CN108503708A - Anti-human CD47 antibody and application thereof - Google Patents
Anti-human CD47 antibody and application thereof Download PDFInfo
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Abstract
This application discloses the antibody or its antigen-binding portion thereof of specific binding people CD47, the application of the polynucleotides, the carrier comprising the polynucleotides, the host cell comprising the polynucleotides or carrier, the method for preparing and purifying the antibody and the antibody or its antigen-binding portion thereof of encoding said antibody or its antigen-binding portion thereof.
Description
Technical field
The application relates generally to genetic engineering and antibody drug field；In particular to anti-human CD47 antibody field and
Its purposes.The application develops new anti-human CD47 antibody, and provides use of the antibody in the disease that treatment CD47 is mediated
On the way.
Background technology
CD47 contactin members are I type transmembrane glycoproteins, including aminoterminal V-type immunoglobulin
Like cell extracellular portion, the transmembrane domain being made of the transmembrane segment of 5 very hydrophobics and hydrophilic c-terminus are tied into the cell
Structure domain, molecular weight is between 47-55kD.Initially, CD47 is because of and integrin alphavβ3It interacts and is recognized by people, therefore
It is also known as integrin associated protein (IAP)1.CD47 passes through extracellular domain and integrin alphavβ3Or αIIbβ3β3Subunit phase
Interaction activates corresponding integrin.
SIRP α (signal adjusting protein alpha (Signal regulatory protein α)), also known as SHPS-1 are (homologous containing Src
- 1 (Src homology 2domain-containing protein tyrosine of 2 domain protein tyrosine kinase substrate
Phosphatase substrate-1))/BIT (brain immunoglobulin-like molecule (brain of the activation motifs with tyrosine
Immunoglobulin-like molecule with tyrosine-based activation motifs))/CD172a,
It is also transmembrane glycoprotein, there are extracellular region 3 immunoglobulin like domain, intracellular domain to have typical immunity receptor
Tyrosine inhibition sequence (ITIM) has 4 tyrosine residues, is potential phosphorylation site.
CD47 is the ligand of SIRP α, and the two is interacted by extracellular domain, forms intercellular communication compound.
After CD47 is combined with SIRP α, SIRP α intracellular ITIM phosphorylations are induced, phosphorylation site combines activation in turn, and (Src is same containing SH2
Source 2 (Src homology 2)) structural domain tyrosine phosphatase SHP-1 and SHP-2, priming signal transmit cascade reaction.
SHP-1 is mainly expressed in hematopoietic cell, the function of these cells of negative regulation2；And SHP-2 expression is very extensive, adjusts small G eggs
White Ras and Rho, forward direction control cell growth and proliferation3。
SIRP α expressed in abundance in neuron and medullary system hematopoietic cell (such as macrophage and dendritic cells)4, and CD47
It is then expressed in most cells.CD47-SIRP alpha signals system is to adjusting macrophage to the phagocytosis of mature blood cell with important
Effect.Normal healthy cells (such as red blood cell5Or blood platelet6) surface CD47 molecules and the receptor SIRP α on macrophage it is mutual
Effect generates inhibition signal, inhibits its phagocytic activity, reaches the life cycle and its quantity in blood for adjusting haemocyte
Purpose.The SIRP α and CD47 on red blood cell on monocyte is acted on, and inhibits Fc γ by myosin-IIA dephosphorylations
The phagocytosis of receptor-independent7.CD47-SIRP alpha signal systems inhibit dendritic cells activation, participate in nervous system development, neutrality
A variety of physiological activities such as the hematopoietic cell generation that granulocyte chemotaxis activates and stroma cell is supported, at the same it is immune in inducing T cell
Tolerance, activation, apoptosis etc. also play a variety of adjustment effects8。
In recent years, CD47-SIRP alpha signals system is sent out by adjusting the phagocytosis of macrophage in cancer immunosurveillance
The effect waved is taken seriously.CD47 is in many malignant tumours, such as oophoroma9, acute myelogenous leukemia (AML)10, B cell leaching
Bar tumor11And solid tumor12In expression significantly raise, Average expression level is 3.3 times of corresponding normal cell or so,
And it is this up-regulation it is directly related with the poor prognosis of malignant tumor patient13.In people's marrow leukaemia cell (in low expression level
Source CD47 cannot be transplanted in Rag2-Il2rg- mouse) in express mouse CD47, Macrophages For Tumor can be inhibited
Phagocytosis promotes tumour cell successful implantation14.It is inferred that the SIRP α on CD47 and macrophage on tumour cell make
With inhibiting the removing of Macrophages For Tumor, promote the growth and transfer of tumour in vivo, height expression CD47 is that tumour is thin
Born of the same parents escape the generalised mechanism of immunosurveillance, block CD47-SIRP α effects, it may be possible to new immunotherapy of tumors strategy.
Anti-cd 47 antibody is used alone10,12,13Or with other anti-tumor antigen antibody use in conjunction11, in people's Acute Meyloid
Good inhibition is all shown on the mouse transplantation model of property leukaemia, non-Hodgkin lymphoma (NHL) and many solid tumors
The effect of tumour growth.The phagocytosis of the anti-CD47 monoclonal antibodies inducing macrophage of humanization AML cells primary to people,
Thoroughly remove people's AML cells in vivo so that transplanting mouse long-term disease-free survival；It is acted on rituximab combination, NHL can be removed
Tumour cures xenograft mouse15.Moreover, the safety of anti-cd 47 antibody is confirmed in monkey body.In addition to anti-CD47 is anti-
Body, the SIRP alpha-mutants (CD172a) of high-affinity antagonism CD47 and then can also block CD47-SIRP alpha signal accesses,
Phagocytosis of the macrophage to AML cells can be dramatically increased on AML models, and inhibits the growth of tumour16。
The development and application of new anti-cd 47 antibody is that this field is required.
Summary of the invention
In a first aspect, this application provides a kind of antibody of specific binding people CD47, it includes containing HCDR1, HCDR2 and
The heavy chain variable region of HCDR3 sequences and light chain variable region containing LCDR1, LCDR2 and LCDR3 sequence, wherein
The HCDR1 sequences are NYWMH, and the HCDR2 sequences are VIAPSDNYTNYNQKFQG, and the HCDR3 sequences are
GGKYSMDY, the LCDR1 sequences are RSSQSIVHSNGNTYLE, and the LCDR2 sequences are KVSNRFS, the LCDR3 sequences
For FQGSHVPFT；Or
The HCDR1 sequences are SYWMH, and the HCDR2 sequences are TIDRSDSYISYNQKFKG, and the HCDR3 sequences are
GGPYGSKMMDN, the LCDR1 sequences are HASQNINVWLS, and the LCDR2 sequences are KASNLHT, and the LCDR3 sequences are
QQGQSYPLT；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPSSGNTKYAQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSLLYSSNKKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as QQFYAYPIS；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPSSGNTKYAQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSVLYSSNQKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as GQYYAYPIT；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPGSGNTRYSQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSLLYSSNKKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as QQFYAYPIS；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPGSGNTRYSQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSVLYSSNQKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as GQYYAYPIT；
Wherein HCDR and LCDR sequences are defined according to Kabat.
In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：23、28、
30, shown in 32 or 33.
In some embodiments, the amino acid sequence of the light chain variable region of the antibody such as SEQ ID NO：24、29、
31, shown in 34 or 35.
In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 23,
The amino acid sequence of the light chain variable region of the antibody such as SEQ ID NO：Shown in 24；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 28, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 29；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 30, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 31；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 32, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 34；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 32, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 35；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 33, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 34；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 33, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 35.
Second aspect, this application provides a kind of antibody of specific binding people CD47, wherein the heavy chain of the antibody can
Become the amino acid sequence and SEQ ID NO in area：23, any one of 28,30,32 or 33 at least 90% consistency,
And the amino acid sequence of the light chain variable region of the antibody and SEQ ID NO：24, any one of 29,31,34 or 35 tool
There is at least 90% consistency.
The third aspect, this application provides a kind of antibody of specific binding people CD47, wherein the antibody and SEQ ID
NO：The combination epitope of 1 leted others have a look at CD47 sections is discontinuous epi-position, including amino acid Q1, E29, A30, Q31, N32, E35,
E97, E100, L101, T102, R103, E104, G105 and E106, above-mentioned residue numbering is with reference to SEQ ID NO：1.
In some embodiments of first aspect to the third aspect, the antibody is monoclonal antibody.
In some embodiments of first aspect to the third aspect, the antibody is inactive type antibody.
In some embodiments of first aspect to the third aspect, the antibody can combine and neutralize people CD47, in turn
Block CD47-SIRP alpha signal accesses.In some embodiments, the antibody can promote gulping down for Macrophages For Tumor
The effect of biting.In some embodiments, the tumor growth of the antibody inhibiting tumor cell.In some embodiments, described
Antibody does not increase phagocytosis of the macrophage to normal plasma cell.
In some embodiments of first aspect to the third aspect, at least one of the antibody has following property：
With affinity combination recombined human CD47 extracellular regions D1 (the SEQ ID NO of the KD values less than 5nM：1)；
With the IC less than 5nM50Value inhibits CD172a high-affinities mutant (SEQ ID NO：4) with the knot of people CD47-Fc
It closes；
Being applied under 0-133nM concentration does not lead to erythrocyte aggregation.
In some embodiments of first aspect to the third aspect, the antibody is whole antibody, Fab segments, F (ab ')2
Segment or Single-Chain Fv Fragment of Murine (scFv).
In some embodiments of first aspect to the third aspect, the antibody is human antibody.
In some embodiments of first aspect to the third aspect, the antibody also includes selected from IgG1 hypotypes, IgG2
The heavy chain constant region of hypotype or IgG4 hypotypes and/or include the constant region of light chain selected from κ hypotypes or λ hypotypes.
Fourth aspect, this application provides nucleic acid molecules, encode antibody described in first aspect to the third aspect or its
Antigen-binding portion thereof.
5th aspect, this application provides pharmaceutical composition, it includes described in first aspect to the third aspect antibody and
Pharmaceutically acceptable excipient, diluent or carrier.
In some embodiments, described pharmaceutical composition is used to treat the disease of CD47 mediations.
In some embodiments, the disease is tumour, such as malignant tumour.
In some embodiments, malignant tumour is Burkitt lymphoma or Huppert's disease.
6th aspect, this application provides the antibody described in first aspect to the third aspect to prepare for preventing or treating
Purposes in the drug for the disease that CD47 is mediated.
In some embodiments, the disease is tumour, such as malignant tumour.
In some embodiments, malignant tumour is Burkitt lymphoma or Huppert's disease.
7th aspect, this application provides the methods prevented or treat the disease that CD47 is mediated, including in need
Body gives the pharmaceutical composition of antibody or the 7th aspect described in first aspect to the third aspect.
In some embodiments, the disease is tumour, such as malignant tumour.
In some embodiments, malignant tumour is Burkitt lymphoma or Huppert's disease.
Description of the drawings
Fig. 1 shows the result of the anti-CD47 monoclonal antibodies and hCD172a competitive bindings hCD47 of the application.
Fig. 2 shows the cause erythrocyte agglutination effect that the anti-CD47 monoclonal antibodies of the application are analyzed based on blood coagulation tests.
Fig. 3 shows that the anti-CD47 monoclonal antibodies that the application is analyzed by flow cytometry promote macrophage phagocytosis Daudi cells
Effect.
Fig. 4 shows the phagocytic rate that the anti-CD47 monoclonal antibodies of the application promote macrophage to Daudi cells.
Fig. 5 shows the effect that the anti-CD47 monoclonal antibodies of the humanization of the application inhibit hCD47 to be combined with hCD172a.
Fig. 6 shows the effect that hS2C3 and its variant inhibit hCD47 and hCD172a-D1M1 to combine.
Fig. 7 shows that the anti-CD47 monoclonal antibodies of the application cause the effect of erythrocyte agglutination degree.
Fig. 8 shows the phagocytic rate that the anti-CD47 monoclonal antibodies of the application promote macrophage to Daudi cells.
Fig. 9 shows that the anti-CD47 monoclonal antibodies of the application inhibit the effect of mouse interior tumor growth.
Figure 10 shows that the anti-CD47 monoclonal antibodies of the application inhibit heterograft human myeloma RPMI in NOD-SCID Mice Bodies
The effect of 8226 tumour growths.
Figure 11 shows that the CD47-Fab composite structures that the application tests, wherein A figures show that CD47-Fab is compound
Object overall structure；B figures show CD47-Fab compound mating surfaces, participate in the amino acid residue club mould of protein-interacting
Type indicates that two main calmodulin binding domain CaMs are marked with dotted line, and C figures show the details that region I interacts in B figures；D figures show B
The details that region II interacts in figure.
Sequence explanation
SEQ ID NO：The amino acid sequence of 1 display people (homo sapiens) CD47 extracellular regions D1 (hCD47).
SEQ ID NO：The amino acid sequence of 2 display mouse (mus musculus) CD47 extracellular regions D1 (mCD47).
SEQ ID NO：The amino acid sequence of 3 display macaque (Macaca mulatta) CD47 extracellular regions D1 (mmCD47).
SEQ ID NO：The high-affinity mutant of 4 display people's CD172a extracellular region immunoglobulin like domain
(hCD172a-D1M1) amino acid sequence.
SEQ ID NO：The amino acid sequence of 5 display His labels (His).
SEQ ID NO：The amino acid sequence of the Fc sections (Fc) of 6 display human antibody IgG1.
SEQ ID NO：The amino acid sequence of the Fc sections (mFc) of 7 display mouse IgG antibody 2a.
SEQ ID NO:The amino acid sequence of 8 display human IgG1's subtype heavy chain constant regions.
SEQ ID NO:The amino acid sequence of 9 display human IgG2's subtype heavy chain constant regions.
SEQ ID NO:The amino acid sequence of 10 display 4 subtype heavy chain constant regions of human IgG.
SEQ ID NO：The amino acid sequence of 11 display mouse IgG1 subtype heavy chain constant regions.
SEQ ID NO:The amino acid sequence of 12 display mouse IgG2a subtype heavy chain constant regions.
SEQ ID NO:The amino acid sequence of 13 display people's κ hypotype constant region of light chain.
SEQ ID NO:The amino acid sequence of 14 display people's λ hypotype constant region of light chain.
SEQ ID NO:The amino acid sequence of 15 display mouse κ hypotype constant region of light chain.
SEQ ID NO：The amino acid sequence of 16 display mouse λ hypotype constant region of light chain.
SEQ ID NO：17 and SEQ ID NO：18 show respectively the anti-CD47 monoclonal antibodies Hu5F9-G4 of humanization VH sequences and
The amino acid sequence of VK sequences.
SEQ ID NO：The full length amino acid sequence of 19 display mouse single-chain antibody S4D12, SEQ ID NO：20 and 21 difference
Show the amino acid sequence of itself VH and VK sequence.
SEQ ID NO：The full length amino acid sequence of 22 display mouse single-chain antibody S2H2, SEQ ID NO：23 and 24 show respectively
Show the amino acid sequence of itself VH and VK sequence.
SEQ ID NO：The full length amino acid sequence of 25 display mouse single-chain antibody S2C3, SEQ ID NO：26 and 27 show respectively
Show the amino acid sequence of itself VH and VK sequence.
SEQ ID NO：28 and 29 show the heavy chain variable amino acid sequence of humanization S4D12 (hS4D12) and light respectively
Chain variable region amino acid sequence.
SEQ ID NO：30 and 31 show the heavy chain variable amino acid sequence and light chain of humanization S2C3 (hS2C3) respectively
Variable region amino acid sequence.
SEQ ID NO：The amino acid sequence of 32 display heavy chain variable region mutant H10C7.
SEQ ID NO：The amino acid sequence of 33 display heavy chain variable region mutant H11E5.
SEQ ID NO：The amino acid sequence of 34 display light chain variable region mutant L25B8.
SEQ ID NO：The amino acid sequence of 35 display light chain variable region mutant L26A6.
Detailed description of the Invention
Present inventor has obtained new anti-human CD47 antibody by antibody engineering technology.In multiple sides of the application
Face provides new anti-human CD47 antibody or its antigen-binding fragment, encodes the antibody or the multinuclear glycosides of its antigen-binding fragment
Acid, the host cell comprising the polynucleotides or carrier, prepares and purifies the antibody at the carrier comprising the polynucleotides
The medicine and biological applications of method and the antibody or its antigen-binding fragment.According to the variable region of antibody provided by the present application
Sequence, the antibody molecule of overall length can be built as drug for treating the disease clinically mediated by CD47.
Unless otherwise specified, implementation of the invention uses molecular biology, microbiology, the cell biological of this field routine
, biochemistry and immunological technique.
Unless otherwise specified, term use herein has the meaning that those skilled in the art are generally understood.
Definition
Term " antibody " as used herein, is referred to via at least one in the variable region of immunoglobulin molecules
Antigen recognition site be specifically bound to the immunoglobulin molecules of target.Target includes but not limited to carbohydrate, more
Polynucleotide, lipid, polypeptide etc.." antibody " used herein includes not only complete (i.e. overall length) antibody, but also is wrapped
Include its antigen-binding fragment (such as Fab, Fab', F (ab')2, Fv), its variant, the fusion protein comprising antibody moiety, people
Source antibody, chimeric antibody, double antibody, linear antibodies, single-chain antibody, multi-specificity antibody (such as bispecific antibody) and appoint
What he includes the modification configuration of the immunoglobulin molecules of the antigen recognition site of required specificity, includes the glycosylation of antibody
The antibody of variant, the amino acid sequence variation of antibody and covalent modification.
In general, complete or overall length antibody includes two heavy chains and two light chains.Each heavy chain contains heavy chain region of variability
(VH) and the first, second and third constant region (CH1, CH2 and CH3).Each light chain contains light chain region of variability (VL) and constant region
(CL).The antibody of overall length can be any kind of antibody, such as IgD, IgE, IgG, IgA or IgM (or above-mentioned subclass), but
Antibody needs not belong to any specific classification.It, can be by immune globulin according to the antibody amino acids sequence of the constant domain of heavy chain
It is appointed as different classifications in vain.In general, there are five types of main classifications for immunoglobulin：IgA, IgD, IgE, IgG and IgM, and
Have in these classifications it is several can be further discriminated between into subclass (homotype) again, such as IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2.It is referred to as α, δ, ε, γ and μ corresponding to the heavy-chain constant domains of different immunoglobulin class.Different classes of exempts from
The sub-unit structure and three-dimensional structure of epidemic disease globulin are well known.
Term " antigen-binding fragment or antigen-binding portion thereof " as used herein refers to the complete antibody being responsible in conjunction with antigen
A part for molecule or region.Antigen binding domain can include heavy chain region of variability (VH), light chain region of variability (VL) or both.
Each of VH and VL usually contain three complementary determining regions CDR1, CDR2 and CDR3.
Well known to those skilled in the art, complementary determining region (CDR usually has CDR1, CDR2 and CDR3) is right in variable region
The maximum region of affinity and specific effect of antibody.There are two types of common definition modes for the CDR sequence of VH or VL, i.e.,
Kabat is defined and Chothia definition.(refering to such as Kabat, " Sequences of Proteins of Immunological
Interest ", National Institutes of Health, Bethesda, Md. (1991)；A1-Lazikani et
Al., J.Mol.Biol.273:927-948(1997)；And Martin et al., Proc.Natl.Acad.Sci.USA86:
9268-9272(1989)).For give antibody variable region sequences, can according to Kabat define or Chothia definition come
Determine CDR region sequence in VH and VL sequences.In the embodiment of the application, CDR sequence is defined using Kabat.
For giving the variable region sequences of antibody, CDR region sequence in variable region sequences, example can be analyzed in several ways
Can such as online software Abysis be utilized to determine (http://www.abysis.org/).
The example of antigen-binding fragment includes but not limited to：(1) Fab segments can be with VL-CL chains and VH-CH1
The monovalent fragment of chain；(2)F(ab')2Segment can be that there are two the bivalent fragment of Fab' segments, two Fab' segments for tool
It is connected by the disulphide bridges (i.e. the dimer of Fab') of hinge area；(3) the Fv segments in the domains VL and VH of the single armed with antibody；(4)
ScFv (scFv), the single victory peptide chain that can be made of via victory peptide connector the domains VH and the domains VL；And (5)
(scFv)2, can include two domains VH and two domains VL by the connection of victory peptide connector, which is via disulphide bridges
It is combined with two domains VH.
Term " specific binding " as used herein, refers to the nonrandom association reaction between two molecules, such as antibody
To the combination of epitope.
Terms used herein " monoclonal antibody " refers to by the antibody of the antibody population acquisition of basic homogeneity, that is, in addition to possible
In a small amount of individual there are abiogenous mutation other than, it is identical to form each antibody of group.Monoclonal described herein
Antibody particularly including " chimeric " antibody, wherein a part for heavy chain and/or light chain with from specific species or belong to specific anti-
Corresponding sequence in the antibody of body class or subclass is identical or homologous, and the remaining part of heavy chain and/or light chain with from another
Species or the corresponding sequence belonged in the antibody of another antibody class or subclass are identical or homologous, and further include such antibody
Segment, as long as they can show desired biological activity (U.S. Patent number 4,816,567；With Morrison etc.,
Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。
Terms used herein " tumour " refers to the neoplasm formed by abnormal cell growth or entity lesion.Tumour can be good
It is property, premalignant or pernicious.
" primary tumor " is the tumour existing for initial site in individual, can be distinguished with " metastatic tumo(u)r ", is turned
Shifting property tumour is present in the distant site of individual middle-range primary tumor.
Terms used herein " malignant tumour " refers to or describes the physiological condition of mammal, is typically featured in that not
Modulated cell growth.Exemplary malignant tumour includes：Cancer, melanoma sarcoma, lymthoma, leukaemia, gonioma and
Enblastoma.More specific examples of malignant tumour include：Squamous cell carcinoma (for example, dermoid cancer), including it is small thin
The gastric cancer of the lung cancer of born of the same parents' lung cancer, non-small cell lung cancer, adenocarcinoma of lung and lung squamous cancer, peritoneal cancer, hepatocellular carcinoma, including human primary gastrointestinal cancers, pancreas
Gland cancer, spongioblastoma, cervix cancer, oophoroma, liver cancer (liver cancer), carcinoma of urinary bladder, urinary tract cancer, hepatoma,
Breast cancer, colon cancer, the carcinoma of the rectum, colorectal cancer, endometrium or uterine cancer, salivary-gland carcinoma, kidney, prostate cancer, vulva
Cancer, thyroid cancer, liver cancer (hepatic carcinoma), cancer of anus, carcinoma of penis, melanoma, Huppert's disease and B cell
Lymthoma, the cancer of the brain and head and neck cancer and associated transitions stove.
Terms used herein " cancer " refers to epithelial cell by making a variation or occurs with unknown histology but have and epithelium
The mutant of the related specific molecular of cell or histologic characteristics' (as generated cytokeratin or intercellular bridge) forms
Aggressive malignant tumour.The exemplary cancer of the application includes oophoroma, carcinoma of vagina, cervix cancer, uterine cancer, prostate cancer, anus
Door cancer, the carcinoma of the rectum, colon cancer, gastric cancer, cancer of pancreas, insulinoma, gland cancer, adenosquamous carcinoma, neuroendocrine tumor, breast cancer, lung cancer,
The cancer of the esophagus, carcinoma of mouth, the cancer of the brain, medulloblastoma, neuroectodermal tumor, glioma, hypophysis cancer and osteocarcinoma.
Terms used herein " lymthoma " refers to the malignant tumour of the lymphocyte of immune system.Lymthoma is usually with solid tumor
In the presence of.Exemplary lymthoma includes：Non-Hodgkin lymphoma, B cell lymphoma, small lymphocytic lymphoma, lymph-plasma are thin
Born of the same parents' lymthoma, primary macroglobulinaemia (Macroglobulinemia), splenic marginal zone lymthoma, slurry
Cytoma, extranodal marginal zone B cell lymphoma, MALT lymthomas, knot inner peripheral area B cell lymphoma (NMZL), follicularis leaching
Bar tumor, lymphoma mantle cell, diffusivity large B cell lymphoid tumor, mediastinum (thymus gland) large B cell lymphoid tumor, the leaching of intravascular large B cell
Bar tumor, lymphoma primary effusion, Burkitt lymphoma, B cell chronic lymphocytic lymphoma, classics Huo Qijin lymphs
Tumor, Nodular lymphocyte are principal mode Hodgkin lymphoma, adult T cell lymphoma, knot external nose type NK/T cell lymphomas, intestines
Sick type t cell lymphoma, liver and spleen t cell lymphoma, mother cell NK cell lymphomas, mycosis fungoides, match thank to syndrome, primary
Property skin CD30 positive T cells lymphocytic hyperplasia disease, lymphoma primary cutaneous anaplastic large cell, lymphomatoid papulosis, blood
Pipe immunoblastic t cell lymphoma non-refers in particular to type lymphoma peripheral T cell and primary cutaneous type.Classics are suddenly
The exemplary form of strange gold lymthoma includes：Nodular sclerosis, mixed cell type, lymphocyte concentration type and lymphocyte disappear
Subtract type or non-abatement type.
Terms used herein " sarcoma " is the mutant in one of Various Tissues for carrying out free foetal mesoderm development
Malignant tumour.Therefore, sarcoma includes the tumour of bone, cartilage, fat, muscle, blood vessel and hematopoietic tissue.For example, the bone from bone
Sarcoma, the chondrosarcoma from cartilage, from fat embryonal-cell lipoma and from the leiomyosarcoma of smooth muscle.Illustrative meat
Tumor includes：Askin's tumor, botryoid sarcoma, chondrosarcoma, ewing's sarcoma-PNET, malignant hemangioma, malignant nerve sheath
Tumor, osteosarcoma, soft tissue sarcoma.The subclass of soft tissue sarcoma includes：Alveolar soft part sarcoma, angiosarcoma, lobate capsule meat
The outer chondrosarcoma of tumor, dermatofibrosarcoma fibroma, desmoplastic small round cell tumor, epithelioid sarcoma bone, the outer bone and flesh of bone
Tumor, fibrosarcoma, hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, embryonal-cell lipoma, lymphatic vessel meat
Tumor, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma and synovial sarcoma.
Terms used herein " leukaemia " is the malignant tumour of blood or marrow, is characterized in that the abnormal increase of leucocyte.
Leukaemia is broad terms, covers a series of diseases.Therefore, leukaemia is widely to be known as the disease kind of neoplastic hematologic disorder
A part for class.Leukaemia is subdivided into multiple major class；The first classification is the acute and chronic form of leukaemia.Acute leukemia
It is characterized in that the rapid growth of immature haemocyte quantity.Since the accumulation of these cells prevents marrow from manufacturing health
Haemocyte.Chronic leukemia is characterized in that relative maturity but still is the excessive generation of abnormal leucocyte.Usually pass through number
The development of the moon or several years, the generation rate of the cell are much higher than normal cell, lead to have many exceptions white thin in blood
Born of the same parents.Leukaemia can also be finely divided by the haemocyte of involvement.It is such boundary by leukaemia be divided into lymphoblast or
Lymphocytic leukemia and myeloide (myeloid) or myeloide (myelogenous) leukaemia.In lymphoblast or lymph
In chronic myeloid leukemia, canceration, which is happened at, to be generally continued in the lymphoblastic myeloid cell type of shape.At myeloide (myeloid)
Or in myeloide (myelogenous) leukaemia, canceration be happened at generally continue to be formed red blood cell, some other types it is white thin
In the myeloid cell type of born of the same parents and blood platelet.Four kinds of primary categories in total are provided in conjunction with both classification.It is main at these four
In each of classification, usually there are some sub-class.There is also the rare types except classification schemes.Illustrative leukaemia packet
It includes：Acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia
(AML), chronic granulocytic leukemia (CML), hairy cell (HCL), T cell prolymphocytic leukemia, big
Granular lymphocyte leukemia, juvenile myelomonocytic leukaemia, B cell prolymphocytic leukemia, the white blood of Hugh Burkitt
Disease and adult T-cell leukemia.
Involved in nucleic acid sequence given herein annex base (other than A, T, C, G conventional base) use,
Meaning is normally understood identical as those skilled in the art.For example, R represents A or G；Y represents C or T, and M represents A or C；K represents G
Or T；S represents C or G；W represents A or T；H represents A or C or T；B represents C or G or T；V represents A or C or G；D represents A or G or T；N
Represent A or C or G or T.
In a first aspect, this application provides a kind of antibody of specific binding people CD47, it includes containing HCDR1, HCDR2 and
The heavy chain variable region of HCDR3 sequences and light chain variable region containing LCDR1, LCDR2 and LCDR3 sequence, wherein
The HCDR1 sequences are NYWMH, and the HCDR2 sequences are VIAPSDNYTNYNQKFQG, and the HCDR3 sequences are
GGKYSMDY, the LCDR1 sequences are RSSQSIVHSNGNTYLE, and the LCDR2 sequences are KVSNRFS, the LCDR3 sequences
For FQGSHVPFT；Or
The HCDR1 sequences are SYWMH, and the HCDR2 sequences are TIDRSDSYISYNQKFKG, and the HCDR3 sequences are
GGPYGSKMMDN, the LCDR1 sequences are HASQNINVWLS, and the LCDR2 sequences are KASNLHT, and the LCDR3 sequences are
QQGQSYPLT；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPSSGNTKYAQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSLLYSSNKKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as QQFYAYPIS；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPSSGNTKYAQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSVLYSSNQKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as GQYYAYPIT；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPGSGNTRYSQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSLLYSSNKKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as QQFYAYPIS；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPGSGNTRYSQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSVLYSSNQKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as GQYYAYPIT；
Wherein HCDR and LCDR sequences are defined according to Kabat.
In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：23、28、
30, shown in 32 or 33.
In some embodiments, the amino acid sequence of the light chain variable region of the antibody such as SEQ ID NO：24、29、
31, shown in 34 or 35.
In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 23,
The amino acid sequence of the light chain variable region of the antibody such as SEQ ID NO：Shown in 24；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 28, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 29；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 30, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 31；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 32, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 34；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 32, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 35；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 33, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 34；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 33, the light chain variable of the antibody
The amino acid sequence in area such as SEQ ID NO：Shown in 35.
Second aspect, this application provides a kind of antibody of specific binding people CD47, wherein the heavy chain of the antibody can
Become the amino acid sequence and SEQ ID NO in area：23, any one of 28,30,32 or 33 at least 90% consistency,
And the amino acid sequence of the light chain variable region of the antibody and SEQ ID NO：24, any one of 29,31,34 or 35 tool
There is at least 90% consistency.
The third aspect, this application provides a kind of antibody of specific binding people CD47, wherein the antibody and SEQ ID
NO：The combination epitope of 1 leted others have a look at CD47 sections is discontinuous epi-position, including amino acid Q1, E29, A30, Q31, N32, E35,
E97, E100, L101, T102, R103, E104, G105 and E106, above-mentioned residue numbering is with reference to SEQ ID NO：1.
In some embodiments of first aspect to the third aspect, the antibody is monoclonal antibody.
In some embodiments of first aspect to the third aspect, the antibody is inactive type antibody.
In some embodiments of first aspect to the third aspect, the antibody can combine and neutralize people CD47, in turn
Block CD47-SIRP alpha signal accesses.In some implementations, the antibody can promote gulping down for Macrophages For Tumor
The effect of biting inhibits the tumor growth of tumour cell, without increasing phagocytosis of the macrophage to normal plasma cell.
In some embodiments of first aspect to the third aspect, at least one of the antibody has following property：
With affinity combination recombined human CD47 extracellular regions D1 (the SEQ ID NO of the KD values less than 5nM：1)；
With the IC less than 5nM50Value inhibits CD172a high-affinities mutant (SEQ ID NO：4) with the knot of people CD47-Fc
It closes；
Being applied under 0-133nM concentration does not lead to erythrocyte aggregation.
In some embodiments of first aspect to the third aspect, the antibody is whole antibody, Fab segments, F (ab ')2
Segment or Single-Chain Fv Fragment of Murine (scFv).
In some embodiments of first aspect to the third aspect, the antibody is human antibody.
In some embodiments of first aspect to the third aspect, the antibody also includes selected from IgG1 hypotypes, IgG2
The heavy chain constant region of hypotype or IgG4 hypotypes and/or include the constant region of light chain selected from κ hypotypes or λ hypotypes.
Fourth aspect, this application provides nucleic acid molecules, encode antibody described in first aspect to the third aspect or its
Antigen-binding portion thereof.
In some embodiments, the nucleic acid molecules are operably connected to regulating and controlling sequence, regulating and controlling sequence can by with
The transformed host cell identification of the carrier.
5th aspect, this application provides pharmaceutical composition, it includes described in first aspect to the third aspect antibody and
Pharmaceutically acceptable excipient, diluent or carrier.
In some embodiments, described pharmaceutical composition is used to treat the disease of CD47 mediations.
In some embodiments, the disease is tumour, such as malignant tumour.
In some embodiments, malignant tumour is Burkitt lymphoma or Huppert's disease.
In some embodiments, pharmaceutical composition also may include it is following in it is one or more：Lubricant, such as talcum
Powder, magnesium stearate and mineral oil；Wetting agent；Emulsifier；Suspending agent；Preservative, such as benzoic acid, sorbic acid and calcium propionate；It is sweetened
Agent and/or flavoring agent etc..
In some embodiments, the pharmaceutical composition in the application can be formulated as tablet, pill, pulvis, pastille, the wine made of broomcorn millet
The forms such as agent, suspension, emulsion, solution, syrup, suppository or capsule.
In some embodiments, any physiologically acceptable administering mode can be utilized to deliver the medicine group of the application
Object is closed, these administering modes include but not limited to：Oral medication, nose administration, rectally, is given in peritonaeum parenteral administration
Medicine, intravascular injection, subcutaneous administration, percutaneous dosing, inhalation etc..
It in some embodiments, can be pharmaceutically acceptable with optionally by mixing reagent with the desired purity
Carrier, excipient etc., the pharmaceutical composition of therapeutical uses is formulated in the form of lyophilized preparation or aqueous solution for storing.
6th aspect, this application provides the antibody described in first aspect to the third aspect to prepare for preventing or treating
Purposes in the drug for the disease that CD47 is mediated.
In some embodiments, the disease is tumour, such as malignant tumour.
In some embodiments, malignant tumour is Burkitt lymphoma or Huppert's disease.
7th aspect, this application provides the methods prevented or treat the disease that CD47 is mediated, including in need
Body gives the pharmaceutical composition of antibody or the 5th aspect described in first aspect to the third aspect.
In some embodiments, the disease is tumour, such as malignant tumour.
In some embodiments, malignant tumour is Burkitt lymphoma or Huppert's disease.
In other respects, the application also provides the nucleic acid of the separation comprising coding the application antibody or its antigen-binding portion thereof
The carrier of molecule and host cell comprising the nucleic acid molecules or carrier.
In other respects, the application also provides the method for generating the application antibody.In some embodiments, antibody is generated
Method include culture host cell in order to express nucleic acid.In some embodiments, the method for generating antibody further include from
Antibody is recycled in host cell culture medium.
It should be appreciated that it is discussed in detail above only for making those skilled in the art more clearly understand present context,
And it is not intended to limit in any way.Those skilled in the art can carry out various changes and change to the embodiment
Change.
Following embodiment is merely to illustrate and the purpose of unrestricted the application range.
Embodiment
Embodiment 1：The preparation of recombinant protein
It needs to use a variety of different recombinant proteins, including people's CD47 extracellular regions D1 during preparing anti-CD47 monoclonal antibodies
(hCD47, SEQ ID NO：1), mouse CD47 extracellular regions D1 (mCD47, SEQ ID NO：2), macaque CD47 extracellular regions D1
(mmCD47, SEQ ID NO：And the high-affinity mutant of people's CD172a extracellular region immunoglobulin like domain 3)
(hCD172a-D1M1, SEQ ID NO：4).These albumen have posttranslational modification (such as glycosylation or disulfide bond), thus
The structure and function for keeping recombinant protein is more advantageous to using mammalian cell expression system.In addition, in order to be conducive to recombinate
The identification of the purifying and monoclonal antibody function of albumen is added to His labels (His, SEQ ID in the C-terminal of these recombinant proteins
NO：Or the Fc of human antibody IgG1 sections (Fc, SEQ ID NO 5)：Or Fc sections (mFc, SEQ the ID NO of mouse IgG antibody 2a 6)：
7).Heavy chain constant region can be human IgG1's hypotype (SEQ ID NO:8), human IgG2's hypotype (SEQ ID NO:9), human IgG 4
Hypotype (SEQ ID NO:Or mouse IgG1 hypotypes (SEQ ID NO 10)：11), mouse IgG2a hypotypes (SEQ ID NO:12)；Light chain
Constant region can be people κ hypotypes (SEQ ID NO:13), people λ hypotypes (SEQ ID NO:Or mouse κ hypotypes (SEQ ID NO 14):
15), mouse λ hypotypes (SEQ ID NO：16).
According to the amino acid sequence of the various purpose recombinant proteins of Uniprot databases, design and synthesize above-mentioned various heavy
The gene (including His labels or Fc, mFc encoding gene) of histone.Using conventional Protocols in Molecular Biology by synthesis
Various recombinant protein gene clonings are then sharp to suitable carrier for expression of eukaryon (such as pcDNA3.1 of invitrogen companies)
It will be prepared with liposome (such as 293fectin of invitrogen companies) or other cationic transfection reagents (such as PEI)
Recombinant protein expression plasmid be transfected into HEK293 cells (such as HEK293F of invitrogen companies), serum free suspension train
It is cultivated 3-4 days under the conditions of supporting.Then culture supernatant is harvested by modes such as centrifugations.
The recombinant protein of His tag fusions expression utilizes metal chelate affinity chromatography column (the HisTrap FF of such as GE companies
Deng) a step purifying is carried out to the recombinant protein in culture supernatant.The recombinant protein of Fc and mFc amalgamation and expressions is close with ProteinA/G
A step purifying is carried out with chromatographic column (the Mabselect SURE of such as GE companies).Then utilize desalting column (such as GE companies
Hitrap desaulting etc.) by recombinant protein preserve buffer exchange be PBS (pH7.0) or other suitable buffer solutions.
When necessary, can antibody samples be filtered with degerming, then packing is stored in -20 DEG C.
Embodiment 2：The preparation of the anti-human CD47 monoclonal antibodies of mouse
1. the preparation of mouse immune and immune antibody library
6-8 week old BALB/c mouses are taken, carrying out tail vein blood to mouse before immune stays background serum.First immunisation takes
HCD47-mFc fusion proteins are emulsified with Freund's adjuvant complete, and every mouse peritoneal injects 50 μ g fusion proteins.Reinforce within two weeks at interval
It is immune, hCD47-mFc fusion proteins are taken to take the emulsification of formula Freund's incomplete adjuvant, and every mouse peritoneal injects 50 μ g fusion proteins, note
Docking blood sampling before penetrating, carries out booster immunization twice altogether.4th time immune, and to use impact instead immune, to be not added with the hCD47-mFc of adjuvant
Recombinant antigen injects 50 μ g fusion proteins as immunogene, every mouse peritoneal, impacts immune latter 3 days and puts to death mouse, collects spleen
Cell.
Mouse spleen lymphocyte is divided using mouse lymphocyte separating liquid (being up to section, #DKW33-R0100)
From using cell total rna extracts kit (Tiangeng, #DP430), by the extraction of the lymphocyte progress total serum IgE of separation.To carry
The total serum IgE taken is template, and weight is respectively synthesized using the first chain cDNA synthetic agent box (Thermo scientific, #K1621)
Chain variable region and light chain variable region, reverse transcription primer take gene-specific primer, primer collochore to be located at heavy chain of antibody
Constant region and antibody light chain constant region, particular sequence are respectively PmCGR:TGCATTTGAACTCCTTGCC and PmCKR:
CCATCAATCTTCCACTTGAC.The cDNA that synthesis is completed is deposited in -70 DEG C immediately to save backup.Then, it is obtained with reverse transcription
The cDNA arrived is template, bibliography (Journal of Immunological Methods, 201 (1997), 35-55) synthesis
Primer, and expand mouse antibody VH and VK respectively using PCR, overlap extension pcr is then utilized, single-chain antibody is built
(scFv).Finally by the mouse ScFv gene cloning of preparation to carrier pADSCFV-S, (experimental technique flow can be found in China
Patent application 201510097117.0), the libraries structure scFv.The storage capacity of this antibody library reaches 1.2 × 10E7, and accuracy is about
85%.
3. the screening and identification in mouse immune library
According to No. 201510097117.0 similar experiment flows of Chinese patent application, to recombinate huCD47-D1-his
For antigen, the bacteriophage of mouse single-chain antibody library is shown using solid-phase screening Policy Filtering, carries out three-wheel screening altogether, it is final to obtain
More plants of sequence differences but the mouse antibody that hCD47 can be specifically bound, including clone S1F9, S2H2, S4D12, S2C3, S2D10.
Bacteriophage-ELISA shows that hCD172a-D1M1 can be tied with wherein four plants of mouse monoclonal antibody (S1F9/S2C3/S2H2/S4D12) competitions
CD47 is closed, and the combination of mouse monoclonal antibody S2D10 and CD47 are not blocked by hCD172a-D1M1.
Embodiment 3：The identification of mouse monoclonal antibody
Choose mouse single-chain antibody S4D12 (SEQ ID NO：19, VH and VK sequences are respectively SEQ ID NO：20 and 21),
S2H2(SEQ ID NO：22, VH and VK sequences are respectively SEQ ID NO：24) and S2C3 (SEQ ID NO 23 and：25, VH and VK
Sequence is respectively SEQ ID NO：26 and 27), utilize molecular biology method Prepare restructuring human IgG 4- κ forms mouse-people's inosculating antibody
Body prepares the anti-CD47 monoclonal antibodies Hu5F9-G4 (VH of humanization referring concurrently to the disclosure of U.S. Patent application US 9017675B2
Sequence and VK sequences are respectively SEQ ID NO：17 and SEQ ID NO：18).
1. 7 MAbs blocking hCD47 combinations hCD172a of recombinant anti-CD 4
Envelope antigen hCD47-mFc, with the hCD172a-D1M1-His of fixed concentration to S2C3/S2H2/S4D12 inosculating antibodies
Body carry out gradient dilution, with HRP- mouse-anti-his IgG (health is century, CW0285M) detection CD172a-D1M1-His and
The combination of hCD47-mFc.Elisa assay result (Fig. 1) shows that monoclonal antibody S2C3, S2H2 and S4D12 block CD47 and CD172a-
The capacity variance of D1M1 interactions is little, IC50About at the range of 2-5nM (table 1).
Table 1：The anti-CD47 monoclonal antibodies of three kinds of differences inhibit the IC that CD47 is combined with CD172a-D1M150
| S2C3-IgG4 | S2H2-IgG4 | S4D12-IgG4 | Hu5F9-IgG4 | |
| IC50(nM) | 5.193 | 2.077 | 3.889 | 9.605 |
2. the affinity analysis of 7 monoclonal antibody of recombinant anti-CD 4
The affinity of IgG4 chimeric antibodies is measured using Biacore X100.Amino coupled kit, human antibody capture examination
The related reagents such as 10 × HBS-EP of agent box, CM5 chips and pH7.4 and consumptive material are purchased from GE healthcare.According to kit
In specification, by anti-human Fc sections of antibody coupling to CM5 chip surfaces, dilution antibody protein to suitable concentration ensures 300RU
The antibody of left and right is by the antibody capture of anti-human Fc.By huCD47-D1-his be arranged a series of concentration gradient (500nM, 167nM,
56nM, 18.5nM, 6.2nM) fixed phase surface is flowed through, the affinity of each monoclonal antibody is measured at 25 DEG C.As a result such as 2 institute of table
Show.
The different anti-CD47 monoclonal antibodies affinity constants of table 2.
| Kon(1/Ms) | Koff(1/s) | KD(M) | |
| S2C3-IgG4 | 8.861E+5 | 2.221E-2 | 2.506E-8 |
| S2H2-IgG4 | 7.169E+5 | 6.959E-3 | 9.707E-9 |
| S4D12-IgG4 | 1.414E+6 | 1.423E-2 | 1.006E-8 |
| Hu5F9-IgG4 | 1.317E+6 | 2.646E-2 | 2.01E-8 |
3. the erythrocyte agglutination capability analysis of 7 monoclonal antibody of recombinant anti-CD 4
Erythrocyte surface height expresses CD47, therefore anti-CD47 monoclonal antibodies can be specifically bound with the CD47 of erythrocyte surface.
Since there are two antigen binding sites for each antibody, thus anti-CD47 monoclonal antibodies may lead to erythrocyte agglutination.Cause red blood cell solidifying
The monoclonal antibody of collection can cause the side reactions such as anaemia, erythrocyte number reduction in vivo.Whether anti-CD47 monoclonal antibodies lead to erythrocyte agglutination
Depend primarily on identification epitope of the monoclonal antibody on CD47 molecules.The present embodiment is led using classical blood coagulation tests confrontation CD47 monoclonal antibodies
The ability of red blood cell condensation is caused to be analyzed.By embodiment before select anti-cd 47 antibody (40 μ g/ml) with PBS dilute into
The a series of doubling dilution of row, then mixes with 1% red cell suspension of preparation in Microhemagglutination reaction plate, is placed in micro shake
Swing oscillation 1min mixings on device.Film recording erythrocyte agglutination journey is used after Microhemagglutination reaction plate is stored at room temperature 1h in 25 DEG C
Degree, and Microhemagglutination reaction plate is tilted into 45° angle last for several minutes, it is further determined that by red blood cell flow rate speed red
Cell agglutination degree.Erythrocyte agglutination degree can be divided into 4 grades：4 congeal into thin uniform layer, and inclination is not flowed；3 congeal into uniformly it is thin
Layer, inclination have slight flow；2 congeal into a small amount of thin layer, and oblique flow is very fast；1 does not have thin layer to be formed, and is flowed with control wells after inclination
Speed is the same.As a result (Fig. 2) is shown, people-mouse is fitted into monoclonal antibody S2C3 and S2H2 and does not cause erythrocyte agglutination, and people-mouse is chimeric single
Anti- S4D12 and humanized antibody Hu5F9 have strong erythroagglutination.
4. 7 monoclonal antibody of recombinant anti-CD 4 can promote macrophage phagocytic tumour cell
PBMC is detached from Whole Blood of Healthy using Ficoll density-gradient centrifugation methods, it is then (anti-human by CD14 magnetic beads
CD14 magnetic-particles-DM, BD) from PBMC sorting obtain CD14+Monocyte.By CD14+Monocyte is with 8 × 105/ hole
Density is inoculated in 12 orifice plates, with 10ng/ml MCSF induce 7 days or so to being divided into ripe macrophage.Tumour cell
(Daudi lymphoma cell lines) is counted after being dyed in advance with CFSE and is resuspended, with 8 × 105Cell concentration and final concentration 10 μ g/ml
Different anti-CD47 monoclonal antibodies or control antibodies (IgG) are incubated 15-30min altogether, are subsequently transferred in the hole containing macrophage.It incubates jointly
Macrophage is collected after educating 3h, macrophage is dyed using anti-human CD14-APC, PBS is washed twice after 30min, is utilized
Flow cytometry (BD AccuriTMC6 APC (FL-4) and CFSE (FL-1) binary channel, and gulping down with macrophage background) are detected
Rate is bitten as starting, calculates the phagocytic rate of each sample.As a result (Fig. 3 and Fig. 4) is shown, three-type-person-mouse of the application is chimeric
Anti- CD47 monoclonal antibodies and Hu5F9 can promote macrophage phagocytic tumour cell.
Embodiment 4：The humanization of mouse monoclonal antibody and identification
1. mouse monoclonal antibody is humanization modified
Humanization research is carried out to reduce its immunogenicity to mouse antibody S2C3 and S4D12.By the heavy chain of S2C3 and S4D12
Respectively compared with the human antibody Germline gene sequences in IMGT databases, selection is closed for variable region (VH) and light chain variable region (VL)
Suitable Germline gene sequences select the suitable areas J gene order to carry to provide the framework region 1 to 3 (FR1+FR2+FR3) of antibody
For framework region 4 (FR4).This template can according to the comparative overall length of such as antibody, CDR size, be located at antibody framework area
(FR) between hypervariable region (CDR) amino acid residue of junction, sequence entirety homology etc. select, selected template can
To be the mixture of multiple sequences or can be shared template, it is therefore an objective to maintain parent's complementary determining region (CDR) as far as possible
Suitable conformation.It is final to determine humanization S4D12 (hS4D12) light and heavy chain variable region amino acid sequence (SEQ ID NO：29, SEQ
ID NO：And light and heavy chain variable region amino acid sequence (the SEQ ID NO of humanization S2C3 (hS2C3) 28)：31, SEQ ID NO：
30).Variable region gene is designed and synthesized according to the amino acid sequence of humanized antibody, and prepares the humanized antibody of IgG4 versions
HS2C3 and hS4D12.
2. the activity analysis of the anti-CD47 monoclonal antibodies of humanization
The energy that the anti-CD47 monoclonal antibodies of humanization inhibit hCD47 to be combined with hCD172a is analyzed with reference to the method in embodiment 3
As a result power shows that (Fig. 5) humanized antibody hS2C3 and hS4D12 can effectively inhibit the combination of hCD47 and hCD172a, IC50It is low
In 5nM (table 3).The affinity of humanized antibody is analyzed using BIAcore, the results show that humanized antibody is basic
Maintain the affinity (table 4) of parent murine antibody.
Table 3：The anti-CD47 monoclonal antibodies of humanization inhibit the IC that CD47 is combined with CD172a50
| Hu5F9-IgG4 | hS4D12-IgG4 | hS2C3-IgG4 | |
| IC50 | 4.161 | 2.68 | 3.364 |
Table 4：The affinity constant of the anti-D47 monoclonal antibodies of humanization
| Kon | Koff | KD | |
| S4D12 | 1.285E+6 | 1.1E-2 | 8.56E-9 |
| hS4D12 | 1.225E+6 | 1.176E-2 | 9.6E-9 |
| S2C3 | 1.111E+6 | 2.055E-2 | 1.85E-8 |
| hS2C3 | 1.088E+6 | 1.099E-2 | 1.01E-8 |
| Hu5F9-IgG4 | 2.095E+6 | 3.142E-2 | 1.499E-8 |
Embodiment 5：The affinity maturation of the anti-CD47 monoclonal antibodies hS2C3 of humanization
To further increase the affinity of humanized antibody, external affinity maturation has been carried out to hS2C3.External affinity
Ripe main policies are to introduce mutation in heavy chain CDR and light chain CDR successively, build light chain or heavy chain mutant library, then
(concrete operations can refer to the Chinese patent application the submitted before applicant to phage display system based on complex carries
201510097117.0 in number
Embodiment 5) respectively screen heavy chain and light chain high-affinity mutant.
The structure in 1.hS2C3 heavy chain mutants library and screening
The mutation library of hS2C3 heavy chain variable regions is built using conventional molecular biological method, storage capacity is 6.5 × 10E5, is dashed forward
The accuracy for becoming library is about 95%, wherein introducing primer needed for mutation in HCDR1 and HCDR2 is shown in Table 5.To recombinate huCD47-
His is antigen, using solid-phase screening strategy to hS2C3-HCDR12 mutation libraries carried out 3 wheel screen, finally obtain affinity into
Two mutant H10C7 (SEQ ID NO that one step improves：And H11E5 (SEQ ID NO 32)：33).
Table 5：Primer needed for the amplification of hS2C3 heavy chain variable region mutation libraries
The structure in 2.hS2C3 light chain mutants library and screening
Similarly, using the degenerate primer in table 6, by overlap extension pcr in hS2C3 light chain variable regions
It is introduced in LCDR1 and LCDR3 and is mutated and builds mutation library.Storage capacity about 3.1 × 10E6 is built, accuracy is about 90%.With weight
Group huCD47-his is antigen, and having carried out 3 wheels to hS2C3-LCDR13 mutation libraries using solid-phase screening strategy screens, and finally obtains
Two mutant L25B8 (SEQ ID NO that affinity further increases：And L26A6 (SEQ ID NO 34)：35)
Table 6：Primer needed for hS2C3 light chain variable region mutation libraries structure
3.hS2C3 the identification of mutant
It is combined, is made using the two kinds of high-affinity light chain mutants and two kinds of high-affinity heavy chain mutants that screen
For the mutant of four kinds of humanized antibody hS2C3：Utilize the two kinds of high-affinity light chain mutants screened and two kinds high parent
It is combined with power heavy chain mutant, is prepared for the mutant of four kinds of humanized antibody hS2C3：H10C7+L25B8、H10C7+
L26A6, H11E5+L25B8 and H11E5+L26A6, and carried out with reference to the mutant of four kinds of hS2C3 of method pair in embodiment 3
Affinity and functional analysis.
It is shown (table 7) using the affinity analysis of BIAcore pairs of four kinds of mutant：Four kinds of mutant are compared with humanized antibody
The affinity of hS2C3 has 5-10 times of raising, and four kinds of mutant can effectively inhibit hCD47 combinations hCD72a-D1M1 (figures
, and IC 6)50Below 5nM (table 8).
Table 7：The affinity constant of hS2C3 mutant
| Antibody | Kon | Koff | KD |
| H10C7+L25B8 | 1.031E+6 | 1.151E-3 | 1.115E-9 |
| H10C7+L26A6 | 8.33E+5 | 1.932E-3 | 2.32E-9 |
| H11E5+L25B8 | 1.189E+6 | 9.597E-4 | 8.072E-10 |
| H11E5+L26A6 | 9.297E+5 | 1.7E-3 | 1.829E-9 |
| hS2C3 | 1.078E+6 | 1.029E-2 | 0.955E-8 |
Table 8：HS2C3 mutant inhibits the IC that hCD47 and hCD172a-D1M1 is combined50
| H10C7+L26A6 | H11E5+L26A6 | H10C7+L25B8 | H11E5+L25B8 | hS2C3 | |
| IC50 | 4.25 | 3.521 | 4.511 | 4.129 | 4.13 |
Erythrocyte agglutination experiment shows the anti-CD47 monoclonal antibodies of humanization of (Fig. 7) four kinds of affinity raising in 0-133nM concentration
It will not result in erythrocyte agglutination in range, and four kinds of Humanized Variants can promote macrophage to CD47+Tumour
The phagocytosis (Fig. 8) of cell (Daudi).
4. inhibiting tumour growth in the anti-CD47 monoclonal antibodies of humanization
Using people's Burkitt lymphoma raji cells, with 1 × 104The amount of cell/mouse inoculates small in male NSG
Mouse grows in gross tumor volume to 100cm3When be randomly divided into 5 groups, every group of 8 mouse：(1) vehicle-control group；(2)20mg/kg
H10C7+L26A6 groups；(3) 10mg/kgH10C7+L26A6 groups；(4) 3mg/kg H10C7+L26A6 groups；(5)10mg/kg
Hu5F9-IgG4 groups.Weekly intraperitoneal administration twice, 3 weeks altogether.In administration process, one week 3 times measurement tumor sizes and animal body
Weight is to evaluate the tumor-inhibiting action of anti-cd 47 antibody.Experimental result is as shown in figure 9, abscissa is that the lotus knurl of raji tumours is small in Fig. 9
Mouse receives the time of drug-treated, and ordinate is gross tumor volume.The result shows that：Compared with vehicle-control group, the anti-CD47 of humanization
Monoclonal antibody H10C7+L26A6 can inhibit the growth of tumour, rejection ability suitable with control antibodies hu5F9-IgG4.
5. the anti-CD47 monoclonal antibodies of humanization are small in people's Huppert's disease RPMI8226 cell Subcutaneous Xenografts NOD/SCID
Inhibition tumour growth effect in rat animal model
In cell inoculation the previous day, mouse is subjected to 60Co treatment with irradiation (150rad).Male NOD/SCID mouse are in the right side
Side back portion inoculates 1 × 107RPMI8226 cells, cell are resuspended in the 1 of PBS and matrigel (matrigel):In 1 suspension
(0.1ml/ is only).Wait for that tumor average volume reaches 80-100mm3When, weigh the weight of all mouse, measure after gross tumor volume with
Machine grouping administration.It is defined as the 0th day on the day of tumor cell inoculation.5 groups are divided into, every group of 8 mouse：(1) vehicle-control group
(i.p.BIW*4, p.o.QD*4)；(2) 10mg/kg reaches thunder wood monoclonal antibody (daratumumab) (i.p.BIW*4) group；(3)10mg/
Kg H10C7+L26A6 (i.p.BIW*4) group；(4) 50mg/kg lenalidomides (lenolidomide) (p.o.QD*4) group；⑤
10mg/kg reaches thunder wood monoclonal antibody (i.p.BIW*4)+50mg/kg lenalidomides (p.o.QD*4) group.In administration process, one week 3 times surveys
Measure tumor size and the weight of animals.It is the monoclonal antibody medicine for Huppert's disease listed, lenalidomide up to thunder wood monoclonal antibody
For clinical multiple myeloma routine administration.Experimental result is as shown in Figure 10, and abscissa is Subcutaneous Xenograft RMPI in Figure 10
The NOD-SCID tumor-bearing mices of 8226 tumours receive the time of drug-treated, and ordinate is gross tumor volume and vehicle-control group phase
Than the anti-CD47 monoclonal antibodies H10C7+L26A6 of humanization can significantly inhibit the growth of tumour, and tumor suppression ability will be significantly higher than up to thunder
The drug combination of the wooden monoclonal antibody, lenalidomide and Da Lei wood monoclonal antibody+lenalidomide.
6. the CD47 combination epitope analysis of the anti-CD47 monoclonal antibodies H10C7+L26A6 of humanization
In order to study the combination epitope of humanization monoclonal antibody H10C7+L26A6 and people CD47, weight is prepared using HEK293 cells
The H10C7+L26A6Fab segments of group and people's CD47 extracellular regions D1 (hCD47, SEQ ID NO of recombination：1) Fab- is formed
HCD47 compounds form compound crystal under the conditions of 2.0M+5% isopropanols of ammonium sulfate, and X-ray is carried out to compound crystal
Diffraction experiment is collected into resolution ratioData (table 9), and it is multiple by molecular replacement technique to have parsed CD47 antigen-antibodies
Close the crystal structure of object.Finally obtained structural model includes CD47 extracellular regions 1-116aa (SEQ ID NO：1), heavy chain of antibody
1-219aa, light chain 1-219aa.Heavy chain of antibody and the last one amino acid residue of L chains are invisible in electron density, prompt it
There is no fixed structure in crystal.In complex crystal structure, CD47 exists with monomeric form, and in N16, N32, N55
There are visible glycosylations with the sites N93.
The interaction area of CD47 and Fab is aboutThe interaction area of wherein CD47 and heavy chain is aboutInteraction area with light chain is aboutInteraction relate generally to the N-terminal of CD47, BC rings, beta chain F,
FG rings and beta chain G, and HCDR1, HCDR2, HCDR3 of heavy chain of antibody variable region, LCDR1, LCDR3 of light chain variable region take part in
Identification (Figure 11) to CD47.Interaction mode includes hydrogen bond, sat linkage, Van der Waals force and hydrophobic interaction (table 10).
Wherein, discontinuous (space) epitope of the anti-CD47 monoclonal antibodies H10C7+L26A6 identification people CD47 of humanization, includes mainly amino
Sour Q1, E29, A30, Q31, N32, E35, E97, E100, L101, T102, R103, E104, G105 and E106.
According to the literature, the antigen binding epitope of anti-human CD47 monoclonal antibodies hu5F9 monoclonal antibodies includes amino acid Q1, E29, K39,
K41, E97, T99, R103 and E104；And the epitope of anti-human CD47 monoclonal antibodies 2A1 includes amino acid Y37, K39, K41, K43,
G44, R45, D46, D51, H90, N93, E97, T99, E104 and E106.Anti-human CD47 monoclonal antibodies H10C7+L26A6, hu5F9 and 2A1
Epitope all overlap, but it is all not exactly the same.
In order to further be verified to the epitope of anti-human CD47 monoclonal antibodies H10C7+L26A6, the part ammonia in epitope is selected
Base acid site is prepared for the mutant of three kinds of hCD47, including Q1A, E35A and L101A using conventional molecular biological method, and
Determine the affinity of humanization anti-CD47 monoclonal antibodies H10C7+L26A6 and these hCD47 mutant using BIAcore, and with day
Right hCD47 is compared (table 11).As a result it shows：L101A point mutation causes not combine completely, and the mutation of Q1A leads to parent
Decline about 100 times with power, and E35A mutation cause affinity to improve about 4 times.Show that these constitute monoclonal antibody H10C7+L26A6 epitopes
The mutation of key amino acid will result directly in the variation (reduce or improve) of antibody-antigene affinity.It analyzes simultaneously anti-human
CD47 monoclonal antibodies hu5F9 and the affinity of these mutant find (table 11), and the Q1A mutation in only hCD47 influence hu5F9 and people
The affinity of CD47, and the E35A and L101A mutation in hCD47 do not change the affinity of hu5F9 significantly.This result with
The epitope of hu5F9 includes Q1, without including E35 consistent with L101.
The X-ray diffraction receipt of 9. compound crystal of table is collected and statistics
* the numerical value in bracket is for highest resolution shell
Table 10：Participate in the amino acid residue that Fab-hCD47 is combined
Table 11：The affinity of different hCD47 mutant and the anti-CD47 monoclonal antibodies of humanization is analyzed based on BIAcore
Bibliography
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Sequence table
<110>Beijing bio tech ltd Zhi Renmeibo
Intelligence Xiang（Shanghai）Pharmaceutical Technology Co., Ltd
Chongqing Zhi Xiangjintai Biology Pharmacy Co., Ltd
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Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
210 215 220
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
260 265 270
Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly Lys
<210> 12
<211> 330
<212> PRT
<213>House mouse (Mus musculus)
<400> 12
Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly
1 5 10 15
Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
100 105 110
Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
115 120 125
Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
130 135 140
Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp
145 150 155 160
Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
165 170 175
Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln
180 185 190
His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn
195 200 205
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
210 215 220
Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu
225 230 235 240
Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met
245 250 255
Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu
260 265 270
Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe
275 280 285
Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
290 295 300
Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr
305 310 315 320
Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
325 330
<210> 13
<211> 107
<212> PRT
<213>Homo sapiens (Homo sapiens)
<400> 13
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 14
<211> 106
<212> PRT
<213>Homo sapiens (Homo sapiens)
<400> 14
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
20 25 30
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
35 40 45
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
65 70 75 80
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210> 15
<211> 107
<212> PRT
<213>House mouse (Mus musculus)
<400> 15
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
1 5 10 15
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 16
<211> 106
<212> PRT
<213>House mouse (Mus musculus)
<400> 16
Gly Gln Pro Lys Ser Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile Thr Asp
20 25 30
Phe Tyr Pro Gly Val Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro
35 40 45
Val Thr Gln Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Met Ala Ser Ser Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu
65 70 75 80
Arg His Ser Ser Tyr Ser Cys Gln Val Thr His Glu Gly His Thr Val
85 90 95
Glu Lys Ser Leu Ser Arg Ala Asp Cys Ser
100 105
<210> 17
<211> 117
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 17
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Thr Ile Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Tyr Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 18
<211> 112
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 18
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 19
<211> 244
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 19
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Ala Pro Ser Asp Asn Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Gln Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Lys Gly Gly Lys Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro
130 135 140
Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
145 150 155 160
Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys
165 170 175
Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
180 185 190
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
195 200 205
Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
210 215 220
Cys Phe Gln Gly Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys
225 230 235 240
Leu Glu Ile Lys
<210> 20
<211> 117
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 20
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Ala Pro Ser Asp Asn Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Gln Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Lys Gly Gly Lys Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 21
<211> 112
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 21
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 22
<211> 242
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 22
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Leu Ser Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Thr Ile Asp Arg Ser Asp Ser Tyr Ile Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Gly Thr Val Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Gly Pro Tyr Gly Ser Lys Met Met Asp Asn Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Asn Gln Ser Pro Ser
130 135 140
Ser Leu Ser Ala Ser Leu Gly Asp Thr Ile Thr Ile Thr Cys His Ala
145 150 155 160
Ser Gln Asn Ile Asn Val Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly
165 170 175
Asn Ile Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gly Phe Thr Leu
195 200 205
Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln
210 215 220
Gln Gly Gln Ser Tyr Pro Leu Thr Phe Gly Thr Gly Thr Lys Leu Glu
225 230 235 240
Leu Lys
<210> 23
<211> 120
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 23
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Leu Ser Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Thr Ile Asp Arg Ser Asp Ser Tyr Ile Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Gly Thr Val Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Gly Pro Tyr Gly Ser Lys Met Met Asp Asn Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 24
<211> 107
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 24
Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 25
<211> 245
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 25
Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asn Pro Ser Ser Gly Asn Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Asp Lys Ala Thr Leu Thr Val Ala Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Arg Glu Glu Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala
130 135 140
Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
145 150 155 160
Leu Leu Tyr Pro Ser Asn Lys Lys Asn Tyr Leu Thr Trp Tyr Gln Gln
165 170 175
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
180 185 190
Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
195 200 205
Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr
210 215 220
Tyr Cys Gln Glu Tyr Tyr Ala Tyr Pro Leu Ser Phe Gly Ala Gly Thr
225 230 235 240
Lys Leu Glu Ile Lys
245
<210> 26
<211> 117
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 26
Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asn Pro Ser Ser Gly Asn Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Asp Lys Ala Thr Leu Thr Val Ala Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Arg Glu Glu Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 27
<211> 113
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 27
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Pro
20 25 30
Ser Asn Lys Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Glu
85 90 95
Tyr Tyr Ala Tyr Pro Leu Ser Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 28
<211> 117
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 28
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Ala Pro Ser Asp Asn Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Lys Gly Gly Lys Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val Ser Ser
115
<210> 29
<211> 112
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 29
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 30
<211> 117
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 30
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Ser Ser Gly Asn Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Glu Glu Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 31
<211> 113
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 31
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Pro
20 25 30
Ser Asn Lys Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Glu
85 90 95
Tyr Tyr Ala Tyr Pro Leu Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 32
<211> 117
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 32
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Arg Tyr Ser Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Glu Glu Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 33
<211> 117
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 33
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Trp Ile Tyr Pro Ser Ser Gly Asn Thr Lys Tyr Ala Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Glu Glu Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 34
<211> 113
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 34
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Lys Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Phe Tyr Ala Tyr Pro Ile Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 35
<211> 113
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 35
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln
85 90 95
Tyr Tyr Ala Tyr Pro Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
Claims (10)
1. a kind of antibody of specific binding people CD47, it includes containing HCDR1, HCDR2 and HCDR3 sequence heavy chain variable region and
Light chain variable region containing LCDR1, LCDR2 and LCDR3 sequence, wherein
The HCDR1 sequences are NYWMH, and the HCDR2 sequences are VIAPSDNYTNYNQKFQG, and the HCDR3 sequences are
GGKYSMDY, the LCDR1 sequences are RSSQSIVHSNGNTYLE, and the LCDR2 sequences are KVSNRFS, the LCDR3 sequences
For FQGSHVPFT；Or
The HCDR1 sequences are SYWMH, and the HCDR2 sequences are TIDRSDSYISYNQKFKG, and the HCDR3 sequences are
GGPYGSKMMDN, the LCDR1 sequences are HASQNINVWLS, and the LCDR2 sequences are KASNLHT, and the LCDR3 sequences are
QQGQSYPLT；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPSSGNTKYAQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSLLYSSNKKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as QQFYAYPIS；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPSSGNTKYAQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSVLYSSNQKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as GQYYAYPIT；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPGSGNTRYSQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSLLYSSNKKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as QQFYAYPIS；Or
The HCDR1 sequences are DYYMH, and the HCDR2 sequences are WIYPGSGNTRYSQKFKD, and the HCDR3 sequences are
REEDYFDY, the LCDR1 sequences are KSSQSVLYSSNQKNYLT, and the LCDR2 sequences are WASTRES, the LCDR3 sequences
It is classified as GQYYAYPIT；
Wherein HCDR and LCDR sequences are defined according to Kabat.
2. antibody according to claim 1, the amino acid sequence such as SEQ ID NO of the heavy chain variable region of the antibody：23、
28, shown in 30,32 or 33.
3. antibody according to claim 1, the amino acid sequence such as SEQ ID NO of the light chain variable region of the antibody：24、
29, shown in 31,34 or 35.
4. antibody according to claim 1, wherein
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 23, the light chain variable region of the antibody
Amino acid sequence such as SEQ ID NO：Shown in 24；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 28, the light chain variable region of the antibody
Amino acid sequence such as SEQ ID NO：Shown in 29；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 30, the light chain variable region of the antibody
Amino acid sequence such as SEQ ID NO：Shown in 31；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 32, the light chain variable region of the antibody
Amino acid sequence such as SEQ ID NO：Shown in 34；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 32, the light chain variable region of the antibody
Amino acid sequence such as SEQ ID NO：Shown in 35；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 33, the light chain variable region of the antibody
Amino acid sequence such as SEQ ID NO：Shown in 34；Or
The amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO：Shown in 33, the light chain variable region of the antibody
Amino acid sequence such as SEQ ID NO：Shown in 35.
5. a kind of antibody of specific binding people CD47, wherein the amino acid sequence of the heavy chain variable region of the antibody and SEQ ID
NO：23, any one of 28,30,32 or 33 at least 90% consistency, and the light chain variable region of the antibody
Amino acid sequence and SEQ ID NO：24, any one of 29,31,34 or 35 at least 90% consistency.
6. a kind of antibody of specific binding people CD47, wherein the antibody and SEQ ID NO：The knot of 1 leted others have a look at CD47 sections
Conjunction epitope is discontinuous epi-position, including amino acid Q1, E29, A30, Q31, N32, E35, E97, E100, L101, T102, R103,
E104, G105 and E106, above-mentioned residue numbering is with reference to SEQ ID NO：1.
7. according to the antibody described in any one of claim 1-6, wherein
The antibody can be with affinity combination recombined human CD47 extracellular regions D1 (the SEQ ID NO of the KD values less than 5nM：1)；With/
Or
The antibody can be with the IC less than 5nM50Value inhibits CD172a high-affinities mutant (SEQ ID NO：4) with people CD47-
The combination of Fc；And/or
The antibody is applied under 0-133nM concentration does not lead to erythrocyte aggregation；And/or
The antibody is whole antibody, Fab segments, F (ab ')2Segment or Single-Chain Fv Fragment of Murine (scFv), it is preferable that the antibody is complete
Human antibody；And/or
The antibody also includes selected from IgG1 hypotypes, the heavy chain constant region of IgG2 hypotypes or IgG4 hypotypes and/or comprising selected from the Asias κ
The constant region of light chain of type or λ hypotypes；And/or
The antibody is monoclonal antibody；And/or
The antibody is inactive type antibody；And/or
The antibody can combine and neutralize people CD47, and then block CD47-SIRP alpha signal accesses；And/or
The antibody can promote the phagocytosis of Macrophages For Tumor；And/or
The antibody can inhibit the tumor growth of tumour cell；And/or
The antibody does not increase phagocytosis of the macrophage to normal plasma cell.
8. nucleic acid molecules encode antibody or its antigen-binding portion thereof described in any one of claim 1-7.
9. pharmaceutical composition, it includes described in any one of claim 1-7 antibody and pharmaceutically acceptable excipient, dilution
Agent or carrier.
10. the antibody described in any one of claim 1-7 is in the drug for preparing the disease for preventing or treating CD47 mediations
In purposes, such as the disease is tumour, such as malignant tumour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2018/100262 WO2019042119A1 (en) | 2017-09-01 | 2018-08-13 | Antibody against human cd47 and use thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710777971 | 2017-09-01 | ||
| CN2017107779710 | 2017-09-01 |
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| Publication Number | Publication Date |
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| CN108503708A true CN108503708A (en) | 2018-09-07 |
| CN108503708B CN108503708B (en) | 2021-07-30 |
Family
ID=63399104
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810375653.6A Active CN108503708B (en) | 2017-09-01 | 2018-04-20 | Anti-human CD47 antibodies and uses thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN108503708B (en) |
| WO (1) | WO2019042119A1 (en) |
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2019042119A1 (en) | 2019-03-07 |
| CN108503708B (en) | 2021-07-30 |
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