CN102027015A - Anti-CXCR4 antibodies - Google Patents
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- CN102027015A CN102027015A CN2009801172303A CN200980117230A CN102027015A CN 102027015 A CN102027015 A CN 102027015A CN 2009801172303 A CN2009801172303 A CN 2009801172303A CN 200980117230 A CN200980117230 A CN 200980117230A CN 102027015 A CN102027015 A CN 102027015A
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Abstract
The present invention provides antibodies that bind human CXCR4 and are characterized as having high affinity and strong neutralizing properties. The antibodies of the invention are useful in the treatment of tumorigenesis, including tumor growth, invasion, angiogenesis, or metastasis.
Description
The present invention relates to monoclonal antibody and their purposes in pathogenetic disease that treatment is mediated by CXCR4 and SDF-1 at CXCR4.
CXCR4, a kind of Chemokine Receptors is 7 transmembrane receptors of G albumen coupling.Similar with other Chemokine Receptors, CXCR4 plays a significant role in immunity and inflammatory reaction by the directional migration and the activation of mediated leucocytes.CXCR4 expresses in kinds of tumor cells system and tissue or crosses and express, and comprises mammary gland, prostate gland, lung, ovary, colon, pancreas, kidney and brain, and non_hodgkin lymphoma and chronic lymphocytic leukemia.Unique known CXCR4 part is stromal cell derived factor-1 (SDF-1, or CXCL12).The interaction of CXCR4 and SDF-1 is comprising that tumor growth, infringement, blood vessel take place and a plurality of tumour emergence period of transfer plays a significant role.
Because CXCR4 relates to multiple serious disease, CXCR4 is studied as the treatment target at present.For example, AMD3100, a kind of bicyclam CXCR4 antagonist can be used for the patient of multiple myelomatosis and non_hodgkin lymphoma.CTCE9908, a kind of peptide CXCR4 antagonist is at present in the clinical trial Ib/II phase that is used for cancer.In addition, (people [Carnec X such as WO 06/089141, US07/0059308 and Carnec in the prior art, Quan L, Olson W, Hazan U, DragicT. (2005) Anti-CXCR4 Monoclonal Antibodies Recognizing OverlappingEpitopes Differ Significantly in Their Ability To Inhibit Entry ofHuman Immunodeficiency Virus Type I.Journal of Virology.Feb.2005:1930-1933])) antibody of target CXCR4 disclosed.
Although the medicament of multiple target CXCR4 in research and development is arranged, still need the medicament of other target CXCR4.Antibody of the present invention is that effective CXCR4 antagonist is gone up in treatment, has the character of multiple expectation.Antibody of the present invention has improved chemistry and physical stability, and solubility.The invention provides with high-affinity in conjunction with people CXCR4 and suppress the CXCR4 antibody of people CXCR4 in conjunction with SDF-1.Efficient allows to use low dosage in treatment plan.In addition, therefore the interaction of these antibody interferes with SDF-1 and CXCR4 reduces tumour and takes place, and comprises that tumor growth, infringement, blood vessel take place and transfer.In addition, the apoptosis of antibody induction tumour cell of the present invention.
The present invention includes people source engineered antibody or its binding fragment in conjunction with people CXCR4, its:
A. suppress the combination of people SDF-1 α (SEQ ID NO:33) and CXCR4, as people CXCR4/ described herein
125I-SDF-1 α is in conjunction with in the inhibition test, to the IC of people CXCR4
50Between 10nM and 0.05nM;
B. suppress the migration that the surface has the cell of CXCR4, in as chemotaxis test described herein, IC
50Between 30nM and 0.3nM.
C. in as surface plasma body resonant vibration described herein (BIAcore) test, showed K between 15nM and 0.05nM
DAvidity.
The present invention preferably provides in conjunction with the people source engineered antibody of people CXCR4 or its binding fragment, and it suppresses the combination of people SDF-1 α (SEQ ID NO:33) and CXCR4, as people CXCR4/ described herein
125I-SDF-1 α is in conjunction with in the inhibition test, to the IC of people CXCR4
50Between 0.5nM and 0.05nM.
The present invention preferably provides in conjunction with the people source engineered antibody of people CXCR4 or its binding fragment, and it suppresses the migration that the surface has the cell of CXCR4, in as chemotaxis test described herein, and IC
50Between 3.0nM and 0.3nM.
The present invention preferably provides in conjunction with the people source engineered antibody of people CXCR4 or its binding fragment, and it has showed K between 1.0nM and 0.05nM in as surface plasma body resonant vibration described herein (BIAcore) test
DAvidity.
The present invention preferably provides in conjunction with the people source engineered antibody of people CXCR4 or its binding fragment, has showed the antitumorgienesis activity by the prevention tumor growth in its tumour heteroplastic transplantation model of describing herein when using with 1mg/kg.
The present invention preferably provides in conjunction with the people source engineered antibody of people CXCR4 or its binding fragment, in the test of its apoptosis of describing herein between with 2 μ g/mL and 10 μ g/mL (dosage) induced the apoptosis of tumour cell when using.
The present invention includes people source engineered antibody or its binding fragment, it comprises light chain and heavy chain, light chain comprises variable region of light chain, variable region of light chain comprises framework region, CDRL1 with aminoacid sequence of SEQ ID NO:8, CDRL2 with aminoacid sequence of SEQ ID NO:9, CDRL3 with aminoacid sequence with SEQ ID NO:10, heavy chain comprises variable region of heavy chain, variable region of heavy chain comprises framework region, CDRH1 with aminoacid sequence of SEQ ID NO:1, have SEQ ID NO:3 aminoacid sequence CDRH3 and have the SEQ of being selected from an ID NO:2, SEQ ID NO:4, SEQ ID NO:5, the CDRH2 of the aminoacid sequence of SEQ IDNO:6 and SEQ ID NO:7, wherein said antibodies people CXCR4.
In addition, the present invention includes antibody in conjunction with people CXCR4, wherein light chain comprises the aminoacid sequence of SEQ ID NO:16, and heavy chain comprises the aminoacid sequence that is selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.
In addition, the present invention includes antibody in conjunction with people CXCR4, wherein light chain comprises the aminoacid sequence of SEQ ID NO:22, and heavy chain comprises the aminoacid sequence that is selected from SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
The present invention also comprises the antibody in conjunction with people CXCR4, and wherein antibody is selected from antibody, the antibody that comprises SEQ ID NO:18 and SEQ ID NO:22, the antibody that comprises SEQ ID NO:19 and SEQ ID NO:22 that comprise SEQ ID NO:17 and SEQ ID NO:22, comprises the antibody of SEQ ID NO:20 and SEQ ID NO:22 and comprise SEQ ID NO:21 and the antibody of SEQ ID NO:22.
As herein the definition antibody of the present invention it is characterized in that, as people CXCR4/ described herein
125I-SDF-1 α is in conjunction with having 10nM or lower IC in the inhibition test
50The preferred antibody of the present invention has the 5.0nM of people CXCR4 or lower binding affinity.The most preferred antibody of the present invention has the 0.5nM of people CXCR4 or lower binding affinity.Preferred, antibody of the present invention as people CXCR4/ described herein
125I-SDF-1 α is in conjunction with having the IC between 10nM and 0.05nM to people CXCR4 in the inhibition test
50Preferred, antibody of the present invention as people CXCR4/ described herein
125I-SDF-1 α is in conjunction with having the IC between 0.5nM and 0.05nM to people CXCR4 in the inhibition test
50
Antibody of the present invention as definition herein is characterized in that having 30nM or lower IC in as chemotaxis test described herein
50The preferred antibody of the present invention has 15nM or lower IC in the chemotaxis test
50The preferred antibody of the present invention has 3.0nM or lower IC in the chemotaxis test
50Preferred, antibody of the present invention has the IC between 30nM and the 0.3nM in as chemotaxis test described herein
50Preferred, antibody of the present invention has the IC between 3nM and the 0.3nM in as chemotaxis test described herein
50
Antibody of the present invention as definition herein is characterized in that having 15nM or lower K in by the test as surface plasma body resonant vibration described herein (BIAcore) assessment antibody binding activity
DThe preferred antibody of the present invention has 10nM or lower K in the BIAcore test
DThe most preferred antibody of the present invention has 1.0nM or lower K in the BIAcore test
DPreferred, antibody of the present invention has the K between 15nM and the 0.05nM in as BIAcore test described herein
DPreferred, antibody of the present invention has the K between 1.0nM and the 0.05nM in as BIAcore test described herein
D
Antibody of the present invention as definition herein is characterized in that, in tumour heteroplastic transplantation model, when using, has the antitumorgienesis activity by the prevention tumor growth with 10mg/kg as use NOD/SCID mouse described herein and people's non_hodgkin lymphoma Namalwa cell.The preferred antibody of the present invention has the antitumorgienesis activity by the prevention tumor growth when using with 10mg/kg.
Antibody of the present invention as definition herein is characterized in that, the apoptosis of inducing tumor cell in as apoptosis test described herein.The preferred antibody of the present invention (dosage) when using between with 2 μ g/mL and 10 μ g/mL, induced the activation of nuclear fragmentation and half Guang L-Aspartase 3 (caspase 3) in the kinds of tumor cells that comprises Namalwa and cem cell, this two be the sign of apoptosis.
The present invention includes pharmaceutical composition, comprise combination as antibody described herein and one or more pharmaceutically useful carriers, thinner or vehicle.In addition, the present invention includes and be used for the treatment of the tumorigenic pharmaceutical composition that comprises that tumor growth, infringement, blood vessel take place or shift, it comprises the combination as the antibody of various description herein and one or more pharmaceutically useful carriers, thinner or vehicle.In addition, the present invention includes pharmaceutical composition, be used for the treatment of and be selected from mammary cancer, carcinoma of the pancreas, melanoma, prostate cancer, kidney, neuroblastoma, non_hodgkin lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme and leukemic cancer, described pharmaceutical composition comprises the combination as the antibody of various description herein and one or more pharmaceutically useful carriers, thinner or vehicle.
The present invention includes as antibody described herein and be used for the treatment of purposes in the tumorigenic medicine in preparation, described tumour comprises that tumor growth, infringement, blood vessel take place or shift.In addition, the present invention includes as antibody described herein and be used for the treatment of purposes in the medicine of cancer in preparation, described cancer is selected from mammary cancer, carcinoma of the pancreas, melanoma, prostate cancer, kidney, neuroblastoma, non_hodgkin lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme and leukemia.
The present invention includes the tumorigenic method that treatment comprises that tumor growth, infringement, blood vessel take place or shift, the patient who comprises needs uses as antibody described herein.In addition, the present invention includes the treatment method for cancer, described cancer is selected from mammary cancer, carcinoma of the pancreas, melanoma, prostate cancer, kidney, neuroblastoma, non_hodgkin lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme and leukemia, and the patient that method comprises needs uses as antibody described herein.
The general structure of " antibody " is well known in the art.The antibody of IgG type has 4 amino acid chains (2 " weight " chain and 2 " gently " chains), by in the chain and the interchain disulfide bond crosslinking.When expressing in some biosystem, the antibody of people Fc sequence with unmodified is in the glycosylation of Fc zone.Antibody also may be in other position glycosylations.The subunit structure of antibody and 3-d modelling are well known in the art.Every heavy chain holds variable region of heavy chain (" HCVR ") and CH (" HCCR ") to form by N-.CH is made up of 3 structural domains (CH1, CH2 and CH3) that are used for IgG, IgD and IgA and 4 structural domains (CH1, CH2, CH3 and CH4) of being used for IgM and IgE.Every light chain is made up of variable region of light chain (herein " LCVR ") and constant region of light chain (" LCCR ").
The variable region pairing of every light/heavy chain forms the binding site of antibody.HCVR and LCVR can further be subdivided into the hypervariable region that is called complementary determining region (CDR), wherein scatter more conservative zone, are called framework region (FR).Each HCVR and LCVR are made up of 3 CDR and 4 FR, hold the C-end with following series arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-.Herein, 3 CDR of heavy chain are called " CDRH1, CDRH2 and CDRH3 ", 3 CDR of light chain are called " CDRL1, CDRL2 and CDRL3 ".CDR comprises and the interactional most of residues of antigen formation specificity.Amino acid to the distribution of each structural domain according to the routine of knowing [for example, Kabat, " Sequences of Proteins of Immunological Interest, " NationalInstitutes of Health, Bethesda, Md.(1991)]。
Antibody of the present invention can have the CH that is selected from any immunoglobulin (Ig) kind (IgA, IgD, IgG, IgM and IgE).In addition, antibody of the present invention comprises the Fc part from human IgG 4 Fc zones, and reason is to compare the ability of the conjugated complement factor of its reduction with other IgG hypotypes.
Antibody can for example comprise from single copy or clone, any eucaryon, protokaryon or phage clone.Preferably, antibody of the present invention with homogeneous or basically the population of homogeneous exist.Antibody can be complete, comprises to comprise the complete of Fc zone or total length constant region, or is the part or the fragment of such antibody, as long as shortening form arbitrarily comprises antigen-binding portion thereof and keeps antigen binding capacity.The form of Suo Duaning for example comprises like this, comprises the CDR of disclosed anti-CXCR4 antibody or Fab fragment, Fab ' fragment or F (ab ') 2 fragments of variable region.In addition, the antibody formation of Suo Duaning can be strand Fv fragment like this, and described fragment can be by connecting the DNA preparation of coding LCVR and HCVR with joint sequence.(seeing Pluckthun, The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, page or leaf 269-315,1994).No matter whether fragment or part are specific, unless otherwise noted, the fragment as the term " antibody " that herein uses comprises or part and single stranded form.As long as protein keeps special or preferentially in conjunction with the ability of CXCR4 and comprise one or more sequence disclosed herein, just is included in the term " antibody ".Antibody of the present invention can use technology preparation well known in the art, for example combination of recombinant technology, display technique of bacteriophage, synthetic technology or these technology or other technology known in the art.
Term " people source engineered antibody " is meant antibody tool somebody source framework, hinge area and constant region and framework identical with constant region or substantially the same (people source basically) from people's gene group sequence.Total man's framework, hinge area and constant region are sequence and the sequence with abiogenous somatic mutation for those ethnic groups.People source engineered antibody can comprise framework, hinge or the constant region from total man's framework, hinge or constant region, and comprises one or more aminoacid replacement, disappearance or interpolation therein.Usually, people source engineered antibody does not preferably have immunogenicity basically in the people.
Can be used alone or in combination the basis of multiple different people's framework sequence as people of the present invention source engineered antibody.Preferably, the framework region behaviour source of antibody of the present invention or (at least 95%, the 97% or 99% people source) in people source basically.People source framework region sequence can be from The Immunoglobulin Factsbook, Marie-Paule Lafranc, and Gerard Lefranc work, Academic Press 2001, ISBN012441351 obtains.
The framework sequence of inventor source engineered antibody is as " donor " variable framework region, can be used for creating other and has and the people source engineered antibody of specified identical CDR the use methods known in the art herein.In addition, be used for inventor source engineered antibody the framework sequence can with other known people's framework sequence comparisons to generate other people source engineered antibody.Therefore, this information can be used for another selected homology people's framework region is transformed the donor amino-acid residue in the people source of these positions " reverse mutation (back-mutate) ".In addition, can in other people's framework, detect arbitrarily " rare " amino acid, thereby can use consistent or amino-acid residue is transformed in donor people source at relevant position.
Term " inhibition " meaning be basically antagonism, stop, avoid, contain, slow down, interrupt, eliminate, stop, reduce or reverse ability in conjunction with the biological effect of CXCR4 acceptor.
" CXCR4 " or " people CXCR4 " is meant any people CXCR4, with and function on activated mutant form.Example including, but not limited to, SEQ ID NO:30, SEQ ID NO:31 and SEQ IDNO:32.
" patient " is Mammals, preferably is the people.
Term " treatment (treating) " (or " treat " or " the treatment ") meaning is process or a seriousness of slowing down, stop, reduce or reverse symptom, disorder, illness or disease.
Term " prevention (preventing) " (or " prevent " or " the prevention ") meaning is generation or the appearance that stops, contains or suppress symptom, disorder, illness or disease.Can treat and prophylaxis of acute incident and chronic disease.In acute events, administration of antibodies when symptom, disorder, illness or disease incidence stops when acute events finishes, and chronic sympton disorder, illness or treatment of diseases need the longer time limit.
Term " treatment significant quantity " is meant after the patient is used separately or repeatedly, and the amount or the dosage of the antibody of the present invention of desired therapeutic or prevention is provided.The treatment significant quantity can comprise every single (for example, group annotate), repeatedly or the amount of continuous administration about 0.001 to 20mg/kg.
The antibody specific of this invention comprises: the antibody that comprises SEQ ID NO:1,2,3,8,9 and 10 aminoacid sequence; The antibody that comprises SEQ ID NO:1,4,3,8,9 and 10 aminoacid sequence; The antibody that comprises SEQ ID NO:1,5,3,8,9 and 10 aminoacid sequence; The antibody that comprises SEQ ID NO:1,6,3,8,9 and 10 aminoacid sequence; The antibody that comprises SEQ ID NO:1,7,3,8,9 and 10 aminoacid sequence.Listed sequence is represented CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 respectively.
The antibody specific of this invention comprises: comprise the LCVR of the aminoacid sequence with SEQ ID NO:16 and the antibody of the HCVR of the aminoacid sequence with SEQ ID NO:11; Have SEQ ID NO:16 aminoacid sequence LCVR and have the antibody of HCVR of the aminoacid sequence of SEQ ID NO:12; Have SEQ ID NO:16 aminoacid sequence LCVR and have the antibody of HCVR of the aminoacid sequence of SEQ ID NO:13; Have SEQ ID NO:16 aminoacid sequence LCVR and have the antibody of HCVR of the aminoacid sequence of SEQID NO:14; Have SEQ ID NO:16 aminoacid sequence LCVR and have the antibody of HCVR of the aminoacid sequence of SEQ ID NO:15.
The present invention includes combination and suppress the active 5 kinds of antibody of CXCR4.Specifically, the present invention includes: comprise the light chain of aminoacid sequence and the antibody of the heavy chain of aminoacid sequence with SEQ ID NO:17 with SEQ ID NO:22; Comprise the light chain of aminoacid sequence and the antibody of the heavy chain of aminoacid sequence with SEQ ID NO:18 with SEQ ID NO:22; Comprise the light chain of aminoacid sequence and the antibody of the heavy chain of aminoacid sequence with SEQ ID NO:19 with SEQ ID NO:22; Comprise the light chain of aminoacid sequence and the antibody of the heavy chain of aminoacid sequence with SEQ ID NO:20 with SEQ ID NO:22; Comprise the light chain of aminoacid sequence and the antibody of the heavy chain of aminoacid sequence with SEQ ID NO:21 with SEQ ID NO:22.
Preferably, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is, as people CXCR4/ described herein
125I-SDF-1 α is in conjunction with having about 10nM or lower IC in the inhibition test
50, preferred about 5.0nM or lower, most preferred 0.5nM or lower.Preferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is, as people CXCR4/ described herein
125I-SDF-1 α is in conjunction with the IC that has in the inhibition test between 10nM and the 0.05nM
50Preferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is, as people CXCR4/ described herein
125I-SDF-1 α is in conjunction with the IC that has in the inhibition test between 0.5nM and the 0.05nM
50
Preferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is to have about 30nM or lower IC in as chemotaxis test described herein
50, preferred about 15nM or lower, even preferred 3.0nM or lower.Preferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is to have the IC between 30nM and the 0.3nM in as chemotaxis test described herein
50Preferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is to have the IC between 3.0nM and the 0.3nM in as chemotaxis test described herein
50
Even it is preferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is to have about 15nM or lower K in as the test by surface plasma body resonant vibration (BIAcore) assessment antibody binding activity described herein
D, preferred about 10nM or lower, most preferred 1.0nM or lower.Preferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is to have the K between 15nM and the 0.05nM in as the test by surface plasma body resonant vibration (BIAcore) assessment antibody binding activity described herein
DPreferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is to have the K between 1.0nM and the 0.05nM in as the test by surface plasma body resonant vibration (BIAcore) assessment antibody binding activity described herein
D
Preferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is, in tumour heteroplastic transplantation model as use NOD/SCID mouse described herein and people's non_hodgkin lymphoma Namalwa cell, when using with 10mg/kg, even preferred when using with 1mg/kg, have the antitumorgienesis activity by stoping tumor growth.
Most preferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQ ID NO limit, its additional features is, the apoptosis of inducing tumor cell in as apoptosis test described herein.Preferred, in the antibody of the present invention all 6 CDR, HCVR, LCVR, HCVR and LCVR, whole piece heavy chain, whole piece light chain or whole piece heavy chain and whole piece light chain by as the concrete sequence that shows of herein SEQID NO limit, its additional features is, (dosage) is when using between with 2 μ g/mL and 10 μ g/mL, induced the activation of nuclear fragmentation and L-Cysteine HCL Anhydrous 3 in the kinds of tumor cells that comprises Namalwa and cem cell, this two be the sign of apoptosis.
Embodiment
Can be as following preparation and antibody purification I, II, III, IV and V.Use optimum default HC: the two the instantaneous or stable transfection proper host cell of the expression system that is used for secretory antibody of single carrier system of LC carrier ratio or coding HC (for example SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27) and LC (for example SEQ ID NO:28), for example HEK 293 EBNA or CHO.Wherein can use any one the purifying secreted clarification substratum that antibody is arranged in many technology of generally using.For example, can easily substratum be applied to compatible buffers for example phosphate buffered saline buffer (pH 7.4) equilibrated a-protein or G sepharose FF post.Clean post to remove the non-specific binding component.The antibody of elution of bound is for example by pH gradient (for example 0.1M sodium phosphate buffer pH6.8 to 0.1M sodium citrate buffer solution pH2.5).Detect the antibody fraction,, merge afterwards for example by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Be further purified and choose wantonly, depend on the purposes of plan.Can use ordinary skill to concentrate and/or sterile filtration antibody.Soluble poly collective and polymer can comprise that size exclusion, hydrophobic interaction, ion-exchange or hydroxyapatite effectively remove by ordinary skill.Antibody purity after these chromatographic step surpasses 99%.But product can be frozen in-70 ℃ or lyophilized at once.The aminoacid sequence of these antibody provides as follows.
SEQ?ID?NO
| Antibody | Heavy chain | Light chain | HCVR | LCVR |
| I | 17 | 22 | 11 | 16 |
| II | 18 | 22 | 12 | 16 |
| III | 19 | 22 | 13 | 16 |
| IV | 20 | 22 | 14 | 16 |
| V | 21 | 22 | 15 | 16 |
| Antibody | ?HCDR1 | HCDR2 | HCDR3 | LCDR1 | LCDR2 | LCDR3 |
| I | ?1 | 2 | 3 | 8 | 9 | 10 |
| II | ?1 | 4 | 3 | 8 | 9 | 10 |
| III | ?1 | 5 | 3 | 8 | 9 | 10 |
| IV | ?1 | 6 | 3 | 8 | 9 | 10 |
| V | ?1 | 7 | 3 | 8 | 9 | 10 |
People CXCR4/
125I-SDF-1 α is in conjunction with inhibition test
SDF-1 is the first step that activates the CXCR4 intracellular signaling pathway in conjunction with CXCR4.For determining whether antibody can block the interaction of SDF-1 and CXCR4,
125The SDF-1 α of I-mark is in conjunction with using the human leukemia CCRF-CEM cell of expressing endogenous CXCR4 in the test.Testing on the untreated polystyrene board at the bottom of the 96 hole U.Use the RPMI1640 medium preparation that contains 10mM N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid (HEPES), pH7.5 and 0.2% bovine serum albumin (BSA) in conjunction with the test damping fluid.In brief, 200 μ L contain 300pM part (60pM
125The cold SDF-1 α of I-SDF-1 α and 240pM), the reaction mixture that detects antibody, 100,000 people CCRF-CEM cells and approaching assay method (SPA) pearl of 0.5mg flicker at the different concns of test in the damping fluid was incubated at room 2 hours.Afterwards with plate on liquid scintillation and luminescent counter with the SPA mode counting.The CXCR4 antagonist reduces the bonded radioactivity in dose-dependent mode in this test.Detect the inhibition ability (IC of antibody
50) use GraphPad Prism computed in software, reduce based on the radioactive dose-dependently of bonded.
The antibody of example has been showed 10nM or lower IC in this test herein
50For example, antibody I II has showed the IC of average 0.45nM in this test
50Digital proof herein the antibody of example with high-affinity in conjunction with people CXCR4, and suppress people CXCR4 in conjunction with SDF-1.
The chemotaxis test
CXCR4/SDF-1 interaction reconciliation statement mask has the migration (chemotaxis) of the cell of CXCR4.For determining to detect the antagonist and the cytoactive of antibody, carried out the chemotaxis test, use human tissue cell's property lymphoma U937 cell of expressing endogenous CXCR4.In brief, the U937 cell cultures is in the Dulbecco MEM (DMEM) that contains 10% foetal calf serum, 1% minimum essential medium (MEM) Sodium.alpha.-ketopropionate solution, 1%MEM non-essential amino acid and 1%L-glutamine, harvested cell is also tested damping fluid (containing 10mM HEPES, the 1xRPMI substratum of pH 7.5 and 0.3%BSA) with chemotaxis and is washed once.After the cleaning, with cell with 5x10
6The concentration of cell/mL is resuspended in the test damping fluid.Test is carried out on 96 porocytes migration plate.Generally speaking, will contain scope from 0.5 μ g/mL to the antibody of 50 μ g/mL or the 50 μ L cell mixtures that do not contain antibody place upper chamber, the SDF-1 α (10ng/mL) that 30 μ L are prepared in 1x chemotaxis test damping fluid adds lower chamber.After the assembling, plate placed under 37 ℃, 5% carbonic acid gas hatched 2.5 hours.After hatching, 5 μ L cell proliferation solution are added lower chamber.Place 37 ℃ to hatch 60 minutes plate, use microwell plate to read the cell that the plate device moves by the absorbance detection of measuring 492nm.The CXCR4 antagonist suppresses cell migration, reduces the absorbancy reading.Detect the inhibition ability (IC of antibody in this test
50) use the GraphPadPrism computed in software, based on the dose-dependently reduction of 492nm absorbancy.
The antibody of example has been showed 30nM or lower IC in this test herein
50Antibody I II has showed the IC of average 5.90nM in this test
50Digital proof herein the antibody of example with high-affinity in conjunction with people CXCR4, and suppress people CXCR4 in conjunction with SDF-1.
Combination by the anti-CXCR4 antibody of surface plasma body resonant vibration (BIAcore) assessment is active
Use
2000 apparatus measures binding kinetics and avidity.
Utilize the change of optical properties detection protein concn of interacting molecule in dextran biosensor matrix of surface plasma body resonant vibration.Unless otherwise noted, all test kit materials available from
AB (Upsala, Sweden).All are measured and ℃ to carry out.In conjunction with testing basically according to people such as Stenlund (Stenlund P, Babcock GJ, Sodroski J, Myszka DG. (2003) Captureand reconstitution of G protein-coupled receptors on a biosensorsurface.Anal Biochem.316 (2): 243-50) and people (NavratilovaI such as Navratilo, Sodroski J, Myszka DG. (2005) Solubilization, stabilization, andpurification of chemokine receptors using biosensor technology.Anal Biochem.339 (2): the carrying out of describing 271-81).Running buffer is 50mM HEPES, 5mM magnesium chloride, 1mM calcium chloride, 150mM sodium-chlor and 2mg/mL BSA, and pH 7.5.People CXCR4 acceptor (SEQ ID NO:29) with C-end line C9 peptide tag is cloned in dog thymocyte Cf2Th cell and is crossed and express, with people such as Mirzabekov (Mirzabekov, N.Bannert, M.Farzan, W.Hofmann, P.Kolchinsky, L.Wu, R.Wyatt and J.Sodroski-Enhanced expression, native purification, and characterization ofCCR5, a principal HIV-1 coreceptor.J.Biol.Chem.274 (1999), pp.2874528750) same way as of describing before.By 1D4 monoclonal antibody identification C-end line C9 peptide tag (D.D.Oprian, R.S.Molday, R.J.Kaufman and H.G.Khorana, Expression of a synthetic bovine rhodopsin gene in monkey kidneycells.Proc.Natl.Acad.Sci.USA 84 (1987), pp.88748878).
Use following multiple analysis cycle assessment combination.By amine coupling (about 10,000-20,000 resonance units antibody) 1D4 Mab (mono-clonal 1D4, University of British Columbia) is cured on the CM5 chip.Cell is resuspended in 20mM Tutofusin tris (pH 7.0), 0.1M ammonium sulfate, 10% glycerine, 5mM magnesium chloride, 1mM calcium chloride and full wafer and does not have in the proteinase inhibitor tablet of ethylenediamine tetraacetic acid (EDTA) (solution).With final concentration 4x10
6Cell/mL (is 2.0x10
6Cell, 0.5mL final volume) is injected to chip.5: 1 ratio (cell is than the ratio of damping fluid volume) transfectional cell and contain washing composition (2% CH, 10% Lauryl.beta.-maltoside and 10%3-[(3-courage amidopropyl) dimethylamino]-the 1-propanesulfonic acid) running buffer be transferred to automixer.Mixtures incubated 10 minutes.After hatching, 150 μ L dissolved acceptors are injected to the 1D4 surface with 20 μ L/ minute flow velocitys.Clean sample loop with running buffer afterwards.Afterwards 20 μ L antibody are injected (to chip) with 100 μ L/ minutes flow velocitys.Afterwards with 10mM sodium hydroxide+1%n-octyl group-β-D-glucopyranoside with 2 times 10 pulse per second (PPS)s of the 100 μ L/ minute flow velocitys chip of living again.Each round-robin association rate constant (" k
On") and the rate constant (" k that dissociates
Off") use " mass transfer in 1: 1 " (" 1: 1 with mass transfer ") combination model in the BIA assessment software to assess." K
D" be dissociation constant, pass through formula: k
Off/ k
On=K
DCalculate.Incorporating parametric is summarized as follows.Digital proof herein the antibody of example with high-affinity in conjunction with people CXCR4, and suppress people CXCR4 in conjunction with SDF-1.
| Antibody | K on(M -1s -1) | K off(s -1) | K D(nM) |
| I | 2.36x10 6 | 1.61x10 -3 | 0.68 |
| II | 2.50x10 6 | 1.81x10 -3 | 0.72 |
| III | 1.12x10 6 | 1.72x10 -3 | 1.54 |
| IV | 7.74x10 5 | 3.98x10 -3 | 5.14 |
| V | 1.91x10 6 | 1.68x10 -3 | 0.88 |
Anti-tumor activity in the SCID/Namalwa heteroplastic transplantation model
As if SDF-1/CXCR4 interact and comprising that tumor growth, infringement, blood vessel take place and tumorigenic a plurality of stages of transfer play a significant role.For assessment detects the anti-tumor in vivo activity of antibody in cancer, used the tumour heteroplastic transplantation model that uses NOD/SCID mouse and people's non_hodgkin lymphoma Namalwa cell.In brief, 200,000Namalwa cell and matrigel (1: 1) mix, the back flank of subcutaneous implantation animal.The growth of tumour cell of implanting becomes solid tumor, can use the clamp monitoring continuously and measure its size.Render a service for measuring to detect in the body of antibody in this model, implant 48 hours aftertreatment animals (10/group) at tumour cell with the detection antibody that is dissolved in salt solution or the phosphate buffer solution of various dose.Subcutaneous administration antibody in 1 μ g/ mouse, 10 μ g/ mouse and 1001 μ g/ mouse scopes was measured gross tumor volume and body weight in per 2 or 3 days.Research is general to continue 3-4 week, depends on growth of tumor.By comparing with the control group gross tumor volume with vehicle treated only, the minimizing per-cent of treatment group gross tumor volume determines to detect the neoplasm growth activity of antibody.
In this test, when using with 10 μ g/ mouse, during promptly about 0.4mg/kg, antibody I suppresses tumor growth.The digital proof antibody I has the antitumorgienesis activity by stoping tumor growth.
SCID/Namalwa blood lymphoma model
Be the further anti-tumor activity of research CXCR4 antibody in lymphoma, by with 200, the 000Namalwa cell has been set up blood lymphoma model through tail vein injection to SCID mouse.Generally speaking, the mouse of injection tumour cell is dead in week at 5-6.Render a service for the antibody that detects in this model, detect antibody treatment animal (10 every group) with 30 μ g/ mouse or 100 μ g/ mouse after 24 hours at tumor cell injection.Per 4 days subcutaneous administrations of antibody once continued for 6 weeks, write down the survival condition of animal every day.
In this blood lymphoma model, when comparing with isotype IgG control group with carrier, the antibody I treatment group has shown statistically evident survival advantage.
Anti-tumor activity in the SCID/CEM heteroplastic transplantation model
For assessment detects the anti-tumor in vivo activity of antibody in cancer, used the tumour heteroplastic transplantation model that uses NOD/SCID mouse and cem cell.In brief, 5.0x10
6Cem cell and matrigel (1: 1) mix, the back flank of subcutaneous implantation animal.The growth of tumour cell of implanting becomes solid tumor, can use the clamp monitoring continuously and measure its size.Render a service for measuring to detect in the body of antibody in this model, detect antibody treatment animal (10 every group) with 10 μ g/ mouse, 30 μ g/ mouse or 100 μ g/ mouse after 24 hours at tumor cell transplantation.Per 4 days subcutaneous administrations of antibody were once measured gross tumor volume and body weight in per 2 or 3 days.
Compare with isotype IgG control group with carrier, in the antibody I treatment group, observe the dose-dependently tumor growth and suppress.The antibody I of all 3 kinds of dosage all significantly suppresses tumor growth.
The apoptosis test
Whether apoptosis-induced for research CXCR4 antibody, the kinds of tumor cells of expressing high-level CXCR4 with the detection antibody treatment is.Contain 1% or the substratum of 10%FBS in, detect antibody treatment cell 2-4 days with different concns.After the processing,, clean with D-PBS with 3.7% formaldehyde fixed cell., clean and sealing the processing of permeableization of cell with the 0.1%Triton X-100 among the D-PBS with the D-PBS that contains 1%BSA.Afterwards cell and the rabbit that the D-PBS that uses 1%BSA dilutes are resisted-(Cat#557135 BD Biosciences NC) is hatched 1 hour to activation half Guang L-Aspartase 3 polyclonal antibodies.Cell cleans 2 times with D-PBS, and (CA) (Invitrogen, Carlsbad CA) are hatched 1 hour with 200ng/mL Hoechst 33342 for Invitrogen, Carlsbad with Alexa Fluor 488 goat anti-rabbit iggs with the D-PBS dilution of 1%BSA afterwards.(Cellomics, Pittsburgh Pennsylvania) scan painted plate, use Target Activation biologic applications software to be used for the quantitative of fluorescent signal to use ArrayScan Vti.
The result proves that antibody I induces the activation of nuclear fragmentation and half Guang L-Aspartase 3 in the kinds of tumor cells that comprises Namalwa and cem cell.The activation of nuclear fragmentation and half Guang L-Aspartase 3 is signs of apoptosis.Therefore, digital proof (dosage) when using when between, the apoptosis of antibody I inducing tumor cell with 2 μ g/mL and 10 μ g/mL.
For further the proof antibody I is apoptosis-induced, in the Namalwa cell that detects antibody or isotype IgG control treatment, studied the variation of annexin V by flow cytometer.Antibody I has induced the dose-dependently of annexin V to improve, and not effect of isotype IgG.In addition, antibody I inductive apoptosis also can be observed in CEM xenotransplantation tumour by TUNEL dyeing.
SEQ ID tabulation
Heavy chain CDR
SEQ?ID?NO：1 GFTSTDYYFS
SEQ?ID?NO：2 FIRTKSKGYTTEYSGSVKG
SEQ?ID?NO：3 EPITTDPRDY
SEQ?ID?NO：4 FIRSKSKGYTTEYSGSVKG
SEQ?ID?NO：5 FIRYKSKGYTTEYSGSVKG
SEQ?ID?NO：6 FIRNKRKGYTTEYSGSVKG
SEQ?ID?NO：7 FIRHKSKGYTTEYSGSVKG
Light chain CDR
SEQ?ID?NO：8 KSSQSLFNSRTRKKYLA
SEQ?ID?NO：9 WASKRKS
SEQ?ID?NO：10 KQSRFLRA
Variable region of heavy chain
SEQ?ID?NO：11
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRTKSKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSS
SEQ?ID?NO：12
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGF?IRSKSKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSS
SEQ?ID?NO：13
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRYKSKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSS
SEQ?ID?NO：14
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWYGFIRNKRKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSS
SEQ?ID?NO：15
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRHKSKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSS
Variable region of light chain
SEQ?ID?NO：16
DIVMTQSPDSLAVSLGERATINCKSSQSLFNSRTRKKYLAWYQQKPGQPPKLLIYWASKRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSRFLRAFGQGTKLEIK
Complete heavy chain
SEQ?ID?NO：17
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGF?IRTKSKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
SEQ?ID?NO：18
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRSKSKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
SEQ?ID?NO：19
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRYKSKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
SEQ?ID?NO：20
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRNKRKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
SEQ?ID?NO：21
EVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRHKSKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
Complete light chain
SEQ?ID?NO：22
DIVMTQSPDSLAVSLGERATINCKSSQSLFNSRTRKKYLAWYQQKPGQPPKLLIYWASKRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSRFLRAFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Nucleotide sequence-variable region of heavy chain
SEQ?ID?NO：23
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGACGAAGTCGAAGGGCTACACCACCGAGTACAGCGGCAGCGTGAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCCCATCACCACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
SEQ?ID?NO：24
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGTCTAAGTCGAAGGGCTACACCACCGAGTACAGCGGCAGCGTGAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCCCATCACCACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
SEQ?ID?NO：25
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGTATAAGTCGAAGGGCTACACCACCGAGTACAGCGGCAGCGTGAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCCCATCACCACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
SEQ?ID?NO：26
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGAACAAGCGGAAGGGCTACACCACCGAGTACAGCGGCAGCGTGAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCCCATCACCACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
SEQ?ID?NO：27
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCACCAGTACCGACTACTACTTTAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCTTCATCCGGCACAAGTCGAAGGGCTACACCACCGAGTACAGCGGCAGCGTGAAGGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTACTGTGCTAGAGAGCCCATCACCACCGACCCTCGGGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA
Nucleotide sequence-variable region of light chain
SEQ?ID?NO：28
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGTTCAACAGCCGGACCCGGAAGAAGTACCTGGCCTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCCAGCAAGAGAAAGAGCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTAAGCAGAGCCGTTTTCTGAGAGCCTTTGGCCAAGGGACCAAGCTGGAGATCAAA
The C-end line C9 peptide tag that is used for people CXCR4 acceptor
SEQ?ID?NO：29 TETSQVAPA
People CXCR4
SEQ?ID?NO：30
MEGISSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS
People CXCR4 isotype A
SEQ?ID?NO：31
MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS
People CXCR4 isotype B
SEQ?ID?NO：32
MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS
People SDF-1 α
SEQ?ID?NO：33
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK
Claims (21)
1. in conjunction with people source engineered antibody or its binding fragment of people CXCR4, its:
A. suppress the combination of people SDF-1 α (SEQ ID NO:33) and CXCR4, as people CXCR4/ described herein
125I-SDF-1 α is in conjunction with in the inhibition test, to the IC of people CXCR4
50Between 10nM and 0.05nM;
B. suppress the migration that the surface has the cell of CXCR4, in as chemotaxis test described herein, IC
50Between 30nM and 0.3nM.
C. in as surface plasma resonance described herein (BIAcore) test, showed K
DAvidity between 15nM and 0.05nM.
2. the antibody of claim 1, it suppresses the combination of people SDF-1 α (SEQ ID NO:33) and CXCR4, to the IC of people CXCR4
50Between 0.5nM and 0.05nM.
3. the antibody of claim 1 or claim 2, it suppresses the migration that the surface has the cell of CXCR4, IC
50Between 3.0nM and 0.3nM.
4. each antibody in the claim 1 to 3, it has showed K
DAvidity between 1.0nM and 0.05nM.
5. each antibody in the claim 1 to 4 has been showed the antitumorgienesis activity by the prevention tumor growth in its tumour heteroplastic transplantation model of describing herein when using with 1mg/kg.
6. each antibody in the claim 1 to 5, the apoptosis of inducing tumor cell when using between with 2 μ g/mL and 10 μ g/mL in its apoptosis of describing herein test.
7. people source engineered antibody or its binding fragment, comprise light chain and heavy chain, light chain comprises variable region of light chain, variable region of light chain comprises framework region, CDRL1 with aminoacid sequence of SEQ ID NO:8, CDRL2 and CDRL3 with aminoacid sequence of SEQ ID NO:9 with aminoacid sequence of SEQ ID NO:10, heavy chain comprises variable region of heavy chain, variable region of heavy chain comprises framework region, CDRH1 with aminoacid sequence of SEQ IDNO:1, have SEQ ID NO:3 aminoacid sequence CDRH3 and have the SEQ of being selected from an ID NO:2, SEQ ID NO:4, SEQ ID NO:5, the CDRH2 of the aminoacid sequence of SEQ ID NO:6 and SEQID NO:7, wherein antibodies people CXCR4.
8. the antibody of claim 7, wherein light chain comprises the aminoacid sequence of SEQ ID NO:16.
9. the antibody of claim 7 or claim 8, wherein heavy chain comprises the aminoacid sequence that is selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.
10. each antibody in the claim 7 to 9, wherein light chain comprises the aminoacid sequence of SEQ ID NO:22.
11. each antibody in the claim 7 to 10, wherein heavy chain comprises the aminoacid sequence that is selected from SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
12. each antibody in the claim 7 to 11, described antibody are selected from antibody, the antibody that comprises SEQ ID NO:18 and SEQ ID NO:22, the antibody that comprises SEQ ID NO:19 and SEQ ID NO:22 that comprise SEQ ID NO:17 and SEQ ID NO:22, comprise the antibody of SEQ ID NO:20 and SEQ ID NO:22 and comprise SEQ ID NO:21 and the antibody of SEQ ID NO:22.
13. each antibody in the claim 7 to 12, described antibody comprise the light chain of 2 SEQ ID NO:22 and the heavy chain of 2 SEQ ID NO:17.
14. pharmaceutical composition comprises in the claim 1 to 13 combination of each antibody and one or more pharmaceutically useful carriers, thinner or vehicle.
15. the purposes of each antibody in treatment in the claim 1 to 13.
16. the purposes of each antibody in the treatment tumour takes place in the claim 1 to 13, described tumour comprises that tumor growth, infringement, blood vessel take place or transfer.
17. each antibody is used for the treatment of purposes in the tumorigenic medicine in preparation in the claim 1 to 13, described tumour comprises that tumor growth, infringement, blood vessel take place or shift.
18. treatment comprises the tumorigenic method that tumor growth, infringement, blood vessel take place or shift, and comprises each antibody in the claim 1 to 13 of patient's administering therapeutic significant quantity of needs.
19. the purposes of each antibody in the treatment cancer in the claim 1 to 13, described cancer is selected from mammary cancer, carcinoma of the pancreas, melanoma, prostate cancer, kidney, neuroblastoma, non_hodgkin lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme and leukemia.
20. each antibody is used for the treatment of purposes in the medicine of cancer in preparation in the claim 1 to 13, described cancer is selected from mammary cancer, carcinoma of the pancreas, melanoma, prostate cancer, kidney, neuroblastoma, non_hodgkin lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme and leukemia.
21. treatment method for cancer, described cancer is selected from mammary cancer, carcinoma of the pancreas, melanoma, prostate cancer, kidney, neuroblastoma, non_hodgkin lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme and leukemia, and method comprises each antibody in the claim 1 to 13 of patient's administering therapeutic significant quantity of needs.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US5319208P | 2008-05-14 | 2008-05-14 | |
| US61/053,192 | 2008-05-14 | ||
| PCT/US2009/043063 WO2009140124A1 (en) | 2008-05-14 | 2009-05-07 | Anti-cxcr4 antibodies |
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| CN102027015A true CN102027015A (en) | 2011-04-20 |
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| CN2009801172303A Pending CN102027015A (en) | 2008-05-14 | 2009-05-07 | Anti-CXCR4 antibodies |
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| EP (1) | EP2297206A1 (en) |
| JP (1) | JP2011520885A (en) |
| KR (1) | KR20100133012A (en) |
| CN (1) | CN102027015A (en) |
| AU (1) | AU2009246683A1 (en) |
| CA (1) | CA2724409A1 (en) |
| EA (1) | EA201071300A1 (en) |
| IL (1) | IL208869A0 (en) |
| MX (1) | MX2010012435A (en) |
| WO (1) | WO2009140124A1 (en) |
| ZA (1) | ZA201007955B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105143259A (en) * | 2013-03-15 | 2015-12-09 | 伊莱利利公司 | Pan-ELR+ CXC Chemokine Antibody |
| CN112521500A (en) * | 2020-12-25 | 2021-03-19 | 暨南大学 | Affinity maturation binding proteins that bind to CXCR4 and uses thereof |
| CN112661845A (en) * | 2020-12-25 | 2021-04-16 | 暨南大学 | Affinity maturation binding proteins that bind to CXCR4 and uses thereof |
Families Citing this family (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2172485A1 (en) * | 2008-10-01 | 2010-04-07 | Pierre Fabre Medicament | Novel anti CXCR4 antibodies and their use for the treatment of cancer |
| US9155780B2 (en) | 2009-02-11 | 2015-10-13 | Yeda Research And Development Co. Ltd. | Short beta-defensin-derived peptides |
| EP2246364A1 (en) * | 2009-04-29 | 2010-11-03 | Pierre Fabre Médicament | Anti CXCR4 antibodies for the treatment of HIV |
| GB201002238D0 (en) | 2010-02-10 | 2010-03-31 | Affitech As | Antibodies |
| EP2371863A1 (en) * | 2010-03-30 | 2011-10-05 | Pierre Fabre Médicament | Humanized anti CXCR4 antibodies for the treatment of cancer |
| MX2013004710A (en) * | 2010-10-27 | 2013-08-29 | Pf Medicament | Antibodies for the treatment of hiv. |
| US20140120555A1 (en) * | 2011-06-20 | 2014-05-01 | Pierre Fabre Medicament | Anti-cxcr4 antibody with effector functions and its use for the treatment of cancer |
| WO2012178137A1 (en) | 2011-06-24 | 2012-12-27 | Gillies Stephen D | Light chain immunoglobulin fusion proteins and methods of use thereof |
| JP2014523745A (en) * | 2011-07-20 | 2014-09-18 | メディミューン リミテッド | Anti-CXCR4 antibody and method of use |
| AR087364A1 (en) | 2011-07-29 | 2014-03-19 | Pf Medicament | ANTI-CXCR4 ANTIBODY AND ITS USE FOR CANCERES DETECTION AND DIANOSTIC |
| EP2776032B1 (en) * | 2011-11-09 | 2018-10-17 | Bristol-Myers Squibb Company | Treatment of hematologic malignancies with an anti-cxcr4 antibody |
| MX2017015811A (en) | 2015-06-12 | 2018-04-10 | Squibb Bristol Myers Co | Treatment of cancer by combined blockade of the pd-1 and cxcr4 signaling pathways. |
| JP7261379B2 (en) | 2016-06-20 | 2023-04-20 | カイマブ・リミテッド | Anti-PD-L1 antibody |
| ES2973728T3 (en) | 2018-03-13 | 2024-06-24 | Fundacion Para La Investigacion Biomedica Del Hospital Univ La Paz | Anti-CXCR4 Antibody Combined with Activated and Expanded Natural Killer Lymphocytes for Cancer Immunotherapy |
| JP7611700B2 (en) | 2018-03-27 | 2025-01-10 | ブリストル－マイヤーズ スクイブ カンパニー | Real-time titer monitoring using UV signal |
| WO2020172658A1 (en) | 2019-02-24 | 2020-08-27 | Bristol-Myers Squibb Company | Methods of isolating a protein |
| KR20220012292A (en) | 2019-05-23 | 2022-02-03 | 브리스톨-마이어스 스큅 컴퍼니 | How to monitor cell culture media |
| CN116406369A (en) | 2020-10-05 | 2023-07-07 | 百时美施贵宝公司 | Method for concentrating proteins |
| US20250188152A1 (en) | 2022-03-09 | 2025-06-12 | Bristol-Myers Squibb Company | Transient expression of therapeutic proteins |
| WO2025049591A1 (en) * | 2023-08-28 | 2025-03-06 | Regeneron Pharmaceuticals, Inc. | Anti-cxcr4 antibodies and uses thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19801265C1 (en) * | 1998-01-15 | 1999-08-12 | Deutsches Krebsforsch | Inhibition of CD95-independent apoptosis in AIDS |
| US20050002939A1 (en) * | 2002-12-23 | 2005-01-06 | Albert Zlotnik | Tumor killing/tumor regression using CXCR4 antagonists |
| US8329178B2 (en) * | 2005-02-18 | 2012-12-11 | Dana-Farber Cancer Institute, Inc. | Antibodies against CXCR4 and methods of use thereof |
| LT2486941T (en) * | 2006-10-02 | 2017-06-12 | E. R. Squibb & Sons, L.L.C. | Human antibodies that bind CXCR4 and uses thereof |
| FR2915102B1 (en) * | 2007-04-23 | 2014-05-16 | Pf Medicament | USE OF ANTI-CXCR4 ANTIBODY FOR THE TREATMENT OF CANCER |
-
2009
- 2009-05-07 EP EP09747210A patent/EP2297206A1/en not_active Ceased
- 2009-05-07 AU AU2009246683A patent/AU2009246683A1/en not_active Abandoned
- 2009-05-07 JP JP2011509562A patent/JP2011520885A/en not_active Withdrawn
- 2009-05-07 CA CA2724409A patent/CA2724409A1/en not_active Abandoned
- 2009-05-07 MX MX2010012435A patent/MX2010012435A/en active IP Right Grant
- 2009-05-07 WO PCT/US2009/043063 patent/WO2009140124A1/en active Application Filing
- 2009-05-07 CN CN2009801172303A patent/CN102027015A/en active Pending
- 2009-05-07 EA EA201071300A patent/EA201071300A1/en unknown
- 2009-05-07 KR KR1020107025445A patent/KR20100133012A/en not_active Ceased
-
2010
- 2010-10-21 IL IL208869A patent/IL208869A0/en unknown
- 2010-11-05 ZA ZA2010/07955A patent/ZA201007955B/en unknown
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105143259A (en) * | 2013-03-15 | 2015-12-09 | 伊莱利利公司 | Pan-ELR+ CXC Chemokine Antibody |
| CN105143259B (en) * | 2013-03-15 | 2018-09-21 | 伊莱利利公司 | Pan-ELR+CXC chemotactic factor (CF) antibody |
| CN109134652A (en) * | 2013-03-15 | 2019-01-04 | 伊莱利利公司 | Pan-ELR+CXC chemotactic factor (CF) antibody |
| CN109134652B (en) * | 2013-03-15 | 2022-05-24 | 伊莱利利公司 | Pan-ELR+ CXC chemokine antibody |
| CN112521500A (en) * | 2020-12-25 | 2021-03-19 | 暨南大学 | Affinity maturation binding proteins that bind to CXCR4 and uses thereof |
| CN112661845A (en) * | 2020-12-25 | 2021-04-16 | 暨南大学 | Affinity maturation binding proteins that bind to CXCR4 and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20100133012A (en) | 2010-12-20 |
| CA2724409A1 (en) | 2009-11-19 |
| EP2297206A1 (en) | 2011-03-23 |
| WO2009140124A1 (en) | 2009-11-19 |
| AU2009246683A1 (en) | 2009-11-19 |
| MX2010012435A (en) | 2011-05-03 |
| ZA201007955B (en) | 2012-04-25 |
| EA201071300A1 (en) | 2011-06-30 |
| JP2011520885A (en) | 2011-07-21 |
| IL208869A0 (en) | 2011-01-31 |
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