CN107955071B - Human anti-human CD47 antibody and coding gene and application thereof - Google Patents

Abstract

The invention provides a human anti-human CD47 antibody and a coding gene and application thereof. The invention screens anti-human CD47 single-chain antibody from a fully synthetic human antibody library by an antibody library technology and a genetic engineering means to obtain the variable region gene thereof, constructs a human monoclonal antibody recombinant vector in an IgG form, expresses and purifies to obtain high-purity antibody protein. The affinity KD of the antibody and human CD47 is not more than 10nM, and the affinity KD of the mutant thereof is not more than 50 nM; the antibody can effectively block the combination of CD47 and SIRP alpha, promotes the phagocytosis of tumor cells by immune cells, has definite therapeutic action on leukemia model mice with positive CD47 expression, and has obvious synergistic action when combined with targeted drugs such as EGFR and the like. The invention provides a specific antibody medicament for preventing and treating tumors and other related diseases such as atherosclerosis and inflammation aiming at the CD47 target.

Description

Human anti-human CD47 antibody and coding gene and application thereof

Technical Field

The invention relates to preparation and application of a human genetic engineering antibody for treatment, and mainly relates to an antibody specifically directed to human CD47, and a coding gene and application thereof.

Background

CD47, also known as integrin-associated protein (IAP), OV-3 (ovarian cancer marker), or MER6, has 305 amino acids in length, an extracellular amino-terminal IgV-like extracellular domain, 5 transmembrane segments with highly hydrophobic extensions, and 1 short, selectively spliced carboxyl-terminal cytoplasmic tail, where the IgV-like domain is its primary ligand binding site. The ligand comprises: signal-regulatory protein alpha chain (SIRP α), Thrombospondin (TSP), and Integrins (Integrins). CD47 is widely expressed on cell membrane surfaces and is present on various cells in all tissues, especially leukocytes, such as polymorphonuclear leukocytes (PMNs), Dendritic Cells (DCs), T cells, erythrocytes, placenta, platelets, normal Hematopoietic Stem Cells (HSCs), and other normal cells. Meanwhile, CD47 is highly expressed in most tumor cells of human body, and it can be used as a standard for tumor diagnosis and prognosis judgment. Over-expression occurs in various solid tumor cells or tumor stem cells (such as leukemia stem cells, leukemia cells) such as acute and chronic myelogenous leukemia, acute lymphocytic leukemia, non-hodgkin lymphoma, bladder cancer, ovarian cancer, breast cancer, rectal cancer, prostate cancer, renal cancer, multiple myeloma, etc., and the high expression is related to the poor clinical prognosis. Studies have shown that CD47 on the surface of tumor cells transmits a "don' T eat me" inhibitory signal to macrophages, DC cells, and other immune cell surface SIRP alpha ligands, thereby evading phagocytosis of these cells (Kershaw MH and Smyth HJ. science.2013, vol 341:41-42), and further evading T cell killing of tumor cells caused by tumor antigen presentation after phagocytosis (Vonderheide RH, Nature Medicine, 1122 2015,21, 1123).

Research shows that (Chao MP, curr, Opin, Immunol.2012,24(2): 225-32; Liu XJ, Nature media, 2015,21(10),1209-15) can promote phagocytosis of tumor cells by macrophages and DC cells, further increase presentation of tumor antigens and activate acquired immune response by blocking the interaction of CD47 on the surface of the tumor cells and SIRP alpha on the surface of the macrophages. anti-CD 47 antibody or anti-SIRP alpha antibody can also promote phagocytosis of tumor cells by macrophages. Secondly, the anti-CD 47 monoclonal antibody can also eliminate tumor cells through Fc receptor mediated cytotoxicity; in addition, anti-CD 47 mab may also eliminate tumor cells by directly inducing apoptosis. Currently, several anti-CD 47 therapeutic antibodies have entered clinical research (NCT02096770, NCT02216409, NCT02367196, NCT02447354, NCT 02488811).

In addition, research shows that the antibody can prevent and treat atherosclerosis by blocking the combination of CD47 and SIRP alpha (Kojima Y, Volkmer JP, McKenna K, et al. Nature,2016,536:86-90), and the anti-CD 47 antibody has good application prospect in the treatment of cardiovascular diseases.

The humanized antibody is the final direction of the development of the therapeutic antibody, the clinical application of the human antibody has good patient compliance, the immunogenicity of the antibody can be reduced to the maximum extent, the half-life of the drug in vivo can be prolonged, the effects of mediated immunoregulation, ADCC, CDC and the like can be realized by virtue of the Fc segment of the human immunoglobulin, and the biological effect of the antibody can be further enhanced. The antibody library technology is an important technical means for obtaining human antibodies, has the characteristics of simplicity, convenience, flexibility and the like, and a plurality of antibodies from the antibody library technology are on the market or in the clinical research stage at present.

Disclosure of Invention

The first purpose of the invention is to provide the amino acid sequence of human anti-human CD47 antibody and its active fragment.

The second object of the present invention is to provide a gene encoding the above antibody or an active fragment thereof.

The third purpose of the invention is to provide the application of the antibody and the active fragment thereof in treating or preventing malignant tumors expressing positive human CD47 and other related diseases.

The invention uses phage surface display technology to screen and obtain single chain gene engineering antibody (scFv) of specific anti-human CD47 from a large-capacity fully-synthesized human single chain antibody library through multiple rounds of biological-panning. Respectively cloning the light chain variable region genes and the heavy chain variable region genes of the antibody into human full-antibody mammal cell transient expression vectors pABL and pABG1, and carrying out transient secretory expression by cotransfecting HEK293T cells to obtain a full antibody of a human antibody ZF1, wherein the amino acid sequence of the ZF1 light chain variable region is characterized as follows: LCDR1 is SGDALGDKYAS (SEQ ID NO.1), LCDR2 is EDSKRPS (SEQ ID NO.2), LCDR3 is QLRAGNAAGW (SEQ ID NO. 3); the heavy chain variable region amino acid sequence is as follows: the HCDR1 is SYAMS (SEQ ID NO.4), the HCDR2 is AISGSGGSTYYADSVKG (SEQ ID NO.5), the HCDR3 is TWWRLFDY (SEQ ID NO.6), the amino acid sequence of the ZF1 light chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8.

The invention also utilizes the yeast display antibody library technology to construct a yeast display mutation library aiming at the heavy chain CDR2 region of the single chain antibody ZF1 and screen the yeast display mutation library to finally obtain the light chain variable region gene and the heavy chain variable region gene of the mutant antibody, and respectively clone the light chain variable region gene and the heavy chain variable region gene into human full antibody mammal cell transient expression vectors pABL and pABG1, and carry out transient secretory expression and purification by cotransfecting HEK293T cells to obtain the full antibody pure product of the mutant antibody.

The amino acid sequence pattern of the antibody variable region provided by the invention is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Here, FR 1-4 represents 4 framework regions, and CDR 1-3 represents 3 hypervariable regions. Wherein the heavy chain is human immunoglobulin germline gene VH III family, the light chain is Lambda III family, further, the light chain framework sequence (FR 1-4) is shown as SEQ ID NO.38-41, and the heavy chain framework amino acid sequence (FR 1-4) is shown as SEQ ID NO. 42-45.

With respect to the CDR regions of light and heavy chains, a large number of mutants were obtained by alanine scanning and other amino acid mutations of the same type, and evaluation of the mutants of CDR1, CDR2 and CDR3 of light chain revealed (here, a large amount of mutation site information is referred to, and when one position is a mutation site, the amino acid sequence of other positions remains unchanged):

LCDR1 sequence: s₁G₁D₁A₁LG₂D₂KYA₂S₂(SEQ ID NO.1) wherein S₁May be replaced by Ala; g₁May be replaced by Ser or Ala; d₁May be replaced by Ala; a. the₁Can be replaced by Asn, Gly or Lys; l may be replaced by Ala; g₂May be replaced by Ala; d₂May be replaced by Met, Leu, Glu, Lys or Trp; y may be replaced by Ala or Gln; a. the₂Can be replaced by Ser or Gly; s₂Ala may be substituted.

LCDR2 sequence: EDS₁KRPS₂(SEQ ID NO.2) where S₁May be replaced by Val, Gly or Ala; k may be replaced with Ala; r may be replaced by Val, Ser or Ala. And S1 and R may be simultaneously replaced by Val.

LCDR3 sequence: QLRA₁G₁NA₂A₃G₂W (SEQ ID NO.3) wherein L may be replaced by Val; g₁May be replaced by Ser or Ala; n may be replaced by Asp, Ala, Ser, Tyr, The, Pro, Glu, Phe, Ile, Trp, Val or Leu, preferably Tyr; a. the₂Can be replaced by Ser, The, Pro, Asp, Lys, Ile or Gly, and is preferably Pro; g₂Ala may be substituted. And when N is replaced by Tyr and The a2 can be simultaneously replaced by Pro, The and Lys.

The evaluation results of the heavy chain CDR1, CDR2 and CDR3 mutants show that:

HCDR1 sequence: s₁YAMS₂(SEQ ID NO.4) wherein S₁May be replaced by Gly or Ala; y may be replaced by Ala or Gly; m may be replaced by Ala or Gly.

HCDR2 sequence: a. the₁IS₁G₁S₂G₂G₃S₃TY₁Y₂A₂DS₄VKG₄(SEQ ID NO.5) where A₁Val can be replaced; i may be replaced by Ala; s1 may be replaced by Asn or Ala; g1 may be replaced by Ser; s₂May be replaced by Gly or Ala; g₂May be replaced by Ser or Ala; g₃Can be replaced by Ala, Ser, Asn, Ile, Lys or Gln; s₃Can be replaced by Ala, Asn, Tyr, Lys, Gln, His or Leu; t may be replaced by Ala; y is₂May be replaced by Ala; a. the₂Can be replaced by Ser; d may be replaced by Ala; s₄May be replaced by Ala; v may be replaced by Ala; k may be replaced with Ala; g₄May be replaced by Ser.

HCDR3 sequence: TW (time-lapse launching) device₁W₂RLFDY (SEQ ID NO.6), wherein W₂Tyr can be replaced.

Alanine scanning of the light and heavy chain CDR regions of the antibody revealed that LCDR3, HCDR3, and partial HCDR2 were critical for binding of the antibody to CD47, and that changes in only a single amino acid could cause several to several tens of fold changes in affinity, as shown in example 3.

In addition, a mutant with improved affinity of multiple strains is obtained by constructing a mutant library on a heavy chain HCDR2 region (SEQ ID NO.5) of the ZF1 antibody and screening, mutation mainly occurs at amino acids 52-57, and the amino acid sequences of the HCDR2 region of the mutant with improved affinity obtained by screening are respectively shown as SEQ ID NO. 9-26.

Furthermore, the HCDR2 is subjected to overlapping mutation on the basis of one mutant antibody ZF1-Y2 (the amino acid sequence of HCDR2 is shown as SEQ ID NO. 10), the affinity of the mutant antibody can be maintained, and the amino acid sequences (49-65 amino acids) at the overlapping part are shown as SEQ ID NO. 27-33. Similarly, the partial mutation of the light chain is subjected to overlapping mutation and is combined with the heavy chain of another mutant ZF1-Y1 (the amino acid sequence of HCDR2 is shown as SEQ ID NO. 9), the affinity of the obtained full-antibody mutant is also maintained or improved, and the information of the overlapping mutation position mutation is as follows: S51V + R53V, 93Y +94P, 93Y +94T, 93Y +94K, 93T +94P, 93T +94K, 93T +94T and 51V53V-93Y, 51V53V-93T, 51V53V-94T, 51V53V-94P, 51V53V-94K, 51V53V-93Y94P, 51V53V-93Y94T, 51V53V-93Y94K, 51V53V-93T94K, 51V53V-93T 94P.

In summary, the present invention provides a human anti-human CD47 antibody, which is:

(1) the light chain variable regions LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the heavy chain variable regions HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; or

(2) In the light chain variable region, any one amino acid mutation of 1-7 and 9-11 of LCDR1 shown in SEQ ID NO.1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; or

(3) In the light chain variable region, any one or more amino acid mutations of 3 rd, 4 th and 5 th of LCDR2 shown in SEQ ID NO.2, LCDR1, LCDR3, HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; or

(4) In the light chain variable region, any one or more amino acid mutations of 2 nd, 5 th to 7 th and 9 th of LCDR3 shown in SEQ ID NO.3, LCDR1, LCDR2, HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; or

(5) In the heavy chain variable region, any one amino acid mutation of 1 st, 2 nd and 4 th positions of HCDR1 shown in SEQ ID NO.4, LCDR1, LCDR2, LCDR3, HCDR2 and HCDR3 are respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO. 6; or

(6) In the heavy chain variable region, any one amino acid mutation of 1-9 and 11-17 of HCDR2 shown in SEQ ID NO.5, LCDR1, LCDR2, LCDR3, HCDR1 and HCDR3 are respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 6; or

(7) In the heavy chain variable region, the 3 rd amino acid mutation of HCDR3 shown in SEQ ID NO.6, LCDR1, LCDR2, LCDR3, HCDR1 and HCDR2 are respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; or

(8) The light chain variable regions LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, the heavy chain variable regions HCDR1 and HCDR3 are respectively shown in SEQ ID NO.4 and SEQ ID NO.6, and the HCDR2 is shown in any one of SEQ ID NO. 9-33.

Wherein, the amino acid mutation at any position 1-7 and 9-11 of LCDR1 shown in SEQ ID NO.1 in (2) refers to any mutation at the following positions, and the mutations are single-point mutations: ser at position 1 to Ala; or a substitution of Gly to Ser or Ala at position 2; or Asp at position 3 is replaced by Ala; or Ala at position 4 with Asn, Gly or Lys; or replacement of Leu at position 5 to Ala; or replacement of Gly to Ala at position 6; or Asp at position 7 is replaced by Met, Leu, Glu, Lys or Trp; or Tyr at position 9 is replaced by Ala or Gln; or Ala at position 10 is replaced by Ser or Gly; or Ser at position 11 with Ala.

Wherein the mutation at any one or more of amino acids 3, 4 and 5 of LCDR2 shown in SEQ ID NO.2 in (3) refers to: ser at position 3 to Val, Gly, or Ala; or Lys at position 4 to Ala; or Arg at position 5 to Val, Ser or Ala; or Ser at position 3 and Arg at position 5 are simultaneously replaced by Val.

Wherein the mutation at any one or more of amino acids 2, 5 to 7 and 9 of LCDR3 shown in SEQ ID NO.3 in (4) refers to: substitution of Leu to Val at position 2; or a substitution of Gly to Ser or Ala at position 5; or Asn at position 6 is replaced by Asp, Ala, Ser, Tyr, The, Pro, Glu, Phe, Ile, Trp, Val or Leu; or Ala at position 7 is replaced by Ser, The, Pro, Asp, Lys, Ile or Gly; or replacement of Gly to Ala at position 9; or Ala at position 7 may be simultaneously replaced by Pro, The or Lys when Asn at position 6 is replaced by Tyr or The.

Wherein, the amino acid mutation at any position 1, 2 or 4 of the HCDR1 shown in SEQ ID NO.4 in (5) refers to any mutation at the following positions, and the mutations are single-point mutations: ser at position 1 replaced by Gly or Ala; or Tyr at position 2 is replaced by Ala or Gly; or Met at position 4 is replaced by Ala or Gly.

Wherein the amino acid mutation at any position 1-9 or 11-17 of HCDR2 shown in SEQ ID NO.5 in (6) refers to any mutation at the following positions, and the mutations are single-point mutations: a substitution of Ala to Val at position 1; or Ile at position 2 to Ala; or Ser at position 3 to Asn or Ala; or the Gly at position 4 is replaced by Ser; or Ser at position 5 to Gly or Ala; or a substitution of Gly to Ser or Ala at position 6; or substitution of Gly to Ala, Ser, Asn, Ile, Lys or Gln at position 7; or Ser at position 8 to Ala, Asn, Tyr, Lys, Gln, His, or Leu; or Thr at position 9 to Ala; or Tyr at position 11 is replaced with Ala; or Ala to Ser at position 12; or Asp at position 13 is replaced by Ala; or Ser at position 14 to Ala; or Val at position 15 to Ala; or Lys at position 16 to Ala; or the Gly at position 17 is replaced by Ser.

Wherein the 3 rd amino acid Trp of HCDR3 shown in SEQ ID NO.6 in (7) above is mutated to Tyr.

Therefore, the invention provides the human anti-CD 47 antibody, wherein the light chain variable region framework regions FR1, FR2, FR3 and FR4 are respectively shown as SEQ ID NO. 38-41; the heavy chain variable region frameworks FR1, FR2, FR3 and FR4 are respectively shown in SEQ ID NO.42-45, and the light chain variable region CDR1-CDR3 and the heavy chain variable region CDR1-CDR3 have the amino acid sequences described above and amino acid mutations at corresponding positions.

The amino acid sequence of the light chain variable region of the humanized anti-CD 47 antibody is shown as SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8.

Further, a series of preferred mutants are provided, wherein the light chain variable region is further preferably represented by SEQ ID NO. 34-36, and the heavy chain variable region is represented by SEQ ID NO. 37.

The antibody provided by the invention can be one of single-chain antibody, Fab, minibody, chimeric antibody or humanized whole antibody immunoglobulin IgG1, IgG2, IgG4, IgA, IgE, IgM or IgD.

The antibody provided by the invention is a whole antibody or various other forms of genetic engineering antibodies. For example, the anti-CD 47 antibody can be a whole antibody or an antibody fragment. The antibody molecules themselves are useful for prophylaxis, therapy and diagnosis. The antibody can be labeled, crosslinked or conjugated and fused with other protein or polypeptide molecules to form a complex (such as cytotoxic substances, radionuclides and/or chemical molecules and the like) for diagnosis and treatment.

The present invention further provides: independent genes for coding the antibody, expression vectors, related control technology for host cell transfection of the vectors, host cells, an antibody expression process and a method for recovering the antibody in cell culture supernatant.

The invention further provides a gene for coding the variable region of the light chain of the antibody, and the corresponding nucleotide sequence of SEQ ID NO.7 is shown in SEQ ID NO. 46. All variable coding genes of the light chain mutant antibody are completed by gene site-directed mutagenesis on the basis of SEQ ID NO.46, and are considered to be in the protection scope of the patent.

The invention further provides a gene for coding the heavy chain variable region of the antibody, and the corresponding nucleotide sequence of SEQ ID NO.8 is shown in SEQ ID NO. 47. All variable coding genes of the heavy chain mutant antibody are completed by gene site-directed mutagenesis on the basis of SEQ ID NO.47, and are considered as the protection scope of the patent.

The invention provides an expression vector containing the gene, and a host bacterium, a host cell or an expression cassette containing the expression vector.

The invention provides a method for preparing a humanized anti-human CD47 antibody, which comprises the steps of screening an anti-CD 47 specific single-chain antibody by utilizing a phage display antibody library technology to obtain an antibody light-heavy chain variable region gene, cloning the antibody light-heavy chain variable region gene into an expression vector of humanized whole antibody IgG, and carrying out whole antibody expression on the antibody by a mammal expression system to obtain the whole antibody protein of the antibody.

The invention also provides a method for performing antibody affinity maturation by using a yeast display antibody library technology, which comprises the steps of constructing a yeast display mutation library of an anti-CD 47 specific single-chain antibody, screening to finally obtain an antibody mutant light-heavy chain variable region gene, cloning the antibody light-heavy chain variable regions into human whole antibody IgG1 mammal cell transient expression vectors pABL and pABG1 respectively, and performing transient secretory expression by co-transfecting HEK293T cells to obtain a mutant of the whole antibody ZF 1.

The sequences disclosed and claimed herein include-conservative sequence modifications, i.e. nucleotide and amino acid sequence modifications that do not significantly affect and alter the binding characteristics of the antibody or an antibody containing the amino acid sequence. Such conservative sequence modifications include nucleotide or amino acid substitutions, additions or deletions. Modifications can be introduced into the antibody coding gene sequence by standard techniques in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, as exemplified by conservative sequence modifications by alanine scanning of the antibody CDR regions and amino acid mutation at some positions as described in example 4 of the present invention. Conservative amino acid substitutions include the replacement of an amino acid residue with an amino acid residue having a similar side chain or with another amino acid residue.

In the art, families of amino acid residues with similar side chains have been defined. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine tryptophan, histidine). Therefore, the substitution of an amino acid residue in an anti-human CD47 antibody with another amino acid residue from the same side chain family does not sometimes affect the activity of the present antibody.

Thus, antibodies encoded by the nucleotide sequences disclosed herein or/and antibodies comprising the amino acid sequences disclosed herein, including antibodies substantially encoded by, or comprising similar sequences modified by, conserved sequences, are considered to be within the scope of the present invention.

In addition, the gene encoding the antibody of the present invention may be, but is not limited to, SEQ ID nos. 46 and 47, taking into consideration the degeneracy of codons. For example, the gene encoding the antibody can be obtained by modifying the sequence of the gene encoding the antibody in the coding region thereof without changing the amino acid sequence. One skilled in the art can artificially synthesize the modified gene according to the codon preference of the host for expressing the antibody to improve the expression efficiency of the antibody.

The invention also provides an antibody targeted drug molecule, which comprises the humanized anti-human CD47 antibody connected with a chemical molecule, a radioactive isotope, a polypeptide molecule, a toxin or a biological macromolecule. The connection mode is that the antibody is marked, crosslinked in vitro or coupled by molecules.

The invention also provides a bispecific or multispecific molecule comprising the above-mentioned human anti-human CD47 antibody or the antigen-binding site of the antibody.

A fusion protein of the antibody and other proteins or/and polypeptides comprises a compound of the human anti-human CD47 antibody and functional protein or polypeptide molecules.

The fusion protein is obtained by connecting the antibody gene with the fusion protein gene to construct a recombinant expression vector and obtaining a recombinant fusion protein molecule through mammalian cells or other expression systems.

The invention also provides a medicament, a preparation or a detection reagent containing the humanized anti-human CD47 antibody.

The invention also provides antibody-containing compositions and pharmacologically acceptable delivery molecules or solutions. The therapeutic components are sterile and can be lyophilized at low temperature.

The invention provides an application of a human anti-human CD47 antibody in preparing a medicament for preventing or treating diseases by taking CD47 as a target. The diseases comprise various blood system tumors, solid tumors, cardiovascular diseases and the like.

In the embodiment of the invention, the applicant utilizes a self-constructed high-capacity fully-synthetic phage single-chain human antibody resource library (ZL.200910091261.8) to obtain a specific single-chain antibody aiming at CD47 by screening with a recombinant human CD47 extracellular domain protein as a target, clones light and heavy chain variable region genes of the specific single-chain antibody into full-antibody mammal cell transient expression vectors pABL and pABG1 respectively, performs transient secretory expression by co-transfecting HEK293-T cells, and performs affinity purification to obtain the full-antibody ZF 1.

Further screening through alanine scanning, site-directed mutagenesis and HCDR2 yeast mutant library to obtain a large amount of ZF1 mutant antibodies with improved affinity or unchanged affinity.

The antibody ZF1 and the mutant antibody thereof have good treatment application prospect, and mainly have the advantages that the specific binding activity with recombinant human CD47 can effectively block the binding of human CD47 and ligand SIRP alpha, and the phagocytosis of macrophages on tumor cells can be remarkably promoted. Most importantly, the ZF1 and the mutant antibody thereof can obviously prolong the survival period of a plurality of transplanted tumor model mice, and also have inhibiting effect on the growth of solid tumors such as SKOV-3 and the like. The application value of the compound is suggested to have potential application value to various tumors clinically.

The BIAcore3000 system is adopted to detect the binding capacity of ZF1 and CD47, and the result shows that the affinity K of ZF1 full antibody and recombinant CD47_D3.50nM, its mutant and recombinant CD47 affinity K_DUp to about 0.5 nM.

The human anti-human CD47 antibody provided by the invention can be specifically combined with natural CD47 on the surface of a cell. The antibody can cause the internalization of CD47 molecules on the surface of tumor cells and promote the phagocytosis of the tumor cells by macrophages; the antibody has definite anti-tumor effect in a nude mouse body; the tumor cells include but are not limited to the following tumor cells positive for CD47 expression: acute lymphoid leukemia cells, acute myeloid leukemia cells, non-Hodgkin's lymphocyte leukemia cells, human colon cancer cells, gastric cancer cells, liver cancer cells, lung cancer cells, kidney cancer cells, ovarian cancer cells, breast cancer cells, prostate cancer cells, bladder cancer cells, pancreatic cancer cells, glioma cells, sarcoma cells, and the like, and programmed cells and the like. Furthermore, all interfering functions possessed by the CD47 antagonists should equally be regarded as objects of the present invention.

The invention obtains 1 strain anti-human CD47 humanized antibody by screening through high-capacity fully-synthesized antibody library technology, and confirms that the invented antibody is specifically combined with CD47 molecules and inhibits the biological function thereof. And a series of mutants with unchanged or improved affinity are obtained.

The antibody product with CD47 inhibiting biological activity is obtained by using the obtained humanized anti-CD 47 gene engineering antibody variable region gene and the whole antibody gene under the antibody sequence characteristic in prokaryotic cell, yeast cell, eukaryotic cell and any recombinant system, or any other gene containing the antibody gene after being reconstructed on the basis of the expression and production of the antibody, or the compound is obtained by using an in vitro labeling or crosslinking method, and the specific antibody medicine for clinically treating CD47 related malignant tumor, cardiovascular diseases and other diseases is prepared.

Drawings

FIG. 1: shown is the result of enzyme-linked immunosorbent assay (ELISA) for identifying the specificity of ZF1 full antibody. The results show that ZF1 can specifically bind to recombinant human CD47 protein, but not to irrelevant proteins such as Vascular Endothelial Growth Factor (VEGF), vascular endothelial growth factor receptor 1(KDR), interferon- γ (IFN- γ), programmed death factor ligand 1(PDL1), human epidermal growth factor receptor 2(HER2), Casein (Casein), Bovine Serum Albumin (BSA), and the like. The concentration of ZF1 was 10. mu.g/ml.

FIG. 2: results of analysis of ZF1 and B6H12 binding activity to recombinant and cell surface human CD 47. Wherein in FIG. 2A: results of solid phase ELISA analysis of ZF1 binding activity to recombinant CD 47. FIG. 2B: results of cell ELISA analysis of ZF1 binding activity to cell surface CD 47. FIG. 2C: flow cytometry analysis results of ZF1 binding activity to cell surface CD 47. Wherein B6H12 is a positive antibody control.

FIG. 3: biacore3000 determined the affinity of ZF1 and B6H12 antibodies to recombinant human CD 47. FIG. 3A: ZF1 affinity assay results plot; FIG. 3B: B6H12 graph of affinity determination results. The curves from top to bottom in the figure represent concentrations of CD47 of 200, 80, 32, 12.8, 5.12, 2.05, 0.82, 0.33, 0.13nM in order.

FIG. 4: competitive inhibition of ZF1 on binding of recombinant human CD47(rhCD47-hFc) to rhsirpa. The rhCD47-hFc concentration was 100ng/ml (corresponding to 1nM CD 47). The antibody concentration was: IC50_ZF1＝130.7nM，IC50_B6H125.1 nM.. Wherein, the Ametumumab is an anti-EGFR monoclonal antibody as an irrelevant control antibody.

FIG. 5: ZF1 promotes the ability of mouse macrophages to phagocytose CCRF and U937 cells. Fig. 5A is the results of a flow cytometry analysis of macrophages engulfed CCRF cells. FIG. 5B is a microscopic count analysis of phagocytosis of U937 cells by mouse macrophages at antibody concentrations of 0.1,1,10and 100. mu.g/ml. The positive and negative controls in the figure are B6H12 and Cetuximab, respectively.

FIG. 6: anti-tumor efficacy of ZF1 on CCRF and U937 leukemia model mice. FIG. 6A: results of the CCRF experimental group. FIG. 6B: u937 experimental group results.

FIG. 7: schematic representation of the modification of the pYD1-831 yeast display vector. FIG. 7A shows the foreign protein displayed on the C-terminal of the Aga2 protein by the original pYD1 vector. FIG. 7B shows the foreign protein on the N-terminal of the Aga2 protein for the modified vector pYD 1-831. FIG. 7C is a diagram showing the structure of the elements of the foreign protein region of pYD 1-831.

FIG. 8: graph of experimental results for the competitive inhibition of rhCD47-hFc/rhSIRP α binding by mutant antibodies. 1, ZF 1; 2, Y1; y2; 4.4-1; 5.4-2; 6.4-6; 7.4-15; B6H 12. The concentration range of No.1 antibody is 0.37-300 mug/ml, the concentration range of other antibodies is 0.04-90 mug/ml, and the concentration of rhCD47 is 200 ng/ml.

FIG. 9: the mutant antibodies ZF1-Y1 and ZF1-Y2 have antitumor effect on U937 leukemia model mice. The administration mode comprises the following steps: 100 ug/2 days, 7 times.

FIG. 10: the mutant antibody binds with relative affinity to native human CD47 on the surface of U937 cells. FIG. 10A: 1, B6H 12; ZF 1; 3, ZF 1-Y2; Y2-64A; y2-49a50V57N64a65A, antibody concentration range: 0.0018-4 ug/ml. FIG. 10B: 1, B6H 12; ZF 1; 3, ZF 1-Y1; 4Y1-L51V53V 93Y; 5Y1-L51V53V93T, antibody concentration range: 0.0018-4 ug/ml. FIG. 10C: Y1-93T 94K-Y1; Y1-93T 94P-Y1; Y1-93Y 94P-Y1; ZF1-Y1, antibody concentration range: 0.0009-2 ug/ml.

FIG. 11: in vitro binding activity of antibodies to human erythrocytes. The concentration range of the antibody is 0.1ng/ml to 2.2 mu g/ml. Hu5F 9; ZF-Y1; Y1-93Y 94P; Y1-93Y 94K; Y1-51V53V93Y 94P.

FIG. 12: anti-tumor effect of the mutant antibody on U937 leukemia model mice. The administration mode comprises the following steps: 50 ug/tub/3 days, 7 times.

FIG. 13: in vitro agglutination activity of antibodies against human erythrocytes. The concentration range of the antibody is 0.6ng/ml to 600 mu g/ml.

Detailed Description

The following examples further illustrate the present invention but should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 specific antibody screening of high Capacity phage antibody libraries

The first, material and method:

1. materials: the high-capacity fully-synthetic phage single-chain antibody library is constructed by the military medical science institute of the people's liberation military (ZL200910091261.8), and the library capacity is 1.35 multiplied by10¹⁰. Recombinant human CD47 extracellular region eggThe common bletilla pseudobulb human Fc fusion protein is produced by ACRO biosystems, and strains XL1-Blue, TOP10 and the like are purchased from holo gene corporation. Eukaryotic expression vectors pABG1, pABL and pABK are stored in the cell, and the vector information is shown in ZL201210533178.3, wherein pABG1 carries a heavy chain constant region (CH1-CH3) coding gene of human IgG1, and the amino acid sequence is shown in SEQ ID No. 52; the coding gene of the human light chain C lambda is carried on pABL, and the amino acid sequence is shown in SEQ ID NO. 53; the pABK carries a coding gene of a human light chain C kappa, and the amino acid sequence is shown in SEQ ID NO. 54. Mammalian cell HEK293T cells are products of Invitrogene.

2. Method of producing a composite material

2.1 panning of anti-CD 47-specific antibodies

1ml of the recombinant protein antigen CD47-his was coated onto an immune tube at 20. mu.g/ml overnight at 4 ℃. The next day, PBS wash 2 times, 2 min/time, 2% BSA blocking at 37 ℃ for 2 h. Phage antibody library displaying single chain antibodies were blocked with blocking solution (2% BSA, 0.1% Tween 20) for 30 min at 37 ℃ and then added to the blocked immune tubes and allowed to bind overnight at 4 ℃. After PBST and PBS were washed well, 1ml of 0.2mol/L glycine-hydrochloric acid (pH2.2) was added to elute, and the mixture was shaken at room temperature for 10 minutes, and immediately after aspiration, the mixture was neutralized to pH7.4 with 1mol/L Tris. The neutralized eluate was aspirated, 9ml of E.coli XL1-Blue in logarithmic growth phase was added thereto, shake-cultured at 37 ℃ and 150rpm for 1 hour, and then plated on 2 XYTCTG plates and cultured overnight at 37 ℃. The next day colonies were scraped for phage display for the next round of screening.

2.2 phage ELISA

Picking single clone in 96-well deep-well plate, shaking culturing 250 μ l2 XYTCTG culture medium at 37 deg.C until OD600 is 0.5, adding 1 × 10⁸The cfu helper phage was allowed to stand at room temperature for 15 minutes, shake-cultured at 37 ℃ and 150rpm for 1 hour, and added with an equal volume of 2 XYTCTKI (kanamycin 50. mu.g/ml, IPTG 0.2mmol/L), and shake-cultured at 30 ℃ overnight. The next day, the supernatant was centrifuged, BSA was added to a final concentration of 2%, tween-20 was added to a final concentration of 0.1%, and the mixture was allowed to stand at 37 ℃ for 15 minutes for ELISA identification. The CD47 antigen was coated in 96-well enzyme-linked plates at 100 ng/well overnight at 4 ℃. The following day, the ELISA plates were blocked with 2% BSA + 0.1% Tween-20 at 200. mu.l/well, 37 ℃ for 2 h. Removing the blocking solution, and adding the blocked phage antibodyStanding at 37 deg.C for 1 h. The liquid was discarded and washed 3 times with PBST for 5 minutes each. HRP-labeled mouse anti-M13 antibody was pre-blocked and added to the enzyme conjugate plate, and allowed to stand at 37 ℃ for 30 minutes. Washing with PBST for 3 times, adding OPD substrate color developing solution, and standing at room temperature for color development. With 2N H₂SO4 stopped the development. And (5) measuring the light absorption value by using an enzyme-labeling instrument. 2.3 phage Single chain antibody conversion to Total antibody

Vectors pABL and pABG1 were used to clone VL and VH variable region genes, respectively, using primers L3F (SEQ ID NO.48) and LR (SEQ ID NO.49) to amplify VL variable region genes; H3F (SEQ ID NO.50) and HR (SEQ ID NO.51) amplified the VH variable region genes. VL variable region genes were cloned into vector PABL using restriction sites BsrG I and Hind III, and VH variable region genes were cloned into vector pABG1 using restriction sites Afl II and NheI. And transforming the recombinant plasmid into escherichia coli Top10, and performing bacterial liquid PCR and sequencing identification on the recombinant plasmid to obtain a correct constructed human full-antibody light and heavy chain expression vector. After greatly extracting the plasmid, the antibody light and heavy chains are mixed in a molar ratio of 1: 1 transfection FreeStyleTMHEK293-T cell, carrying out transient expression of human whole antibody, purifying expression supernatant through a ProteinA affinity chromatography column, and carrying out electrophoresis analysis, affinity and specificity determination and the like on the purified whole antibody.

Second, result in

Acquisition of ZF1 phage antibodies

Identifying the monoclonal obtained by the third round of screening to obtain a specific phage antibody ZF1, wherein the amino acid sequence of the light chain variable region is shown as SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 46; the heavy chain variable region amino acid sequence is shown as SEQ ID NO.8, and the nucleotide sequence is shown as SEQ ID NO. 47. The light and heavy chain variable region gene of the phage antibody ZF1 is respectively cloned into expression vectors pABL and pABG1 (ZL201210533178.3), a whole antibody recombinant expression vector is constructed, and the transient secretory expression of mammalian cells is realized by a HEK293T cell transient expression system. And purifying by a Protein A column to obtain a whole antibody Protein sample.

Characterization of ZF1 specificity

ZF1 specificity was analyzed at the full antibody level. Enzyme-linked plates were coated with various recombinant proteins commonly used in this laboratory as control antigens along with recombinant CD47 for routine ELISA experiments. The primary antibody is 10 mu g/ml of purified ZF1 full antibody, and the secondary antibody is an HRP-labeled anti-human IgG antibody. The results are shown in figure 1, ZF1 binds specifically only to recombinant human CD47 and not to other proteins.

Example 2ZF1 affinity and in vitro Activity assays

The first, material and method:

1.1 materials

CM5 chips and other related consumables are available from GE Healthcare Life Sciences. Cell lines SKOV-3(ATCC NO. HTB-77), U937(ATCC NO. CRL-1593.2) and CCRF (ATCC No. CCL-120) which express positive human CD47 on the cell surface are respectively given to Phlebox, Phlebox and Sunbao Philippir in the institute of biological engineering; newborn bovine serum NCS was purchased from PAA corporation. RPMI 1640 cell culture medium was purchased from Gibco. Other sources of material were the same as in example 1.

1.2 construction, expression and purification of full antibody expression vector of B6H12

B6H12 is a humanized anti-CD 47 functional antibody, the sequence information of which is found in patent US 2015/0183874. Synthesizing the coding gene of the amino acid sequence of the light chain and heavy chain variable region of the antibody, introducing restriction sites Xba I and Kas I at two ends of the light chain gene, introducing restriction sites Afl II and Nhe I at two ends of the heavy chain, and cloning the two into vectors pABK (the vector contains a human light chain constant region Ck) and pABG1 (the vector contains a human light chain constant region CH) and pABG1 respectively by using restriction enzyme ligation operation_1-3) (see patent ZL201210533178.3 for specific information on the carrier). And transforming the recombinant plasmid into escherichia coli Top10, and performing bacterial liquid PCR and sequencing identification on the recombinant plasmid to obtain a correct constructed light and heavy chain expression vector of the full antibody. After greatly extracting the plasmid, the antibody light and heavy chains are mixed in a molar ratio of 1: 1 to transfect HEK293-T cells, performing transient expression of a whole antibody, purifying an expression supernatant through a ProteinA affinity chromatography column, and performing subsequent research by taking the expression supernatant as a control antibody.

2. Method of producing a composite material

2.1 cell ELISA and flow cytometry analysis

Binding of ZF1 to cell surface CD47 was analyzed using cell ELISA and flow cytometry. Cell ELISA: SKOV-3 cells are inoculated to a 96-well culture plate at 5000 cells/wellAfter overnight culture, after ice-cold PBS washing, 5% skimmed milk powder is sealed for 1h at 4 ℃, diluted antibodies with different concentrations to be detected are taken as primary antibodies and combined for 2h at 4 ℃, after PBST washing, an HRP-labeled anti-human IgG antibody is added as a secondary antibody, and ELISA experiment under conventional low temperature is carried out. Flow cytometry analysis: pre-closed 5X 10⁵The cells were incubated with anti-CD 47 antibody in 100. mu.l system for 1 hour at 4 ℃, after PBST washing, pre-blocked FITC-labeled goat anti-human IgG secondary antibody was added and incubated for 30 minutes on ice; after 3 times of washing, precooled PBS is added for resuspension, and fluorescence analysis is carried out on a flow cytometer. The sample without the antibody to be detected was used as a control. Half maximal binding effect concentration was calculated from the assay results (EC 50). And the EC50 value and the maximum binding signal intensity are used as relative affinity evaluation basis.

2.2Biacore 3000 System determination of antibody affinity

The affinity of the antibody was determined by anti-human antibody capture. The amino group of the capture Antibody (AHC) of the Fc fragment of the anti-human antibody was coupled to the surface of the CM5 chip in advance according to the kit instructions. In the assay, purified antibody was diluted to 1ug/mL with HBS-EP buffer at 25 ℃ and a flow rate of 5. mu.L/min, and passed over the AHC precoated chip surface for an adjusted time to achieve a capture target of about 500 RU. Recombinant CD47 antigen was used as the mobile phase and diluted in HBS-EP in a two-fold concentration gradient ranging from 200nM to 3.1 nM. And (3) testing conditions are as follows: the temperature is 25 ℃, and the flow rate is 30 mu L/min; the binding time was 3 minutes and the dissociation time was 15 minutes. After completing a reaction, regenerating the chip under the following conditions: 3M MgCl2, 30. mu.L/min. times.30 seconds, after regeneration, the antibody to be tested of the same target value is captured continuously, and the next reaction is carried out. At the end of the experiment, blank control and irrelevant antibody control response values were subtracted and 1: 1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated.

2.3 competitive inhibition assay

The ability of the antibodies to inhibit the binding of recombinant human Fc fused human CD47(rhCD47-hFc, ACRO biosystems, CD7-H5256) to recombinant human SIRP alpha (rhSIRP alpha, ACRO biosystems, SIA-H5225) was evaluated by a competitive inhibition ELISA. The working concentration of 100ng/ml hFc-CD47 was mixed with antibody in a gradient concentration, incubated at 37 ℃ for 1 hour, and then added to the pre-coated rhSIRP α enzyme plates for 1 hour at 37 ℃. The subsequent steps are conventional ELISA reaction steps. From the measurement results, the half maximal inhibitory concentration (IC50) was calculated.

2.4 cell phagocytosis assay

Kunming mice were injected intraperitoneally with 1.5ml of starch broth, macrophages were harvested after 3 days, stained with FarRed and plated in culture plates for overnight culture. Macrophages were starved for 2 hours in serum-free medium prior to the experiment. Leukemia cells (CCRF or U937) were harvested by centrifugation, stained for CFSE, added with antibody and incubated for 30 min at 37 ℃. Then adding the mixture into a culture plate inoculated with macrophages in advance, and incubating for 2-4 hours at 37 ℃. Finally, the suspended leukemia cells are washed away, observed under a microscope and the experimental results are recorded, or the cells are digested for flow cytometry analysis.

Second, result in

1. Binding Activity of antibody ZF1 with recombinant and cell surface human CD47

The ELISA results showed (fig. 2A) that ZF1 bound to recombinant human CD47 with a clear dose dependence with approximately 2.5-fold difference between EC50 and the control antibody B6H12 (EC50)_ZF1＝45ng/ml,EC50 _B6H1218 ng/ml); both the cell ELISA (fig. 2B) and flow cytometric results showed (fig. 2C), ZF1 specifically bound to CD47 on the surface of SKOV-3 cells and showed a good dose relationship, with EC50 comparable to the control antibody B6H12 (flow EC50)_ZF1＝166ng/ml,EC50_B6H12112 ng/ml; cell ELISA, EC50_ZF1＝311ng/ml,EC50_B6H12339ng/ml), but the maximum binding signal value of ZF1 was significantly lower than B6H 12.

Biacore3000 determination of affinity of ZF1 to recombinant human CD47

Antibodies were coated on the surface of a CM5 chip, recombinant CD47 at different concentrations was used as a mobile phase, the reaction curves of ZF1 (FIG. 3A) and a control antibody B6H12 (FIG. 3B) with recombinant human CD47 were determined by a Biacore3000 system as shown in FIG. 3, and the affinity was calculated by fitting the curves, resulting in ZF1 with an affinity (KD) of 3.50nM and B6H12 with an affinity (KD) of 5.27 nM. The detailed kinetic parameters are shown in table 1 below.

TABLE 1 Biacore determination of ZF1 affinity constant for B6H12

Dose-dependent inhibition of binding of recombinant human CD47 to human SIRP alpha by ZF1

Competition inhibition ELISA results showed that ZF1 could dose-dependently inhibit the binding of recombinant rhCD47-hFc to rhsirpa (fig. 4), while the control antibody, Ametumumab (anti-EGFR monoclonal antibody, ZL201210465918.4), had no inhibitory effect on the binding of both. IC50 when the concentration of rhCD47-hFc was 100ng/ml (corresponding to 1nM CD 47)_ZF1This blocking activity was much lower than the positive control antibody B6H12 (IC50) at 130.7nM_B6H125.1 nM). ZF1 promoting phagocytosis of tumor cells by macrophages

Macrophage phagocytosis experiment results show that the ZF1 can effectively promote phagocytosis of mouse macrophages on human CD47 positive CCRF (figure 5A) and U937, and has a good dose-effect relationship (figure 5B). The irrelevant control antibody Cetuximab (anti-EGFR monoclonal antibody drug, Germany Merck) does not promote phagocytosis of tumor cells by macrophages.

Example 3 evaluation of ZF1 antitumor Effect in vivo

Female Babl/c nude mice, 5-6 weeks old, 7-9 mice per group. 3mg of cyclophosphamide is injected into abdominal cavity, and the next day, the tail vein of the mouse is injected and inoculated with 1 × 10⁷And 100. mu.g/mouse of the anti-CD 47 antibody ZF1 or B6H12 was intraperitoneally administered to each mouse on day 2, 1 time per day, for 2 weeks. The control group was given the same dose of human IgG. Mice were recorded for time to morbidity and mortality. Mice are moribund and will be given euthanasia treatment.

The experimental results show that: ZF1 had significant therapeutic effects on nude mice CCRF and U937 leukemia models (fig. 6A and 6B). In the CCRF experiment, mice in the control group developed disease and died sequentially at weeks 3 and 4 after the start of administration, and the antibody-treated group survived healthily to week 6. The group 1 mice developed morbidity and subsequently died at week 7, and mice developed morbidity at week 10, week 2. Subsequently, other mice gradually became sick and died until week 17 (fig. 6A). The results of the U937 experiment were similar to CCRF (fig. 6B), with the control group morbiding and dying at weeks 3 and 4 after inoculation, and the administration group morbiding and dying at weeks 6 to 13. Compared with B6H12, the final treatment effect of the two medicines is not significantly different.

Example 4 ZF1 light and heavy chain CDR alanine scanning and directed mutagenesis

First, experiment method

1. Mutant construction

The construction of the mutant expression vector is carried out by adopting a plasmid site-directed mutagenesis method, and can be specifically referred to documents [ Ronghao, Chenruichuan and Liu runzhong. An optimization method of rapid point mutation. Xuesheng university bulletin (Nature science edition), 2008, Vol 47, sup 2, 282-. Mutant antibody expression and purification methods were as above.

2. Comparison of relative affinities of mutants

The affinity comparison of the mutants to the parent antibody was done using the biacore3000 system. The specific method is the same as above, except that only a single line was measured for all mutant affinities to assess relative affinities. The same target value of the test antibody was first coated and a relative affinity assay was performed with 100nM recombinant human CD47(ACRO biosystems, CD7-H5227) as the mobile phase.

Second, experimental results

Alanine scanning of 6 CDR regions obtains a large number of mutants, and the result of measuring the affinity of the mutant antibody shows that the affinity of the antibody is greatly changed after mutation of a plurality of sites, and the information of the mutation sites and the change of the affinity are shown in the following tables 2 and 3.

As can be seen from the data in the table, after the amino acid mutation of individual position, the affinity is greatly influenced, and the affinity of some sites is reduced by more than 10 times. Meanwhile, The affinity of The antibody is improved after mutation of some sites, for example, after mutation of The 51 th site of a light chain into Ser to Ala or Val, mutation of The 53 th Arg to Ala or Val, mutation of The 93 th Asn to Tyr, The or Ser, and mutation of The 94 th Asn to Lys, Pro or The, The affinity of The antibody can be improved to a certain extent, and The mutation sites can be further subjected to superposition mutation, so that The affinity is kept unchanged or further improved. HCDR3 is crucial to binding, most of the affinity after mutation is reduced; the HCDR2 region may also have several amino acid site mutations to raise the affinity of the antibody.

TABLE 2 statistical table of affinity changes of ZF1 light chain mutants

TABLE 3 statistical table of affinity changes of ZF1 heavy chain CDR region mutants

Note: compared with the parent antibody, the mutant antibody is characterized in that the affinity is improved by more than 2 times by means of ↓; +/-indicates no more than a 2-fold change in affinity; ↓ represents the decrease of affinity between 2 and 5 times; ↓ represents the decrease of affinity by more than 5 times; ↓ ↓ ↓ represents the decrease of affinity by more than 10 times.

Example 5 ZF1 heavy chain CDR2 region Yeast mutant library screening for high affinity mutant antibodies

First, experiment method

1. Mutation library construction

Based on ZF1, a yeast mutant library of ZF1 heavy chain CDR2 region is constructed by adopting a yeast display single chain antibody (scFv) method, and then a method combining magnetic bead affinity screening and flow cytometry screening is carried out to screen high-affinity mutants. Specifically, first, a heavy chain CDR2 region single chain antibody library gene fragment is obtained: the 50-58 amino acids in CDR2 region are selected as mutation parts, random primers, splicing primers and yeast library recombination primers are designed, and the complete heavy chain variable region gene with homologous sequences at the upstream and downstream is obtained by splicing extension PCR. Secondly, obtaining a heavy chain CDR2 region single-chain antibody library display carrier: the ZF1 single-chain antibody gene is cloned into a yeast display vector pYD1-831 (which is transformed by an Invitrogen commercial vector pYD1, and the map is shown in figure 7), then the heavy chain of ZF1 is cut by utilizing the enzyme cutting site SacI in a single-chain antibody linker and the enzyme cutting site EcoRI at the downstream of the heavy chain variable region, and the large fragment of the vector is recovered. Finally, the heavy chain variable region gene of the electrotransformation saccharomyces cerevisiae EBY100 (submitted by doctor Liuming of institute of Chinese academy of sciences, ATCC: MYA-4941) competent cell prepared by the common electric shock transformation of the single chain antibody library gene and the heavy chain library display vector is subjected to homologous recombination in the yeast cell to form the mutant library. The method is a conventional electric shock transformation method (Current Protocols in Molecular Biology (2002)), and a constructed yeast mutant library (Boder and Wittrup,1997) is frozen and stored in SD-CAA medium containing 15% of glycerol at-70 ℃.

2. Mutation library screening

The mutation library screening is carried out by adopting a screening mode combining magnetic bead affinity screening and flow cytometry sorting. Inoculating the frozen yeast mutant library to a culture medium SD-CAA for overnight culture, and performing induced display for 12-16h by the culture medium SG-CAA. The displayed mutant library was induced for magnetic bead affinity screening and Flow cytometry sorting (Flow cytometry protocols, Terea S.Hawlery and Robert G.Hawlery), and was incubated with biotin (Thermo, 21343) -labeled recombinant human CD47 for 30 minutes at room temperature, washed 3 times with PBS containing 0.5% BSA and 2mM EDTA, and acted on by streptavidin-coated immunomagnetic beads (Order No.130-^TMseparator, Miltenyi Biotec) was positively sorted and yeast cells were screened for binding to bio-rhCD 47. Two rounds of magnetic bead affinity screening were performed to obtain yeast cells, which were subjected to flow cytometry screening and similarly induced to obtain yeast cells for bead affinity screening, wherein the displayed yeast cells were previously added with an antibody against a V5-tagged protein displayed on the surface of the yeast cells in a fusion manner (MB2020, bioprold Technology, Inc), incubated at room temperature for 30 minutes, added with biotin-labeled recombinant human CD47, incubated at room temperature for 1 hour, washed 3 times with PBS containing 0.5% BSA and 2mM EDTA, further added with a PE-labeled goat anti-mouse IgG antibody (sc-3738, Santa Cruz biotechnology, Inc) and APC-labeled streptavidin (SA, 554067, BD) for 30 minutes, and after washing sufficiently, the yeast cells with a dual fluorescence signal were sorted by flow cytometry.

3. Identification of monoclonal

(1) The single clones were picked from the culture plate and inoculated into 3ml SD-CAA medium, respectively, and cultured at 30 ℃ until saturation, and the cells were collected and transferred to SG-CAA induction medium at an initial concentration of 0.5OD600/ml and cultured at 30 ℃ for 12 hours. (2) The cells were collected by centrifugation and analyzed by flow cytometry, the specific method being the same as the method for sorting the mutant library by flow cytometry in method 2.

(3) The positive clone obtained by flow analysis is transferred to a fresh SD-CAA culture medium for overnight culture, Lyticase muramidase (L4025, Sigma) is added to digest the cell wall, the digested product is used as a template, 1 pair of primers (LMCX-F: 5-TATACTTTAACGTCAAGGAGAAAAA-3; LMCX-R: 5-TCTAAAGTTGGTGAGGGGATTTGCT-3) on a display carrier are used for carrying out PCR reaction, the ScFv gene displayed by the monoclonal yeast is captured, and the PCR product is sequenced.

(4) After obtaining the gene sequence of the positive clone ScFv, the positive clone ScFv is cloned into full antibody expression vectors pABL and pABH, and is transiently expressed by H293T cells, and Protein A is purified.

Second, result in

1. High affinity mutant antibody screening

Through 2 rounds of immunomagnetic bead screening and 2 rounds of flow cytometry screening, multiple clones with improved binding activity are finally obtained through flow cytometry identification, and the clones are subjected to whole antibody modification and eukaryotic expression to finally obtain 18 mutants with mutation in HCDR2 region. The names, HCDR2 region sequences and corresponding sequence numbers are shown in Table 4.

TABLE 4 screening of the yeast display mutant library to obtain the sequence of mutant antibody HCDR2

2. Mutant affinity assay

The primary affinity assays were performed on the screened monoclonal antibodies using the Biacore3000 system using method 2 of example 4, and showed varying degrees of improvement in the affinity of the mutant antibodies (Table 5), where Y4-2 and Y4-6 reached 0.5nM after precision measurements.

TABLE 5 affinity assay results for mutant antibodies obtained by screening of yeast display mutant libraries

4. Comparison of mutants with ZF1 blocking Activity

The competitive ELISA assay of example 2 was used to compare the differences in the ability of the mutant antibodies with the parent antibody ZF1 to block binding to rhCD 47-hFc/rhsirpa. The experimental results show (fig. 8) that the activity of the mutant antibody competitively inhibiting the binding of rhCD 47-hFc/rhsirpa is improved to a different extent compared to the parent antibody ZF 1. 5. Mutant antibody with improved in vivo anti-tumor activity

In vivo antitumor activities of the mutant antibodies ZF1-Y1 and ZF1-Y2 having partially improved blocking activity were compared, and the experimental procedure was the same as in example 3 except that the administration frequency was adjusted to 2 days/time for 7 times. The dose administered was 100. mu.g/mouse.

The results show that: under the experimental condition, the antitumor effect of the mutant antibody with improved affinity and blocking activity is improved compared with that of the parent antibody ZF1, is slightly lower than that of a control antibody B6H12 (figure 9), the median survival time is taken as an index, a model control group is 23 days, a ZF1 group is 45 days, ZF1-Y1 and ZF1-Y2 are both 55 days, and the same dosage of B6H12 group is 80 days. It is suggested that the improvement of affinity and blocking activity can further improve the antitumor effect of the antibody.

Example 6 overlay mutant

The amino acids observed in the research and having the effect of improving the affinity are subjected to superposition mutation to obtain a series of mutant antibodies with the affinity kept unchanged or further improved, and the experimental method for constructing the mutants is the same as that in example 4. Wherein, the mutation of the light chain mainly occurs in LCDR2 and LCDR3, the mutation of the heavy chain occurs in HCDR2, and the amino acid sequences of the CDR regions of the light chain and the heavy chain corresponding to the mutants are shown in Table 6. The relative affinity was determined by cell ELISA, as described in example 2. The binding activity of each antibody to SKOV-3 cell surface CD47 under the conditions of this experiment is statistically shown in table 6, and the relative binding activity curves for the partial mutants are shown in fig. 10.

The results show that the affinity of each stacked mutant is obviously higher than that of the original antibody ZF1(EC50 ═ 311ng/ml), and the affinity of the mutant on the basis of ZF1-Y2 is equal to or slightly higher than that of ZF1-Y2, and the affinity of the mutant on the basis of ZF1-Y1 is further improved or kept unchanged.

TABLE 6 mutant antibody sequences and changes in relative affinity Activity

Example 7 blocking Activity of mutant antibodies against cell surface native CD47/rhSIRP alpha binding

First, experiment method

Obtaining high affinity mutants of SIRP alpha

A high-affinity mutant FD6 amino acid sequence reference document report sequence (Weiskopf K, et al science,2013,341(6141),88-91.) of SIRP alpha is designed and synthesized, a coding gene of the high-affinity mutant of the human SIRP alpha is cloned into the upstream of a coding gene of a human Fc segment of a eukaryotic expression vector pABH, an SIRP alpha-hFc fusion Protein expression vector is constructed, transient expression is carried out through HEK293T cells, Protein A is used for purification, a pure product of the recombinant SIRP alpha-hFc fusion Protein is obtained, and FITC (Sigma, F4274) labeling is carried out on the pure product.

2. Flow cytometry-based binding blockade assay

The antibodies diluted in the gradient are respectively mixed with equal amount of U937 cells, 0.1 mu g/ml of FITC-labeled rhSIRP alpha-hFc is added after 30 minutes of incubation at 4 ℃, the reaction is continued for 1 hour, after PBS washing and full washing, the blocking activity of the antibodies on the cell surface CD47 and the recombinant SIRP alpha is observed by adopting flow cytometry.

Second, experimental results

The results show that the mutant antibody with improved affinity has correspondingly improved capability of blocking the combination of the recombinant SIRPa-hFc and the natural CD47 on the cell surface. The statistical results of the blocking activity IC50 for each antibody under the present experimental conditions are shown in table 7 below. The results show that the blocking effect of the mutant antibody on SIRP alpha-hFc and CD47 on the cell surface is obviously improved compared with that of the parent antibody ZF1, and the antitumor activity of the mutant antibody is further improved.

TABLE 7 inhibitory Activity of Y1 and Y2 series mutant antibodies against native CD47 and rhSIRP α -hFc

Example 8 anti-tumor Activity of mutant antibodies

The results of example 5 suggest that the increase in blocking activity is accompanied by a further increase in antitumor activity in vivo. Therefore, the mutant antibody Y1-93Y94P with further improved blocking activity was further tested for in vivo antitumor activity by the same experimental method as in example 3, wherein the administration frequency and dose were further reduced, the administration time was adjusted to 3 days/dose, and the administration dose was adjusted to 50. mu.g/dose for 7 times.

The results show that: under the experimental conditions, the antitumor effect of the mutant antibody Y1-93Y94P with improved blocking activity is further improved, and the median survival time can still reach 55 days under the condition of reducing the dosage and the administration frequency (figure 12). It is suggested that the improvement of affinity and blocking activity is positively correlated with the improvement of antitumor effect. The invention also carries out similar observation on the effect of the anti-tumor drugs on other mutants and obtains the same conclusion.

In addition, the pharmacodynamic research result of the mutant antibody on the SKOV-3 mouse transplantation tumor model indicates that anti-CD 47 antibodies such as Y1-93Y94P and the like have obvious inhibition effect on the tumor growth of the transplantation tumor model mouse.

Example 9 binding of antibody to human Red blood cells and coagulation assay

CD47 is widely expressed in human body, especially in human erythrocyte. Thus, anti-CD 47 antibodies are used in humans at risk of causing erythrocyte agglutination. In vitro erythrocyte concentration experiments prove that the antibody does not have the problem of causing erythrocyte agglutination.

Materials and methods

1. Materials: human red blood cells from doctor hong Kong Dai in the laboratory. The preparation method of the kit (Tianjin tertiary amino-layer, LZS11131) for separating the human neutrophil can be seen in the product instruction. Other sources of experimental materials were as above.

2. Human erythrocyte binding assay: the assay was performed using flow cytometry. The method comprises the following specific steps:

1) the red blood cells are treated, collected by a stirring fiber method and diluted to 2 x 10^7/ml by using 1640 culture medium.

2) The antibody was diluted from 2.2. mu.g/ml with a 3-fold gradient of 1640 medium.

3) Binding, 100. mu.l cells and 100. mu.l antibody per well were mixed and incubated at 4 ℃ for 1 hour, during which time resuspension was performed every 20 minutes with shaking to prevent red blood cells from settling.

4) Washing, centrifugation at 1200g for 5 minutes at 4 ℃ to remove supernatant, resuspension of cells in 300. mu.l of pre-cooled 1640 medium per well, and centrifugation at 1200g for 5 minutes at 4 ℃ to remove supernatant.

5) And (3) binding the secondary antibody, diluting the FITC-labeled goat anti-human IgG antibody with 1640 culture medium by 2500 times, precooling, resuspending the cells by 200 mu l per well, binding at 4 ℃ for 40 minutes, and resuspending by shaking at 20 minutes to prevent the erythrocyte from settling.

6) Flow analysis, 4 degrees C1200 g centrifugation for 5 minutes to remove the supernatant, each hole 300 u l precooled 1640 medium heavy suspension cells, 4 degrees C1200 g centrifugation for 5 minutes to remove the supernatant. Cells were resuspended in 200. mu.l of pre-cooled 1640 medium. Post up flow cytometry analysis.

3. Human erythrocyte agglutination assay:

uniformly diluting the antibody to a concentration of 600 mu G/mL by taking a round bottom 96-well plate (Costar 3799, Corning, NY 14831) and a PBS buffer solution (Neuronbc, 500mL, LOT:9116015G, EXP:2017/07/15, Shijiazhuang), diluting by a gradient of 10 times, fully blowing and adding the antibody into each well; taking 400 mu L of red blood cells for later use after centrifugation, adding 10mL of PBS and mixing uniformly to prepare 4% red blood cell suspension; adding equal volume of 4% erythrocyte suspension into each well with a row gun until the final concentration of erythrocyte is 1%, placing in an incubator at 37 deg.C for 4hr, photographing, and determining the result.

Second, experimental results

The results of erythrocyte binding experiments show that the antibody of the present invention has a dose-dependent binding activity to human erythrocytes (fig. 11), but the results of agglutination experiments show that the antibody of the present invention does not cause agglutination of erythrocytes even at extremely high antibody concentrations (fig. 13).

Sequence listing

<110> Shanghai Sailon Biotechnology GmbH, institute of bioengineering, military medical science institute of the liberation force of Chinese people

<120> human anti-human CD47 antibody and coding gene and application thereof

<130> KHP161116759.4

<160> 54

<170> PatentIn version 3.5

<210> 1

<211> 11

<212> PRT

<213> LCDR1 of CD47 antibody

<400> 1

Ser Gly Asp Ala Leu Gly Asp Lys Tyr Ala Ser

1 5 10

<210> 2

<211> 7

<212> PRT

<213> LCDR2 of CD47 antibody

<400> 2

Glu Asp Ser Lys Arg Pro Ser

1 5

<210> 3

<211> 10

<212> PRT

<213> LCDR3 of CD47 antibody

<400> 3

Gln Leu Arg Ala Gly Asn Ala Ala Gly Trp

1 5 10

<210> 4

<211> 5

<212> PRT

<213> HCDR1 of CD47 antibody

<400> 4

Ser Tyr Ala Met Ser

1 5

<210> 5

<211> 17

<212> PRT

<213> HCDR2 of CD47 antibody

<400> 5

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 6

<211> 8

<212> PRT

<213> HCDR3 of CD47 antibody

<400> 6

Thr Trp Trp Arg Leu Phe Asp Tyr

1 5

<210> 7

<211> 109

<212> PRT

<213> ZF1 light chain variable region

<400> 7

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Gly Asp Lys Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Glu Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Leu Arg Ala Gly Asn Ala Ala Gly

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 8

<211> 117

<212> PRT

<213> ZF1 heavy chain variable region

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Trp Trp Arg Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 9

<211> 17

<212> PRT

<213> ZF1-Y1 HCDR2

<400> 9

Ala Ile Asn Tyr Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 10

<211> 17

<212> PRT

<213> ZF1-Y2 HCDR2

<400> 10

Ala Ile Ser Tyr Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 11

<211> 17

<212> PRT

<213> ZF1-Y3 HCDR2

<400> 11

Ala Ile Ser Tyr Asp Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 12

<211> 17

<212> PRT

<213> ZF1-Y4 HCDR2

<400> 12

Ala Ile Ser Tyr Asp Gly Ser Gly Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 13

<211> 17

<212> PRT

<213> ZF1-Y9 HCDR2

<400> 13

Ala Ile Asn Tyr Glu Gly Asp Lys Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 14

<211> 17

<212> PRT

<213> ZF1-Y15 HCDR2

<400> 14

Ala Ile Ser Tyr Asp Gly Lys Lys Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 15

<211> 17

<212> PRT

<213> ZF1-Y18 HCDR2

<400> 15

Ala Ile Ser Tyr Asp Gly Asn Gly Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 16

<211> 17

<212> PRT

<213> ZF1-Y21 HCDR2

<400> 16

Ala Ile Ser Tyr Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 17

<211> 17

<212> PRT

<213> ZF1-Y24 HCDR2

<400> 17

Val Ile Ser Tyr Asp Gly Arg Glu Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 18

<211> 17

<212> PRT

<213> ZF1-Y4-1 HCDR2

<400> 18

Ala Ile Ser Tyr Asp Gly Glu Lys Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 19

<211> 17

<212> PRT

<213> ZF1-Y4-2 HCDR2

<400> 19

Ala Ile Ser Tyr Asp Gly Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 20

<211> 17

<212> PRT

<213> ZF1-Y4-6 HCDR2

<400> 20

Ala Ile Ser Tyr Asp Gly Gly Lys Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 21

<211> 17

<212> PRT

<213> ZF1-Y4-15 HCDR2

<400> 21

Val Ile Ser Tyr Asp Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 22

<211> 17

<212> PRT

<213> ZF1-Y4-16 HCDR2

<400> 22

Ala Ile Thr Tyr Asp Gly Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 23

<211> 17

<212> PRT

<213> ZF1-Y4-28 HCDR2

<400> 23

Ala Ile Asn Tyr Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 24

<211> 17

<212> PRT

<213> ZF1-Y4-31 HCDR2

<400> 24

Ala Ile Asn Tyr Ala Gly Asp Lys Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 25

<211> 17

<212> PRT

<213> ZF1-A2 HCDR2

<400> 25

Val Ile Asn Tyr Asp Gly Lys Ala Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 26

<211> 17

<212> PRT

<213> ZF1-A1 HCDR2

<400> 26

Ala Ile Ser Tyr Asp Gly Lys Arg Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 27

<211> 18

<212> PRT

<213> HCDR2

<400> 27

Ala Val Ile Ser Tyr Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly

<210> 28

<211> 18

<212> PRT

<213> HCDR2

<400> 28

Ala Val Ile Ser Tyr Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

Ala Gly

<210> 29

<211> 18

<212> PRT

<213> HCDR2

<400> 29

Ala Val Ile Ser Tyr Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Ala

1 5 10 15

Lys Gly

<210> 30

<211> 18

<212> PRT

<213> HCDR2

<400> 30

Ala Val Ile Ser Tyr Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly

<210> 31

<211> 18

<212> PRT

<213> HCDR2

<400> 31

Ala Val Ile Ser Tyr Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Ala

1 5 10 15

Ala Gly

<210> 32

<211> 18

<212> PRT

<213> HCDR2

<400> 32

Ala Val Ile Ser Tyr Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

Ala Gly

<210> 33

<211> 18

<212> PRT

<213> HCDR2

<400> 33

Ala Val Ile Ser Tyr Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Ala

1 5 10 15

Ala Gly

<210> 34

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> misc_feature

<222> (93)..(93)

<223> Xaa can be Tyr or The

<221> misc_feature

<222> (94)..(94)

<223> Xaa can be Pro or The or Lys

<400> 34

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Gly Asp Lys Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Glu Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Leu Arg Ala Gly Xaa Xaa Ala Gly

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 35

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> misc_feature

<222> (93)..(93)

<223> Xaa can be Tyr or The

<400> 35

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Gly Asp Lys Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Glu Asp Val Lys Val Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Leu Arg Ala Gly Xaa Ala Ala Gly

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 36

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> misc_feature

<222> (94)..(94)

<223> Xaa can be Pro or The or Lys

<400> 36

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Gly Asp Lys Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Glu Asp Val Lys Val Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Leu Arg Ala Gly Asn Xaa Ala Gly

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 37

<211> 117

<212> PRT

<213> Artificial sequence

<400> 37

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Asn Tyr Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Trp Trp Arg Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 38

<211> 22

<212> PRT

<213> LFR1

<400> 38

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys

20

<210> 39

<211> 15

<212> PRT

<213> LFR2

<400> 39

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

1 5 10 15

<210> 40

<211> 32

<212> PRT

<213> LFR3

<400> 40

Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr

1 5 10 15

Leu Thr Ile Ser Gly Thr Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys

20 25 30

<210> 41

<211> 12

<212> PRT

<213> LFR4

<400> 41

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

1 5 10

<210> 42

<211> 30

<212> PRT

<213> HFR1

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210> 43

<211> 14

<212> PRT

<213> HFR2

<400> 43

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

<210> 44

<211> 32

<212> PRT

<213> HFR3

<400> 44

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 45

<211> 11

<212> PRT

<213> HFR4

<400> 45

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 46

<211> 327

<212> DNA

<213> ZF1 light chain variable region

<400> 46

agctacgaac tgacccagcc gccgagcgtg tcggtggcgc cgggtcagac cgcgcgtatc 60

acctgctcgg gcgatgcgct gggcgataaa tacgcgagct ggtatcagca gaaaccgggt 120

caggcaccgg tgctggtgat ttacgaagat tctaaacgcc cgtctggcat cccggaacgc 180

tttagcggct cgaattcggg caacaccgcg accctgacca ttagcggcac ccaggcggag 240

gatgaggcgg actattactg ccagttgcgg gctggcaacg cggctggttg ggtgtttggc 300

ggtggcacca agcttaccgt cctaggt 327

<210> 47

<211> 351

<212> DNA

<213> ZF1 heavy chain variable region

<400> 47

gaagtgcaat tggtggaaag cggtggcggt ctggtgcagc cgggtggcag cctgcgtctg 60

agctgcgcag cgagcggctt cacctttagc agctacgcga tgagctgggt gcgccaggca 120

ccgggtaaag gtctggaatg ggtgagcgcg attagcggta gcggcggcag cacctactat 180

gcggatagcg tgaaaggccg ttttaccatc tcgcgtgata actcgaaaaa caccctgtac 240

ctgcagatga acagcctgcg tgcggaagat accgcggtgt attattgcgc acgtacgtgg 300

tggcggttgt tcgattactg gggtcagggc actctggtga ccgtgtcgag c 351

<210> 48

<211> 47

<212> DNA

<213> Artificial sequence

<400> 48

gaaggatcct taatggtgat gatggtgatg gctccattcg ttggaca 47

<210> 49

<211> 45

<212> DNA

<213> Artificial sequence

<400> 49

gcgcccctta agggcgtgca gtgcgaagtt caactggttc aaagc 45

<210> 50

<211> 45

<212> DNA

<213> Artificial sequence

<400> 50

gcgcccctta agggcgtgca gtgcgaagtg caattggtgg aaagc 45

<210> 51

<211> 44

<212> DNA

<213> Artificial sequence

<400> 51

cgcgtgtaca ggaagctggg ccgatatcgt tctgactcaa cctc 44

<210> 52

<211> 330

<212> PRT

<213> heavy chain constant region of human IgG1

<400> 52

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 53

<211> 105

<212> PRT

<213> human light chain C lambda

<400> 53

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 54

<211> 105

<212> PRT

<213> human light chain C.kappa.

<400> 54

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

1 5 10 15

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

20 25 30

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

35 40 45

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

50 55 60

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

65 70 75 80

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

85 90 95

Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

Claims (5)

1. A human anti-human CD47 antibody, characterized in that,

the light chain variable regions LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the heavy chain variable regions HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

2. The human anti-human CD47 antibody of claim 1, wherein the amino acid sequences of FR1-FR4 in the light chain variable region are shown in SEQ ID Nos. 38-41, respectively, and the amino acid sequences of FR1-FR4 in the heavy chain variable region are shown in SEQ ID Nos. 42-45, respectively.

3. The human anti-human CD47 antibody according to any one of claims 1 to 2, which is a single chain antibody.

4. A gene encoding the human anti-human CD47 antibody according to any one of claims 1 to 3.

5. A biological material comprising the gene of claim 4, wherein the biological material is an expression vector, a host bacterium or an expression cassette.

Images