CN1326878C - Anti human non-Hodgkin's lymphoma chimeric antibody and its derivative and application - Google Patents
Anti human non-Hodgkin's lymphoma chimeric antibody and its derivative and application Download PDFInfo
- Publication number
- CN1326878C CN1326878C CNB031230547A CN03123054A CN1326878C CN 1326878 C CN1326878 C CN 1326878C CN B031230547 A CNB031230547 A CN B031230547A CN 03123054 A CN03123054 A CN 03123054A CN 1326878 C CN1326878 C CN 1326878C
- Authority
- CN
- China
- Prior art keywords
- ser
- chimeric antibody
- leu
- sequence
- thr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 title claims abstract description 41
- 102100038080 B-cell receptor CD22 Human genes 0.000 claims abstract description 18
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims description 19
- 125000000539 amino acid group Chemical group 0.000 claims description 17
- 201000010099 disease Diseases 0.000 claims description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 9
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 4
- 229960004316 cisplatin Drugs 0.000 claims description 4
- 231100000614 poison Toxicity 0.000 claims description 4
- 239000002574 poison Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 208000023275 Autoimmune disease Diseases 0.000 claims description 3
- -1 I 131 Chemical compound 0.000 claims description 3
- 238000012412 chemical coupling Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 claims description 2
- 229960004768 irinotecan Drugs 0.000 claims description 2
- 206010025135 lupus erythematosus Diseases 0.000 claims description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 2
- 229960000641 zorubicin Drugs 0.000 claims description 2
- FBTUMDXHSRTGRV-ALTNURHMSA-N zorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(\C)=N\NC(=O)C=1C=CC=CC=1)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 FBTUMDXHSRTGRV-ALTNURHMSA-N 0.000 claims description 2
- 229910021173 SMO3 Inorganic materials 0.000 claims 16
- 150000001413 amino acids Chemical group 0.000 claims 2
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 16
- 239000013604 expression vector Substances 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 10
- 238000002474 experimental method Methods 0.000 abstract description 7
- 230000005847 immunogenicity Effects 0.000 abstract description 7
- 238000010276 construction Methods 0.000 abstract 2
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 abstract 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 abstract 1
- 230000003053 immunization Effects 0.000 abstract 1
- 238000002649 immunization Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 35
- 241001529936 Murinae Species 0.000 description 32
- 238000000034 method Methods 0.000 description 20
- 210000003719 b-lymphocyte Anatomy 0.000 description 19
- 206010028980 Neoplasm Diseases 0.000 description 16
- 201000011510 cancer Diseases 0.000 description 14
- 125000003275 alpha amino acid group Chemical group 0.000 description 13
- 235000015097 nutrients Nutrition 0.000 description 12
- 229960004641 rituximab Drugs 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 10
- 108010076504 Protein Sorting Signals Proteins 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 238000002512 chemotherapy Methods 0.000 description 10
- 102220023256 rs387907547 Human genes 0.000 description 10
- 238000010367 cloning Methods 0.000 description 9
- 108060003951 Immunoglobulin Proteins 0.000 description 8
- 102000018358 immunoglobulin Human genes 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 230000000875 corresponding effect Effects 0.000 description 7
- 231100000135 cytotoxicity Toxicity 0.000 description 7
- 230000003013 cytotoxicity Effects 0.000 description 7
- 229940079593 drug Drugs 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 238000002560 therapeutic procedure Methods 0.000 description 5
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 206010025323 Lymphomas Diseases 0.000 description 4
- 102220369446 c.1274G>A Human genes 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000004520 electroporation Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 230000002637 immunotoxin Effects 0.000 description 4
- 229940051026 immunotoxin Drugs 0.000 description 4
- 239000002596 immunotoxin Substances 0.000 description 4
- 231100000608 immunotoxin Toxicity 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 230000010534 mechanism of action Effects 0.000 description 4
- 102220023257 rs387907546 Human genes 0.000 description 4
- 230000009182 swimming Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 230000003073 embolic effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- 230000010412 perfusion Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 108010029157 Sialic Acid Binding Ig-like Lectin 2 Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 238000010322 bone marrow transplantation Methods 0.000 description 2
- 102220369447 c.1352G>A Human genes 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000000973 chemotherapeutic effect Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 229960004679 doxorubicin Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 229950009760 epratuzumab Drugs 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 201000009277 hairy cell leukemia Diseases 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000000527 lymphocytic effect Effects 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 210000003519 mature b lymphocyte Anatomy 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 210000002741 palatine tonsil Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000000306 recurrent effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 102220023258 rs387907548 Human genes 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 102000019260 B-Cell Antigen Receptors Human genes 0.000 description 1
- 108010012919 B-Cell Antigen Receptors Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010010144 Completed suicide Diseases 0.000 description 1
- 108010053187 Diphtheria Toxin Proteins 0.000 description 1
- 102000016607 Diphtheria Toxin Human genes 0.000 description 1
- MWWSFMDVAYGXBV-RUELKSSGSA-N Doxorubicin hydrochloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 MWWSFMDVAYGXBV-RUELKSSGSA-N 0.000 description 1
- 241000406668 Loxodonta cyclotis Species 0.000 description 1
- NPBGTPKLVJEOBE-IUCAKERBSA-N Lys-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N NPBGTPKLVJEOBE-IUCAKERBSA-N 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- 206010042971 T-cell lymphoma Diseases 0.000 description 1
- 208000027585 T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000001055 blue pigment Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 210000001728 clone cell Anatomy 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000011254 conventional chemotherapy Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000001085 cytostatic effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229960002918 doxorubicin hydrochloride Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 229960005420 etoposide Drugs 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 201000003444 follicular lymphoma Diseases 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 230000004987 nonapoptotic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 229940116176 remicade Drugs 0.000 description 1
- 229940107685 reopro Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229940115586 simulect Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention discloses an antihuman non-Hodgkin lymphoma chimeric antibody, a construction method thereof and an application thereof. The present invention aims to provide the antihuman non-Hodgkin lymphoma chimeric antibody and a derivative thereof, wherein the antihuman non-Hodgkin lymphoma chimeric antibody can maintain the original specificity, the original affinity and low immunogenicity. The antihuman non-Hodgkin lymphoma chimeric antibody is specifically different from a chimeric antibody SM03 of CD22, and the humanized constant region of the antihuman non-Hodgkin lymphoma chimeric antibody is of the isotype human IGg1/ human Kappa. In the construction method for expression vectors of the chimeric antibody SM03, the DNA sequences of the variable regions of light chains and the variable regions of heavy chains of the SM03 are respectively connected to the corresponding constant regions of the expression vectors of humanized light chains and humanized heavy chains so as to obtain the expression vectors of the light chains and the heavy chains of the chimeric antibody SM03. The chimeric antibody SM03 has extremely low immunogenicity, and an IgG1 humanized Fc fragment also strengthens the curative effect of immunization, which is initiated by the antibody; experiments verify that the purified chimeric antibody SM03 and the mother antibody of a mouse source have the same specificity and affinity.
Description
Technical field
The present invention relates to a kind of anti-people's non Hodgkin lymphoma chimeric antibody and derivative and application, particularly the chimeric antibody of a kind of CD22 of being specific to and derivative thereof and be the medicine of activeconstituents with them.
Background technology
Non Hodgkin lymphoma (non-Hodgkin ' slymphoma, NHL) be the Lymphoid tissue tumour of a group heterogeneous (heterogeneous).In the U.S., there is 6 percent to eight cancer to be diagnosed as NHL, 5.5 ten thousand new cases are arranged every year approximately.Estimate that according to U.S. carninomatosis association (The American Cancer Society) NHL male sex's sickness rate has 2.6 ten thousand people to die from this disease (Cancer Statistics a little more than the women every year approximately, CA:Cancer J.forClinicians, Jan/Feb 2000, Vol.50, No.1).Though existing a lot of effectively chemotherapeutics can be treated NHL, NHL remains the fifth-largest killer of the U.S..Most common therapeutic method comprises high dose chemotherapy, radiotherapy, and self-bone marrow transplantation etc.But these therapies all fail to make most of patient to obtain comparatively persistent disease alleviation.
In general, made a definite diagnosis the patient who suffers from slight folliculus (low grade follicular) NHL, mean survival time probably has five to seven years (Dana et al.1990.J.Clin.Oncol.8:1155-1162).But except the I phase does not also spread the lymphoma patient of (Stage I localized), concerning most of patient, conventional chemotherapy means can not reach the purpose of healing.Multinomial research points out that various conventional embolic chemotherapy and means fail to prolong follicular lymphoma patient's life-span (Klimo et al.1987.Semin.Hemat.24:26-34; Fisheret al.1993.N.Eng.J.Med.328:1002-1006; Longo et al.1991.J.Clin.Oncol.9:25-38).
Also there is report to point out, treats preliminary complete reaction (initial complete response) (the Dana et al.1990.J.Clin.Oncol.8:1155-1162 that the patient who suffers from extensive diffusive NHL has 60-80% with the means of multiple different chemotherapy combinations; Klimo et al.1987.Semin.Hemat.24:26-34; Fisher et al.1993.N.Eng.J.Med.328:1002-1006).However, but still have the patient of 20-40% to fail to obtain preliminary complete reaction, and obtain to have among the preliminary complete reaction patient 20-40% to recur.The embolic chemotherapy (conventional salvage chemotherapy) of the still available tradition rescue property of the patient of these recurrences is cured, and wherein this class of 30-40% patient can reach preliminary complete reaction; But major part has the patient of reaction for the second time can recur (Velasquez et al.1988.Blood 71:117-122 in a short time; Cabanillas et al.1982.Blood60:693-697; Cabanillas et al.1987.J.Clin.Oncol.5:407-412).
By high dose chemotherapy (high-dose chemotherapy), add autologous bone marrow transplantation (autologousbone marrow transplantation) or peripheral stem cell transplantation (ABMT/PSCT), can make the recurrence of 25-40% or high-risk NHL patient reach existing state (the Velasquez et al.1988.Blood71:117-122 that avoids morbid state for a long time; Cabanillas et al.1982.Blood 60:693-697; Cabanillas et al.1987.J.Clin.Oncol.5:407-412).But the above-mentioned patient of remaining 60-75% also can be recurred after too high dose chemotherapy and the ABMT/PSCT treatment accepting.Concerning attempting embolic chemotherapy that tradition rescues property and/or high dose chemotherapy and ABMT/PSCT but the invalid recurrent NHL patient, extremely lack brand-new effective methods of treatment.
The appearance of monoclonal antibody provides another kind of treatment means.From Milstein﹠amp; Kohler has invented hybridoma technology in 1975, monoclonal antibody is in clinical diagnosis, and the applying value and the DEVELOPMENT PROSPECT of treatment and prevention aspect have obtained general affirming.For example, monoclonal antibody-radioactivity conjugate is used as treatment t cell lymphoma (Rosenet al.1989.Nucl.Med.Biol.16:667-668), pernicious melanosarcoma (Larson et al.1983.J.Clin.Invest.72:2102-2114), ovarian cancer (Epenetos et al.1987.J.Clin.Oncol.5:1890-1899), and Hodgkin (Lenhard et al.1985.J.Clin.Oncol.3:1296-1300).But, the clinical application of monoclonal antibody, subject matter is that most monoclonal antibody all is the B cell hybridoma from mouse.The antibody in these mouse sources singly can not cause " human anti-mouse antibody " immune response (HAMA) in human body, and also shortage can guide the human body autoimmune function as with the complement being the cytotoxicity (CMC) of media or the cytotoxicity (ADCC) of antibody dependent cellular mediation etc.Until middle 1990s, united states drug Surveillance Authority (FDA) just for the first time ratifies a kind of curative monoclonal antibody, for NHL patient brings new hope.Getting permission in 1997 at " Rituximab " of U.S. listing (Rituxan), is a chimeric mAb (chimericantibody), also is that first is got permission the antibody with the form of therapy cancer of naked antibody since the dawn of human civilization, and its special target is a CD20 antigen.CD20 antigen only finds on normal B cell and Malignant B cell surface.Rituximab is used for the treatment of recurrence or traditional treatment is lost slight (low grade) NHL of reaction the most effective.The method of standard care NHL is to slight NHL, the effect that the middle rank (intermediate) of recurrent and high-level (high-grade) lymphoma are not cured (Czuczman et al.1992.Contemporary Oncology, Septemberp.45-52).It is the B cell tumour that 80% NHL is arranged approximately, and all expresses distinctive CD20 antigen above this class B cancer cells surface of 95%.CD20 antigen is only at sophisticated B cell surface expression; Greatly/little pre B cell, early/organize the B cell late period, the stem cell of hematopoiesis, general plasmocyte, or the healthy tissues of non-lymph etc. can not expressed CD20.So in the category of immunotherapy, CD20 is a target spot that attracts very much the investigator to note.And CD20 antigen can't come off from cell surface, also not can with antibodies after and regulate its expression amount.Under suitable condition, test tube experiment showed, that Rituximab can bring out ADCC, functional effects such as CDC (Reff et al.1994.Blood83:435-445).In addition, the Rituximab effect that strain has growth to restrain to bone-marrow-derived lymphocyte in the test tube experiment, also the cancer cells that chemotherapeutic is produced resistibility can be become to chemotherapeutic, as diphtheria toxin, Ricin, cisplatin (cisplatinum), doxorubicin hydrochloride (doxorubicin), with rely on pool sweet (etoposide) etc., more responsive, and cause the apoptosis effect (Demidem et al.1997.Cancer Biother.Radiopharmacol.12:177-185) of dose response (dose-response).
Though it is effective to non Hodgkin lymphoma that Rituximab is extensively confirmed, most patient finally can be recurred.Wherein the patient of part recurrence has lost reaction to Rituximab.And, be not that each patient who suffers from non Hodgkin lymphoma responds to Rituximab.Concerning these unfortunate patients, they are badly in need of a kind of the elephant as the Rituximab effectively and the appearance of the another kind of antibody of same safety.In fact, except anti-CD20 antibodies, also have much and can and be specific to the antibody of B cell at different qualities.Just the antibody with anti-CD22 is example, and these antibody do not have cross reaction except having the specific combination with normal B cell with other healthy tissues.Because the B cell upgrades metabolism hastily in human body, and the specific antibody of B cell does not influence the first ancestor stem cell, so these antibody only can be of short duration and be transition natures in the intravital toxicity of people, can not constitute clinical problem (Stein et al.1993.Cancer Immunol.Immunother.37:1293-298).
CD22 antigen can find in all B cells, comprise pre B cell and ancestral B cell (Dorkin et al.1989.In:Leukocyte Typing IV:White Cell Differentiation Antigens, Knapp etal., Eds.P.63-64.Oxford University Press, New York, New York).But the surface expression of CD22 only finds on the surface of difference mature B cell stage by stage.CD22 plays a part certain (Tedder et al.1997.Annu.Rev.Immunol.15:481 to the reaction that adjusting B cell causes because of antigen; Law et al.1994.Immunol.Today 15:442).Sherbina etc. (Sherbina et al.1996.J.Immunol.157:4390-4398) have proved that working as B cell antigen receptor senses stimulation, the intravital CD22 of cell can move to plasma membrane from tenuigenin rapidly, thereby makes it express and increase 50-100% at be upset back 5 minutes internal surfaces of cell.And the CD22 of surface expression is also constantly with t
1/2Less than one hour speed internalization (Shanet al.1995.J.Immunol.154:4466) rapidly.Also can be brought into (Shan et al.1995.J.Immunol.154:4466 in the cell paste with CD22 at cell surface bonded antibody with same speed; Shih et al.1994.Int.J.Cancer 56:538; Leung et al.1995.Mol.Immunol.32:1413).And the original murine antibody of anti-NHL is obviously bigger than normal B cell to the avidity (avidity) of Malignant B cell.
Scientific circles are also unclear to the mechanism of action of the treatment effective non-coupling antibody of cancer (naked antibody).In general, the mechanism of action of suggestion is: (1) apoptosis (apoptosis); (2) cell growth inhibitory effect (growtharrest); (3) cytotoxicity (ADCC) of antibody dependent cellular mediation; (4) cytotoxicity of complement-mediated (CDC) etc.Owing to lack the animal model of a rational people NHL, prove or find out very difficulty of the lymphadenomatous mechanism of action of anti-CD22 Antybody therapy.
The humanized antibody hLL2 (epratuzumab) of anti-CD22 is once at clinical I, and the II phase is used in testing, with treatment NHL patient.Usage is the form with naked antibody, with similar sharp method medication (once a week, with the method medication of intravenous infusion, being total to the four stars phase) of holding in the palm former times, curative effect highly significant.Have the patient of half that medicine is had objective curative effect reaction (objective response) approximately, and wherein half conditions of patients that responds all obtain to alleviate fully (complete remission) (Leonard et al.2000.Proc.Am.Soc.Clin.Oncol.19:17a; Leonard et al.2000.Blood 96:578a; Leonard et al.2002.Semin Oncol 29:81-86; Press et al.2001.Hematology 221-240).It is as safe as a house that humanized antibody that it should be noted that this anti-CD22 is used for human body.Test in the clinical I/II phase that the New of USA New York York Weil Cornell Medical Center is carried out, the patient infused the 120-1000 milligram/square metre/week, totally four times, toxic reaction all is with the process of transfusion but not relevant with medicine basically, and only belongs to Grade I toxicity.Symptom comprises heating, feels cold and ypotension etc.The same with Rituximab, its mechanism of action also fails to be proved till now.
The mouse source antibody of anti-NHL (Campana et al.1985.J.Immunol.134:1524-1530), once be widely used as immunotoxin, and obtain to a certain degree anti-NHL and hairy cell leukemia (hairy cellleukemia) clinical before and clinical efficacy (Amlot et al.1993.Blood 82:2624-2633; Sausvilleet al.1995.Blood 85:3457-3465; Mansfield et al.1996.Bioconjugate Chem.7:557-563; Mansfield et al.1997.Blood 90:2020-2026).Because immunotoxin intracellular toxin part all is the biological extraction from bacterium or inhuman source, the immunogenicity of itself is very high, and in this case, no matter whether humanization or chimericization of antibody moiety is little to the whole immunogenicity influence of immunotoxin.So, never will resist chimericization of original murine antibody or humanized trial or the report of NHL.
Summary of the invention
The purpose of this invention is to provide a kind of energy and keep original specificity and avidity, the anti-people's non Hodgkin lymphoma chimeric antibody and the derivative thereof of reduced immunogenicity.
Anti-people's non Hodgkin lymphoma chimeric antibody provided by the invention is the chimeric antibody SM03 that is specific to CD22, and its Ren Yuan Heng distinguishes the Kappa into isotype (isotype) people IGg1/ people surely.Its light chain (VK) and heavy chain (VH) variable region amino acid sequence (variable region sequences) are derived from original murine antibody variable region amino acid sequence, and as shown in Figure 1, wherein the variable region of light chain of original murine antibody is SEQ ID №: 1; The variable region of heavy chain of original murine antibody is SEQ ID №: 2; Among the figure, the sequence in the frame is CDR; The light chain of described SM03 is SEQ ID № in the sequence table: 7 amino acid residue sequence: the heavy chain of described SM03 is SEQ ID № in the sequence table: 8 amino acid residue sequence.
The light chain of described SM03 (VK) and heavy chain (VH) variable region amino acid sequence (variable regionsequences) have comprised at N-end and/or C-end introduces the human antibody amino-acid residue different with original murine antibody, as shown in Figure 2, wherein the variable region of light chain of chimeric antibody SM03 is SEQ ID №: 3; The variable region of heavy chain of chimeric antibody SM03 is SEQ ID №: 4; The amino-acid residue of line part is the sequence different with the original murine antibody variable region sequences, and the sequence in the frame is CDR.The light chain of described SM03 is SEQ ID № in the sequence table: 9 amino acid residue sequence; The heavy chain of described SM03 is SEQ ID № in the sequence table: 10 amino acid residue sequence.
The derivative of chimeric antibody SM03 provided by the present invention is SM03 fragment (Fab, Fab ', F (ab ') 2, Deng), single-chain antibody (scFv, diabodies), the antibody/antibody fragment-factor is (as, antibody/antibody fragment-interleukin-, antibody/antibody fragment-Interferon, rabbit, antibody/antibody fragment-toxin) fusion rotein, antibody/antibody fragment-chemical coupling thing (as, antibody/antibody fragment-chemical poison, antibody/antibody fragment-active nucleus) etc.
Described chemical poison is Zorubicin (doxorubicin), irinotecan, and cisplatin (cisplatin), etc.; Described active nucleus is I
125, I
131, Y
90, In
111, Re
188Deng.
Another object of the present invention provides a kind of method that makes up chimeric antibody SM03 expression vector.
The method of structure chimeric antibody SM03 expression vector provided by the present invention is that the dna sequence dna with the light chain of SM03 and variable region of heavy chain is connected to corresponding separately people's endogenous light chain and heavy chain expression carrier De Heng distinguishes surely, obtains light chain and the heavy chain expression carrier of chimeric antibody SM03.Specifically, it can may further comprise the steps:
(1) VK of clone SM03 and the dna sequence dna of VH;
(2) dna sequence dna of the VK of the SM03 after will cloning and VH inserts the stage carrier;
(3) dna sequence dna with VK in the stage carrier and VH inserts corresponding separately people's endogenous light chain (CK) and heavy chain (IgG respectively
1) expression vector De Heng distinguishes surely, obtains light chain and the heavy chain expression carrier of SM03.
Described stage carrier is VKpSTAGE and VHpSTAGE.
Described light chain and heavy chain expression carrier are respectively SM03pEkappa and SM03pEgammal.
The medicine that the 3rd purpose of the present invention provides a kind of effective treatment and CD22 expresses diseases related, this drug source is lower from the immunogenicity of female immunoglobulin (Ig).
Treatment provided by the present invention and CD22 express the medicine of diseases related, and its activeconstituents is a chimeric antibody SM03 or derivatives thereof.
Medicine provided by the invention is the naked antibody of chimeric antibody SM03, also can be the antibody coupling matter of chimeric antibody SM03, and the mode that can inject is used for human body.
When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant of pharmaceutical field routine etc., can also add flavouring agent, sweeting agent etc. in case of necessity.
Medicine of the present invention can be made various ways such as injection liquid, tablet, pulvis, granula, capsule, oral liquid, paste, creme.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
The consumption of said medicine is generally 100-400mg/m
2(injection), be the four stars phase course of treatment, the per week medication is once.
Because chimeric antibody SM03 also is the antibody of anti-CD22 (B epi-position), the same with epratuzumab, it also is human IgG1/kappa isotype, and its original murine antibody widely with the form of immunotoxin carry out clinical before and clinical trial, proved that it only combines with mature B cell or B cancer cells specifically.With naked antibody, or being applied on one's body the NHL patient with conjugate (as active nucleus, toxin etc.) bonded mode, especially for those anti-CD20 antibodies such as profit holder former times etc. being lost the patient of curative effect reaction, is safe and effective therapeutic method.
Chimeric antibody SM03 is except can be with the form of naked antibody as medicine, and its derivative also can be used for treating different and CD22 and expresses diseases related (as, NHL, rheumatoid arthritis, lupus erythematosus, and other autoimmune disease etc.).
The present inventor is according to the sequence in its original murine antibody variable region, with gene synthetic method, the dna sequence dna of VL and VH is connected to corresponding separately people's endogenous light chain (CK) and heavy chain (IgG
1) Heng distinguishes surely.The light chain (VK-CK) and the heavy chain (VH-CH1-hinge-CH that have chimeric antibody SM03
2-CH
3) carrier by electroporation (electroporation) transfection to SP2/0 myeloma cell (myeloma cell line) and after expressing, it is the IgG in people source that isolated chimeric antibody has 2/3 aminoacid sequence
1/ k isotype (isotype).Similar IgG
1/ k isotype chimeric antibody is as Rituximab (rituximab) (IDEC/Genentech, CA, USA), Reopro (Centocor, PA, USA), Remicade (Centocor, PA, USA), Simulect (Novartis, NJ, USA) etc., be widely used, and confirmed its security and few side effect.So the immunogenicity of chimeric antibody SM03 is extremely low.IgG wherein
1People source Fc section also can add the immune curative effect that powerful antibody causes.Chimeric antibody SM03 behind the purifying the experiment proved that to have with its original murine antibody the same specificity and avidity.
Have the chimeric antibody SM03 that Ren Yuan Heng distinguishes surely and have the cytotoxicity (ADCC) that biological effect is active as antibody dependent cellular mediates, or the cytotoxicity of complement-mediated (CMC) etc.
Chimeric mAb SM03 can discern CD22 antigen and do combining of specificity with it.Chimeric mAb SM03 has specific the combination with the B cell, thereby instrument/means that B cell NHL had medical potentiality are provided.
With immunoglobulin (Ig) of the present invention is that the medicine of activeconstituents can suffer from the cancer of CD22 height expression amount or the diseases such as rheumatic arthritis that disease of immune system causes in treatment and is used widely.
Description of drawings
Fig. 1 is the VK of chimeric antibody SM03 original murine antibody and the aminoacid sequence of VH
Fig. 2 is the VK of chimeric antibody SM03 and the aminoacid sequence of VH
Fig. 3 is the VK of chimeric antibody SM03 and the dna sequence dna of VH
Fig. 4 A is the aminoacid sequence that the chimeric antibody SM03 people Kappa Heng of variable region sequences unmodified distinguishes chimeric light chain (VK-CK) immunoglobulin (Ig) surely
Fig. 4 B is the aminoacid sequence of chimeric antibody SM03 human IgG1 heavy chain (VH-CH1-hinge-CH2-CH3) immunoglobulin (Ig) of variable region sequences unmodified
Fig. 5 A is the aminoacid sequence that the chimeric antibody SM03 people Kappa Heng of variable region sequences after modified distinguishes chimeric light chain (VK-CK) immunoglobulin (Ig) surely
Fig. 5 B is the aminoacid sequence of chimeric antibody SM03 human IgG1 heavy chain (VH-CH1-hinge-CH2-CH3) immunoglobulin (Ig) of variable region sequences after modified
Fig. 6 A is the physical map of VK stage carrier
Fig. 6 B is the physical map of VH stage carrier
Fig. 7 A is the physical map of light chain expression vector
The attach most importance to physical map of chain expression vector of Fig. 7 B
Fig. 8 is typical 3 liters of Celligen bio-reactor production data charts
Fig. 9 is that the SDS-PAGE of the antibody behind expression and the purifying analyzes
Figure 10 is that the flow cytometry (flow cytometry) of chimeric antibody SM03 is analyzed
Figure 11 is the competitive flow cytometry of chimeric antibody SM03 and mouse source antibody
Figure 12 A is the cytotoxic effect that chimeric antibody SM03 brings out in the glass-tube experiment
Figure 12 B is that stagnant the giving birth to of cell that chimeric antibody SM03 brings out in the glass-tube experiment acts on
Figure 13 is the original murine antibody VK of the chimeric antibody SM03 after cloning and the dna sequence dna of VH
Embodiment
The VK of the original murine antibody of embodiment 1, clone's chimeric antibody SM03 and the dna sequence dna of VH
The manufacture method of the original murine antibody of chimeric antibody SM03 is earlier in Balb/c mouse peritoneal injection 5 * 10
6Human tonsil's lymphocyte.After eight days, append 5 * 10
6The lymphocytic intravenous injection of human tonsil.After four days, splenocyte of mouse and P3-NSI/1-Ag4-1 myeloma cell are merged.Thereafter according to conventional hybridization knurl technology, find a strain cell strain, the original murine antibody of its excretory chimeric antibody SM03 is through the frozen section screening, only confirm and responding property of bone-marrow-derived lymphocyte, with other healthy tissues such as not reactive (Campana D such as kidney, Janossy G, Bofill M, et al.1985.J.Immunol.134:1524-1530).Utilize Track mRNA IsolationKit (Invitrogen, San Diego, CA), from 3 * 10
7Hybridoma is isolated PolyA
+RNA uses cDNA cycle kit (Invitrogen) to extract cDNA again.Briefly, the polyA of 1 microgram
+RNA cooperates the primer CH1B (5 '-ACA GTC ACT GAG CTG G-3 ') of the CH1 part of a special rat immune globulin heavy chain IgG; Or the primer Ck3BH1 (5 '-GCC GGA TCC TCA CTG GATGGT GGG AAG ATG GAT ACA-3 ') of a special rat immune globulin light chain Ck part extracts relevant weight chain first chain (first-stranded) cDNA with reverse transcriptase (Reverse Transcriptase), and then with polyG oligos+Ck3BH1 or polyG oligos+CH1B weight chain two strands (double-stranded) cDNA that is correlated with is increased respectively with the method that conventional RACE adds PCR and to extract. VH after the amplification and VK dna fragmentation are cloned in TA Cloning Vector (Invitrogen) carrier according to ordinary method.Dna fragmentation behind the clone is with the method for Sanger go the to decode dna sequence dna of VK and VH, VK behind the clone and the dna sequence dna of VH as shown in figure 13, among the figure, the line part be a restriction site, the variable region of light chain dna sequence dna after wherein cloning is SEQ ID №: 11; The variable region of heavy chain dna sequence dna is SEQ ID №: 12.
The structure of embodiment 2, VK and VH stage carrier (Staging Vector)
Want chimeric antibody expression SM03, the VK and the VH segment DNA of its original murine antibody coding must be inserted into earlier in the expression vector that is implanted with human light chain and heavy chain regional code DNA.For this reason, earlier VK or VH segment DNA are inserted suitable stage carrier.Stage carrier after synthetic connects the expression promotor (Promoter) of an IgG in the upstream of VK or VH segment DNA, and a segment signal peptide (Signal Peptide) sequence.
VK stage carrier comes from the M13VKPCR1 (Orlandi et al.1989.PNAS86:3833-3837) of improvement.The BamH1/HindIII section that will wherein be loaded with IgG expression promotor (Promoter) sequence and segment signal peptide (SignalPeptide) sequence is cloned in pBR322 (Δ PvuII) carrier.PBR322 (Δ PvuII) be originally a pBR322 carrier (Strategene, La Jolla, CA, USA), with restriction enzyme Accl/Ball PvuII enzyme point of contact deletion unique in the carrier after, just become pBR322 (Δ PvuII) carrier.The pBR322 (Δ PvuII) that contains the BamH1/HindIII section among the M13VKPCR1 is a VK stage carrier (VKpSTAGE), and its structural representation as shown in Figure 6A.The VK section of the original murine antibody of chimeric antibody SM03 is connected on corresponding expression promotor/signal peptide sequence and finishes by following steps: (1) is template with the VK dna sequence dna of the original murine antibody of chimeric antibody SM03, with 5 '-GAC ATC CAG CTG ACC CAG ACT ACA TCC-3 ' and 5 ' TTA GAT CTC CAGCTT GGT GCC TCC-3 ' is primer, PCR method by standard, a PvuII and a BglII limiting enzyme point have been introduced, (2) cut the PCR product of VK gene of the original murine antibody of chimeric antibody SM03 with the PvuII/BglII enzyme, the PCR product cloning of the VK gene of the original murine antibody of the chimeric antibody SM03 that (3) cut the PvuII/BglII enzyme is to the corresponding position of VKpSTAGE.The chimeric antibody SM03 PCR product sequence of being cloned into VKpSTAGE through the method proof of Sanger is with original design the same, the result as shown in Figure 3, among the figure, the runic base represents and the original different dna sequence dna of original murine antibody that the base of line is a restriction enzyme site.The aminoacid sequence (as Fig. 5 A) that the N-of chimeric antibody after the expression in the VK section holds and C-holds is variant with the VK section (as Fig. 4 A) in original mouse source, among Fig. 5 A, and the amino-acid residue after the runic amino-acid residue is represented to modify.
VH stage carrier is that improvement is from M13VHPCR1 (Orlandi et al.1989.PNAS 86:3833-3837).The BamH1/HindIII section that will wherein be loaded with IgG expression promotor (Promoter) sequence and a segment signal peptide (Signal Peptide) sequence is cloned in the pBS carrier (Stratagene), obtain VH stage carrier (VHpSTAGE), its structural representation is shown in Fig. 6 B.The VK section of the original murine antibody of SM03 antibody is connected on corresponding expression promotor/signal peptide sequence and finishes by following steps: (1) is template with the VH dna sequence dna of the original murine antibody of chimeric antibody SM03, with 5 '-CAG GTC CAA CTG CAG GAG TCT GGG GGA GGC-3 ' and 5 '-TGA GGAGAC GGT GAC CAG AGT CCC TTG GCC CCA GTA-3 ' is primer, PCR method by standard, a PstI and a BstEII restriction point of contact have been introduced, (2) cut the PCR product of VK gene of the SM03 original murine antibody of chimeric antibody with the PstI/BstEII enzyme, the PCR product cloning of the VK gene of the SM03 original murine antibody of the chimeric antibody that (3) cut the PvuII/BglII enzyme is to the corresponding position of VHpSTAGE.The chimeric antibody SM03 PCR product sequence of being cloned into VHpSTAGE through the method proof of Sanger is with original design the same, and the result as shown in Figure 3.The aminoacid sequence (as Fig. 5 B) that the N-of chimeric antibody after the expression in the VH section holds and C-holds is variant with the VH section (as Fig. 4 B) in original mouse source, among Fig. 5 B, and the amino-acid residue after the runic amino-acid residue is represented to modify.
The structure of embodiment 3, chimeric antibody SM03 expression vector
The light chain expression vector pEkappa of chimeric antibody SM03 is the expression vector of an about 10kb.It has comprised the IgG enhanser (Enhancer) of a upstream, the gene order (genomic sequence) that the people Kappa Qing Lian Heng that is used for inserting the HindIII/BamH1 cloning site of VK section and the intron of ining succession (intron) distinguishes surely.Surely distinguish the downstream of gene order at Qing Lian Heng, placed a hygromycin selective marker with the SV40 promoter expression (selection marker), its structure is shown in Fig. 7 A.IgG promotor/signal peptide/SM03 VK sequence is extracted with the HindIII/BamH1 restriction endonuclease from SM03VKpSTAGE, insert the HindIII/BamH1 cloning site of pEkappa again, just become chimeric antibody SM03 light chain expression vector (SM03pEkappa) (as Fig. 7 A).
Heavy chain expression carrier pEgammal is the expression vector of an about 10kb.It has comprised the IgG enhanser (Enhancer) of a upstream, the gene order (genomic sequence) that the human IgG1 Chong Lian Heng that is used for inserting the HindIII/BamH1 cloning site of VH section and the intron of ining succession (intron) distinguishes surely.Surely distinguish the downstream of gene order at Chong Lian Heng, inserted a gpt selective marker with the SV40 promoter expression (selection marker), its structure is shown in Fig. 7 B.IgG promotor/signal peptide/SM03 VH sequence is extracted with the HindIII/BamH1 restriction endonuclease from SM03VHpSTAGE, insert the HindIII/BamH1 cloning site of pEgammal again, just become SM03 heavy chain expression carrier (SM03pEgammal) (as Fig. 7 B).
The preparation and the production thereof of embodiment 4, chimeric antibody SM03 express cell
About 10 micrograms are added about 30 micrograms by the SM03pEgammal of PvuI restriction endonuclease linearize by the SM03pEkappa of BstXI restriction endonuclease linearize (linearized), and (electroporation) goes transfection 5 * 10 with electroporation
6The SP2/0 cell.Cell after the transfection, in normal nutrient solution through two days later rehabilitation period, just begin in nutrient solution, to add 0.5g/L hygromycin (Calbiochem, SanDiego, CA).The cell strain that is subjected to transfection and survives can occur after week in 2-3.The cell strain of survival is cultivated respectively, and after the expansion, available enzyme linked immunosorbent adsorption test (ELISA) method goes to detect the concentration of the people source heavy chain Fc section in the nutrient solution.Positive findings and three the highest strain clone cells of Fc concentration can be amplified stores.After positive cell was trained through about 500 milliliters suicide group, available Protein A chromatography column was purified the humanized antibody in the nutrient solution.The antibody producing amount of cell strain is according to " mode of the cumulative volume of the nutrient solution that the antibody amount of can purifying ÷ is used to purify is calculated " (method of calculation of following antibody producing amount are all as standard).Select high-throughput, the most stable, the fastest SM03 of growth velocity produces the production of cell strain as bio-reactor.Adopt three liters of Celligen fermentor tanks of New Brunswick Scientific.About 2 * 10
9SM03 produce cell strain and insert fermentor tank as seed culture (seedculture).The tight cell growing state that detects, as the oxygen depletion amount, pH value, glucose consumption amount, antibody concentration etc.Glucose content in nutrient solution is lower than 1 grams per liter, and fresh nutrient solution is just imported fermentor tank in the mode of perfusion, and the old nutrient solution of equivalent also can be exported simultaneously in the mode of perfusion.Because the Celligen fermentor tank is to utilize fiber small thin slices (fiber disc) to remove to pin cell, cell can not run off in a large number with the old nutrient solution of perfusion output.The speed of perfusing rate is decided on the glucose content in the nutrient solution.Target is to keep glucose content in the nutrient solution about 1 grams per liter.Fig. 8 represents the antibody producing amount of a standard fermentation.Cultured nutrient solution can be purified with the method for Protein A chromatography column.Antibody after the purification is with the Quality Control of SDS-PAGE electrophoretic method, electrophoresis result as shown in Figure 9, among the figure, swimming lane 1 is a weight stamp, and swimming lane 2 is the human antibody standard control, and swimming lane 3 is test-manufactured first for SM03, swimming lane 4 is second batch of SM03 test manufacture, and as can be seen from the figure, the purity of antibody is very high.
Embodiment 5, usefulness flow cytometer (flow cytometry) are analyzed the stability of SM03
For the SM03 that guarantees different production lots still keeps its specificity (specificity) and avidity (affinity), four batches of different productions and storage time are (the longest 1 year, the shortest fortnight) purifying SM03 flow cytometry, the specific combination of testing itself and Raji human lymphoma cell.The result shows that four crowdes of SM03 are consistent in conjunction with the fluorescence intensity that the back is produced with the Raji human lymphoma cell as shown in figure 10, illustrates that different productions and storage time can not influence specificity and the avidity of SM03.
The comparison of embodiment 6, SM03 and original murine antibody avidity
Present embodiment goes to analyze the avidity of comparison original murine antibody and SM03 with competitive flow cytometry.The practice is the FITC-original murine antibody conjugate with fixed concentration, after competition antibody (mouse VS SM03 test-manufactures first antibody) mixing with different concns, again with the Raji cell response.Then, analyze under the influence of competition antibody, also have how many residual FITC-original murine antibodies to combine with the Raji cell with fluorescent activation cell divide scanning device (FACScan).The result as shown in figure 11, show that original murine antibody follows chimeric antibody SM03 as competition antibody close avidity to be arranged, the antibody SM03 specificity after chimericization and avidity do not weaken because of the aminoacid sequence (the runic amino-acid residue among Fig. 5 A and Fig. 5 B) of changing V section a small amount of in the process of chimericization.Though the competitive power of chimeric antibody SM03 seems a little more than original murine antibody, it is very slight respectively, within normal experimental error, may come from the trickle difference of antibody concentration assessment.Generally speaking, the SM03 avidity of chimeric antibody also is not less than, or basic identical in original murine antibody.
Cytotoxicity/cell that embodiment 7, SM03 bring out in the glass-tube experiment stagnates to give birth to and acts on (cytostatic effect)
Be stained with SM03 or be stained with in the 96-orifice plate (96-well plate) of incoherent contrast humanized antibody, adding 4 * 10
5Raji cell cultures, the ratio with Trypan Blue pigment assessment non-viable non-apoptotic cell and healthy cell again in the different periods.The result shows chimeric antibody SM03 energy inducing cytotoxic shown in Figure 12 A, with after hatching 24 hours, the mortality ratio of cell reaches 30 percent especially.Similarly, have in the 96-of the incoherent contrast humanized antibody orifice plate (96-well plate) sticking chimeric antibody SM03 to be arranged or stick, add 1 * 10
5Ramos people's lymphocytic cancer cell is cultivated, and assesses the total quantity of cells again in the different periods.The result shows that chimeric antibody SM03 can restrain the cell growth shown in Figure 12 B.
Sequence table
<160>12
<210>1
<211>107
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
Asp?Ile?Gln?Met?Thr?Gln?Thr?Thr?Ser?Ser?Leu?Ser?Ala?Ser?Leu
1 5 10 15
Gly?Asp?Arg?Val?Thr?Ile?Ser?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Ser
20 25 30
Asn?Tyr?Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Asp?Gly?Thr?Val?Lys
35 40 45
Leu?Leu?Ile?Tyr?Tyr?Thr?Ser?Ile?Leu?His?Ser?Gly?Val?Pro?Ser
50 55 60
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Tyr?Ser?Leu?Thr?Ile
65 70 75
Ser?Asn?Leu?Glu?Gln?Glu?Asp?Phe?Ala?Thr?Tyr?Phe?Cys?Gln?Gln
80 85 90
Gly?Asn?Thr?Leu?Pro?Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu
95 100 105
Ile?Lys
107
<210>2
<211>123
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>2
Glu?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Lys?Pro?Gly
1 5 10 15
Gly?Ser?Leu?Lys?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Ala?Phe?Ser
20 25 30
Ile?Tyr?Asp?Met?Ser?Trp?Val?Arg?Gln?Trp?Pro?Glu?Lys?Arg?Leu
35 40 45
Glu?Trp?Val?Ala?Tyr?Ile?Ser?Ser?Gly?Gly?Gly?Thr?Thr?Tyr?Tyr
50 55 60
Pro?Asp?Thr?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala
65 70 75
Lys?Asn?Thr?Leu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Lys?Ser?Glu?Asp
80 85 90
Thr?Ala?Met?Tyr?Tyr?Cys?Ala?Arg?His?Ser?Gly?Tyr?Gly?Ser?Ser
95 100 105
Tyr?Gly?Val?Leu?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
110 115 120
Val?Ser?Ala
123
<210>3
<211>107
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>3
Asp?Ile?Gln?Leu?Thr?Gln?Thr?Thr?Ser?Ser?Leu?Ser?Ala?Ser?Leu
1 5 10 15
Gly?Asp?Arg?Val?Thr?Ile?Ser?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Ser
20 25 30
Asn?Tyr?Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Asp?Gly?Thr?Val?Lys
35 40 45
Leu?Leu?Ile?Tyr?Tyr?Thr?Ser?Ile?Leu?His?Ser?Gly?Val?Pro?Ser
50 55 60
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Tyr?Ser?Leu?Thr?Ile
65 70 75
Ser?Asn?Leu?Glu?Gln?Glu?Asp?Phe?Ala?Thr?Tyr?Phe?Cys?Gln?Gln
80 85 90
Gly?Asn?Thr?Leu?Pro?Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu
95 100 105
Ile?Lys
107
<210>4
<211>123
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>4
Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Gly?Gly?Leu?Val?Lys?Pro?Gly
1 5 10 15
Gly?Ser?Leu?Lys?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Ala?Phe?Ser
20 25 30
Ile?Tyr?Asp?Met?Ser?Trp?Val?Arg?Gln?Trp?Pro?Glu?Lys?Arg?Leu
35 40 45
Glu?Trp?Val?Ala?Tyr?Ile?Ser?Ser?Gly?Gly?Gly?Thr?Thr?Tyr?Tyr
50 55 60
Pro?Asp?Thr?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala
65 70 75
Lys?Asn?Thr?Leu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Lys?Ser?Glu?Asp
80 85 90
Thr?Ala?Met?Tyr?Tyr?Cys?Ala?Arg?His?Ser?Gly?Tyr?Gly?Ser?Ser
95 100 105
Tyr?Gly?Val?Leu?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
110 115 120
Val?Ser?Ser
123
<210>5
<211>321
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
gacatccagc?tgacccagac?tacatcctcc?ctgtctgcct?ctctgggaga?cagagtcacc 60
attagttgca?gggcaagtca?ggacattagc?aattatttaa?actggtatca?gcagaaacca 120
gatggaactg?ttaaactcct?gatctactac?acatcaatat?tacactcagg?agtcccatca 180
aggttcagtg?gcagtgggtc?tggaacagat?tattctctca?ccattagcaa?cctggagcaa 240
gaagattttg?ccacttactt?ttgccaacag?ggtaatacgc?ttccgtggac?gttcggtgga 300
ggcaccaagc?tggagatcaa?a 321
<210>6
<211>369
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
caggtccaac?tgcaggagtc?tgggggaggc?ttagtgaagc?ctggagggtc?cctgaaactc 60
tcctgtgcag?cctctggatt?cgctttcagt?atctatgaca?tgtcttgggt?tcgccagact 120
ccggagaaga?ggctggagtg?ggtcgcatac?attagtagtg?gtggtggtac?cacctactat 180
ccagacactg?tgaagggccg?attcaccatc?tccagagaca?atgccaagaa?caccctgtac 240
ctgcaaatga?gcagtctgaa?gtctgaggac?acagccatgt?attactgtgc?aagacatagt 300
ggctacggta?gtagctacgg?ggttttgttt?gcttactggg?gccaagggac?tctggtcacc 360
gtctcctca 369
<210>7
<211>213
<2?12>PRT
＜213〉artificial sequence
<220>
<223>
<400>7
Asp?Ile?Gln?Met?Thr?Gln?Thr?Thr?Ser?Ser?Leu?Ser?Ala?Ser?Leu
1 5 10 15
Gly?Asp?Arg?Val?Thr?Ile?Ser?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Ser
20 25 30
Asn?Tyr?Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Asp?Gly?Thr?Val?Lys
35 40 45
Leu?Leu?Ile?Tyr?Tyr?Thr?Ser?Ile?Leu?His?Ser?Gly?Val?Pro?Ser
50 55 60
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Tyr?Ser?Leu?Thr?Ile
65 70 75
Ser?Asn?Leu?Glu?Gln?Glu?Asp?Phe?Ala?Thr?Tyr?Phe?Cys?Gln?Gln
80 85 90
Gly?Asn?Thr?Leu?Pro?Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu
95 100 105
Ile?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro
110 115 120
Ser?Asp?Glu?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu
125 130 135
Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp
140 145 150
Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Gln?Gln
155 160 165
Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu
170 175 180
Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Leu?Tyr?Ala?Cys?Glu?Val
185 190 195
Thr?His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg
200 205 210
Gly?Glu?Cys
213
<210>8
<211>452
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>8
Glu?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Lys?Pro?Gly
1 5 10 15
Gly?Ser?Leu?Lys?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Ala?Phe?Ser
20 25 30
Ile?Tyr?Asp?Met?Ser?Trp?Val?Arg?Gln?Trp?Pro?Glu?Lys?Arg?Leu
35 40 45
Glu?Trp?Val?Ala?Tyr?Ile?Ser?Ser?Gly?Gly?Gly?Thr?Thr?Tyr?Tyr
50 55 60
Pro?Asp?Thr?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala
65 70 75
Lys?Asn?Thr?Leu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Lys?Ser?Glu?Asp
80 85 90
Thr?Ala?Met?Tyr?Tyr?Cys?Ala?Arg?His?Ser?Gly?Tyr?Gly?Ser?Ser
95 100 105
Tyr?Gly?Val?Leu?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
110 115 120
Val?Ser?Ala?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala
125 130 135
Pro?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu
140 145 150
Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser
155 160 165
Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln
170 175 180
Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser
185 180 195
Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?ASn?His?Lys
200 205 210
Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Ala?Glu?Pro?Lys?Ser?Cys
215 220 225
Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu
230 235 240
Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr
245 250 255
Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp
260 265 270
Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp
275 280 285
Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln
290 295 300
Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His
305 310 315
Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn
320 325 330
Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys
335 340 345
Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg
350 355 360
Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys
365 370 375
Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly
380 385 390
Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser
395 400 405
Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser
410 415 420
Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu
425 430 435
Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro
440 445 450
Gly?Lys
452
<210>9
<211>213
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>9
Asp?Ile?Gln?Leu?Thr?Gln?Thr?Thr?Ser?Ser?Leu?Ser?Ala?Ser?Leu
1 5 10 15
Gly?Asp?Arg?Val?Thr?Ile?Ser?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Ser
20 25 30
Asn?Tyr?Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Asp?Gly?Thr?Val?Lys
35 40 45
Leu?Leu?Ile?Tyr?Tyr?Thr?Ser?Ile?Leu?His?Ser?Gly?Val?Pro?Ser
50 55 60
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Tyr?Ser?Leu?Thr?Ile
65 70 75
Ser?Asn?Leu?Glu?Gln?Glu?Asp?Phe?Ala?Thr?Tyr?Phe?Cys?Gln?Gln
80 85 90
Gly?Asn?Thr?Leu?Pro?Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu
95 100 105
Ile?Lys-Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro
110 115 120
Ser?Asp?Glu?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu
125 130 135
Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp
140 145 150
Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Gln?Gln
155 160 165
Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu
170 175 180
Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Leu?Tyr?Ala?Cys?Glu?Val
185 190 195
Thr?His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg
200 205 210
Gly?Glu?Cys
213
<210>10
<211>452
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>10
Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Gly?Gly?Leu?Val?Lys?Pro?Gly
1 5 10 15
Gly?Ser?Leu?Lys?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Ala?Phe?Ser
20 25 30
Ile?Tyr?Asp?Met?Ser?Trp?Val?Arg?Gln?Trp?Pro?Glu?Lys?Arg?Leu
35 40 45
Glu?Trp?Val?Ala?Tyr?Ile?Ser?Ser?Gly?Gly?Gly?Thr?Thr?Tyr?Tyr
50 55 60
Pro?Asp?Thr?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala
65 70 75
Lys?Asn?Thr?Leu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Lys?Ser?Glu?Asp
80 85 90
Thr?Ala?Met?Tyr?Tyr?Cys?Ala?Arg?His?Ser?Gly?Tyr?Gly?Ser?Ser
95 100 105
Tyr?Gly?Val?Leu?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
110 115 120
Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala
125 130 135
Pro?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu
140 145 150
Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser
155 160 165
Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln
170 175 180
Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser
185 190 195
Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys
200 205 210
Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Ala?Glu?Pro?Lys?Ser?Cys
215 220 225
Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu
230 235 240
Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr
245 250 255
Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp
260 265 270
Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp
275 280 285
Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln
290 295 300
Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His
305 310 315
Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn
320 325 330
Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys
335 340 345
Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg
350 355 360
Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys
365 370 375
Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly
380 385 390
Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser
395 400 405
Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser
410 415 420
Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu
425 430 435
Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro
440 445 450
Gly?Lys
452
<210>11
<211>321
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>11
gatatccaga?tgacccagac?tacatcctcc?ctgtctgcct?ctctgggaga?cagagtcacc 60
attagttgca?gggcaagtca?ggacattagc?aattatttaa?actggtatca?gcagaaacca 120
gatggaactg?ttaaactcct?gatctactac?acatcaatat?tacactcagg?agtcccatca 180
aggttcagtg?gcagtgggtc?tggaacagat?tattctctca?ccattagcaa?cctggagcaa 240
gaagattttg?ccacttactt?ttgccaacag?ggtaatacgc?ttccgtggac?gttcggtgga 300
ggcaccaagc?tggaaatcaa?a 321
<210>12
<211>369
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>12
gaagtgcagc?tggtggagtc?tgggggaggc?ttagtgaagc?ctggagggtc?cctgaaactc 60
tcctgtgcag?cctctggatt?cgctttcagt?atctatgaca?tgtcttgggt?tcgccagact 120
ccggagaaga?ggctggagtg?ggtcgcatac?attagtagtg?gtggtggtac?cacctactat 180
ccagacactg?tgaagggccg?attcaccatc?tccagagaca?atgccaagaa?caccctgtac 240
ctgcaaatga?gcagtctgaa?gtctgaggac?acagccatgt?attactgtgc?aagacatagt 300
ggctacggta?gtagctacgg?ggttttgttt?gcttactggg?gccaagggac?tctggtcact 360
gtctctgca 369
Claims (11)
1, a kind of chimeric antibody SMO3 of anti-people's non Hodgkin lymphoma is made up of light chain and heavy chain, and it is specific to CD22, and the constant region of described heavy chain is the isotype human IgG
1Constant region, the constant region of described light chain is the constant region of isotype people Kappa; The variable region of described heavy chain has the amino acid residue sequence of sequence 2 in the sequence table or 4, and the variable region of described light chain has the amino acid residue sequence of sequence 1 in the sequence table or 3.
2, chimeric antibody SMO3 according to claim 1 is characterized in that: the constant region of described heavy chain has the sequence of forming from the 124th-452 amino acids residue of aminoterminal by sequence in the sequence table 8 or sequence 10; The constant region of described light chain has the sequence of forming from the 108th-213 amino acids residue of aminoterminal by sequence in the sequence table 7 or sequence 9.
3, chimeric antibody SMO3 according to claim 2 is characterized in that: the light chain of described SMO3 is SEQ ID № in the sequence table: 7 amino acid residue sequence; The heavy chain of described SMO3 is SEQ ID № in the sequence table: 8 amino acid residue sequence.
4, chimeric antibody SMO3 according to claim 2 is characterized in that: the light chain of described SMO3 is SEQ ID № in the sequence table: 9 amino acid residue sequence; The heavy chain of described SMO3 is SEQ ID № in the sequence table: 10 amino acid residue sequence.
5, the derivative of the chimeric antibody SMO3 of the described anti-people's non Hodgkin lymphoma of arbitrary claim in the claim 1 to 4 is chemical coupling thing, single-chain antibody, Fab, Fab ' or the F (ab ') of SMO3
2
6, the derivative of chimeric antibody SMO3 according to claim 5 is characterized in that: the chemical coupling thing of described chimeric antibody SMO3 is the conjugate of described chimeric antibody SMO3 and chemical poison or active nucleus.
7, the derivative of chimeric antibody SMO3 according to claim 6 is characterized in that: described chemical poison is a Zorubicin, irinotecan or cisplatin; Described active nucleus is I
125, I
131, Y
90, In
111Or Re
188
8, the application of the chimeric antibody SMO3 or derivatives thereof of the described anti-people's non Hodgkin lymphoma of arbitrary claim in the medicine of preparation treatment and CD22 expression diseases related in the claim 1 to 7.
9, application according to claim 8 is characterized in that: it is autoimmune disease that described and CD22 expresses diseases associated.
10, application according to claim 9 is characterized in that: described autoimmune disease is NHL, rheumatoid arthritis and lupus erythematosus.
11, the medicine of a kind of treatment and CD22 expression diseases related, its activeconstituents is the chimeric antibody SMO3 or derivatives thereof of the described anti-people's non Hodgkin lymphoma of arbitrary claim in the claim 1 to 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031230547A CN1326878C (en) | 2003-04-29 | 2003-04-29 | Anti human non-Hodgkin's lymphoma chimeric antibody and its derivative and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031230547A CN1326878C (en) | 2003-04-29 | 2003-04-29 | Anti human non-Hodgkin's lymphoma chimeric antibody and its derivative and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1542019A CN1542019A (en) | 2004-11-03 |
| CN1326878C true CN1326878C (en) | 2007-07-18 |
Family
ID=34321198
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031230547A Expired - Lifetime CN1326878C (en) | 2003-04-29 | 2003-04-29 | Anti human non-Hodgkin's lymphoma chimeric antibody and its derivative and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1326878C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10370447B2 (en) | 2014-07-16 | 2019-08-06 | Ucb Biopharma Sprl | Molecules with specificity for CD79 and CD22 |
| US11472879B2 (en) | 2015-07-16 | 2022-10-18 | UCB Biopharma SRL | Antibody molecules which bind CD22 |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL2564695T3 (en) * | 2009-07-08 | 2015-10-30 | Kymab Ltd | Animal models and therapeutic molecules |
| CN103214578B (en) * | 2013-05-10 | 2014-05-28 | 北京东方百泰生物科技有限公司 | Novel humanized anti-CD22 antibody |
| EP3867277A4 (en) * | 2018-10-18 | 2022-09-07 | Sinomab Bioscience Limited | Method of modulating autoimmunity by disrupting cis-ligand binding of siglec type antigens |
| CN112816709A (en) * | 2021-02-03 | 2021-05-18 | 杏联药业(苏州)有限公司 | Activity detection method of SM03 monoclonal antibody and application of activity detection method in quality monitoring of SM03 monoclonal antibody |
| AU2022369457A1 (en) * | 2021-10-18 | 2024-05-02 | Sinomab Bioscience Limited | Aqueous formulations of an anti-cd22 antibody and uses thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1149299A (en) * | 1993-09-02 | 1997-05-07 | 达特茅斯学院理事 | Anti-GP39 antibodies and uses therefor |
-
2003
- 2003-04-29 CN CNB031230547A patent/CN1326878C/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1149299A (en) * | 1993-09-02 | 1997-05-07 | 达特茅斯学院理事 | Anti-GP39 antibodies and uses therefor |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10370447B2 (en) | 2014-07-16 | 2019-08-06 | Ucb Biopharma Sprl | Molecules with specificity for CD79 and CD22 |
| US11261252B2 (en) | 2014-07-16 | 2022-03-01 | UCB Biopharma SRL | Molecules with specificity for CD79 and CD22 |
| US11472879B2 (en) | 2015-07-16 | 2022-10-18 | UCB Biopharma SRL | Antibody molecules which bind CD22 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1542019A (en) | 2004-11-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20230348589A1 (en) | Antibodies That Bind Human Cannabinoid 1 (CB1) Receptor | |
| CN108472349A (en) | LAG-3 antibody, its antigen-binding fragment and its medical usage | |
| CN108779179A (en) | CD47 antibody, its antigen-binding fragment and its medical usage | |
| CN108883164A (en) | For the antibody of TIGIT | |
| TW202039563A (en) | Chimeric antigen and t cell receptors and methods of use | |
| US20230159634A1 (en) | Antibodies that bind human cannabinoid 1 (cb1) receptor | |
| EA015009B1 (en) | Antigen binding molecules with increased fc receptor binding affinity and effector function | |
| JP2002514421A (en) | Antibodies to CD23, their derivatives, and their therapeutic use | |
| TW201132356A (en) | Antagonist anti-IL-7 receptor antibodies and methods | |
| TWI793325B (en) | Antibodies specific for cd3 and uses thereof | |
| EP3178484B1 (en) | Therapeutic agent for cancer which comprises combination of il-18 and molecule-targeting antibody | |
| CN109053892A (en) | The monoclonal antibody and the preparation method and application thereof of specific bond people and monkey CD38 antigen | |
| EP3992211A1 (en) | Anti-cea antibody and application thereof | |
| RU2261723C2 (en) | Silent anti-cd28-antibody and their applying | |
| CN1326878C (en) | Anti human non-Hodgkin's lymphoma chimeric antibody and its derivative and application | |
| CN104987420B (en) | The full human monoclonal antibody of anti-CD20 and its application | |
| US20040116675A1 (en) | Silenced anti-cd28 antibodies and use thereof | |
| CN105143269A (en) | Fully humanized anti-CD20 monoclonal antibody and uses thereof | |
| CN102863531A (en) | CD20-resistant monoclonal antibody as well as preparation method and application thereof | |
| US20240076396A1 (en) | Anti-gitr antibodies and uses thereof | |
| CN113912721B (en) | Anti-human CD47 monoclonal antibody and application thereof | |
| WO2023143444A1 (en) | Anti-dkk1 antibody, pharmaceutical composition thereof and use thereof | |
| WO2024073435A2 (en) | Anti-lag-3/il-2 fusion proteins, encoding polynucleotides, pharmaceutical compositions, and therapeutic applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20070718 |
|
| CX01 | Expiry of patent term |