anti-CD47 monoclonal antibody and application thereof

文档序号：931902 发布日期：2021-03-05 浏览：2次中文

阅读说明：本技术 抗cd47的单克隆抗体及其用途 (anti-CD47 monoclonal antibody and application thereof ) 是由张鹏李百勇夏瑜王忠民于 2020-09-03 设计创作，主要内容包括：本发明涉及抗CD47的单克隆抗体及其用途,其由保藏号为CCTCC NO：C2018135的杂交瘤细胞株分泌。(The invention relates to a monoclonal antibody of anti-CD47 and application thereof, wherein the monoclonal antibody is prepared from the following components in percentage by weight with a preservation number of CCTCC NO: c2018135.)

1. An antibody or antigen-binding fragment thereof that specifically binds CD47, wherein:

the antibody comprises CDR sequences contained in a heavy chain variable region and a light chain variable region selected from

(1) SEQ ID NO: 2 comprises HCDR1, HCDR2 and HCDR3, and

SEQ ID NO: 4 comprises LCDR1, LCDR2 and LCDR 3; or

(2) SEQ ID NO: 12 comprises HCDR1, HCDR2 and HCDR3, and

SEQ ID NO: 14 comprises LCDRl, LCDR2 and LCDR 3; or

(3) SEQ ID NO: 16 comprises HCDR1, HCDR2 and HCDR3, and

SEQ ID NO: 18 comprises LCDR1, LCDR2 and LCDR 3; or

(4) SEQ ID NO: 20 comprises HCDR1, HCDR2 and HCDR3, and

SEQ ID NO: 22 comprises LCDR1, LCDR2 and LCDR 3;

preferably, the antibody comprises:

HCDR1 comprising SEQ ID NO: 5, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting thereof,

HCDR2 comprising SEQ ID NO: 6, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consists thereof, and

HCDR3 comprising SEQ ID NO: 7, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting thereof, and said antibody further comprises:

LCDR1 comprising SEQ ID NO: 8, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting thereof,

LCDR2 comprising SEQ ID NO: 9, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consists thereof, and

LCDR3 comprising SEQ ID NO: 10, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting thereof.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody further comprises a framework region FR of a heavy chain variable region and a framework region FR of a light chain variable region selected from the group consisting of:

(1) the framework regions FR of the heavy chain variable region comprise FR-H1, FR-H2, FR-H3 and FR-H4, wherein FR-H1 comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence identical to SEQ ID NO: 23, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 23, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no; FR-H2 comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence corresponding to SEQ ID NO: 24, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a sequence set forth in SEQ ID NO: 24 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H3 comprises the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence substantially identical to SEQ ID NO: 25, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a sequence as set forth in SEQ ID NO: 25, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no; FR-H4 comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence identical to SEQ ID NO: 26, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a sequence set forth in SEQ ID NO: 26, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in (b),

the framework regions FR of the light chain variable region comprise FR-L1, FR-L2, FR-L3 and FR-L4, wherein FR-L1 comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence identical to SEQ ID NO: 27, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 27, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (a); FR-L2 comprises the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence identical to SEQ ID NO: 28, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 28, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L3 comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence substantially identical to SEQ ID NO: 29, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 29 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L4 comprises the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence corresponding to SEQ ID NO: 30, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 30, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b);

(2) the framework regions FR of the heavy chain variable region comprise FR-H1, FR-H2, FR-H3 and FR-H4, wherein FR-H1 comprises the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence substantially identical to SEQ ID NO: 31, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence as set forth in SEQ ID NO: 31, or consists of, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H2 comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence substantially identical to SEQ ID NO: 32, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 32, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H3 comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence identical to SEQ ID NO: 33, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 33, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H4 comprises the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence corresponding to SEQ ID NO: 34, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 34, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions),

the framework regions FR of the light chain variable region comprise FR-L1, FR-L2, FR-L3 and FR-L4, wherein FR-L1 comprises the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence corresponding to seq id NO: 35, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 35, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L2 comprises the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence corresponding to SEQ ID NO: 36, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence as set forth in SEQ ID NO: 36, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (a); FR-L3 comprises the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence identical to SEQ ID NO: 37, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 37, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b); FR-L4 comprises the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence substantially identical to SEQ ID NO: 38, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 38, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

(3) the framework regions FR of the heavy chain variable region comprise FR-H1, FR-H2, FR-H3 and FR-H4, wherein FR-H1 comprises the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence substantially identical to SEQ ID NO: 39, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 39, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (a); FR-H2 comprises the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence substantially identical to SEQ ID NO: 40, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 40, or consists of, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H3 comprises the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence substantially identical to SEQ ID NO: 41, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 41, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H4 comprises the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence corresponding to SEQ ID NO: 42, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 42, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in (b),

the framework regions FR of the light chain variable region comprise FR-L1, FR-L2, FR-L3 and FR-L4, wherein FR-L1 comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence substantially identical to SEQ ID NO: 43, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 43, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b); FR-L2 comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence identical to SEQ ID NO: 44, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 44 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L3 comprises the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence substantially identical to SEQ ID NO: 45, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 45, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L4 comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence substantially identical to SEQ ID NO: 46, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 46 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions);

(4) the framework regions FR of the heavy chain variable region comprise FR-H1, FR-H2, FR-H3 and FR-H4, wherein FR-H1 comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence identical to SEQ ID NO: 47, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 47, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no; FR-H2 comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence substantially identical to SEQ ID NO: 48, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 48 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H3 comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that is identical to SEQ ID NO: 49, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 49 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H4 comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence substantially identical to SEQ ID NO: 50, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 50 with one or more, preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 conservative amino acid mutations, preferably substitutions, insertions or deletions,

the framework regions FR of the light chain variable region comprise FR-L1, FR-L2, FR-L3 and FR-L4, wherein FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence identical to SEQ ID NO: 51, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to seq id no: 51 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence identical to SEQ ID NO: 52, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 52, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L3 comprises the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence identical to SEQ ID NO: 53, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 53 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L4 comprises the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence identical to SEQ ID NO: 54, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 54, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody comprises:

(1) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO: 2, or

And SEQ ID NO: 2, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

And SEQ ID NO: 2 (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions), and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO: 4, or

And SEQ ID NO: 4 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

And SEQ ID NO: 4 (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no;

(2) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO: 12, or

And SEQ ID NO: 12 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

And SEQ ID NO: 12, and (c) an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in (c), and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO: 14, or

And SEQ ID NO: 14 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

And SEQ ID NO: 14, and (b) an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) as compared to the amino acid sequence set forth in (b);

(3) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO: 16, or

And SEQ ID NO: 16 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

And SEQ ID NO: 16, and one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in seq id No. 16, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO: 18, or

And SEQ ID NO: 18, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

And SEQ ID NO: 18, and/or a variant thereof, wherein the variant has one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) as compared to the amino acid sequence set forth in (18);

(4) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO: 20, or

And SEQ ID NO: 20 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

And SEQ ID NO: 20, and one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in seq id No. 20, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO: 22, or

And SEQ ID NO: 22 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

And SEQ ID NO: 22, preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof comprises HCDR1-3 and LCDR1-3 as shown below:

the amino acid sequences of the 3 CDR regions of the heavy chain variable region are as follows:

HCDR1：GYTFTSYW(SEQ ID NO：5)，

HCDR2：IDPSDSET(SEQ ID NO：6)，

HCDR 3: ARLYRWYFDV (SEQ ID NO: 7); and

the amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:

LCDR1：EIVGTY(SEQ ID NO：8)，

LCDR2：GAS(SEQ ID NO：9)，

LCDR3：GQSYNFPYT(SEQ ID NO：10)。

5. the antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody further comprises a heavy chain constant region and a light chain constant region, and the constant regions are from a species other than murine, such as from a human antibody, preferably from a human IgG or IgM, more preferably IgG1, preferably the heavy chain constant region is Ig gamma-1chain C region, ACCESSION: p01857(SEQ ID NO: 58) or Ig gamma-4chain C region, ACCESSION: p01861.1(SEQ ID NO: 56); the light chain constant region is Ig kappa chain C region, ACCESSION: p01834(SEQ ID NO: 57).

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody comprises a heavy chain and a light chain selected from the group consisting of:

(1) SEQ ID NO: 59 and SEQ ID NO: a light chain represented by 60;

(2) SEQ ID NO: 61 and SEQ ID NO: a light chain represented by 62;

(3) SEQ ID NO: 63 and SEQ ID NO: a light chain represented by 64;

(4) SEQ ID NO: 65 and SEQ ID NO: a light chain represented by 66;

(5) SEQ ID NO: 67 and SEQ ID NO: 68, a light chain; and

(6) SEQ ID NO: 69 and the heavy chain of SEQ ID NO: 70, or a light chain.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody further comprises an amino acid mutation introduced at position 234 and/or 235 according to the EU numbering system.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody comprises mutations L234A and/or L235A according to the EU numbering system.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the antibody comprises a heavy chain and a light chain selected from the group consisting of:

(1) SEQ ID NO: 59 and SEQ ID NO: a light chain represented by 60;

(2) SEQ ID NO: 61 and SEQ ID NO: a light chain represented by 62; and

(3) SEQ ID NO: 63 and SEQ ID NO: 64, and a light chain.

10. An isolated polypeptide comprising:

(1) SEQ ID NO: 5, 6 and 7, wherein said polypeptide specifically binds to human CD47 as part of an anti-human CD47 antibody further comprising the amino acid sequence set forth in SEQ ID NO: 8, 9 and 10;

(2) SEQ ID NO: 8, 9 and 10, wherein said polypeptide specifically binds human CD47 as part of an anti-human CD47 antibody further comprising the amino acid sequence set forth in SEQ ID NO: 5, 6 and 7;

(3) selected from the group consisting of SEQ ID NO: 2, 12, 16 or 20 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is part of an antibody against human CD47 specifically binding to human CD47, said antibody further corresponding to a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 4, 14, 18 or 22 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; or

(4) Selected from the group consisting of SEQ ID NO: 4, 14, 18 or 22 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is part of an antibody against human CD47 that specifically binds to human CD47, said monoclonal antibody further comprising a heavy chain variable region selected from the group consisting of SEQ ID NO: 2, 12, 16 or 20 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

11. The antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab')₂Fd, Fv, dAb, Fab/c, Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody is a humanized antibody, a chimeric antibody, a multispecific antibody (e.g., bispecific antibody).

13. The antigen of any one of claims 1-12, or an antigen-binding fragment thereof, wherein the antibody is present at less than about 10^- ⁵M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or less binds to human CD47 protein.

14. The antigen of any one of claims 1-13, or an antigen-binding fragment thereof, wherein the antibody binds human CD47 protein with an EC50 of less than about 100nM, e.g., less than about 10nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, or less.

15. An isolated polynucleotide encoding a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 5, 6 and 7, wherein the polypeptide specifically binds to human CD47 as part of an antibody against human CD47 further comprising the amino acid sequence set forth in SEQ ID NO: 8, 9 and 10;

(2) comprises the amino acid sequence of SEQ ID NO: 8, 9 and 10, wherein said polypeptide specifically binds human CD47 as part of an antibody against human CD47 further comprising the amino acid sequence set forth in SEQ ID NO: 5, 6 and 7;

(3) comprises a sequence selected from SEQ ID NO: 2, 12, 16 or 20 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or a polypeptide having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) of the amino acid sequence as compared to said sequence, wherein said polypeptide is part of an anti-human CD47 antibody that specifically binds to human CD47, said antibody further corresponding to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 14, 18 or 22 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; or

(4) Comprises a sequence selected from SEQ ID NO: 4, 14, 18 or 22 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or a polypeptide having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) of the amino acid sequence as compared to said sequence, wherein said polypeptide is part of an anti-human CD47 antibody that specifically binds to human CD47, said antibody further corresponding to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 12, 16 or 20 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

16. The isolated polynucleotide of claim 15, wherein

(1) The polynucleotide molecule comprises or consists of the sequence: SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 15 or SEQ ID NO: 19, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to said sequence;

(2) the polynucleotide molecule comprises or consists of the sequence: SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to said sequence.

17. A vector comprising the polynucleotide molecule of claim 15 or 16.

18. A host cell comprising the polynucleotide molecule of claim 15 or 16, or the vector of claim 17.

19. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-14, comprising the steps of the host cell of claim 18 under suitable conditions, and recovering the antibody or antigen-binding fragment thereof from the cell culture.

20. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14, and a conjugate moiety conjugated to the antibody or antigen-binding fragment thereof, the conjugate moiety being a purification tag (e.g., a His-tag), a cytotoxic agent, or a detectable label. Preferably, the coupling moiety is a radioisotope, a luminescent substance, a coloured substance, an enzyme or polyethylene glycol.

21. A multispecific antibody, preferably a bispecific antibody, comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14, and an antibody or antigen-binding fragment directed to another antigen and/or other epitope.

22. A fusion protein comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14.

23. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14, or comprising the antibody conjugate of claim 20, the multispecific antibody of claim 21, or the fusion protein of claim 22.

24. The kit of claim 23, further comprising a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

25. The hybridoma cell strain is a hybridoma cell strain LT012 with a preservation number of CCTCC NO: 2018135.

26. use of the antibody or antigen-binding fragment thereof of any one of claims 1-14, the antibody conjugate of claim 20, the multispecific antibody of claim 21, or the fusion protein of claim 22 to detect the presence or level of human CD47 in a sample, or in the manufacture of a kit to detect the presence or level of human CD47 in a sample.

27. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14, the antibody conjugate of claim 20, the multispecific antibody of claim 21, or the fusion protein of claim 22; optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.

28. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the antibody conjugate of claim 20, the multispecific antibody of claim 21, or the fusion protein of claim 22 in the preparation of a medicament comprising:

a drug for blocking the combination of human CD47 and human SIRP alpha,

a drug which blocks or down-regulates the activity of human CD47, or

An agent that blocks the cellular response mediated by the binding of human sirpa to CD 47.

29. The antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the antibody conjugate of claim 20, the multispecific antibody of claim 21, or the fusion protein of claim 22 for use in, or in the manufacture of a medicament for, the treatment of a tumor, preferably a tumor that expresses CD47, preferably a cancer, such as a hematological malignancy or a solid tumor, more preferably a lymphoma, colon cancer, or breast cancer; more preferably non-hodgkin's lymphoma, and even more preferably B-cell lymphoma cells.

30. The use of claim 29, the medicament being in a form suitable for injection, preferably in a form suitable for administration by subcutaneous, intradermal, intravenous, intramuscular or intralesional injection.

31. An in vivo or in vitro method comprising the step of applying a cell comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the antibody conjugate of claim 20, the multispecific antibody of claim 21 or the fusion protein of claim 22, or the step of administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, the antibody conjugate, the multispecific antibody or the fusion protein, the method being selected from the group consisting of:

a method of blocking the binding of CD47 to human SIRPa,

a method of blocking human CD47 activity or down-regulating the level thereof, or

A method of blocking a cellular response mediated by the binding of human sirpa to CD 47.

Technical Field

The present invention relates to the field of autoimmune disease treatment and molecular immunology. Relates to an anti-CD47 antibody, a pharmaceutical composition containing the same and application thereof. In particular, the invention relates to a monoclonal antibody against CD 47.

Background

CD47 is also known as an Integrin Associated Protein (IAP). CD47 is a 5-transmembrane protein with a molecular weight of about 50kDa, and belongs to the immunoglobulin superfamily. The extracellular N-terminal is IgV structural domain which is connected with alpha v beta 3(CD51/CD61) and alpha IIb beta 3(CD41/CD61) integrin. CD47 is involved in a variety of physiological functions, such as cell transfer, T cell and Dendritic Cell (DC) activation, axonal development, and the like.

CD47 is expressed on all cell types including erythrocytes, while CD47 is highly expressed on tumor cells. CD47 has two ligands, Signal Regulatory Protein alpha (SIRP alpha) and Thrombospondin-1 (Thrombospondin-1, TSP 1). SIRP α is a class of immunoglobulin domain-containing receptor-type transmembrane glycoproteins that belong to the SIRP family and are expressed primarily on macrophages and neural cells. In the CD 47-SIRPa pathway, binding of CD47 protein to SIRPa phosphorylates its immunoreceptor tyrosine inhibitory motif ITIM, followed by intracellular recruitment of SHP-1 protein to produce a cascade of inhibition of macrophage phagocytosis (Matozaki T, Murata Y, Okazawa H, et al. functions and molecular mechanisms of the CD 47-SIRPa signalling. trends in cell biology, 2009, 19 (2): 72-80.). While normal erythrocytes are not phagocytosed due to the inhibitory signal generated by the combination of CD47 on the surface of the cell membrane and sipra of macrophages. (Oldenborg P A, ZHeleznyak A, FangY F, et al. role of CD47 as armker of self on red blood cells. science, 2000, 288 (5473): 2051-. TSP1 Is a homotrimer composed of 3 peptide chains, and Is involved in cell proliferation, apoptosis, Adhesion, migration, angiogenesis, etc. by interacting with other cell surface receptors, matrix components and growth factors (Jiang P, Lagenaur CF, Narayanan V.Integrin-associated Protein Is a Ligand for the P84 Neural addition molecular. journal of biological Chemistry 1999.274: 559-62).

Macrophages (Macrophages) are derived from monocytes, which in turn are derived from precursor cells in the bone marrow. The main functions are to phagocytize cell debris and pathogens in the form of fixed cells or free cells and to activate lymphocytes or other immune cells to respond to pathogens. It is considered that tumor cells have a mechanism of escape macrophage phagocytosis, and specific proteins such as calreticulin are formed on the surface during the growth of tumor cells, exposing tumor identity and attracting macrophage phagocytosis, but at the same time, macrophages with SIRP alpha and tumor cells with high expression of CD47 inhibit phagocytosis of macrophages due to activation of CD47-SIRP alpha pathway, and macrophages are misjudged as normal cells to cause tumor cells to escape phagocytosis of macrophages (CD47 is regulated on circulating biochemical stem cells and leucocytes to involved in pathological phagocytosis. Jaiswal S, Jamieson C H M, Pang W, et al. cell, 2009, 138(3) 271-285).

It has now been found that anti-CD47 antibodies kill tumor cells primarily by two mechanisms. 1. Binding of anti-CD47 antibodies to CD47 blocks the CD 47-sirpa pathway allowing macrophages to exert phagocytosis. 2. anti-CD47 antibodies exert tumor killing effects by DC cells and CD8+ T cells. The DC cells phagocytize tumor cells through the synergistic effect of anti-CD47 antibody and phagocytophilic molecules such as calreticulin, and present tumor-associated antigens to CD8+ T cells, thereby exerting the specific killing effect of CD8+ T cells on tumors (CD47 Block as animal cancer therapy for cancer. Vonderheide R H. Nature Medicine, 2015, 21 (10): 1122). The combination of these two mechanisms indicates that the anti-CD47 antibody is most likely to have the ability to activate both non-specific and specific immunity.

At present, the CD 47-resistant monoclonal antibody medicine has wide application prospect and good tumor treatment effect, and can be used for treating various tumors. An anti-CD47 monoclonal antibody drug Hu5F9-G4 can effectively inhibit The growth and metastasis of malignant blood diseases and solid tumors in preclinical experiments (Abstract PR 13: The anti-CD47 antibody Hu5F9-G4 is a novel immune cancer inhibitor with synthetic activity in combination with clinical active cancer targeting antibodies [ J ] Chao M P, McKenna K M, Cha A, et al, 2016).

Therefore, the development of an antibody drug with high affinity with CD47 for tumor treatment has better treatment effect and lower toxic and side effects, and has very important significance.

Disclosure of Invention

The inventor utilizes a mammalian cell expression system to express recombinant human CD47, immunizes a mouse as an antigen, and obtains hybridoma cells by fusing mouse spleen cells and myeloma cells. The following hybridoma cell lines were obtained by screening a large number of samples.

The inventor finds that:

the hybridoma cell line LT012 can secrete a monoclonal antibody (named 6F7) specifically bound with CD47, and the monoclonal antibody can compete with a receptor SIRP alpha ECD-hFc-Biotin to bind with CD47, effectively block the binding of SIRP alpha and CD47, and further promote the phagocytosis of tumor cells by macrophages.

Further, the present inventors produced humanized antibodies of monoclonal antibody 6F7 (designated 6F7H1L1, 6F7H2L2 and 6F7H3L 3).

The present invention thus provides the following inventions:

one aspect of the invention relates to an antibody or antigen-binding fragment thereof, wherein:

the antibody comprises CDR sequences contained in a heavy chain variable region and a light chain variable region selected from

(1) SEQ ID NO: 2 comprises HCDR1, HCDR2 and HCDR3, and

SEQ ID NO: 4 comprises LCDR1, LCDR2 and LCDR 3; or

(2) SEQ ID NO: 12 comprises HCDR1, HCDR2 and HCDR3, and

SEQ ID NO: 14 comprises LCDR1, LCDR2 and LCDR 3; or

(3) SEQ ID NO: 16 comprises HCDR1, HCDR2 and HCDR3, and

SEQ ID NO: 18 comprises LCDR1, LCDR2 and LCDR 3; or

(4) SEQ ID NO: 20 comprises HCDR1, HCDR2 and HCDR3, and

SEQ ID NO: 22 comprises LCDR1, LCDR2 and LCDR 3;

preferably, the antibody comprises:

In an embodiment of the invention, the antibody comprises:

(1) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO: 2, or

And SEQ ID NO: 2, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO: 4, or

And SEQ ID NO: 4 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

(2) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO: 12, or

And SEQ ID NO: 12 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO: 14, or

And SEQ ID NO: 14 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

(3) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO: 16, or

And SEQ ID NO: 16 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO: 18, or

And SEQ ID NO: 18, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

(4) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO: 20, or

And SEQ ID NO: 20 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO: 22, or

And SEQ ID NO: 22 has at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity, or

The amino acid sequences of the CDR regions of the antibody sequences in (1) to (12) above are analyzed by technical means well known to the person skilled in the art, for example by the VBASE2 database:

antibodies 6F7, 6F7H1L1, 6F7H2L2 and 6F7H3L3 of the invention have the same HCDR1-3 and LCDR 1-3:

the amino acid sequences of the 3 CDR regions of the heavy chain variable region are as follows:

HCDR1：GYTFTSYW(SEQ ID NO：5)，

HCDR2：IDPSDSET(SEQ ID NO：6)，

HCDR3：ARLYRWYFDV(SEQ ID NO：7)；

the amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:

LCDR1：EIVGTY(SEQ ID NO：8)，

LCDR2：GAS(SEQ ID NO：9)，

LCDR3：GQSYNFPYT(SEQ ID NO：10)。

in some embodiments, the antibodies of the invention are selected from the group consisting of:

6F7H1L1(G1M) heavy chain (SEQ ID NO: 59)

6F7H1L1(G1M) light chain (SEQ ID NO: 60)

6F7H2L2(G1M) heavy chain (SEQ ID NO: 61)

6F7H2L2(G1M) light chain (SEQ ID NO: 62)

6F7H3L3(G1M) heavy chain (SEQ ID NO: 63)

6F7H3L3(G1M) light chain (SEQ ID NO: 64)

6F7H1L1(hG4) heavy chain (SEQ ID NO: 65)

6F7H1L1(hG4) light chain (SEQ ID NO: 66)

6F7H2L2(hG4) heavy chain (SEQ ID NO: 67)

6F7H2L2(hG4) light chain (SEQ ID NO: 68)

6F7H3L3(hG4) heavy chain (SEQ ID NO: 69)

6F7H3L3(hG4) light chain (SEQ ID NO: 70)

In an embodiment of the invention, the antibody (preferably the 6F7 antibody) further comprises the framework region FR of the heavy chain variable region, preferably the framework region FR comprises FR-H1, FR-H2, FR-H3 and FR-H4, wherein FR-H1 comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence corresponding to SEQ ID NO: 23, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 23, or consists of, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no; FR-H2 comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence corresponding to SEQ ID NO: 24, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a sequence set forth in SEQ ID NO: 24 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H3 comprises the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence substantially identical to SEQ ID NO: 25, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a sequence as set forth in SEQ ID NO: 25, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no; FR-H4 comprises the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence corresponding to SEQ ID NO: 26, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a sequence set forth in SEQ ID NO: 26, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions).

In an embodiment of the invention, the antibody (preferably the 6F7 antibody) further comprises the framework region FR of the light chain variable region, preferably the framework region FR comprises FR-L1, FR-L2, FR-L3 and FR-L4, wherein FR-L1 comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence identical to SEQ ID NO: 27, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 27, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (a); FR-L2 comprises the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence identical to SEQ ID NO: 28, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 28, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L3 comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence substantially identical to SEQ ID NO: 29, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 29 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L4 comprises the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence corresponding to SEQ ID NO: 30, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 30, or consists of one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In an embodiment of the invention, the antibody (preferably the 6F7H1L1 antibody) further comprises the framework region FR of the heavy chain variable region, preferably the framework region FR comprises FR-H1, FR-H2, FR-H3 and FR-H4, wherein FR-H1 comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that is identical to SEQ ID NO: 31, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence as set forth in SEQ ID NO: 31, or consists of, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H2 comprises the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence identical to SEQ ID NO: 32, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 32, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H3 comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence identical to SEQ ID NO: 33, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 33, or consists of an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no; FR-H4 comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence identical to SEQ ID NO: 34, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 34, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In an embodiment of the invention, the antibody (preferably the 6F7H1L1 antibody) further comprises the framework region FR of the light chain variable region, preferably the framework region FR comprises FR-L1, FR-L2, FR-L3 and FR-L4, wherein FR-L1 comprises the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence that is identical to SEQ ID NO: 35, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence set forth in SEQ ID NO: 35, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L2 comprises the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence identical to SEQ ID NO: 36, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to a sequence as set forth in SEQ ID NO: 36, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (a); FR-L3 comprises the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence identical to SEQ ID NO: 37, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 37, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (b); FR-L4 comprises the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence identical to SEQ ID NO: 38, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 38, or consists of one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In an embodiment of the invention, the antibody (preferably the 6F7H2L2 antibody) further comprises the framework region FR of the heavy chain variable region, preferably the framework region FR comprises FR-H1, FR-H2, FR-H3 and FR-H4, wherein FR-H1 comprises the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence substantially identical to SEQ ID NO: 39, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 39, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in (a); FR-H2 comprises the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence substantially identical to SEQ ID NO: 40, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 40, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions), as compared to the amino acid sequence set forth in SEQ ID NO: 41, or an amino acid sequence identical to SEQ ID NO: 41, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 41, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H4 comprises the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence identical to SEQ ID NO: 42, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 42 compared to an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions or deletions).

In an embodiment of the invention, the antibody (preferably the 6F7H2L2 antibody) further comprises the framework region FR of the light chain variable region, preferably the framework region FR comprises FR-L1, FR-L2, FR-L3 and FR-L4, wherein FR-L1 comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence substantially identical to SEQ ID NO: 43, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 43, or consists of, an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence; FR-L2 comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence identical to SEQ ID NO: 44, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 44 compared to an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L3 comprises the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence substantially identical to SEQ ID NO: 45, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 45, or consists of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L4 comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence substantially identical to SEQ ID NO: 46, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 46, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In an embodiment of the invention, the antibody (preferably the 6F7H3L3 antibody) further comprises the framework region FR of the heavy chain variable region, preferably the framework region FR comprises FR-H1, FR-H2, FR-H3 and FR-H4, wherein FR-H1 comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence identical to SEQ ID NO: 47, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence set forth in SEQ ID NO: 47, or consists of, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) as compared to the amino acid sequence set forth in seq id no; FR-H2 comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence substantially identical to SEQ ID NO: 48, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 48 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H3 comprises the amino acid sequence of SEQ ID NO: 49 or, or an amino acid sequence substantially identical to SEQ ID NO: 49, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 49 compared to an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-H4 comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence substantially identical to SEQ ID NO: 50, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 50 with one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In an embodiment of the invention, the antibody (preferably the 6F7H3L3 antibody) further comprises the framework region FR of the light chain variable region, preferably the framework region FR comprises FR-L1, FR-L2, FR-L3 and FR-L4, wherein FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence identical to SEQ ID NO: 51, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 51, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence identical to SEQ ID NO: 52, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 52, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions); FR-L3 comprises the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence identical to SEQ ID NO: 53, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 53, or consists of, an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no; FR-L4 comprises the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence identical to SEQ ID NO: 54, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to the sequence set forth in SEQ ID NO: 54, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In one aspect of the invention, isolated polypeptides are contemplated comprising SEQ ID NOs: 5, 6 and 7, wherein said polypeptide specifically binds to human CD47 as part of an anti-human CD47 antibody further comprising the amino acid sequence set forth in SEQ ID NO: 8, 9 and 10.

In one aspect of the invention, isolated polypeptides are contemplated comprising SEQ ID NOs: 8, 9 and 10, wherein said polypeptide specifically binds human CD47 as part of an anti-human CD47 antibody further comprising the amino acid sequence set forth in SEQ ID NO: 5, 6 and 7.

In one aspect the invention relates to an isolated polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 12, 16 or 20 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is part of an antibody against human CD47 specifically binding to human CD47, said antibody further corresponding to a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 4, 14, 18 or 22 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; or

In one aspect the invention relates to an isolated polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 18 or 22 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is part of an antibody against human CD47 that specifically binds to human CD47, said monoclonal antibody further comprising a heavy chain variable region selected from the group consisting of SEQ ID NO: 2, 12, 16 or 20 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In an embodiment of the invention, the antigen binding fragment is selected from the group consisting of Fab, Fab ', F (ab')₂Fd, Fv, dAb, Fab/c, Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, domain antibodies.

In an embodiment of the invention, the antibody is a humanized antibody, a chimeric antibody, a multispecific antibody (e.g., bispecific antibody).

In an embodiment of the invention, the antibody is present in an amount less than about 10^-5M, e.g. less thanAbout 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or less binds to human CD47 protein. Preferably, the KD is measured by Fortebio molecular interaction.

In embodiments of the invention, the antibody binds human CD47 protein with an EC50 of less than about 100nM, e.g., less than about 10nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, or less. Specifically, the EC50 was measured by indirect ELISA method.

In an embodiment of the invention, the antibody comprises a constant region, and the constant region is from a species that is not murine, for example from a human antibody, preferably from a human IgG, more preferably IgG1 or IgG 4.

In an embodiment of the invention, the constant region of the antibody is of human origin, e.g. the heavy chain constant region is the Ig gamma-1chain C region, more preferably GenBank ACCESSION No. access: the Ig gamma-1chain C region of P01857(SEQ ID NO: 58), or with Ig gamma-4chain C region, more preferably GenBank ACCESSION No: ig gamma-4chain C region of P01861.1(SEQ ID NO: 56); the light chain constant regions all adopt Ig kappa chain C region, more preferably GenBank ACCESSION No. ACCESSION: ig kappa chain C region of P01834(SEQ ID NO: 57). The antibodies of the invention employ the following constant regions on the basis of the variable regions of 6F7H1L1, 6F7H2L2 and 6F7H3L 3: the heavy chain constant region is Ig gamma-1chain C region, ACCESSION: p01857(SEQ ID NO: 58) or heavy chain constant region Ig gamma-4chain C region, ACCESSION: p01861.1(SEQ ID NO: 56); the light chain constant region is Ig kappa chain C region, ACCESSION: p01834(SEQ ID NO: 57). Another aspect of the invention relates to an isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 5, 6 and 7, wherein said polypeptide specifically binds to human CD47 as part of an anti-human CD47 antibody further comprising the amino acid sequence set forth in SEQ ID NO: 8, 9 and 10.

In one aspect the invention relates to an isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, 9 and 10, wherein said polypeptide specifically binds human CD47 as part of an anti-human CD47 antibody further comprising the amino acid sequence set forth in SEQ ID NO: 5, 6 and 7.

In one aspect the invention relates to an isolated polynucleotide encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 12, 16 or 20 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is part of an antibody against human CD47 specifically binding to human CD47, said antibody further corresponding to a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 4, 14, 18 or 22 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; or

In one aspect the invention relates to an isolated polynucleotide encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 18 or 22 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is part of an antibody against human CD47 specifically binding to human CD47, said antibody further corresponding to a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 2, 12, 16 or 20 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

in particular, the polynucleotide molecule comprises or consists of the sequence: SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 15 or SEQ ID NO: 19, or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to said sequence.

In particular, the polynucleotide molecule comprises or consists of the sequence: SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21 or a sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% sequence identity to said sequence.

Yet another aspect of the invention relates to a vector comprising a polynucleotide molecule according to any one of the above-described aspects of the invention.

Yet another aspect of the invention relates to a host cell comprising a polynucleotide molecule of any one of the above described in the present invention, or a vector of the present invention.

Yet another aspect of the present invention relates to a method of making an antibody or antigen-binding fragment thereof of any one of the above-described in the present invention, comprising the steps of culturing a host cell of the present invention under suitable conditions, and recovering the antibody or antigen-binding fragment thereof from the cell culture.

In one aspect of the invention, there is also provided an antibody conjugate comprising said anti-human CD47 antibody or antigen-binding fragment thereof, and a conjugate moiety conjugated thereto, said conjugate moiety being a purification tag (e.g. a His-tag), a cytotoxic agent, or a detectable label. Preferably, the coupling moiety is a radioisotope, a luminescent substance, a coloured substance, an enzyme or polyethylene glycol.

In one aspect of the invention, there is also provided a multispecific, preferably bispecific antibody comprising said anti-human CD47 antibody or antigen-binding fragment thereof, and an antibody or antigen-binding fragment directed against another antigen and/or other epitope.

In one aspect of the invention, there is also provided a fusion protein comprising an antibody or antigen-binding fragment thereof against human CD47 as described in any one of the above.

In one aspect of the invention, there is also provided a kit comprising an antibody or antigen-binding fragment thereof of any one of the invention as described above, or an antibody conjugate, multispecific antibody or fusion protein of the invention.

Preferably, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

In one aspect of the invention, hybridoma cell lines selected from the following and monoclonal antibodies produced therefrom are also provided: the hybridoma cell strain LT012 has the preservation number of CCTCC NO: 2018135.

in a further aspect the invention relates to the use of an antibody or antigen-binding fragment thereof according to any one of the above described invention or an antibody conjugate or multispecific antibody or fusion protein of the invention in the detection of the presence or level of human CD47 in a sample, or in the preparation of a kit for detecting the presence or level of human CD47 in a sample.

In a further aspect the present invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of the above in the present invention or an antibody conjugate, multispecific antibody or fusion protein according to the present invention; optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.

In a further aspect the present invention relates to the use of an antibody or antigen-binding fragment thereof according to any one of the above or an antibody conjugate or multispecific antibody or fusion protein of the invention in the manufacture of a medicament for:

a drug for blocking the combination of human CD47 and human SIRP alpha,

a drug which blocks or down-regulates the activity of human CD47, or

An agent that blocks a cellular response mediated by binding of human sirpa to CD 47;

in one aspect the invention relates to the use of an antibody or antigen-binding fragment thereof according to any one of the above or an antibody conjugate or multispecific antibody or fusion protein of the invention in the treatment of a tumour or in the manufacture of a medicament for the treatment of a tumour.

Yet another aspect of the invention relates to an in vivo or in vitro method comprising the step of administering a cell comprising an antibody or antigen-binding fragment thereof according to the invention, an antibody conjugate, a multispecific antibody or fusion protein according to the invention, or the step of administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof according to any one of the above or an antibody conjugate or multispecific antibody or fusion protein according to the invention, said method being selected from the following:

a method of blocking the binding of CD47 to human SIRPa,

a method of blocking human CD47 activity or down-regulating the level thereof, or

A method of blocking a cellular response mediated by the binding of human sirpa to CD 47.

In an embodiment of the invention, the in vitro method is of non-therapeutic or diagnostic purpose.

A further aspect of the present invention relates to the use of an antibody or antigen-binding fragment thereof according to any one of the above-mentioned aspects of the present invention or an antibody conjugate or multispecific antibody or fusion protein according to the present invention in the prevention and/or treatment and/or adjuvant therapy and/or diagnosis of an associated tumor or in the manufacture of a medicament for the prevention and/or treatment and/or adjuvant therapy and/or diagnosis of an associated tumor.

In one aspect of the invention, the tumor is preferably a tumor expressing CD47, preferably a cancer, such as a hematological malignancy or a solid tumor, more preferably a lymphoma, colon cancer or breast cancer, more preferably a non-hodgkin lymphoma, even more preferably a B-cell lymphoma cell.

In an embodiment of the invention, the medicament is in a form suitable for injection, preferably in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in the present invention are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "antigen-binding region" means a protein or portion of a protein that specifically binds to a specified antigen. For example, the portion of an antibody that contains amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen is referred to as an "antigen-binding region". The antigen-binding region typically includes one or more "complementary-determining regions" (CDRs). Certain antigen binding regions also include one or more "framework" regions ("FRs"). CDRs are amino acid sequences that contribute to antigen binding specificity and affinity.

As used herein, the term "antibody" refers to an intact immunoglobulin of any isotype or an antigen-binding fragment thereof that can compete with an intact antibody for specific binding to a target antigen, and includes, for example, chimeric, humanized, fully humanized, and bispecific antibodies or antigen-binding fragments thereof. Such "antibodies" are a class of antigen binding proteins. A full antibody typically comprises at least two full length heavy chains and two full length light chains, but in some cases fewer chains may be included, such as an antibody naturally occurring in a camelid that may comprise only heavy chains. An antibody or antigen-binding fragment thereof may be derived from only a single source, or may be "chimeric," i.e., different portions of an antibody may be derived from two different sources as described further below. The antibodies or antigen-binding fragments thereof can be produced in hybridomas by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes derivatives, variants and fragments thereof, in addition to antibodies comprising two full-length heavy chains and two full-length light chains.

As used herein, the term "antibody" or "antigen-binding fragment" (or simply "fragment") of an "immunoglobulin chain (heavy or light chain)" comprises a portion of an antibody (regardless of how the portion is obtained or synthesized) that lacks at least some of the amino acids present in the full-length chain of the antibody, but is capable of specifically binding an antigen. Such fragments are biologically active in that they specifically bind to a target antigen and can compete with other antibodies or antigen binding fragments thereof for specific binding to a given epitope. In one aspect, such fragments will retain at least one CDR present in the full length light or heavy chain of the antibody, and in some embodiments will comprise a single heavy and/or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques, or can be produced, for example, by enzymatic or chemical cleavage of an intact antibody. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab ', F (ab')₂Fab/c, dAb, Fv, domain antibodies and single chain antibodies, and may be derived from any mammalian source, including but not limited to human, mouse, rat, camelid, or rabbit. It is also contemplated that a functional portion of an antibody disclosed herein, e.g., one or more CDRs, can be covalently bound to a second protein or small molecule to generate a therapeutic agent that targets a particular target in the body, thereby having bifunctional therapeutic properties or having an extended serum half-life, e.g., a fusion protein.

As used herein, the terms "full-length antibody," "intact antibody (antibody)" and "whole antibody (antibody)" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or a heavy chain having an Fc region as defined herein.

The term "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequence to confer binding specificity. The full-length light chain includes a variable region domain V_LAnd constant region Domain C_L. The variable region domain of the light chain is at the amino terminus of the polypeptide. Light chains include kappa and lambda chains.

The term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. Full-length heavy chain includes variable region domain V_HAnd 3 constant region domains C_H1、C_H2And C_H3。V_HThe structural domain is at the amino terminal end of the polypeptide, and C_HThe structural domain is at the carboxyl terminal, C_H3Closest to the carboxy terminus of the polypeptide. The heavy chain may be of any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

As used herein, the term "Fab fragment" consists of one light chain and C_H1And the variable region of one heavy chain. The heavy chain of a Fab molecule is unable to form a disulfide bond with another heavy chain molecule.

As used herein, the term "Fc" region contains two antibody-containing Cs_H1And C_H2A heavy chain fragment of a domain. Two heavy chain fragments through two or more disulfide bonds and through C_H3The hydrophobic interactions of the domains remain together.

As used herein, the term "Fab' fragment" contains one light chain and one heavy chain portion (which contains V)_HDomains and C_H1Domains and also C_H1And C_H2Part of the region between the domains) so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab 'fragments to form F (ab')₂A molecule.

As used herein, the term "F (ab')₂Fragment "contains two light chains and two contain C_H1And C_H2Heavy chains that are part of a constant region between domains, such that an interchain disulfide bond is formed between the two heavy chains. F (ab')₂The fragment thus consists of two Fab' fragments held together by the disulfide bond between the two heavy chains.

As used herein, the term "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

As used herein, the term "Fd" fragment means a fragment consisting of V_HAnd C_H1Antibody fragments consisting of domains (Ward et al, Nature 341: 544-546 (1989)).

As used herein, the term "dAb" fragment (Ward et al, Nature 341: 544-546(1989)) is derived from V_HDomain composition.

As used herein, the term "Fab '-SH" is the designation herein for Fab', wherein one or more cysteine residues of the constant domain carry a free thiol group.

As used herein, the term "Fab/c" fragment is an intermediate in the cleavage of an immunoglobulin by pepsin digestion, which combines the advantages of the Fab and Fc regions, i.e., strong diffusion capacity and slow metabolic clearance in vivo, while maintaining high affinity (Liujian Jun, J. cell & molecular immunology, 1989 (4): 29-29).

As used herein, the term "single chain antibody" is an Fv molecule in which the heavy and light chain variable regions are joined by a flexible linker to form a single polypeptide chain (which forms the antigen binding region) (see, e.g., Bird et al, science.242: 423-58426 (1988) and Huston et al, Proc. Natl. Acad. Sci. USA.90: 5879-5883 (1988)). Single chain antibodies are described in detail in international patent application publication No. WO 88/01649 and U.S. patent nos. 4,946,778 and 5,260,203 (the disclosures of which are incorporated by reference).

As used herein, the term "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain,including multivalent domain antibodies or bivalent domain antibodies. In some cases, two or more V_HThe regions are covalently linked by a peptide linker to generate multivalent domain antibodies (particularly bivalent domain antibodies). Two V of bivalent domain antibody_HThe regions may target the same or different antigens.

As used herein, the term "bivalent antigen binding protein" or "bivalent antibody" comprises two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. The diabody can be bispecific.

As used herein, the term "multispecific antigen-binding protein" or "multispecific antibody" is an antigen-binding protein or antibody that targets more than one antigen or epitope.

As used herein, the term "bispecific", "dual specificity" or "bifunctional" antigen binding proteins or antibodies are hybrid antigen binding proteins or antibodies, respectively, having two different antigen binding sites. A bispecific antibody is a multispecific antigen-binding protein or multispecific antibody and may be produced by a variety of methods, including, but not limited to, fusion of hybridomas or attachment of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, clin. exp. immunol.79: 315- > 321; kostelny et al, 1992, j.immunol.148: 1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes that are present on the same or different protein targets.

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using hybridoma technology first reported by Kohler et al (Nature, 256: 495, 1975), but can also be obtained using recombinant DNA technology (see, e.g., U.S. P4, 816, 567).

As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or a portion of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody of the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the Framework Region (FR) of the acceptor antibody may be substituted with those of the corresponding non-human antibody, or with those of other antibodies, to further refine or optimize the performance of the antibody. For more details on humanized antibodies, see, e.g., Jones et al, Nature, 321: 522525 (1986); reichmann et al, Nature, 332: 323329 (1988); presta, curr, op.struct.biol., 2: 593596 (1992); and Clark, immunol. today 21: 397402(2000).

As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to in the art as an "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrates or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996). In a linear epitope, the points of all interactions between a protein and an interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the protein. In conformational epitopes, the point of interaction exists across protein amino acid residues that are separated from each other.

The terms "polypeptide" or "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term may also include amino acid polymers that have been modified or phosphorylated, for example by the addition of saccharide residues to form glycoproteins. Polypeptides and proteins can be produced by naturally occurring cells and non-recombinant cells; or it is produced by genetically engineered or recombinant cells and comprises a molecule having the amino acid sequence of a native protein, or a molecule having a deletion, addition and/or substitution of one or more amino acids of the native sequence.

In some embodiments, the terms "polypeptide" and "protein" specifically include antibodies, such as anti-human CD47 antibodies (also referred to as CD47 antibodies), CD47 binding proteins, or variants thereof, such as antibodies or sequences having deletions, additions, and/or substitutions of one or more amino acids.

The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxy-terminal deletion, and/or an internal deletion as compared to a full-length protein. Such fragments may also contain modified amino acids compared to the full-length protein. In certain embodiments, fragments are about 5 to 500 amino acids in length. For example, a fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of the human CD47 antibody, useful fragments include, but are not limited to, CDR regions, variable domains of heavy or light chains, portions of antibody chains or variable domains thereof comprising exactly 2 CDRs, and the like.

A "derivative" of a polypeptide is a polypeptide (e.g., an antigen binding protein or antibody) that is chemically modified in a different manner than an insertion, deletion, or substitution variant, e.g., by conjugation of another chemical moiety, e.g., a PEG-conjugated polypeptide.

As used herein, the term "isolated" or "isolated" refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" or "isolated" does not exclude the presence of substances mixed artificially or synthetically or other impurities which do not affect the activity of the substance.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide may be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody is present in an amount less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or less affinity (K)_D) Binding the antigen.

As used in the present invention, the term "K_D"refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. Among several parameters of molecular binding kinetics assays, K_DThe value is dissociation equilibrium constant, in the research of antibody drugs, the parameter is used for representing the strength of the affinity action of the tested antibody and the target antigen molecule, the calculation method is KD (kdis/kon), the smaller the dissociation equilibrium constant, the tighter the antibody-antigen binding is, and the higher the affinity between the antibody and the antigen is. Kon, the binding rate constant-the rate of formation of antigen-antibody complexes, the smaller Kon suggests faster binding of antibody to antigen; kdis, the dissociation rate constant is the rate at which an antibody dissociates from an antigen-antibody complex, and the smaller kdis, the slower the antibody is detached from the antigen, the more firmly the antibody binds to the antigen. Typically, the antibody is present in an amount less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10Dissociation equilibrium constant (K) of M or less_D) Binding to an antigen (e.g., L1 protein), for example, as determined in a BIACORE or Fortebio molecular interaction instrument using Surface Plasmon Resonance (SPR).

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the terms "hybridoma" and "hybridoma cell line" are used interchangeably, and when referring to the terms "hybridoma" and "hybridoma cell line," they also include subclones and progeny cells of the hybridoma.

As used herein, the terms "percent sequence identity" and "percent sequence homology" are used interchangeably.

As used herein, the terms "similarity" or "sequence similarity", "identity" refer to the relationship between the sequences of two or more protein or polypeptide molecules, as determined by aligning and comparing the sequences. "percent identity" means the percentage of identical residues between amino acids in the molecules being compared, and can be calculated based on the size of the smallest molecule to be compared. In order to perform these calculations, gaps in the alignment (if any) must be addressed by a specific mathematical model or computer program (i.e., an "algorithm"). The term "substantial identity", when applied to polypeptides, means that two peptide sequences, when optimally aligned, for example using the programs GAP or BESTFIT, using default GAP weights provided by the programs, share at least 70%, 75% or 80% sequence identity, at least 90% or 95% sequence identity, and at least 97%, 98% or 99% sequence identity. In some cases, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain R group that possesses similar chemical properties (e.g., charge or aqueous). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be upregulated to correct for the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, e.g., Pearson, Methods mol. biol. 243: 307-31(1994). Examples of groups of amino acids having side chains with similar chemical properties include 1) aliphatic hydroxyl side chains: glycine, alanine, valine, leucine, and isoleucine: 2) aliphatic hydroxyl side chain: serine and threonine: 3) amide-containing side chain: asparagine and glutamine: 4) aromatic side chain: phenylalanine, tyrosine and tryptophan: 5) basic side chain: lysine, arginine and histidine: 6) acidic side chain: aspartic acid and glutamic acid; and 7) sulfur containing side chains: cysteine and methionine. For example, the conservative amino acid substitution group is valine-leucine-isoleucine-glycine-alanine, phenylalanine-tyrosine, threonine-serine, lysine-arginine, glutamic acid-aspartic acid and asparagine-glutamine

Alternatively, conservative substitutions are described in Gonnet et al, Science 256: 1443-45(1992) (incorporated herein by reference) has any variation in positive values in the PAM250 log-likehood matrix (PAM250 log-likehood matrix). A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence analysis software is commonly used to measure the sequence identity of polypeptides. Protein analysis software uses a measure of similarity assigned to different substitutions, deletions and other modifications (including conservative amino acid substitutions) to match sequences. For example, GCG includes programs such as "Gap" and "Bestfit" which (using default parameters specified by the program) can be used to determine sequence homology or sequence identity between closely related polypeptides (e.g., homologous polypeptides from different biological species) or between a wild-type protein and its mutein. See, for example, GCG Version 6.1(University of Wisconsin, WI). Polypeptide sequences can also be compared using FASTA using default or recommended parameters, see GCG Version 6.10 FASTA (e.g., FASTA2 and FASTA3) to provide alignments and percent sequence identities of regions of optimal overlap between the challenge sequence and the search sequence (Pearson, Methods enzymol.183: 63-98 (1990): Pearson, Methods Mo l.biol.132: 185-219 (2000)). Another preferred algorithm when comparing sequences to a database containing a large number of sequences from different organisms is the computer program BLAST, in particular blastp or tblastn (using default parameters provided by the program). See, e.g., Altschul et al, mol. biol. 215: 403-410(1990): al tschul et Al, Nucleic Acids Res.25: 3389-402(1997).

Compared with the prior art, the invention has the following advantages:

the anti-CD47 monoclonal antibody can effectively block the combination of SIRP alpha and CD47 by specifically binding CD47, thereby promoting the phagocytosis of tumor cells by macrophages. The affinity of the antibody (such as 6F7H1L 1) of the invention to the tumor cell CD47 is equivalent to that of the control antibody Hu5F9-G4, while the affinity to the normal human RBC is lower than that of the control antibody Hu5F9-G4, so the antibody of the invention has better tumor treatment potential on the basis of avoiding influencing the normal human erythrocyte as much as possible.

Drawings

FIG. 16F 7H1L1(hG4) shows the results of detection of the binding activity to human CD47 IgV TEV-His.

FIG. 26F 7H1L1(G1M) shows the results of detection of the binding activity to human CD47 IgV TEV-His. In the figure, 6F7H1L1(hG 1DM) is 6F7H1L1 (G1M).

FIG. 36F 7H1L1(hG4) and human SIRP α ECD-hFc-Biotin compete for binding to human CD47 IgV TEV-His activity assay results.

FIG. 46F 7H1L1(G1M) and human SIRP α ECD-hFc-Biotin compete for binding to human CD47 IgV TEV-His activity assay results. In the figure, 6F7H1L 1(hGlDM) is 6F7H1L1 (G1M).

FIG. 5 is a graph showing the result of detecting the affinity constant of murine antibody 6F7 with human CD 47.

FIG. 66F 7H1L1(hG4) is a graph showing the result of affinity constant assay for human CD 47.

FIG. 7 is a graph of affinity constant assay results for Hu5F9-G4 and human CD 47.

FIG. 86F 7 binding curves (FACS) of H1L1(G1M) to human RBC.

FIG. 96F 7H1L1(G1M) binding activity to tumor cells Raji (FACS).

FIG. 106 Activity assay (FACS) of F7H1L1(G1M) competing with SIRP for binding to LOVO.

FIG. 116F 7H1L1(G1M) agglutination of human erythrocytes with the antibody.

FIG. 126F 7H1L1(hG4) binding curves to human RBC (FACS).

FIG. 136F 7H1L1(hG4) shows the binding activity to tumor cells Raji (FACS).

FIG. 146F 7H1L1(hG4) competes with SIRP for competitive binding activity to tumor cells Raji (FACS).

FIG. 156F 7H1L1(hG4) binding curves to tumor cell LOVO (FACS).

FIG. 166F 7H1L1(hG4) Activity detection (FACS) competed with SIRP for binding to LOVO.

FIG. 17 agglutination of human erythrocytes by anti-CD47 antibody.

FIG. 186F 7H1L1(hG4) therapeutic effect on MDA-MB-231 subcutaneous transplantable tumors.

FIG. 196F 7H1L1(hG4) and Hu5F9-G4 cynomolgus monkey show changes in hemoglobin concentration following a single administration.

FIG. 206F 7H1L1(hG4) and Hu5F9-G4 cynomolgus monkey change in hematocrit after a single administration.

Description of biological Material preservation:

the hybridoma cell strain LT012 is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 6 and 21 months, and the preservation number is CCTCC NO: c2018135, the preservation address is China, Wuhan university, postcode: 430072.

the specific implementation mode is as follows:

embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature of the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, but are conventional products available on the market.

In the following examples of the present invention, BALB/C mice used were purchased from the Guangdong provincial animal center for medical experiments.

The control antibody drug used was Hu5F9-G4 (synthesized by Zhongshan Kangfang biomedical Co., Ltd., prepared from Hu5F9-G4 sequence of CD47 antibody of form Seven, Inc., namely, SEQ ID NO: 37 of US20150183874 as the heavy chain variable region, SEQ ID NO: 42 as the light chain variable region, and Ig gamma-4chain constant region (GenbankID: P01861.1)).

Example 1: preparation of anti-human CD47 antibody 6F7

1. Preparation of hybridoma cell line 6F7

The antigen used for preparing the anti-CD47 antibody of the hybridoma cell strain 6F7 is CD47 IgV TEV-His (including GenbankID: NP 942088.1 position: 19-141 human CD47 mature peptide and TEV (amino acid sequence: ENLYFQG, SEQ ID NO: 74) -His labeled fusion protein, synthesized by Zhongshan Kangfang biomedical Limited company) and 3T3-CD47 cells (NIH/3T3, manufacturer: ATCC, cat # CRL-1658, the human CD47 mature peptide is transfected into the cells on the basis of NIH/3T3, and a 3T3-CD47 stable expression system is constructed). Taking spleen cells of immunized mice and myeloma cells of the mice to fuse to prepare hybridoma cells, respectively taking CD47 IgV TEV-His and 3T3-CD47 cells as antigens, and screening the hybridoma cells by an indirect ELISA method to obtain the hybridoma cells capable of secreting antibodies specifically combined with CD 47. And (3) screening the hybridoma cells obtained by ELISA screening by competitive ELISA to obtain a hybridoma cell strain which can secrete a monoclonal antibody capable of being combined with a human CD47 IgV TEV-His in a competitive mode and can be in competition with a receptor human SIRP alpha ECD-hFc-Biotin (wherein the SIRP alpha ECD represents an extracellular region of SIRP alpha, and the position 31-373 of a protein GenBank accession number NP-542970.1; and hFc is a human IgG Fc purification label, specifically Ig gamma-1chain C region, GenbankID: P01857 position 114-330), and obtaining the stable hybridoma cell strain by a limited dilution method. The hybridoma cell line was designated as hybridoma cell line LT012, and the monoclonal antibody secreted therefrom was designated as 6F 7.

The hybridoma cell line LT012(CD47-6F7) is preserved in China Center for Type Culture Collection (CCTCC) in 6 and 21 months in 2018, and the preservation number is CCTCC NO: c2018135, the preservation address is China, Wuhan university, postcode: 430072.

2. preparation of anti-CD47 antibody 6F7

The LT011, LT012 and LT015 cell lines prepared above were fractionated with CD Medium (Chemical Defined Medium)Separately, the culture was carried out (CD medium containing 1% streptomycin in 5% CO)₂Cultured in a cell culture chamber at 37 ℃) and after 7 days, the cell culture supernatant was collected and purified by high-speed centrifugation, microfiltration membrane vacuum filtration and HiTrap protein A HP column to obtain antibody 6F 7.

Example 2: sequence analysis of anti-CD47 antibody 6F7

mRNA was extracted from the LT012 cell line cultured in example 1 according to the method of the kit for extracting total RNA from cultured cells (Tiangen, cat # DP 430).

According to InvitrogenIII First-Strand Synthesis System for RT-PCR kit instructions Synthesis of cDNA, and PCR amplification.

The PCR amplification product was directly subjected to TA Cloning, and the specific operation was carried out with reference to the pEASY-T1 Cloning Kit (Transgen CT101) Kit instruction.

The products of the TA clones were sequenced directly, with the following results:

the nucleic acid sequence of the heavy chain variable region is shown as SEQ ID NO: 1, and the length is 351 bp.

The coded amino acid sequence is shown as SEQ ID NO: 2, 117aa in length, wherein the sequence of heavy chain CDR1 is as shown in SEQ ID NO: 5, the sequence of the heavy chain CDR2 is shown in SEQ ID NO: 6, the sequence of the heavy chain CDR3 is shown in SEQ ID NO: shown at 7.

The nucleic acid sequence of the light chain variable region is shown as SEQ ID NO: 3, the length is 321 bp.

The coded amino acid sequence is shown as SEQ ID NO: 4, 107aa in length, wherein the light chain CDR1 has the sequence shown in SEQ ID NO: 8, the light chain CDR2 has the sequence shown in SEQ ID NO: 9, the light chain CDR3 has the sequence shown in SEQ ID NO: shown at 10.

Example 3: design and preparation of light chain and heavy chain of humanized antibodies 6F7H1L1(hG4), 6F7H2L2(hG4) and 6F7H3L3(hG4) against human CD47

1. Light chain and heavy chain design of humanized antibodies 6F7H1L1(hG4), 6F7H2L2(hG4) and 6F7H3L3(hG4) against human CD47

Based on the three-dimensional crystal structure Of human CD47 protein (Hage T, Reinier P, Sebald W. Crystals Of a 1: 1 complex between human interferon-4 and the extracellular domain Of its receptor alpha. char. Eur. J biochem.1998; 258 (2): 831-6.) and the sequence Of antibody 6F7 obtained in example 2, mutations were designed from the model by computer simulation Of an antibody model to obtain variable region sequences Of antibodies 6F7H1L1, 6F7H2L2 and 6F7H3L3 (antibody constant region sequences, from the NCBI database, heavy chain constant region sequences are Ig gamma-4 chamin C region, ACCESSION: P01861.1; light chain constant region sequences are kappa in C region, ACCESSION: Ig P01834).

The variable region sequences were designed as follows:

(1) heavy chain variable region and light chain variable region sequences of humanized monoclonal antibody 6F7H1L1

The nucleic acid sequence of the heavy chain variable region is shown as SEQ ID NO: 11, 351bp in length.

The coded amino acid sequence is shown as SEQ ID NO: 12, 117aa in length, wherein the sequence of heavy chain CDR1 is as set forth in SEQ ID NO: 5, the sequence of the heavy chain CDR2 is shown in SEQ ID NO: 6, the sequence of the heavy chain CDR3 is shown in SEQ ID NO: shown at 7.

The nucleic acid sequence of the light chain variable region is shown as SEQ ID NO: 13, and the length is 321 bp.

The coded amino acid sequence is shown as SEQ ID NO: 14, 107aa in length, wherein the light chain CDR1 has the sequence shown in SEQ ID NO: 8, the light chain CDR2 has the sequence shown in SEQ ID NO: 9, the light chain CDR3 has the sequence shown in SEQ ID NO: shown at 10.

(2) Heavy chain variable region and light chain variable region sequences of humanized monoclonal antibody 6F7H2L2

The nucleic acid sequence of the heavy chain variable region is shown as SEQ ID NO: 15, 351bp in length.

The coded amino acid sequence is shown as SEQ ID NO: 16, 117aa in length, wherein the sequence of heavy chain CDR1 is as set forth in SEQ ID NO: 5, the sequence of the heavy chain CDR2 is shown in SEQ ID NO: 6, the sequence of the heavy chain CDR3 is shown in SEQ ID NO: shown at 7.

The nucleic acid sequence of the light chain variable region is shown as SEQ ID NO: 17, and the length is 321 bp.

The coded amino acid sequence is shown as SEQ ID NO: 18, 107aa in length, wherein the light chain CDR1 has the sequence shown in SEQ ID NO: 8, the light chain CDR2 has the sequence shown in SEQ ID NO: 9, the light chain CDR3 has the sequence shown in SEQ ID NO: shown at 10.

(3) Heavy chain variable region and light chain variable region sequences of humanized monoclonal antibody 6F7H3L3

The variable region in the heavy chain has the nucleic acid sequence shown in SEQ ID NO: 19 and the length of 351 bp.

The coded amino acid sequence is shown as SEQ ID NO: 20, 117aa in length, wherein the sequence of heavy chain CDR1 is as set forth in SEQ ID NO: 5, the sequence of the heavy chain CDR2 is shown in SEQ ID NO: 6, the sequence of the heavy chain CDR3 is shown in SEQ ID NO: shown at 7.

The nucleic acid sequence of the light chain variable region is shown as SEQ ID NO: 21, and the length is 321 bp.

The coded amino acid sequence is shown as SEQ ID NO: 22, 107aa in length, wherein the light chain CDR1 has the sequence shown in SEQ ID NO: 8, the light chain CDR2 has the sequence shown in SEQ ID NO: 9, the light chain CDR3 has the sequence shown in SEQ ID NO: shown at 10.

2. Preparation of humanized antibodies 6F7H1L1(hG4), 6F7H2L2(hG4) and 6F7H3L3(hG4)

The heavy chain constant regions are all formed by Ig gamma-4chain C region, ACCESSION: p01861.1; light chain constant regions were all replaced with Ig kappa chain C region, access: p01834.

Cloning 6F7H1L1(hG4) heavy chain cDNA and light chain cDNA, 6F7H2L2(hG4) heavy chain cDNA and light chain cDNA, and 6F7H3L3(hG4) heavy chain cDNA and light chain cDNA into pUC57simple (provided by Kinsyry company) vector to obtain pUC57simple-6F7H1 and pUC57simple-6F7L 1; pUC57simple-6F7H2, pUC57simple-6F7L2, pUC57simple-6F7H3 and pUC57simple-6F7L 3. Referring to the standard technique introduced in molecular cloning instruction (second edition), EcoRI & HindIII enzyme-cleaved genes synthesized heavy and light chain full-length genes were subcloned into expression vector pcDNA3.1 by restriction enzyme cleavage of restriction enzymes (EcoRI & HindIII); expression plasmids pcDNA3.1-6F7H1, pcDNA3.1-6F7L1, pcDNA3.1-6F7H2 pcDNA3.1-6F7L2, pcDNA3.1-6F7H3 and pcDNA3.1-6F7L3 are obtained, and the heavy-light chain gene of the recombinant expression plasmid is further subjected to sequencing analysis. Then co-transfecting 293F cells with corresponding light and heavy chain recombinant plasmid design gene combinations (pcDNA3.1-6F7H1/pcDNA3.1-6F7L1, pcDNA3.1-6F7H2/pcDNA3.1-6F7L2, and pcDNA3.1-6F7H3/pcDNA3.1-6F7L3), and collecting and purifying culture solution. After the sequencing verification is correct, an expression plasmid with endotoxin removed level is prepared, the plasmid is transiently transfected into HEK293 cells for antibody expression, cell culture solution is collected after 7 days of culture, and a Protein A column (MabSelect SURE (GE)) is adopted for affinity purification to obtain the humanized antibody.

Example 4: design and preparation of light chain and heavy chain of humanized antibodies 6F7H1L1(G1M), 6F7H2L2(G1M) and 6F7H3L3(G1M) against human CD47

1. Light chain and heavy chain design of humanized antibodies 6F7H1L1(G1), 6F7H2L2(G1) and 6F7H3L 3(G1) against human CD47

Based on the three-dimensional crystal structure of the human CD47 protein (Hage T, Reinier P, Sebald W. Crystals of a 1: 1 complex between human interferon-4 and the extracellular domain of its receptor alpha chain. Eur.J biochem.1998; 258 (2): 831-6.) and the sequence of antibody 6F7 obtained in example 2, mutations were designed according to the model by computer modeling to obtain variable region sequences of antibodies 6F7H1L1, 6F7H2L2 and 6F7H3L3 (antibody constant region sequence, from the database of NCBI, heavy chain constant region Ig gamma-1chain C region, ACCESSION: P01857; light chain constant region named kappa in C region, ACCESSION: P01637, humanized antibody P01857, humanized antibody H6323, humanized antibody H639G 7, humanized antibody H2G 7, humanized antibody H3G 7H 2H 639, IgG 6H 3G 7, humanized antibody Ig 7H 2H 3, and IgG 6F7H3L3, respectively).

The variable region sequences of humanized antibodies 6F7H1L1(G1), 6F7H2L2(G1) and 6F7H3L 3(G1) designed in this example were identical to those of 6F7H1L1(hG4), 6F7H2L2(hG4) and 6F7H3L3(hG4) in example 3.

2. Preparation of humanized antibodies 6F7H1L1(G1), 6F7H2L2(G1) and 6F7H3L 3(G1)

The heavy chain constant regions are all formed by Ig gamma-1chain C region, ACCESSION: p01857; light chain constant regions were all replaced with Ig kappa chain C region, access: p01834.

Cloning cDNA of 6F7H1L1 heavy chain and light chain, cDNA of 6F7H2L2 heavy chain and light chain, and cDNA of 6F7H3L3 heavy chain and light chain into pUC57simple (provided by Kinsley company) vector to obtain pUC57simple-6F7H1 and pUC57simple-6F7L 1; pUC57simple-6F7H2, pUC57simple-6F7L2, pUC57simple-6F7H3 and pUC57simple-6F7L 3. Referring to the standard technique introduced in molecular cloning instructions (second edition), EcoRI & HindIII digested full-length heavy and light chain genes were subcloned into expression vector pcDNA3.1 by restriction of restriction enzymes (EcoRI & HindIII) to obtain expression plasmids pcDNA3.1-6F7H1, pcDNA3.1-6F7L1, pcDNA3.1-6F7H2, pcDNA3.1-6F7L2, pcDNA3.1-6F7H3, and pcDNA3.1-6F7L3, and further the heavy and light chain genes of the recombinant expression plasmids were subjected to sequencing analysis. Then co-transfecting 293F cells with corresponding light and heavy chain recombinant plasmid design gene combinations (pcDNA3.1-6F7H1/pcDNA3.1-6F7L1, pcDNA3.1-6F7H2/pcDNA3.1-6F7L2, and pcDNA3.1-6F7H3/pcDNA3.1-6F7L3), and collecting and purifying culture solution. After the sequencing verification is correct, an expression plasmid with endotoxin removed level is prepared, the plasmid is transiently transfected into HEK293 cells for antibody expression, cell culture solution is collected after 7 days of culture, and a Protein A column (MabSelect SURE (GE)) is adopted for affinity purification to obtain the humanized antibody.

3. Design of non-variable region amino acid mutations based on humanized antibodies 6F7H1L1(G1), 6F7H2L2(G1) and 6F7H3L 3(G1)

Based on 6F7H1L1(G1), 6F7H2L2(G1) and 6F7H3L 3(G1), the inventors obtained a new humanized antibody by introducing a point mutation of leucine to alanine at position 234 (L234A) and a point mutation of leucine to alanine at position 235 (L235A) in the hinge region of the heavy chain, and named 6F7H1L1(G1M), 6F7H2L2(G1M) and 6F7H3L3(G1M), respectively.

Example 5 detection of binding Activity of antibodies to antigens by ELISA method

Determination of the binding Activity of antibody 6F7H1L1(hG4) with the antigen human CD47 IgV TEV-His by ELISA method

The experimental steps are as follows: human CD47 IgV TEV-His, 2. mu.g/mL coated ELISA plate, was incubated at 4 ℃ for 12 hours. After incubation, the antigen-coated elisa plate was rinsed 3 times with PBS, and then blocked for 2 hours with 1% BSA PBS solution as elisa plate blocking solution. After blocking the microplate, the plate was rinsed 3 times with PBS. The antibody diluted in PBST solution in gradient is added into the wells of the enzyme label plate, and the antibody dilution gradient is detailed in Table 2. The ELISA plate added with the antibody to be detected is placed at 37 ℃ for incubation for 30 minutes, and after the incubation is finished, the plate is washed 3 times by PBST. After washing the plate, a 1: 5000 dilution of HRP-labeled goat anti-human IgG (H + L) (purchased from Jackson ImmunoResearch Inc., cat # 109-. After incubation, the plate was washed 3 times with PBST, then TMB (Neogen, 308177) was added and developed for 5min in the dark, and stop solution was added to terminate the color development reaction. Immediately putting the ELISA plate into an ELISA reader, and reading the OD value of each hole of the ELISA plate by selecting the wavelength of 450 nm. The data were analyzed using SoftMax Pro 6.2.1 software.

The results of detecting the binding of antibody 6F7H1L1(hG4) to the antigen human CD47 IgV TEV-His are shown in FIG. 1. The OD values for each dose are shown in table 1. Curve fitting is carried out by taking the concentration of the antibody as an abscissa and the absorbance value as an ordinate, and the combined EC of the antibody is calculated₅₀The results are shown in table 1 below.

The results show that 6F7H1L1(hG4) binds human CD47 IgV TEV-His with an EC50 of 0.078nM and a binding activity comparable to Hu5F 9-G4.

TABLE 16F 7H1L1(hG4) results of measurement of binding activity to human CD47 IgV TEV-His

Determination of the binding Activity of antibody 6F7H1L1(G1M) with the antigen human CD47 IgV TEV-His by ELISA method

The results of detecting the binding of antibody 6F7H1L1(G1M) to the antigen human CD47 IgV TEV-His are shown in FIG. 2. The OD values for each dose are shown in Table 2. Curve fitting is carried out by taking the concentration of the antibody as an abscissa and the absorbance value as an ordinate, and the combined EC of the antibody is calculated₅₀The results are shown in Table 2 below.

TABLE 26F 7H1L1(G1M) results of measurement of binding Activity to human CD47 IgV TEV-His

The results show that the binding EC50 of 6F7H1L1(G1M) and human CD47 IgV TEV-His is 0.037nM, and the binding activity is slightly better than Hu5F 9-G4.

3. Competitive ELISA method for respectively determining activity of antibody 6F7H1L1(hG4) and human SIRP alpha ECD-hFc-Biotin for competitive binding to human CD47 IgV TEV-His

The experimental steps are as follows: the plate was coated with 2. mu.g/mL human CD47 IgV TEV-His, 50. mu.L per well and incubated at 4 ℃ for 16 hours. Plates were rinsed once and blocked by adding 300 μ L of 1% BSA solution (dissolved in PBS) to each well, incubated for 2 hours at 37 deg.C, and plates were rinsed three times. The antibody was diluted to 3. mu.g/mL (final concentration 1.5. mu.g/mL) as the starting concentration, and a gradient dilution of 1: 3 was performed on the microplate for 7 concentrations, while blank controls were set, each in 2 duplicate wells with 50. mu.L volume, and incubated at room temperature for 10 minutes. 0.2. mu.g/mL (final concentration of 0.1. mu.g/mL) of human SIRP. alpha. ECD-hFc-Biotin (synthesized by Zhongshan Kangfang biomedical Co., Ltd.) was added to the microplate, 50. mu.L per well volume was gently mixed with the antibody in a volume ratio of 1: 1, and incubated at 37 ℃ for 30 minutes. The plates were rinsed three times, 50. mu.L of SA-HRP (KPL, 14-30-00) working solution was added to each well, and incubated at 37 ℃ for 30 minutes. And (3) washing the plate for four times, adding 50 mu L of TMB color development solution into each hole, and after 5 minutes of light-shielding color development at room temperature, adding 50 mu L of stop solution into each hole to stop the color development reaction. Immediately putting the ELISA plate into an ELISA reader, selecting the wavelength of light with 450nm to read the OD value of each hole of the ELISA plate, and analyzing and processing the data by using SoftMax Pro 6.2.1 software.

The results of the detection are shown in FIG. 3. The OD values for each dose are shown in Table 3. Bound EC50 was obtained by performing absorbance intensity quantification of bound antibody and the curve simulates the binding efficiency of the antibody (table 3).

The results show that 6F7H1L1(hG4) can effectively block the combination of antigen human CD47 IgV TEV-His and receptor human SIRP alpha ECD-hFc-Biotin, the blocking efficiency shows a dose-dependent relation, 6F7H1L1(hG4) has the blocking EC50 of 0.194nM, and the blocking activity is the same as Hu5F 9-G4.

Table 3.6F 7H1L1(hG4) and human SIRP α ECD-hFc-Biotin compete for binding to human CD47 IgV TEV-His activity assay results

4. Determination of antibody 6F7H1L1(G1M) and human SIRP alpha by competitive ELISA method

Activity of ECD-hFc-Biotin to compete for binding to human CD47 IgV TEV-His

The experimental steps are as follows: the plate was coated with 2. mu.g/mL human CD47 IgV TEV-His, 50. mu.L per well and incubated at 4 ℃ for 16 hours. Plates were rinsed once and blocked by adding 300. mu.L of 1% BSA solution (dissolved in PBS) to each well, incubated for 2 hours at 37 ℃ and rinsed three times. The antibody was diluted to 3. mu.g/mL (final concentration 0.5. mu.g/mL) as the starting concentration, and a gradient dilution of 1: 3 was performed on the microplate for 7 concentrations, while blank controls were added, each in 2 duplicate wells with 50. mu.L volume, and incubated at room temperature for 10 minutes. 0.2 mu g/mL (final concentration is 0.1 mu g/mL) of human SIRP alpha ECD-hFc-Biotin is added into the ELISA plate, 50 mu L of human SIRP alpha ECD-hFc-Biotin is gently mixed with the antibody volume of 1: 1 in each pore volume, and the mixture is incubated for 30 minutes at 37 ℃. The plates were rinsed three times, 50. mu.L of SA-HRP (KPL, 14-30-00) working solution was added to each well, and incubated at 37 ℃ for 30 minutes. And (3) washing the plate for four times, adding 50 mu L of TMB color development solution into each hole, and after 5 minutes of light-shielding color development at room temperature, adding 50 mu L of stop solution into each hole to stop the color development reaction. Immediately putting the ELISA plate into an ELISA reader, selecting the wavelength of light with 450nm to read the OD value of each hole of the ELISA plate, and analyzing and processing the data by using SoftMax Pro 6.2.1 software.

The results of the detection are shown in FIG. 4. The OD values for each dose are shown in Table 4. Bound EC50 was obtained by performing absorbance intensity quantification of bound antibody and the curve simulates the binding efficiency of the antibody (table 4).

TABLE 46F 7H1L1(G1M) Activity assay results for human SIRP α ECD-hFc-Biotin competing for binding to human CD47 IgV TEV-His

The results show that 6F7H1L1(G1M) can effectively block the combination of antigen human CD47 IgV TEV-His and receptor human SIRP alpha ECD-hFc-Biotin, the blocking efficiency shows a dose-dependent relation, 6F7H1L1(G1M) has the blocking EC50 of 0.274nM, and the blocking activity is equivalent to Hu5F 9-G4.

Example 6: determination of affinity constant of murine antibody 6F7 with human CD47

Kinetic parameters of the binding of murine antibody 6F7 to the antigen human CD47 IgV TEV-His were determined using a Fortebio molecular interaction apparatus (Forteio, model: QKe).

EDC/NHS was used to activate the AR2G sensor (Forteio, cat # 18-5092), the antibody was immobilized in an amino-coupled manner to the activated AR2G sensor, the sensor was equilibrated in PBST for 300s, the antigen immobilized on the sensor was bound to the antibody at an antigen concentration of 3.125-100nM (two-fold gradient dilution) for a binding time of 420s, and the antigen antibody was dissociated in PBST for a time of 600 s.

The results of the determination of the affinity constants of murine antibodies 6F7 and Hu5F9-G4 (as control antibodies) and human CD47 IgV TEV-His are shown in Table 5, and the results are shown in FIG. 5.

TABLE 5 detection results of affinity constants of murine antibody 6F7 and human CD47

KD is the affinity constant; KD-kdis/kon.

The results show that: as shown in Table 5 and FIG. 5, the affinity constants of murine antibodies 6F7 and Hu5F9-G4 and human CD47 IgV TEV-His were 6.52E-10M and 6.38E-10M, respectively, and the results of the two affinity constants were comparable. It was suggested that the CDR regions of 6F7 have binding ability to CD47 comparable to Hu5F9-G4, and all have stronger binding ability.

Example 7.6F7 determination of the affinity constant of H1L1(hG4) for human CD47

According to the instructions, the affinity constants of antibody 6F7H1L1(hG4) and human CD47 IgV TEV-His were determined using a Biacore molecular interaction apparatus (Forteio, model: QKe). The buffer solution is PBST, and the human CD47 IgV-TEV-His is fixed on the surface of a CM5 chip in an amino coupling mode, and the fixed signal value is 171.6 RU. The antibody bound to human CD47 at an antibody concentration of 0.78-12.5nM (two-fold dilution), a flow rate of 30 μ L/min, an association time of 120s, and a dissociation time of 300 s. Chip uses 3M MgCl₂Regeneration was carried out at a flow rate of 30. mu.L/min for a period of 30 s. The data were analyzed by fitting a 1: 1 model to obtain affinity constants. Data acquisition was performed using Biacore Control 2.0 software and data analysis was performed using Biacore T200 Evaluation 2.0 software. The results of the detection of affinity constants of 6F7H1L1(hG4) and Hu5F9-G4 (as control antibody) and human CD47 IgV TEV-His are shown in Table 6, and are shown in FIGS. 6 and 7.

The results show that: as shown in the figure, the affinity constant of 6F7H1L1(hG4) and human CD47 IgV TEV-His is 1.52E-10M, and the affinity constant of Hu5F9-G4 and human CD47 IgV TEV-His is 4.42E-11M, which indicates that 6F7H1L1(hG4) has stronger binding ability with human CD 47.

TABLE 6.6F 7H1L1(hG4) and human CD47 IgV TEV-His affinity constant assay results

Name of antibody

KD(M)

ka(1/Ms)

SE(ka)

kd(1/s)

SE(kd)

Rmax(RU)

6F7H1L1(hG4)

1.52E-10

2.54E+06

1.48E+04

3.88E-04

1.14E-06

253.21-272.60

Hu5F9-G4

4.42E-11

3.00E+06

8.49E+03

1.32E-04

5.69E-07

238.81-327.23

KD is the affinity constant; KD-kdis/kon.

Example 8: 6F7H1L1(G1M) cell biological Activity Studies

FACS method for detecting 6F7H1L1(G1M) and normal human RBC binding condition

The separation operation of normal human red blood cells is carried out in a biological safety cabinet: uniformly mixing the blood buffer solution A and the blood buffer solution B according to the proportion of 1: 9 to obtain a blood buffer solution; uniformly mixing 20mL of fresh blood with 60mL of blood buffer solution; adding 15mL of Ficoll plain separating medium into a 50mL centrifuge tube, and slowly adding diluted fresh blood on the liquid surface of the separating medium according to the volume ratio of 3: 4, namely adding 20mL of diluted blood into each tube; centrifuging at 1550rpm for 30 min; carefully sucking up RBC at the bottom of the centrifuge tube and centrifugally washing 3 times by PBS; resuspend the cell pellet with 500 μ L of 1% PBSA and count; adjusting the concentration of RBC cells, transferring the cells to a 1.5mL centrifuge tube according to 30 ten thousand cells per tube; centrifuging at 5600rpm/min for 5min, and removing the supernatant; adding antibodies with corresponding concentrations (final concentration is 100, 10, 1, 0.1, 0.01, 0.001, nM) according to experimental design, each tube is 100 muL, and Blank (PBSA + cells) and isotype control (hIgG) groups are designed and incubated for 1h on ice; adding 1% PBSA 500 μ L, centrifuging at 5600rpm/min for 5min, and removing supernatant; adding 100 μ L FITC goat anti-human IgG (1: 500) into each tube, mixing, and incubating on ice in dark for 30 min; adding 500 μ L of 1% PBSA, centrifuging at 5600rpm/min for 5min, and removing supernatant; 200 μ L of 1% wash buffer/tube resuspended cells and the fluorescence signal detected on a flow cytometer using a FITC channel.

Results of Floweng software analysis, GraphPad prism 5 were curve-fitted with MFI and sample concentration, respectively, to calculate EC 50.

The results of 6F7H1L1(G1M) binding to CD47 on the cell membrane surface of normal human RBC are shown in FIG. 8. The results show that both 6F7H1L1(G1M) and the homologous point marketed drug Hu5F9-G4 can be specifically combined with CD47 on the cell membrane surface of normal human RBC, the combined EC50 is respectively 0.077nM and 0.057nM, and the combined activities of the two are equivalent.

FACS method for detecting binding activity of 6F7H1L1(G1M) and Raji

Collecting Raji cells in logarithmic phase, centrifuging, washing, resuspending cell pellet with 1% PBSA, counting cell number and survival rate at 3.0 × 10⁵Cells/500. mu.L/tube cells were transferred to a 1.5mL centrifuge tube; centrifuging at 5600rpm for 5min, and removing the supernatant; the corresponding antibodies were added in a gradient dilution of 100. mu.L per tube according to the experimental design, and a blank (PBSA + fine particles) was designedCell) and isotype control group (hIgG), incubated on ice for 1 hr; after 1hr, adding 1% PBSA, centrifuging at 5600rpm for 5min, and removing supernatant; adding 100 μ L FITC goat anti-human IgG (1: 500) into each tube, mixing, and incubating on ice in dark for 30 min; adding 500 μ L of 1% PBSA, centrifuging at 5600rpm for 5min, and removing supernatant; 1% PBSA/tube heavy suspension cells, flow cytometry using FITC channel detection of fluorescence signal. EC50 was calculated by curve fitting MFI and sample concentration.

The results of the binding activity of 6F7H1L1(G1M) to Raji are shown in FIG. 9. As shown in the figure, the binding test results show that both 6F7H1L1(G1M) and Hu5F9-G4 can specifically bind to Raji cell membrane surface CD47, and the binding EC50 is 0.013nM and 0.012nM respectively, and the binding activities of the two are equivalent.

FACS method for detecting biological activity of 6F7H1L1(G1M) and SIRP competing for LOVO binding

Logarithmic phase LOVO cells (China academy of sciences cell center, code: bio-73085) were collected routinely and washed by centrifugation. 1% PBSA resuspends the cell pellet, counts the cell number and the survival rate; adjusting the cell concentration to a proper range by 1% PBSA, transferring the cells to a 1.5mL centrifuge tube according to groups, centrifuging the cells at 5600rpm for 5min after 500 mu L of cells in each tube, and discarding the supernatant; antibodies after gradient dilution (final concentrations: 300, 100, 10, 1, 0.3, 0.1, 0.01, 0.001, 0.0001nM from high to low) were added, while a blank control (100. mu.L of 1% PBSA plus cells) and an isotype control (human IgG) were set, and incubated on ice for 30 min. Adding 100 mu L SIRP alpha-mFc into each tube, mixing uniformly, keeping the final concentration at 20nM, and continuously incubating for 1h on ice; adding 1% PBSA, centrifuging at 5600rpm for 5min, and removing supernatant; adding FITC goat anti-mouse IgG, diluting with 100 μ L/tube (1: 500), and incubating on ice in dark for 40 min; 1% PBSA was added, centrifuged at 5600rpm for 5min, and the supernatant was discarded. Adding 200. mu.L of 1% PBSA to resuspend the cell pellet, and transferring to a flow-type sample loading tube; fluorescence signals were detected on a flow cytometer using the FITC channel. EC50 was calculated by curve fitting MFI and sample concentration.

Activity assay of 6F7H1L1(G1M) competed with SIRP for binding to LOVO. The results are shown in FIG. 10. As shown in the figure, both 6F7H1L1(G1M) and Hu5F9-G4 can compete with SIRP for binding to the surface CD47 of the LOVO membrane, so that the binding of SIRP to CD47 is blocked, and the competitive binding of the SIRP to EC50 is respectively 0.16nM and 0.24nM, and the binding activities of the two are equivalent.

4.6F 7H1L1(G1M) Effect on RBC agglutination in Normal humans

Preparation of normal human RBCs: human blood PBMC were isolated according to the Ficoll-Paque Plus reagent instructions and the bottom erythrocytes were precipitated for this experiment; the red blood cells were diluted with PBS to a red blood cell concentration of 1 x 10⁷Per mL, obtaining a red blood cell suspension; adding the erythrocyte suspension into a round bottom 96-well plate, adding a positive antibody with corresponding concentration, adding 0.1g/mL Dextran T500 for control, adding corresponding hIgG or PBS for negative control, and culturing at 37 ℃ for 4 hours; the agglutination of the red blood cells was observed and photographed.

The effect of 6F7H1L1(G1M) on agglutination of normal human erythrocytes is shown in FIG. 11. As shown in the figure, 6F7H1L1(G1M) and the control antibody Hu5F9-G4 have no influence on erythrocyte agglutination when the antibody concentration is lower than 20 mu G/mL, but when the antibody concentration is higher than 20 mu G/mL, the obvious erythrocyte agglutination phenomenon can be seen in Hu5F9-G4, and 6F7H1L1(G1M) has no influence on erythrocyte agglutination.

Example 9: 6F7H1L1(hG4) cell biological activity study

FACS method for detecting 6F7H1L1(hG4) and normal human RBC binding condition

The experimental steps are as follows: adding 1g of blood buffer solution A (D- (+) -glucose, CaCl₂：0.0056g，MgCl₂6H 2O: 0.1992g, KCl: 0.4026g, Tris: 17.5650g, the above reagents were dissolved in 1L of ultrapure water) and B (NaCl: 8.19g of the buffer solution was dissolved in 1L of ultrapure water) was mixed uniformly in a ratio of 1: 9 to obtain a blood buffer solution. Fresh blood was mixed well with blood buffer (dilution ratio of concentrated blood 1: 3). 15mL of Ficoll plane plus fraction (GE, cat # 17-1440-02) was added to a 50mL centrifuge tube, and diluted fresh blood was slowly added to the surface of the fraction at a volume ratio of 3: 4, i.e., 20mL of diluted blood was added to each tube. Centrifuge 1550rpm for 30min after each tube was trimmed. The cells PBMC of the middle leucocyte layer were aspirated. Adding blood buffer solution according to the ratio of cell volume to blood buffer solution of 1: 4, mixing uniformly, centrifuging at 950rpm, discarding supernatant after 15 min. The PBMC were suspended by adding 20mL of blood buffer, centrifuged and the supernatant discarded. Centrifuged and washed twice. With 10mL RPMI-1640 (F-free)BS) cells were washed once. Centrifuging to remove supernatant, resuspending cells with 5mL RPMI-1640 (containing 10% FBS), counting, adding 3 × 105 cells/sample, adding 500 μ L1% PBSA per tube, centrifuging at 5600rpm for 5min, and removing supernatant; after adding the corresponding concentration of antibody (final concentration of 300, 100, 10, 1, 0.1, 0.01, 0.001nM) 100. mu.L/tube, Blank (PBSA + cells) and isotype control (human IgG) were designed and incubated on ice for 1 h; adding 1% PBSA, centrifuging at 5600rpm for 5min, and removing supernatant; each tube was added with 100. mu.L of FITC goat anti-human IgG (Jacson, cat # 109-; adding 1% PBSA, centrifuging at 5600rpm for 5min, and removing supernatant; 200 μ L wash buffer/tube, resuspend cells, and detect fluorescent signal with FITC channel on a flow cytometer. Results of Floweng software analysis, GraphPad prism 5 were curve-fitted with MFI and sample concentration, respectively, to calculate EC 50.

The results of 6F7H1L1(hG4) binding to CD47 on the cell membrane surface of normal human RBCs are shown in fig. 12 and table 7. The results show that both 6F7H1L1(hG4) and the homologous point marketed drug Hu5F9-G4 can be specifically combined with CD47 on the cell membrane surface of normal human RBC, the combined EC50 is 0.60nM and 0.06nM respectively, and the affinity of Hu5F9-G4 and RBC is about 10 times higher than that of 6F7H1L1(hG 4).

TABLE 7 results of FACS detection of binding of anti-CD47 antibody to human RBC

Concentration (nM)/MFI	0.00123	0.0123	0.123	1.23	3.7	11.1	33.3	EC50
									Hu5F9-G4	19.48	59.35	132.68	212.25	207.52	219.34	219.02	0.06
6F7 H1L1(hG4)	10.25	19.55	47.29	134.58	152.50	185.31	190.18	0.60

FACS method for detecting binding activity of 6F7H1L1(hG4) and Rajji

The biological activity of the CD47 antibody combined with tumor cells Raji (cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences, Cat. No.: TCHU44) was detected by flow cytometry.

Raji cell counts and viability, 3 x 10⁵Cells/sample, 500. mu.L of 1% PBSA per tube, centrifuged at 5600rpm for 5min, discarding the supernatant; adding corresponding antibodies diluted in a gradient manner according to the experimental design, designing a blank (PBSA + cells) and an isotype control group (human IgG), and incubating for 1h on ice; adding 1% PBSA, centrifuging at 5600rpm for 5min, and removing supernatant; adding 100 μ L FITC goat anti-human IgG (1: 500) or FITC goat anti-mouse IgG (1: 500) into each tube, mixing, and incubating on ice in dark for 30 min; adding 500 μ L of 1% PBSA, centrifuging at 5600rpm for 5min, and removing supernatant; 200 μ L wash buffer/tube, resuspend cells, and detect fluorescent signal with FITC channel on a flow cytometer.

The results of the binding activity of 6F7H1L1(hG4) to Raji are shown in fig. 13 and table 8. As shown in the figure and the table, the binding test results show that both 6F7H1L1(hG4) and Hu5F9-G4 can be specifically bound with Raji cell membrane surface CD47, and the binding EC50 is 0.32nM and 0.22nM respectively, and the activities of the two are equivalent.

TABLE 8 results of FACS detection of the binding of anti-CD47 antibody to Raji tumor cells

Detection of competitive binding Activity of 6F7H1L1(hG4) and SIRP for competitive binding to Raji by FACS method

Log phase Raji cells were collected routinely and washed by centrifugation. 1% PBSA resuspends the cell pellet, counts the cell number and the survival rate; adjusting the cell concentration to a proper range by 1% PBSA, transferring the cells to a 1.5mL centrifuge tube according to groups, centrifuging the cells at 5600rpm for 5min after 500 mu L of cells in each tube, and discarding the supernatant; antibodies after gradient dilution (final concentrations from high to low: 1, 0.3, 0.1, 0.01, 0.001, 0.0001nM) were added, while a blank control (100. mu.L of 1% PBSA plus cells) and an isotype control (human IgG) were set, and incubated on ice for 30 min. Adding 100 μ L SIRP α -ECD-mFc (sequence of mFc is shown in SEQ ID NO: 71) into each tube to make final concentration 20nM, mixing, and further incubating on ice for 1 h; adding 1% PBSA, centrifuging at 5600rpm for 5min, and removing supernatant; adding FITC goat anti-mouse IgG, diluting with 100 μ L/tube (1: 500), and incubating on ice in dark for 40 min; add 500. mu.L of 1% PBSA, centrifuge at 5600rpm for 5min, and discard the supernatant. Add 200. mu.L of 1% PBSA to resuspend the cell pellet and detect the fluorescent signal on a flow cytometer using the FITC channel.

The results of 6F7H1L1(hG4) competing with SIRP for binding to tumor cells Raji are shown in fig. 14 and table 9. As shown in the figure and table, 6F7H1L1(hG4) and Hu5F9-G4 can compete with SIRP to bind to Raji membrane surface CD47, block the binding of SIRP to CD47, and compete with EC50 at 0.017nM and 0.014nM respectively, and the competitive binding activity of the two is equivalent.

TABLE 9 results of FACS detection of anti-CD47 antibody competing with SIRP for binding to Raji cells

Concentration (nM)/MFI	0.0001	0.001	0.01	0.1	0.3	1	EC50
								Hu5F9-G4	54.83	49.37	37.16	10.77	10.34	11.4	0.014
6F7 H1L1(hG4)	50.27	54.37	42.64	16.80	15.66	14.83	0.017

FACS method for detecting binding activity of 6F7H1L1(hG4) and LOVO

Log phase LOVO cells (China academy of sciences cell center, code: bio-73085) were collected, washed by centrifugation, 500. mu.L of 1% PBSA was resuspended in the cell pellet, and the cell count and viability were counted at 3.0 × 10⁵Cells/500. mu.L/tube cells were transferred to a 1.5mL centrifuge tube; centrifuging at 5600rpm for 5min, and discarding the supernatant; add corresponding antibody diluted in gradient according to the experimental design, each tube 100 u L, and design blank (PBSA + cell) and isotype control group (human IgG, its heavy chain sequence is SEQ ID NO: 72, light chain sequence is SEQ ID NO: 73), incubate 1 hour on ice; then, 500. mu.L of 1% PBSA was added, centrifuged at 5600rpm for 5 minutes, and the supernatant was removed; adding 100 μ L FITC goat anti-human IgG (1: 500) into each tube, mixing, and incubating for 30min on ice in dark; adding 500 μ L1% PBSA, centrifuging at 5600rpm for 5min, and removing supernatant; 200 μ L1% PBSA/tube heavy suspension cells, transferred to the flow-through loading tube; detecting by an up-flow cytometer BD FACSCalibur; results of Floweng software analysis, GraphPad prism 5 were curve-fitted with MFI and sample concentration, respectively, to calculate EC 50.

The results of binding of 6F7H1L1(hG4) to tumor cell LOVO are shown in fig. 15 and table 10. As shown in the figure and the table, both 6F7H1L1(hG4) and Hu5F9-G4 can be specifically combined with the LOVO cell membrane surface CD47, the combination EC50 is 0.02nM and 0.06nM respectively, and 6F7H1L1(hG4) is slightly better than Hu5F 9-G4.

TABLE 10 results of FACS detection of binding of anti-CD47 antibody to LOVO

Concentration (nM)/MFI	0.0001	0.001	0.01	0.1	0.3	1	EC50
								Hu5F9-G4	11.35	13.04	30.44	102.94	145.13	150.57	0.06
6F7 H1L1(hG4)	15.26	25.41	48.15	135.95	146.67	149.81	0.02

FACS method for detecting biological activity of 6F7H1L1(hG4) and SIRP in competition binding to LOVO

Experimental procedure as in example 3, only Raji cells were changed to LOVO cells.

The results of the activity assay of 6F7H1L1(hG4) in competition with SIRP for binding to LOVO are shown in fig. 16 and table 11. As shown in the figure and the table, both 6F7H1L1(hG4) and Hu5F9-G4 can compete with SIRP to bind to the surface CD47 of the LOVO membrane, so that the combination of SIRP and CD47 is blocked, the competitive binding of EC50 is 0.10nM and 0.24nM respectively, and 6F7H1L1(hG4) is slightly better than that of Hu5F 9-G4.

TABLE 11 results of FACS detection of anti-CD47 antibody competing with SIRP for binding to LOVO cells

Concentration (nM)/MFI	0.0001	0.001	0.01	0.1	0.3	1	10	100	300	EC50
											Hu5F9-G4	69.81	62.47	64.45	47.30	38.98	11.63	10.51	9.59	9.93	0.24
6F7 H1L1(hG4)	56.79	59.59	64.52	34.21	23.76	26.52	17.46	11.35	9.44	0.10

6. Effect of anti-CD47 antibodies on RBC agglutination in Normal humans

Preparation of normal human RBCs: human blood PBMC were isolated according to the isolate Ficoll-Paque _ Plus (GE, cat # 17-1440-02) reagent instructions and the bottom red blood cells were pelleted for this experiment; the red blood cells were diluted with PBS to a red blood cell concentration of 1 x 10⁷Per mL, obtaining a red blood cell suspension; adding the erythrocyte suspension into a round-bottom 96-well plate, adding a positive antibody with corresponding concentration, and adding 0.1g/mL Dextran T500 as a controlAdding corresponding human IgG1 (Kangfang organism) or PBS (PBS) into the negative control, and culturing for 4 hours at 37 ℃; the agglutination of the red blood cells was observed and photographed.

The effect of 6F7H1L1(hG4) on hemagglutination in normal human erythrocytes is shown in FIG. 17. As shown, 6F7H1L1(hG4) did not cause hemagglutination at all concentrations tested, and the control antibody Hu5F9-G4 at concentrations equal to less than 3.3 μ G/mL did not cause hemagglutination. When the concentration is higher than or equal to 10 mu G/mL, the obvious hemagglutination phenomenon can be seen in Hu5F 9-G4.

Example 10: therapeutic effect of 6F7H1L1(hG4) on MDA-MB-231 subcutaneous transplantation tumor

The in vivo activity of 6F7H1L1(hG4) was studied by measuring the volume of human breast cancer cell MDA-MB-231 subcutaneously transplanted tumors on SCID/beige mice after 6F7H1L1(hG4) administration. The collected MDA-MB-231(ATCC, Cat: HTB-26) cells were cultured at 5X 10⁶One cell/mouse was inoculated subcutaneously into the right flank of the SCID/beige mouse, and a total of 40 mice were inoculated. When the tumor volume reaches 100-³On the left and right, mice were divided into 5 groups on the average according to tumor volume, a model group, a Hu5F9-G4 high dose group, a Hu5F9-G4 low dose group, a 6F7H1L1(hG4) high dose group, and a 6F7H1L1(hG4) low dose group, the high dose group was administered at 0.2mg/kg, the low dose group was administered at 0.02mg/kg, and the divided day was designated as D0, and were administered at D0, D3, D7, D10, D14, and D17, respectively, and 7 mice per group were administered.

Tumor size was measured twice weekly after grouping using a vernier caliper and according to the calculation formula TV-0.5 xab²Tumor volume was calculated, where a is the major diameter of the tumor, b is the minor diameter of the tumor, TV is the tumor volume, TGI (%) was calculated from the tumor volume (tumor growth inhibition ratio), formula: % TGI ═ 100% (1- (Ti-T0)/(Ci-C0)), Ti and Ci are mean tumor volumes at day i of the administered group and the model group, respectively, and T0 and C0 are mean tumor volumes at day 0 of the administered group and the model group, respectively. And (4) performing single-factor analysis of variance and evaluation on results after the group comparison is performed by GraphPad statistical processing software.

The results are shown in fig. 18, at day 24 after grouping, the control antibody Hu5F9-G4 high dose group and 6F7H1L1(hG4) high dose group were both able to effectively inhibit the growth of MDA-MB-231 tumors (P < 0.01), and Hu5F9-G4 and 6F7H1L1(hG4) had dose-effect relationship on the inhibition of MDA-MB-231 tumor growth, TGI (%) of the control antibody Hu5F9-G4 high dose group, 6F7H1L1(hG4) high dose group and 6F7H1L1(hG 9) low dose group were 67%, 63% and 25%, respectively, and compared with the control antibody group, the 6F7H1L1(hG4) low dose group was significantly better than hu5F9-G4, and the high dose was equivalent to 6F7H1L1(hG 1) high dose (3605) and the control antibody group, the efficacy of the high dose was equivalent to hui (%) of the control antibody group (3605).

Example 11: effect of a Single dose of 6F7H1L1(hG4) and Hu5F9-G4 cynomolgus monkey on hemoglobin and hematocrit

4 cynomolgus monkeys, according to weight, sex random classification into 2 groups, each group of 2, male and female half. The test was carried out in 6F7H1L1(hG4) group and Hu5F9-G4 group at a dose of 10mg/kg, administered intravenously. Hematology analyzers detect Hemoglobin (Hemoglobin), Hematocrit (Hematocrit).

The results are shown in FIGS. 19 and 20, and Table 12.

The results show that after single intravenous injection of 10mg/kg, 6F7H1L1(hG4) and Hu5F9-G4 in cynomolgus monkeys, hemoglobin and hematocrit are reduced to different degrees after administration, and the lowest point of anemia appears in 2-7 days, wherein the anemia of Hu5F9-G4 is more serious than that of 6F7H1L1(hG 4); anemia in both antibodies can return spontaneously, to baseline levels approximately 20 days after dosing.

TABLE 12.6F 7H1L1(hG4) and Hu5F9-G4 cynomolgus monkeys individual data for hemoglobin and hematocrit after a single administration

While the embodiments of the present invention have been described in detail, the invention is not limited to the embodiments, and those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the invention, and such equivalent modifications or substitutions are included in the scope of the claims of the present application.

A sequence table:

6F7 heavy chain variable region:

CAGGTGCAGCTGCAGCAGCCAGGAGCAGAGCTGGTGAGGCCAGGAGCATCCGTGAAGCTGTCTTGTAAGGCCAGCGGCTACACCTTCACATCCTATTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGACTGGAGTGGATCGGCATGATCGACCCAAGCGATTCCGAGACCCACAACAATCAGATGTTTAAGGACAAGGCCACCCTGACAGTGGATAAGAGCTCCAATACCGCCTACATGCACCTGTCTAGCCTGACATCTGAGGACAGCGCCGTGTATCACTGCGCCCGGCTGTACAGATGGTATTTTGACGTGTGGGGAGCAGGAACCACAGTGACCGTGTCCTCT(SEQ ID NO：1)

QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIDPSDSETHNNQMFKDKATLTVDKSSNTAYMHLSSLTSEDSAVYHCARLYRWYFDVWGAGTTVTVSS(SEQ ID NO：2)

6F7 light chain variable region:

AACATCGTGATGACCCAGTCCCCCAAGTCTATGAGCATGTCCCTGGGCGAGAGGGTGACCCTGTCCTGTAAGGCCTCTGAGATCGTGGGCACATACGTGTCTTGGTTTCAGCAGAAGCCACACCAGAGCCCCAAGCTGCTGATCTACGGCGCCTCCAATCGGTATACAGGCGTGCCTGACAGATTCACCGGCTCTGGCAGCGCCACAGACTTCACCCTGACAATCTCTAACGTGCAGGCCGAGGACCTGGCCGATTATCACTGCGGCCAGAGCTACAATTTCCCTTATACCTTTGGCGGCGGCACAAAGCTGGAGATCAAG(SEQ ID NO：3)

NIVMTQSPKSMSMSLGERVTLSCKASEIVGTYVSWFQQKPHQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISNVQAEDLADYHCGQSYNFPYTFGGGTKLEIK(SEQ ID NO：4)

6F7CDR

HCDR1：GYTFTSYW(SEQ ID NO：5)

HCDR2：IDPSDSET(SEQ ID NO：6)

HCDR3：ARLYRWYFDV(SEQ ID NO：7)

LCDR1：EIVGTY(SEQ ID NO：8)

LCDR2：GAS(SEQ ID NO：9)

LCDR3：GQSYNFPYT(SEQ ID NO：10)

6F7H1：

CAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGCTGAGCTGTAAGGCCTCCGGCTACACCTTCACAAGCTATTGGATGAACTGGGTGCGGCAGAGACCAGGACAGGGACTGGAGTGGATCGGAATGATCGACCCTTCCGATTCTGAGACCCACAATGCCCAGAAGTTTCAGGGCAAGGCCACCCTGACAGTGGACAAGAGCACCTCCACAGCCTACATGCACCTGAGCTCCCTGCGGTCCGAGGACACAGCCGTGTACTATTGCGCCAGGCTGTACCGCTGGTATTTTGACGTGTGGGGAGCAGGAACCACAGTGACCGTGTCTAGC(SEQ ID NO：11)

QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMNWVRQRPGQGLEWIGMIDPSDSETHNAQKFQGKATLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSS(SEQ ID NO：12)

6F7L1：

AACATCGTGATGACCCAGTCCCCAGCCACAATGTCTATGAGCCCAGGAGAGAGGGTGACCCTGTCCTGTAGAGCCTCTGAGATCGTGGGCACATACGTGTCTTGGTTTCAGCAGAAGCCAGGACAGGCACCTAGGCTGCTGATCTACGGAGCAAGCAACAGGTATACCGGAGTGCCAGCACGCTTCTCCGGCTCTGGCAGCGGCACAGACTTTACCCTGACAATCAGCTCCGTGCAGCCTGAGGACCTGGCCGATTATCACTGCGGCCAGTCTTACAATTTCCCATATACCTTTGGCGGCGGCACAAAGCTGGAGATCAAG(SEQ ID NO：13)

NIVMTQSPATMSMSPGERVTLSCRASEIVGTYVSWFQQKPGQAPRLLIYGASNRYTGVPARFSGSGSGTDFTLTISSVQPEDLADYHCGQSYNFPYTFGGGTKLEIK(SEQ ID NO：14)

6F7H2：

CAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGGTGAGCTGTAAGGCCTCCGGCTACACCTTCACATCCTATTGGATGAACTGGGTGCGGCAGAGACCAGGACAGGGACTGGAGTGGATCGGAATCATCGACCCTTCCGATTCTGAGACCTCTAATGCCCAGAAGTTTCAGGGCCGGGTGACCCTGACAGTGGACAAGAGCACCTCCACAGCCTACATGCACCTGAGCTCCCTGAGGAGCGAGGACACAGCCGTGTACTATTGCGCCAGGCTGTACCGCTGGTATTTTGACGTGTGGGGAGCAGGAACCACAGTGACCGTGTCTAGC(SEQ ID NO：15)

QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYWMNWVRQRPGQGLEWIGIIDPSDSETSNAQKFQGRVTLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSS(SEQ ID NO：16)

6F7L2：

AACATCGTGATGACCCAGTCCCCAGCCACACTGTCTCTGAGCCCAGGAGAGAGGGTGACCCTGTCCTGTAGAGCCTCTGAGATCGTGGGCACATACGTGTCTTGGTTTCAGCAGAAGCCAGGACAGGCACCTAGGCTGCTGATCTATGGCGCCAGCAACAGGGCAACCGGCATCCCCGCACGCTTCTCCGGCTCTGGCAGCGGCACAGACTTTACCCTGACAATCAGCTCCCTGCAGCCTGAGGACCTGGCCGATTACTATTGCGGCCAGTCTTACAATTTCCCATATACCTTTGGCGGCGGCACAAAGCTGGAGATCAAG(SEQ ID NO：17)

NIVMTQSPATLSLSPGERVTLSCRASEIVGTYVSWFQQKPGQAPRLLIYGASNRATGIPARFSGSGSGTDFTLTISSLQPEDLADYYCGQSYNFPYTFGGGTKLEIK(SEQ ID NO：18)

6F7H3：

CAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGGTGAGCTGTAAGGCCTCCGGCTACACCTTCACATCCTATTGGATGAACTGGGTGCGGCAGGCACCAGGACAGGGACTGGAGTGGATCGGCATCATCGACCCTTCCGATTCTGAGACCTCTTACGCCCAGAAGTTTCAGGGCAGGGTGACCCTGACAGTGGACAAGAGCACCTCCACAGCCTATATGGAGCTGAGCTCCCTGCGCAGCGAGGACACAGCCGTGTACTATTGCGCCCGGCTGTACAGATGGTATTTTGACGTGTGGGGAGCAGGAACCACAGTGACCGTGTCTAGC(SEQ ID NO：19)

QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWIGIIDPSDSETSYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSS(SEQ ID NO：20)

6F7L3：

AACATCGTGATGACCCAGTCCCCAGCCACACTGTCTCTGAGCCCAGGAGAGAGGGTGACCCTGTCCTGTAGAGCCTCTGAGATCGTGGGCACATACCTGTCTTGGTATCAGCAGAAGCCAGGACAGGCACCTAGGCTGCTGATCTACGGAGCCAGCACCAGGGCAACAGGCATCCCCGCACGCTTCTCCGGCTCTGGCAGCGGCACCGACTTTACCCTGACAATCAGCTCCCTGCAGCCTGAGGATTTTGCCGTGTACTATTGCGGCCAGTCTTACAATTTCCCATATACCTTTGGCGGCGGCACAAAGCTGGAGATCAAG(SEQ ID NO：21)

NIVMTQSPATLSLSPGERVTLSCRASEIVGTYLSWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCGQSYNFPYTFGGGTKLEIK(SEQ ID NO：22)

6F7 heavy chain framework region

FR-H1：QVQLQQPGAELVRPGASVKLSCKAS(SEQ ID NO：23)

FR-H2：MNWVKQRPGQGLEWIGM(SEQ ID NO：24)

FR-H3：HNNQMFKDKATLTVDKSSNTAYMHLSSLTSEDSAVYHC(SEQ ID NO：25)

FR-H4：WGAGTTVTVSS(SEQ ID NO：26)

6F7 light chain framework region

FR-L1：NIVMTQSPKSMSMSLGERVTLSCKAS(SEQ ID NO：27)

FR-L2：VSWFQQKPHQSPKLLIY(SEQ ID NO：28)

FR-L3：NRYTGVPDRFTGSGSATDFTLTISNVQAEDLADYHC(SEQ ID NO：29)

FR-L4：FGGGTKLEIK(SEQ ID NO：30)

6F7H1 framework region

FR-H1：QVQLVQSGAEVVKPGASVKLSCKAS(SEQ ID NO：31)

FR-H2：MNWVRQRPGQGLEWIGM(SEQ ID NO：32)

FR-H3：HNAQKFQGKATLTVDKSTSTAYMHLSSLRSEDTAVYYC(SEQ ID NO：33)

FR-H4：WGAGTTVTVSS(SEQ ID NO：34)

6F7L1 framework region

FR-L1：NIVMTQSPATMSMSPGERVTLSCRAS(SEQ ID NO：35)

FR-L2：VSWFQQKPGQAPRLLIY(SEQ ID NO：36)

FR-L3：NRYTGVPARFSGSGSGTDFTLTISSVQPEDLADYHC(SEQ ID NO：37)

FR-L4：FGGGTKLEIK(SEQ ID NO：38)

6F7H2 framework region

FR-H1：QVQLVQSGAEVVKPGASVKVSCKAS(SEQ ID NO：39)

FR-H2：MNWVRQRPGQGLEWIGI(SEQ ID NO：40)

FR-H3：SNAQKFQGRVTLTVDKSTSTAYMHLSSLRSEDTAVYYC(SEQ ID NO：41)

FR-H4：WGAGTTVTVSS(SEQ ID NO：42)

6F7L2 framework region

FR-L1：NIVMTQSPATLSLSPGERVTLSCRAS(SEQ ID NO：43)

FR-L2：VSWFQQKPGQAPRLLIY(SEQ ID NO：44)

FR-L3：NRATGIPARFSGSGSGTDFTLTISSLQPEDLADYYC(SEQ ID NO：45)

FR-L4：FGGGTKLEIK(SEQ ID NO：46)

6F7H3 framework region

FR-H1：QVQLVQSGAEVVKPGASVKVSCKAS(SEQ ID NO：47)

FR-H2：MNWVRQAPGQGLEWIGI(SEQ ID NO：48)

FR-H3：SYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYC(SEQ ID NO：49)

FR-H4：WGAGTTVTVSS(SEQ ID NO：50)

6F7L3 framework region

FR-L1：NIVMTQSPATLSLSPGERVTLSCRAS(SEQ ID NO：51)

FR-L2：LSWYQQKPGQAPRLLIY(SEQ ID NO：52)

FR-L3：TRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYC(SEQ ID NO：53)

FR-L4：FGGGTKLEIK(SEQ ID NO：54)

IgG1M heavy chain constant region

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：55)

Heavy chain constant region Ig gamma-4chain C region

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：56)

Light chain constant region Ig kappa chain C region

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：57)

Heavy chain constant region Ig gamma-1chain Cregion

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：58)

Amino acid sequence of heavy chain of 6F7H1L1(G1M)

QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMNWVRQRPGQGLEWIGMIDPSDSETHNAQKFQGKATLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：59)

Amino acid sequence of 6F7H1L1(G1M) light chain

NIVMTQSPATMSMSPGERVTLSCRASEIVGTYVSWFQQKPGQAPRLLIYGASNRYTGVPARFSGSGSGTDFTLTISSVQPEDLADYHCGQSYNFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：60)

Amino acid sequence of heavy chain of 6F7H2L2(G1M)

QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYWMNWVRQRPGQGLEWIGIIDPSDSETSNAQKFQGRVTLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：61)

Amino acid sequence of 6F7H2L2(GlM) light chain

NIVMTQSPATLSLSPGERVTLSCRASEIVGTYVSWFQQKPGQAPRLLIYGASNRATGIPARFSGSGSGTDFTLTISSLQPEDLADYYCGQSYNFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：62)

Amino acid sequence of heavy chain of 6F7H3L3(GlM)

QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWIGIIDPSDSETSYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：63)

Amino acid sequence of 6F7H3L3(G1M) light chain

NIVMTQSPATLSLSPGERVTLSCRASEIVGTYLSWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCGQSYNFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：64)

Amino acid sequence of heavy chain of 6F7H1L1(hG4)

QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMNWVRQRPGQGLEWIGMIDPSDSETHNAQKFQGKATLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：65)

Amino acid sequence of 6F7H1L1(hG4) light chain

Amino acid sequence of heavy chain of 6F7H2L2(hG4)

QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYWMNWVRQRPGQGLEWIGIIDPSDSETSNAQKFQGRVTLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYToKSLSLSLGK(SEQ ID NO：67)

Amino acid sequence of 6F7H2L2(hG4) light chain

Amino acid sequence of heavy chain of 6F7H3L3(hG4)

QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWIGIIDPSDSETSYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：69)

Amino acid sequence of 6F7H3L3(hG4) light chain

Sequence of the mFc tag: (SEQ ID NO: 71)

PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

heavy chain sequence of hIgG (SEQ ID NO: 72)

EVQLEQSGAELMKPGASVKISCKATGYTFTTYWIEWIKQRPGHSLEWIGEILPGSDSTYYNEKVKGKVTFTADASSNTAYMQLSSLTSEDSAVYYCARGDGFYVYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Light chain sequence of hIgG (SEQ ID NO: 73)

DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYTSQSMSGIPSRFSGSGSGTDFTLSINSVETEDFGVYFCQQSGSWPRTFGGGTKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The TEV amino acid sequence is ENLYFQG, SEQ ID NO: 74

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