7185) DMEM (Mediatech, CRL 10013, contain high concentration glucose, L-glutaminate and Sodium.alpha.-ketopropionate) in contain 10% foetal calf serum (Hyclone, Logan, UT), 10%P388DI (ATCC, CRL TIB-63) condition matrix, 3-5%origen (IGEN).After one to two week, culturing cell in the substratum of having replaced HAT with HT.Then by ELISA and FACS (as mentioned above) for each hole of the anti-O8E mono-clonal of people IgG antibody screening.Then by ELISA use the O8E recombinant protein or by FACS use express O8E cell for example the cell of CHO-O8E transfection for O8E positive antibody screening positive clone.In brief, results are expressed the cell of O8E from tissue culture flasks, and the preparation single cell suspension.Express the cell suspension first antibody substantive dyeing of O8E, perhaps with the fixing poststaining of the PBS solution of 1% paraformaldehyde.About 1,000,000 cells are resuspended in the PBS that contains 0.5%BSA and 50-500 μ g/ml first antibody, and hatched on ice 30 minutes.With containing 0.1%BSA, 0.01%NaN 3PBS washed cell twice, it is resuspended in 100 μ l1: in the mountain goat anti-human igg (Jackson ImmunoResearch, WestGrove, PA) of FITC couplings of 100 dilutions, and hatched again on ice 30 minutes.Washed cell twice again, is resuspended in the 0.5ml lavation buffer solution, with the dyeing of FACSScalibur hematimeter (Becton-Dickinson, San Jose, CA) analysis of fluorescence.
In case hybridoma growth widely occurs, usually monitored substratum after 10-14 days.With the hybridoma of secretory antibody plating again, screening again and if remain positive for human IgG, will resist O8E monoclonal antibody subclone at least twice by limiting dilution.Then at the stable subclone of vitro culture, be used for further characterizing to produce a small amount of antibody in tissue culture medium (TCM).
Select hybridoma clone 1G11,2A7,2F9,12E1 and 13D12 further to analyze.
Embodiment 2
The structural characterization of human monoclonal antibodies 1G11,2A7,2F9,12E1 and 13D12
The present embodiment discloses the sequential analysis of five species specificity in conjunction with the human monoclonal antibodies of O8E.
Utilize Standard PC R technology to obtain respectively the heavy chain of coding 1G11,2A7,2F9,12E1 and 13D12 monoclonal antibody and the cDNA sequence of variable region of light chain from 1G11,2A7,2F9,12E1 and 13D12 hybridoma, and utilize the standard DNA sequencing technologies to check order.
The Nucleotide of the variable region of heavy chain of 1G11 and aminoacid sequence are shown in Figure 1A and SEQ ID NO:41 and 1.
The Nucleotide of the variable region of light chain of 1G11 and aminoacid sequence are shown in Figure 1B and SEQ ID NO:46 and 6.
It is the VH section of VH 4-34 that the relatively proof of 1G11 heavy chain immunoglobulin sequence and known person racial immunity sphaeroprotein sequence of heavy chain, 1G11 heavy chain have been used from ethnic group.The 1G11VH sequence is that comparing of VH 4-34 sequence is presented in Fig. 6 with planting.Utilize Kabat CDR district mensuration system to delineate out heavy chain CDR1, CDR2 and CDR3 district to the further analysis of 1G11 VH sequence, respectively as Figure 1A and 6 and SEQ ID NO:11,16 and 21 as shown in.
It is the VL section of VK A27 that the relatively proof of 1G11 light chain immunoglobulin sequences and known person racial immunity sphaeroprotein sequence of light chain, 1G11 light chain have been used from ethnic group.1G11 VL sequence is that comparing of VK A27 sequence is shown in Fig. 9 with planting.Utilize Kabat CDR district mensuration system to delineate out light chain CDR1, CDR2 and CDR3 district to the further analysis of 1G11 VL sequence, respectively as Figure 1B and 9 and SEQ ID NO:26,31 and 36 as shown in.
The Nucleotide of the variable region of heavy chain of 2A7 and aminoacid sequence are shown in Fig. 2 A and SEQID NO:42 and 2.
The Nucleotide of the variable region of light chain of 2A7 and aminoacid sequence are shown in Fig. 2 B and SEQID NO:47 and 7.
The relatively proof of 2A7 heavy chain immunoglobulin sequence and known person racial immunity sphaeroprotein sequence of heavy chain, 2A7 heavy chain have been used from ethnic group and have been the VH section of VH 3-53 and are the JH section of JH 6b from ethnic group.2A7 VH sequence is that comparing of VH 3-53 sequence is shown in Fig. 7 with planting.Utilize Kabat CDR district mensuration system to delineate out heavy chain CDR1, CDR2 and CDR3 district to the further analysis of 2A7 VH sequence, respectively as Fig. 2 A and 7 and SEQ ID NO:12,17 and 22 as shown in.
It is the VL section of VK A27 that the relatively proof of 2A7 light chain immunoglobulin sequences and known person racial immunity sphaeroprotein sequence of light chain, 2A7 light chain have been used from ethnic group.The 2A7VL sequence is that comparing of VK L18 sequence is shown in Fig. 9 with planting.Utilize Kabat CDR district mensuration system to delineate out light chain CDR1, CDR2 and CDR3 district to the further analysis of 2A7 VL sequence, respectively as Fig. 2 B and 9 and SEQ ID NO:27,32 and 37 as shown in.
The Nucleotide of the variable region of heavy chain of 2F9 and aminoacid sequence are shown in Fig. 3 A and SEQID NO:43 and 3.
The Nucleotide of the variable region of light chain of 2F9 and aminoacid sequence are shown in Fig. 3 B and SEQID NO:48 and 8.
The relatively proof of 2F9 heavy chain immunoglobulin sequence and known person racial immunity sphaeroprotein sequence of heavy chain, 2F9 heavy chain have been used from ethnic group and have been the VH section of VH 3-53 and are the JH section of JH 6b from ethnic group.The 2F9VH sequence is that comparing of VH 3-53 sequence is shown in Fig. 7 with planting.Utilize Kabat CDR district mensuration system to delineate out heavy chain CDR1, CDR2 and CDR3 district to the further analysis of 2F9VH sequence, respectively as Fig. 3 A and 7 and SEQ ID NO:13,18 and 23 as shown in.
It is the VL section of VK A27 that the relatively proof of 2F9 light chain immunoglobulin sequences and known person racial immunity sphaeroprotein sequence of light chain, 2F9 light chain have been used from ethnic group.The 2F9VL sequence is that comparing of VK A27 sequence is shown in Fig. 9 with planting.Utilize Kabat CDR district mensuration system to delineate out light chain CDR1, CDR2 and CDR3 district to the further analysis of 2F9VL sequence, respectively as Fig. 3 B and 9 and SEQ ID NO:28,33 and 38 as shown in.
The Nucleotide of the variable region of heavy chain of 12E1 and aminoacid sequence are shown in Fig. 4 A and SEQ ID NO:44 and 4.
The Nucleotide of the variable region of light chain of 12E1 and aminoacid sequence are shown in Fig. 4 B and SEQ ID NO:49 and 9.
The relatively proof of 12E1 heavy chain immunoglobulin sequence and known person racial immunity sphaeroprotein sequence of heavy chain, 12E1 heavy chain used from ethnic group be VH 3-9 the VH section, be the D section of 3-10 and be the JH section of JH 6b from ethnic group from ethnic group.12E 1VH sequence is that comparing of VH3-9 sequence is shown in Fig. 8 with planting.Utilize Kabat CDR district mensuration system to delineate out heavy chain CDR1, CDR2 and CDR3 district to the further analysis of 12E1 VH sequence, respectively as Fig. 3 A and 8 and SEQ ID NO:14,19 and 24 as shown in.
The relatively proof of 12E1 light chain immunoglobulin sequences and known person racial immunity sphaeroprotein sequence of light chain, 12E1 light chain have been used from ethnic group and have been the VL section of VK L6 and are the JK section of JK 1 from ethnic group.The 12E1VL sequence is that comparing of VK L6 sequence is shown in Figure 10 with planting.Utilize Kabat CDR district mensuration system to delineate out light chain CDR1, CDR2 and CDR3 district to the further analysis of 12E1 VL sequence, respectively as Fig. 3 B and 10 and SEQ ID NO:29,34 and 39 as shown in.
The Nucleotide of the variable region of heavy chain of 13D12 and aminoacid sequence are shown in Fig. 5 A and SEQ ID NO:45 and 5.
The Nucleotide of the variable region of light chain of 13D 12 and aminoacid sequence are shown in Fig. 5 B and SEQ ID NO:50 and 10.
It is the VH section of VH 4-34 that the relatively proof of 13D12 heavy chain immunoglobulin sequence and known person racial immunity sphaeroprotein sequence of heavy chain, 13D12 heavy chain have been used from ethnic group.The 13D12VH sequence is that comparing of VH 4-34 sequence is shown in Fig. 6 with planting.Utilize Kabat CDR district mensuration system to delineate out heavy chain CDR1, CDR2 and CDR3 district to the further analysis of 13D 12VH sequence, respectively as Fig. 5 A and 6 and SEQ ID NO:15,20 and 25 as shown in.
It is the VL section of VK A27 that the relatively proof of 13D12 light chain immunoglobulin sequences and known person racial immunity sphaeroprotein sequence of light chain, 13D12 light chain have been used from ethnic group.13D12 VL sequence is that comparing of VK A27 sequence is shown in Fig. 9 with planting.Utilize Kabat CDR district mensuration system to delineate out light chain CDR1, CDR2 and CDR3 district to the further analysis of 13D12 VL sequence, respectively as Fig. 5 B and 9 and SEQ ID NO:30,35 and 40 as shown in.
Embodiment 3
The sign of the binding specificity of anti-O8E human monoclonal antibodies
The present embodiment discloses the combination by the O8E of the more anti-O8E antibody of standard ELISA and Immunological purification, is used for detecting the binding specificity for O8E.
Spend the night with the coated flat board of O8E restructuring myc-mark with restructuring His-mark, then detect the combination with anti-O8E human monoclonal antibodies 2A7,12E1 and 13D12.Carry out the standard ELISA program.Concentration with 1 μ g/ml adds anti-O8E human monoclonal antibodies, and with the downward titration of serial dilution degree of 1: 2.Mountain goat anti-human igg (Fc or the κ chain specificity) polyclonal antibody of use and horseradish peroxidase (HRP) coupling is as second antibody.
By albumin A chromatography purification of Recombinant B7H4-Ig from the supernatant liquor of the 293T cell of use B7H4-Ig construct transfection.Then employment antibody sandwich elisa plate adds the protein of purifying, then detects with the anti-B7H4 antiserum(antisera) of rabbit.Referring to Figure 11 A.Use the 2A7 affinity column by chromatography from have the restructuring Penta-B7H4 of C-9 label with purifying the supernatant liquor of the 293T cell of Penta-B7H4-C9 construct transfection.With the coated elisa plate of anti-mouse Fc, then add monoclonal anti-C9 (0.6ug/ml), then Penta-B7H4 titration shown in then using is people's antibody of 1ug/ml.Then coated anti-mouse Fc is that M-resists-C9 (0.6ug/ml), and then Penta-O8E titration shown in then using is people's antibody of 1ug/ml.See Figure 11 B.
Anti-O8E human monoclonal antibodies 2A7,12E1 and 13D 12 are combined with the O8E high specific.
Embodiment 4
The sign that the O8E that anti-O8E antibody is expressed on the breast cancer cell line surface is combined
The present embodiment discloses by the anti-O8E antibody of Flow cytometry and CHO-O8E (a/k/a B7H4, B7S1 and B7x) transfectant and expressed the combination of the mammary gland cell cancer cells of O8E on its cell surface.
Detect with the Chinese hamster ovary celI system of O8E transfection and the antibodies of mammary gland cell cancerous cell line SKBR3 (ATCC preserving number HTB-30).The anti-O8E human monoclonal antibodies of HuMAb 2A7 in conjunction with following evaluation: with 1 * 10 5Individual cell is hatched together with 2A7 that concentration is 1 μ g/ml.Washed cell detects its combination with the anti-human IgG Ab of FITC mark.Use FACScan flow cytometer (Becton Dickinson, San Jose, CA) to carry out flow cytometry.Result is presented in Figure 12 and 13.
The anti-O8E HuMAb of these digital proofs is combined with the Chinese hamster ovary celI of expressing O8E, and is combined with the mammary gland cell cancerous cell line of example.
Embodiment 5
The Scatchard of the binding affinity of anti-O8E monoclonal antibody analyzes
The present embodiment discloses and has adopted Scatchard analyzing and testing human monoclonal antibodies 1G11,2F9,2A7,12E1 and 13D12 to the binding affinity of the HEK clone of O8E transfection.
Application standard technology total length O8E transfection HEK cell, and it is grown in the RPMI substratum that contains 10% foetal calf serum (FBS).(Figure 12 demonstration utilizes the anti-O8E monoclonal antibody of 2A7 people to the facs analysis of these HEK-O8E cells) with this cell of tryptic digestion, uses binding buffer liquid (24mM Tris pH 7.2,137mM NaCl, 2.7mMKCl, 2mM glucose, 1mM CaCl based on Tris 2, 1mM MgCl 2, 0.1%BSA) wash once, with binding buffer liquid with Cell regulate to 2 * 10 6Cell/ml.Millipore plate (MAFB NOB) is coated with 1% skim milk powder aqueous solution, and stores under 4 ℃ and spend the night.Wash plate three times with 0.2ml binding buffer liquid.Add independent 50 microlitre damping fluids (total binding) in the maximum combined hole.Add independent 25 microlitre damping fluids (non-specific binding) in control wells.To add volume in porose be the different concns of 25 μ l 125I-resists-O8E antibody.(in some cases, carry out titration with the antibody of FITC mark, because the non-marked material can't obtain, in these cases in conjunction with being affected.) to add volume in the control wells be the unlabelled antibody of 100 times of excessive different concns of 25 μ l, and to add the Chinese hamster ovary celI (2 * 10 of the O8E transfection of 25 μ l in binding buffer liquid in porose 6Cell/ml).Plate was hatched 2 hours with 200RPM on 4 ℃ of shaking tables.When hatching end, wash damping fluid (24mM Tris pH 7.2,500mMNaCl, 2.7mM KCl, 2mM glucose, 1mM CaCl with the 0.2ml cold wash 2, 1mM MgCl 2, 0.1%BSA) wash the Millipore plate three times.Take out filter membrane, count with gamma counter.Use Prism software (SanDiego, CA) to carry out the evaluation of balance combination with the unit point incorporating parametric.
Use S shape dose response (PRIZM TM) by the nonlinear regression analysis data, calculate thus EC50, sort with the EC50 antagonist, as shown in table 2.The EC50 value of calculating in these experiments is the qualitative value of affinity of antibody, does not represent the absolute avidity for O8E.
Table 2
Antibody EC50 95％CI
2F9.E6-FITC 407pM 250-663pM
13D12.G10 746pM 569-979pM
2A7.C11 750pM 519pM-1nM
1G11.H11-FITC 1.69nM 1.4-2.0nM
12E1.G9 * 19.8pM 14-27.6nM
*Low value and high value are as the constant adjustment, to compensate incomplete curve.
Embodiment 6
The internalization of anti-O8E monoclonal antibody
The present embodiment proof uses the test of Hum-Zap internalization to detect the ability of anti-O8E HuMAb internalization in the CHO that expresses O8E and the breast cancer cell.The Hum-Zap test is by the internalization in conjunction with the first antibody of detection of second antibody, and this second antibody has being coupled to the affinity of the human IgG on toxin saporin (saporin).
To express the breast cancer cell line SKBR3 of O8E with 1.25 * 10 4The density of cells/well is inoculated in 100 μ l holes spends the night.It is anti-O8E HuMAb antibody 1G11,2F9,2A7,12E1 or the 13D12 of 10pM that Xiang Kongzhong adds concentration.Use to the nonspecific isotype control antibodies of O8E as negative control.Concentration with 11nM adds Hum-Zap (AdvancedTargeting Systems, San Diego, CA, IT-22-25), and plate was hatched 72 hours.Then use 1.0Ci 3The H-thymidine carried out pulse 24 hours to plate, and results are with Top Count scintillation counter (Packard Instruments, Meriden, CT) reading.Result is presented in following table 3 and Figure 14-15.Anti-O8E antibody 1G11,2F9,2A7,12E 1 or 13D 12 show, in the SKBR3 breast cancer cell of expressing O8E 3The H-thymidine mixes and depends on the antibody concentration reduction.
The anti-O8E antibody of these digital proofs 1G11,2F9,2A7,12E1 and 13D12 internalization are in breast cancer cell line.
Table 3
Figure S2006800460653D00941
The order of the internalization efficient in two experiments of three experiments of SKBR3 and CHO-O8E is averaged.Internalization order, and the EC50 of being combined with CHO-O8E show in table 4 and table 5.Result shows that internalization efficient is not directly related with binding affinity, and the prompting internalization depends on epi-position.
Table 4 is according to the internalization efficient of the internalization sorting in the SBKR3 breast cancer cell line
Figure S2006800460653D00951
Table 5 is according to the internalization efficient of the internalization sorting in CHO-O8E clone
Figure S2006800460653D00952
End user's monoclonal antibody 2A7,2F9 and 1G11 measure the internalization of saporin conjugate in CHO-O8E with dose response active on the scope of about 500pM to 1pM.As shown in figure 14, internalization is very effective, and EC50 is in low pM scope.CHO parental cell system and Hu IgG-SAP are as negative control, and showing does not have remarkable background toxicity or non-specific internalization.Anti-O8E uses together with the SKBR3 cell with the direct conjugate of SAP.Figure 15 shows the internalization per-cent (with respect to contrast) as the Ig-SAP dose function.
Embodiment 7
The evaluation of toxin conjugated anti-O8E antibody to the cell killing of mammary gland cell cancerous cell line
This embodiment discloses and has utilized cell proliferation test detection and toxin conjugated anti-O8E monoclonal antibody to kill and wound O8E +The ability of mammary gland cell cancerous cell line.
Anti-O8E HuMAb antibody 1G11,2F9,2A7,12E 1 or 13D12 can be with toxin by linker couplings such as peptide, hydrazone or disulphide linker.To express the breast cancer cell line of O8E such as SKBR3 with about 1-3 * 10 4The density of cells/well is seeded in 100 μ l holes 3 hours.It is the anti-O8E antibody-toxin conjugated thing of 30nM that Xiang Kongzhong adds starting point concentration, and with the downward titration of serial dilution degree in 1: 3.Use for the nonspecific isotype control antibodies of O8E as negative control.Plate was hatched 69 hours.Then use 1.0Ci 3The H-thymidine carried out pulse 24 hours to plate, results, and with Top Count scintillometer (Packard Instruments, Meriden, CT) reading.Be expected in the breast cancer cell of expressing O8E, anti-O8E antibody shows that antibody-toxin concentration is dependent 3The H-thymidine mixes minimizing.The anti-O8E antibody of this digital proof 1G11,2F9,2A7,12E1 and 13D12 are when having potential cytotoxicity for breast cancer cell line when toxin conjugated.
Embodiment 8
The evaluation of the ADCC activity of anti-O8E antibody
This embodiment discloses and has utilized the anti-O8E monoclonal antibody of fluorocyte toxicity test detection to kill and wound O8E by the cytotoxicity (ADCC) that antibody relies under the effector cell exists +The ability of clone.
Followingly prepare people effector cell with whole blood.By standard Ficoll-paque partition method Purification of Human peripheral blood lymphocytes from the whole blood of heparinization.This cell is resuspended in the RPMI1640 substratum that contains 10%FBS and 200U/ml human IL-2, and 37 ℃ of lower overnight incubation.Next day, collecting cell is washed four times with substratum, and with 2 * 10 7The concentration resuspension of cell/ml.O8E +Target cell and BATDA reagent (Perkin Elmer, Wellesley, MA) are with every 1 * 10 6Individual target cell/mL uses the ratio of 2.5 μ l BATDA to hatch under 37 ℃ 20 minutes.Target cell is washed four times, centrifugal, and to make final concentration be 1 * 10 5Cell/ml.
Use Delfia fluorescent emission analytical method as described below detects O8E +The antibodies specific ADCC of the SKOV3 clone of clone SKBR3 and O8E transfection to the anti-O8E monoclonal antibody of people.Every kind of target cell system (target cells of 100 μ l marks) hatches together with 50 μ l effector cells and 50 μ l antibody.Use the target of 1: 50 in whole experimentation: the effect ratio.In all researchs, contrast as negative control with human IgG1's isotype.Centrifugal with the 2000rpm pulse and 37 ℃ hatch one hour after, collect supernatant liquor, again centrifugal fast, and 20 μ l supernatant liquors are transferred in flat underside, add wherein 180 μ l Eu solution (Perkin Elmer, Wellesley, MA), read plate device (BMG Labtech) reading with RubyStar.The following calculating of cracking %: (sample release-spontaneous release * 100)/(maximum release-spontaneous release), wherein spontaneous release is the fluorescence from the hole that only contains target cell, and maximum release is fluorescence from the hole of containing target cell and processing with 2%Triton-X.Anti-O8E antibody 1G11,2F9 and 2A7 are presented in Figure 17 the cytotoxicity % cracking of SKBR3 cell; Anti-O8E antibody 1G11,2F9 and 2A7 are presented in Figure 18 the cytotoxicity % cracking of the clone of SKOV3-O8E transfection; Anti-O8E antibody 2F9 and 2A7 are presented in Figure 19 the concentration dependent cytotoxicity % cracking of SKBR3 cell.For the anti-O8E antibody of HuMAb 1G11,2F9 and 2A7, express O8E +Clone SKBR3 and SKOV3-O8E all show antibody-mediated cytotoxicity.The anti-O8E antibody of this digital proof HuMAb shows the SC to the O8E+ express cell.
Embodiment 9
Use the anti-O8E Antybody therapy in-vivo tumour heteroplastic transplantation model of naked anti-O8E antibody and cytotoxin coupling
The present embodiment discloses with toxin conjugated anti-O8E antibody and has treated in vivo the mouse that is implanted with the mammary gland cell tumor, to detect this antibody to effect in the body of tumor growth.
With the standard test program at amplification in vitro SKBR3 or other suitable mammary gland cell cancer cells.Use the 6-8 male Ncr nude mouse (Taconic, Hudson, NY) in age in week, every mouse is right flank is subcutaneous 7.5 * 10 in being implanted in 0.2ml PBS/Matrigel (1: 1) 6ACHN or A-498 cell.After implantation, mouse is weighed, use electronic caliper to measure tumour on three dimensions, twice weekly.Gross tumor volume is calculated as height * width * length.Lotus there is average 270mm 3ACHN tumour or average 110mm 3The mouse of A498 tumour be randomized into treatment group.At the 0th day, give and to use PBS carrier, toxin conjugated isotype control antibodies or toxin conjugated anti-O8E HuMAb in mouse peritoneum.Can describe in being numbered the unsettled U.S. Patent application of MEDX-0034US4 with the example of the toxin compound of antibody coupling of the present invention.Detect with three kinds of different toxin compounds the mouse of accepting anti-O8E HuMAb.In 60 days after administration, the tumor growth of monitoring mouse.When tumour reaches tumour terminal point (2000mm 3) time to mouse row euthanasia.Extended with toxin conjugated suitable anti-O8E antibody and reached tumour terminal point volume (2000mm 3) mean time, and delayed the progress of tumor growth.Therefore, with this anti-O8E antibody-toxin conjugated thing treatment, tumor growth had inhibition in direct body.
Embodiment 10
Use the immunohistochemical study of anti-O8E HuMAb 2A7
The present embodiment discloses use normal mouse tissue array (IMGENEX Histo-Array, Imgenex Corp., San Diego, CA) and has detected anti-O8E HuMAb2A7 to the identification of O8E by ImmunohistochemistryMethods Methods.
For immunohistochemical methods, use the tissue block of 5 μ m.After dry 30 minutes, cut into slices (at room temperature 10 minutes) and air-dry 5 minutes with acetone is fixing.With PBS rinsing slide, then with PBS in 10% normal goats serum preincubate 20 minutes, at room temperature hatched 30 minutes with the 2A7 of 10 μ g/ml fitcization in the PBS that contains 10% normal goats serum subsequently.Then, wash slide three times with PBS, at room temperature hatched 30 minutes with the anti-FITC of mouse (10 μ g/ml, DAKO).Wash slide with PBS again, at room temperature hatched 30 minutes with goat anti-mouse HRP conjugate (DAKO).Wash slide three times with PBS.As substrate, produce brown colouring with diaminobenzidine (Sigma).After distilled water wash, slide is used haematoxylin redyeing 1 minute.Then 10 seconds kinds of washing slide in the distilled water that flows, fixing in (DAKO) at glycerogel (glycergel).The result of these tests is presented in table 6.
The immunoreactivity of table 6O8E in the normal mouse tissue array
Figure S2006800460653D00981
Other parts - - -
Stomach surface and other parts of glandular epithelium neuroplexus 1+，2+，ocas - - 1+，2+，freq ±，1+ - 1+，2+，ocas - -
Other parts of pancreas bubble epithelium pancreas islet 1+ - - 2+ ± - ±，1+ - -
Other parts of sialisterium acinus epithelium ± - 1+ - - -
Other parts of liver liver cell - - ±，- - - -
Cerebral neuron neuropil/fiber ± -，± 2+，1+，freq 2+，2+，ocas ±，- -
Pons neurone neuropil/fiber ± ± ± 2+，1+，freq ± -
Other parts of Cerebellar Cortex Purkinje Cell white matter ±，1+ - - 1+ 1+，2+ - ±，- - -
Other parts of macrolymphocyte in splenic red pulp - - 1+，2+，rare -，± - -
Thymus gland - - -
Skeletal muscle - - -
Tongue - - -
Heart - -，± -
Lung - - -
Renal cortex - -，± -
Kidney medulla - - -
Bladder
Figure S2006800460653D01001
These data and the corresponding data of collecting for anti-O8E antibody 1G11 and 2F9 prove, exist strong to violent O8E immunoreactivity (3+, 4+) in the intestines Studies of Endocrine-like Cells in colon and small intestine and in the liquid of the chamber of seminal vesicle; In Crypt Cells in the neuropil and fiber of cerebral neuron, brain and pons, in cerebellar white matter, at small intestine, in a small amount of macrolymphocyte of spleen, show weak O8E immunoreactivity (1+, 2+) to moderate; Proof has weak O8E immunoreactivity (1+) in the acinus epithelium of colon surface epithelium, Cerebellar Cortex Purkinje Cell, sialisterium and pancreas; Show suspicious to weak O8E immunoreactivity in the transitional epithelium of bladder, the primary spermatocyte of testis (primary spermotocyte), stomogastric nerve clump; Every other organ shows negative to suspicious dyeing, comprises skin, liver, heart, lung, thymus gland, kidney, uterus, ovary, epididymis, tongue and skeletal muscle.
Embodiment 11
Take off the generation of fucosylation HuMAb
The present embodiment has illustrated the generation of the anti-O8E HuMAb that lacks the fucosido residue.
Proved that the antibody of fucosido residue reduced number improves the ADCC ability of antibody.Be that Ms704-PF (Biowa, Inc., Princeton, NJ) carries out electroporation with the carrier of expressing anti-O8E HuMAb heavy chain and light chain to the Chinese hamster ovary celI that lacks fucosyl transferase gene FUT 8.By growth screening resistance clone in the Ex-Cell 325-PF CHO substratum (JRH Biosciences, Lenexa, KS) that contains 6mM L-glutaminate and 500 μ g/ml G418 (Invitrogen, Carlsbad, CA).Measure according to IgG by standard ELISA and express screening and cloning.Produced two independent clones, B8A6 and B8C11, its productive rate scope is every cell 1.0 to 3.8 piks every day.
Embodiment 12
Take off the evaluation of the ADCC activity of the anti-O8E antibody of fucosylation
This embodiment disclose utilize the fluorocyte toxicity test to detect to take off fucosylation kill and wound O8E by the cytotoxicity (ADCC) that antibody relies on non-anti-O8E monoclonal antibody of taking off fucosylation under the effector cell exists +The ability of cell.
As mentioned above the anti-O8E monoclonal antibody of people is taken off fucosylation.As described belowly prepare people effector cell with whole blood.By standard Ficoll-paque partition method Purification of Human peripheral blood lymphocytes from the whole blood of heparinization.This cell is resuspended in the RPMI1640 substratum (substratum) that contains 10%FBS and 200U/ml human IL-2, and 37 ℃ of lower overnight incubation.Next day, collecting cell is washed once with substratum, and with 2 * 10 7The concentration resuspension of cell/ml.O8E+ target cell and BATDA reagent (Perkin Elmer, Wellesley, MA) are with every 1 * 10 6Target cell/mL substratum (being added with the 2.5mM probenecid) (mensuration substratum) uses the ratio of 2.5 μ lBATDA to hatch under 37 ℃ 20 minutes.Target cell is washed four times with the PBS that contains 20mM HEPES and 2.5mM probenecid, centrifugal, and to make the final concentration of measuring in matrix be 1 * 10 5Cell/ml.
Use Delfia fluorescent emission analytical method as described below detects O8E+ clone ARH-77 (people B lymphoblast leukemia; ATCC preserving number CRL-1621) for the antibodies specific ADCC of the anti-O8E monoclonal antibody of people of taking off fucosylation and Fei Tuo fucosylation.Target cell is ARH-77 (target cells of 100 μ l marks) with 50 μ l effector cells and 50 μ l 1G11 or takes off and hatch together with the 1G11 antibody of fucosylation.Use the target of 1: 50 in whole experimentation: the effect ratio.Contrast as negative control with human IgG1's isotype.Centrifugal with the 2100rpm pulse and 37 ℃ hatch one hour after, collect supernatant liquor, again centrifugal fast, and 20 μ l supernatant liquors are transferred in flat underside, add wherein 180 μ l Eu solution (Perkin Elmer, Wellesley, MA), read plate device (Perkin Elmer) reading with Fusion Alpha TRF.The following calculating of cracking %: (sample release-spontaneous release * 100)/(maximum release-spontaneous release), wherein spontaneous release is the fluorescence from the hole that only contains target cell, and maximum release is fluorescence from the hole of containing target cell and processing with 3%Lysol.The clone ARH-77 that expresses O8E+ shows the antibody-mediated cytotoxicity for the anti-O8E antibody of HuMAb 1G11, and the specificity cracking per-cent of demonstration and the relevant increase of the fucosylation form of taking off of anti-O8E antibody 1G11.Therefore, the anti-O8E antibody of HuMAb that takes off fucosylation has strengthened the SC to the O8E+ express cell.
Embodiment 13
Estimate the internalization of the anti-O8E antibody of HuMab by immuning fluorescent dyeing analysis
Target cell is O8E +SKBR3 (human breast carcinoma, ATCC#HTB-30) and ZR-75 (human breast carcinoma, ATCC#CRL-1500) be used for utilizing immunofluorescence dyeing detect the anti-O8E antibody of HuMab 2A7C11,1G11H1 and 2F9E6 with Cell binding after internalization.
SKBR3 and ZR-75 cell (the every 100 μ l 10 in every hole in 96 orifice plates 4) gather in the crops from tissue culture flasks by processing with 0.25% trypsinase/EDTA, the anti-O8E antibody of every kind of HuMab with 5 μ g/ml in FACS damping fluid (PBS+5%FBS, medium) was hatched on ice 30 minutes together.The contrast of human IgG1's isotype is as negative control.After medium washing 2 times, cell is resuspended in (every hole 100 μ l) in medium, then hatched on ice 30 minutes together with the anti-human second antibody of goat that is coupled to PE (JacksonImmunoResearch Lab) of diluting at 1: 00.After the medium washing, in the time of 0 minute, perhaps after hatching the different time under 37 ℃, with cell imaging under fluorescent microscope (Nikon) immediately.Gather the image of morphocytology and the immune fluorescence intensity of staining cell in the different time points shown in figure below.Only observe fluorescence in the cell of the anti-O8E antibody staining with HuMab.Use the IgG1 control antibodies fluorescence not detected.Use in test the anti-O8E antibody of HuMab of the direct coupling of FITC-also to obtain similar result
Imaging data shows, uses the anti-O8E antibody of whole three kinds of HuMab, all occurs fluorescence in the time of 0 minute on cytolemma.In 30 minutes hatched, the film fluorescence intensity significantly reduced, and cell inner dyeing strengthens.When 120 minute hour, fluorescence on film disappears, and appears in the intracellular region chamber.This digital proof, the anti-O8E antibody of HuMab is can be by the specificity internalization after the endogenous tumour cell of expressing O8E is combined.
Embodiment 14
The effect of anti-O8E antibody to HEK-B7H4 tumour in the SCID mouse
In this embodiment, process in vivo the SCID mouse of having implanted the HEK-B7H4 tumour with naked anti-O8E antibody, detect antibody to effect in the body of tumor growth.
Use and lack functional B and the lymphocytic severe severe combined immunodeficiency of T (SCID) mice study tumor growth.In the future the cell of the HEK tumor cell line of personal B7H4 transfection in matrigel (50%v/v) with the subcutaneous implantation of the density of 500 ten thousand cell/mouse.Every mouse was accepted the inoculation of 0.2ml cell at the 0th day.Check the tumor growth of mouse since the 0th day, and monitor weekly tumor growth twice, continue about 6 weeks.When tumour reaches about 130mm 3The time, according to gross tumor volume, mouse is randomized into 3 groups.Process mouse with the naked anti-O8E antibody 2A7 of 10mg/kg, use isotype control antibodies or preparation damping fluid as negative control.Give animal by every 5 days peritoneal injections, inject altogether 5 times.Use electronic caliper measure tumour (high * wide * long) and calculate gross tumor volume on three dimensions.
When tumour reaches 1500mm 3Volume or lose weight greater than 15% the time to mouse row euthanasia.Result is presented in Figure 20.Anti-O8E antibody 2A7 processes and has suppressed tumor growth.It was 63% at the 34th day that the tumor growth of the group of processing with 2A7 suppresses intermediate value.Tumour regrows after stopping administration.It is effective that the anti-O8E antibody of these results proof is treated the tumour aspect of expressing O8E in vivo.
Embodiment 15
Use the immunohistochemical study of anti-O8E antibody
Use detects the ability of anti-B7H4HuMAb 2A7 identification B7H4 from the clinical biopsy of ovarian cancer, lung cancer, mammary cancer and head and neck cancer by Immunohistochemical Method.
For immunohistochemistry, use the freezing section (Ardais Inc, USA) of 5 μ m.After dry 30 minutes, will cut into slices and fix (at room temperature 10 minutes) and air-dry 5 minutes with acetone.With PBS rinsing slide, then with PBS in 10% normal goats serum preincubate 20 minutes, at room temperature hatched 30 minutes with the fitcization antibody of 10 μ g/ml in the PBS that contains 10% normal goats serum subsequently.Then, wash slide three times with PBS, at room temperature hatched 30 minutes with the anti-FITC of mouse (10 μ g/ml, DAKO).Wash slide with PBS again, at room temperature hatched 30 minutes with goat anti-mouse HRP conjugate (DAKO).Wash slide three times with PBS.As substrate, produce brown colouring with diaminobenzidine (Sigma).After distilled water wash, slide is used haematoxylin redyeing 1 minute.Then 10 seconds kinds of washing slide in the distilled water that flows, fixing in glycerogel (DAKO).Clinical biopsy immunohistochemical staining shows positive dyeing in lung cancer, mammary cancer, ovarian cancer and head and neck cancer sample.
Embodiment 16
The quantitative RT-PCR of normal and cancerous tissue
Utilize quantitative ThermoScript II PCR (RT-PCR) normal and cancerous tissue samples various according to the expression screening of O8E mRNA.The expression indication O8E protein expression of mRNA.
For quantitative RT-PCR, use following O8E primer: B7-H4.3:AGGATGGAATCCTGAGCTGCACTT; B7-H4.4:TCCGACAGCTCATCTTTGCCTTCT is provided by Operon (Huntsville, AL).Application standard reaction conditions (the cDNA template of 5 μ l 1ng/ μ l, 0.1ul the upstream primer of 40 μ M, 0.1 the downstream primer of μ l 40 μ M, 6 μ l 2 * SYBR Green PCR mixtures (AppliedBiosystems#4367659), and 0.8 μ l water).Utilize Standard PC R condition amplification cDNA totally 40 circulations in ABI Prism 7900HT (AppliedBiosystems, Foster City, CA).The quantitative RT-PCR result is presented in following table 7.Do not determine that the sample representative of counting is lower than the value of fluorescence threshold.Mammary gland, ovary and neck tumour Explicit Expression O8E see the expression of highest level in some ovarian cancers and head and neck cancer sample.O8E expression ratio healthy tissues in this proof mammary cancer, ovarian cancer and neck tumor sample increases.
The quantitative RT-PCR of table 7 in normal and cancerous tissue expressed
Tissue Counting Amount
Normal adipocyte (#301) 28.953062 25.57793
Normal artery (#303) 31.856901 3.0423617
Normal bladder (#257) 30.620392 7.5326214
Normal bone marrow (#342) Do not determine 0
Normal brain activity (#258) 34.33955 0.49280354
Normal breast (#259) 25.63064 292.28528
Normal Colon (#261) Do not determine 0
Normal esophagus (#262) 32.27514 2.2388945
Normal heart (#125) Do not determine 0
Normal kidney (#264) 33.599422 0.8479082
Normal hepatocytes (#266) Do not determine 0
Normal lung (#268) 32.44523 1.9763907
Normal Lymph Nodes (#315) Do not determine 0
Normal ovarian (#270) 35.045704 0.29364112
Normal pancreas (#271) 28.446985 37.06916
Normal circumference blood leukocytes (#302) 34.652363 0.39180183
Normal prostatic (#272) 32.635994 1.7184163
Normal retina (#256) 34.70426 0.37717298
Normal bone flesh (#119) Do not determine 0
Normal bone flesh (#126) Do not determine 0
Normal skin (#273) Do not determine 0
Normal spinal cord (#129) 39.383526 0.01220525
Normal spleen (#274) Do not determine 0
Normal gastric (#275) Do not determine 0
Normal tongue (#324) 30.956758 5.886249
Normal tonsilla (#325) Do not determine 0
Normal tracheae (#314) 29.771343 14.03797
Breast tumor (#176) 33.798374 0.7328206
Breast tumor (#177) 25.759022 266.02777
Breast tumor (#178) 28.572468 33.81085
Breast tumor (#179) 25.31508 368.374
Breast tumor (#180) 29.323488 19.494516
Head/neck tumour (larynx, #402) 28.116425 47.23582
Head/neck tumour (pharynx, #403) 25.776083 262.72076
Head/neck tumour (tongue, #403) 26.950275 111.07142
Head/neck tumour (tonsilla, #404) 23.03704 1957.3722
Tumor of kidney (#167) 27.029814 104.77927
Ovarian tumor (#187) 25.321087 366.75525
Ovarian tumor (#188) 22.846964 2250.0833
Ovarian tumor (#189) 25.079527 437.81958
Ovarian tumor (#190) 27.964441 52.80399
Ovarian tumor (#191) 22.686525 2530.9656
Scope of the present invention is not limited by specific embodiments as herein described.In fact, according to above description and accompanying drawing, except as herein described, be also apparent to the various modifications of present disclosure to those skilled in the art.Within these modifications drop on the scope of appended claims.
Quoted in this application patent, patent application, publication, the description of product and scheme, its open file is complete being incorporated herein by reference for all purposes all.
Figure ISB00000493106100011
Figure ISB00000493106100021
Figure ISB00000493106100031
Figure ISB00000493106100041
Figure ISB00000493106100051
Figure ISB00000493106100081
Figure ISB00000493106100091
Figure ISB00000493106100101
Figure ISB00000493106100111
Figure ISB00000493106100121
Figure ISB00000493106100131
Figure ISB00000493106100141
Figure ISB00000493106100151

Claims (20)

1. the monoclonal antibody of a separation or its antigen-binding portion thereof, it comprises: contain the variable region of heavy chain of CDR1, CDR2 and CDR3 structural domain and contain CDR1, CDR2 and the variable region of light chain of CDR3 structural domain, the CDR1 of wherein said variable region of heavy chain and variable region of light chain, CDR2 and CDR3 structural domain are respectively:
The variable region of heavy chain CDR1 of the aminoacid sequence as shown in SEQ ID NO:12; The variable region of heavy chain CDR2 of the aminoacid sequence as shown in SEQID NO:17; The variable region of heavy chain CDR3 of the aminoacid sequence as shown in SEQ ID NO:22; The variable region of light chain CDR1 of the aminoacid sequence as shown in SEQ ID NO:27; The variable region of light chain CDR2 of the aminoacid sequence as shown in SEQ ID NO:32; With the variable region of light chain CDR3 of aminoacid sequence as shown in SEQ ID NO:37,
Wherein said antibody or its antigen-binding portion thereof are specifically in conjunction with O8E.
2. the monoclonal antibody of a separation or its antigen-binding portion thereof, it comprises variable region of heavy chain and variable region of light chain, and wherein said variable region of heavy chain and variable region of light chain are respectively:
The variable region of light chain of the variable region of heavy chain of the aminoacid sequence as shown in SEQ ID NO:2 and the aminoacid sequence as shown in SEQ ID NO:7,
Wherein said antibody or its antigen-binding portion thereof are specifically in conjunction with O8E.
3. antibody as claimed in claim 1 or 2 or its antigen-binding portion thereof, wherein said antibody be combined with its antigen rear by internalization.
4. antibody as claimed in claim 1 or 2 or its antigen-binding portion thereof, wherein said antibody is combined with mammary gland cell tumor clone.
5. antibody as claimed in claim 4 or its antigen-binding portion thereof, wherein said mammary gland cell tumor cell is SKBR3 clone.
6. antibody as claimed in claim 1 or 2 or its antigen-binding portion thereof, wherein said antibody deficiency fucosyl residues.
7. immune conjugate, it comprises the described antibody of claim 1 or 2 or its antigen-binding portion thereof that is connected with therapeutical agent.
8. immune conjugate as claimed in claim 7, wherein said therapeutical agent is cytotoxin.
9. immune conjugate as claimed in claim 7, wherein said therapeutical agent is radio isotope.
10. immune conjugate, it comprises antibody claimed in claim 2 or its antigen-binding portion thereof that is connected with therapeutical agent.
11. a composition, it contains immune conjugate claimed in claim 7 and medicine acceptable carrier.
12. the nucleic acid molecule of a separation, its coding described antibody of claim 1 or 2 or its antigen-binding portion thereof.
13. an expression vector, it comprises the described nucleic acid molecule of claim 12.
14. a host cell, it comprises the described expression vector of claim 13.
15. a method for preparing anti-O8E antibody is included in and expresses this antibody in the described host cell of claim 14, and separates this antibody from this host cell.
16. in the described antibody of claim 1 or 2 or its antigen-binding portion thereof or claim 7-10, the described immune conjugate of any one is in the purposes in take the growth of tumour cell of expressing O8E as the medicine of the disease of feature for the preparation for the treatment of or prevention.
17. purposes as claimed in claim 16, wherein said disease are cancer.
18. purposes as claimed in claim 17, wherein said cancer is selected from mammary gland cell cancer, ovarian cancer, kidney and head and neck cancer.
19. purposes as claimed in claim 16, wherein said antibody or its antigen-binding portion thereof are connected with therapeutical agent.
20. purposes as claimed in claim 19, wherein said therapeutical agent are cytotoxin or radio isotope.
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